RU2520808C2 - Method of identifying improved protein versions - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a method of identifying improved protein versions. The method includes testing a multitude of protein versions with single substitutions in the first test of the first property and in the second test of the second property. The parent protein property is given a value 1.0 in each test. The favourable first or second property possesses a value, exceeding 1.0, and extremely unfavourable first or second property has a value less than approximately 0.80. The said parent protein represents protease. The first property is stability, the second property is washing efficiency. Identification of substitution and introduction of the substitution into the protein to obtain the protein with multiple substitutions are carried out. The substitution does not interact in a three-dimensional structure of the said parent protein, and one of the substitutions contains a change of the total charge, equal to 0, -1 or -2 with respect to the parent protein, and, at least, one of the substitutions contains the change of the total charge, equal to +1 or +2 with respect to the parent protein. The protein version with multiple changes in the first and second tests is tested and the said protein version is identified.

EFFECT: invention makes it possible to obtain the protein version with improved stability and washing efficiency.

14 cl, 6 dwg, 6 tbl, 7 ex

Description

Cross references to related applications

This application claims the priority of provisional US patent applications Nos. 60/933307, 60/933331 and 60/933312, filed June 6, 2007, included, therefore, by reference in full.

Field of Invention

The present invention relates to methods for constructing proteins in order to optimize their effectiveness under certain environmental conditions of interest. In some embodiments, the present invention relates to methods for constructing enzymes to optimize their catalytic activity under specific environmental conditions. In some preferred embodiments, the present invention relates to methods for constructing enzymes to optimize their catalytic activity and / or stability under adverse environmental conditions. In some preferred embodiments, the present invention relates to methods for constructing enzymes to optimize their storage stability, particularly under adverse environmental conditions. In some preferred embodiments, the present invention relates to methods for changing the total surface charge and / or distribution of surface charges of enzymes (e.g. metalloproteases) to produce enzyme variants that exhibit improved efficiency and / or stability in detergent compositions compared to the initial or parent enzyme .

BACKGROUND OF THE INVENTION

The properties of proteins that function outside their natural environment are often suboptimal. For example, enzymes (e.g., proteases, lipases, amylases, cellulases, etc.) are often used to clean matter from paint in laundry detergents, which typically include a complex combination of active ingredients. In fact, most cleaning products contain a surfactant system, bleaches, detergents, soap foam dampers, dirt-suspending components, dirt-repellent components, optical brighteners, softeners, dispersants, compounds that inhibit dye transfer, abrasives, bactericides and perfumes, as well as enzymes for the cleaning. Thus, despite the complexity of modern detergents, there are many paints that are completely difficult to remove, partly due to the suboptimal efficiency of the enzyme. Despite many studies aimed at the development of enzymes, in this area there remains a need for methods of creating proteins for specific applications and conditions. In fact, there remains a need in the art for methods for quickly and systematically adapting the electrostatic properties of others in order to optimize their effectiveness in commercial applications. In particular, there remains a need in the art for methods for creating industrially suitable enzymes, including but not limited to lipase, amylase, cutinase, mannanase, oxidoreductase, cellulase, pectinase, protease and other enzymes, in order to provide improved activity, stability and solubility in cleaning solutions.

SUMMARY OF THE INVENTION

The present invention relates to methods for constructing proteins in order to optimize their effectiveness under specific environmental conditions of interest. In some embodiments, the present invention relates to methods for constructing enzymes to optimize their catalytic activity under specific environmental conditions. In some preferred embodiments, the present invention relates to methods for constructing enzymes to optimize their catalytic activity and / or stability under adverse environmental conditions. In some preferred embodiments, the present invention relates to methods for constructing enzymes to optimize their storage stability, particularly under adverse environmental conditions. In some preferred embodiments, the present invention relates to methods for changing the total surface charge and / or distribution of the surface charges of enzymes (e.g. metalloproteases) in order to obtain enzyme variants that exhibit improved efficiency and / or stability in detergent compositions compared to the initial or parent an enzyme.

The present invention relates to methods for producing improved protein variants, comprising: testing the first property of a plurality of protein variants with single substitutions in the first test and the second property in the second test, where the property of the parent protein is a predetermined value of 1.0, in each test, favorable the first or second property has a value greater than 1.0, and the excessively unfavorable first or second property has a value of less than about 0.80, or in some preferred Rianta embodiment, less than about 0.60; identifying a substitution in at least one protein variant with single substitutions that is associated with a favorable first property and which is not associated with an excessively unfavorable second property; identifying a substitution in at least one protein variant with single substitutions that is associated with a favorable second property and which is not associated with an overly unfavorable first property; introducing a substitution from a previous step into a protein to produce a multiple-protein variant. In some embodiments, the methods further comprise testing a protein variant with multiple substitutions in a first test and a second test, wherein the improved protein variant reaches a value in excess of 1.0 in both the first and second specified tests, or a value in excess of 1.0 , in the first test, and values from 0.80 to 1.0 in the second test. In some further embodiments, the methods further comprise producing an improved variant of the protein (s). In some embodiments, the first and second properties are negatively correlated. In some further embodiments, the favorable first or second property has a value in excess of about 1.2. In some additional embodiments, the overly unfavorable first or second property has a value of approximately less than 0.40. In some preferred embodiments, the first property is stability, and the second property is washing efficiency. In some particularly preferred embodiments, stability includes stability in the detergent and washing efficiency includes the efficiency of washing off blood-milk-ink (CMP) in the detergent. In some further preferred embodiments, the protein is a neutral metalloprotease. In some additional embodiments, the parent protein is a mature form of a wild-type neutral metalloprotease, while in other embodiments, the variant is derived from a neutral metalloprotease from the Bacillaceae family. In some particularly preferred embodiments, the embodiment is derived from a neutral metalloprotease of the genus Bacillus . In still further embodiments, the washing efficiency is tested in a powder or liquid detergent composition having a pH between 5 and 12.0. In some further embodiments, the washing efficiency is tested in cold water with a liquid detergent having a basic pH. In still further embodiments, at least one of the substitutions comprises a change in the total charge equal to 0, -1, or -2 with respect to the parent neutral metalloprotease, while in some alternative embodiments, at least one of the substitutions contains a change total charge equal to +1 or +2 with respect to the parent neutral metalloprotease. It is not intended that the steps be limited to the exact order listed above, since any suitable order finds use in the present invention. In some preferred embodiments, the improved protease variant has a total charge change of +1 or +2 with respect to the parent neutral metalloprotease. In still further embodiments, the substitutions are at positions in the parent neutral metalloprotease having a solvent accessible surface (DRP) of approximately greater than 50%. In other further embodiments, one or more positions in the parent neutral metalloprotease are positions having a solvent accessible surface (DRP) of greater than about 65%.

The present invention relates to methods for producing improved protease variants, comprising: testing the first property of a plurality of protease variants with single substitutions in the first test and the second property in the second test, where the property of the parent protease is a predetermined value of 1.0, in each test, favorable the first or second property has a value in excess of 1.0, and the excessively unfavorable first or second property has a value of less than about 0.80, or in some preferences nnny options for implementation of approximately less than 0.60; identifying a substitution in at least one protease variant with single substitutions that are associated with a favorable first property and which are not associated with an excessively unfavorable second property; identifying a substitution in at least one protease variant with single substitutions that are associated with a favorable second property and which are not associated with an overly unfavorable first property; introducing a substitution from a previous step into a protease to obtain a protease variant with multiple substitutions. In some embodiments, the methods further comprise testing a protease variant with multiple substitutions in the first test and second test, where the improved protease variant reaches a value in excess of 1.0 in both the first and second tests, or a value in excess of 1.0, in the first test, and values from 0.80 to 1.0 in the second test. In some further embodiments, the methods further comprise producing an improved variant of the protease (h). In some embodiments, the first and second properties are negatively correlated. In some further embodiments, the favorable first or second property has a value of approximately greater than 1.2. In some additional embodiments, the overly unfavorable first or second property has a value of approximately less than 0.40. In some preferred embodiments, the first property is stability, and the second property is washing efficiency. In some particularly preferred embodiments, the stability comprises stability in the detergent and the washing efficiency comprises the efficiency of washing off blood-milk-ink (CMP) in the detergent. In some further preferred embodiments, the protease is a neutral metalloprotease. In some additional embodiments, the parent protease is a mature form of a wild-type neutral metalloprotease, while in other embodiments, the variant is derived from a neutral metalloprotease of the Bacillaceae family. In some particularly preferred embodiments, the embodiment is derived from a neutral metalloprotease of the genus Bacillus . In still further embodiments, the washing efficiency is tested in a powder or liquid detergent composition having a pH between 5 and 12.0. In some further embodiments, the washing efficiency is tested in cold water with a liquid detergent having a basic pH. In still further embodiments, at least one of the substitutions comprises a change in the total charge equal to 0, -1, or -2 with respect to the parent neutral metalloprotease, while in some alternative embodiments, at least one of the substitutions contains a change total charge equal to +1 or +2 with respect to the parent neutral metalloprotease. It is not intended that the steps be limited to the exact order listed above, since any suitable order finds use in the present invention. In some preferred embodiments, the improved protease variant has a total charge change of +1 or +2 with respect to the parent neutral metalloprotease. In still further embodiments, the substitutions are at positions in the parent neutral metalloprotease having a solvent accessible surface (DRP) of approximately greater than 50%. In other further embodiments, one or more positions in the parent neutral metalloprotease are positions having a solvent accessible surface (DRP) of greater than about 65%. The present invention also relates to proteins with multiple substitutions made using the methods described herein. In some preferred embodiments, the present invention relates to neutral metalloprotease variants produced by the method described herein. In some particularly preferred embodiments, the present invention provides protease variants comprising a substitution at the residue position corresponding to the residue position 83 of the Bacillus neutral metalloprotease shown as SEQ ID NO: 3. In some further preferred embodiments, the replacement comprises an L83K replacement. NprE variants are also provided containing combinations of substitutions selected from the group consisting of: i) 4K-45K-50R-54K-59K-90K-129I-138L-179P-190L-199E-214Q-220E-244S-265P-269H- 285R-296E; 45K-50R-59K-90K-1291-138L-179P-190L-199E-214Q-220E-244S-265P-285R; 45K-59K-90K-129I-138L-179P-190L-199E-214Q-220E-265P-285R; and 59K-90K-129I-179P-190L-199E-214Q-220E-265P-285R.

The present invention also relates to isolated polynucleotides encoding the protease variants set forth herein. In addition, the present invention relates to expression vectors containing the polynucleotide set forth herein, in functional combination with a promoter. The present invention also provides host cells transformed with the expression vector (s) provided herein. The present invention also relates to cleaning compositions containing protease variants produced by the methods of the present invention.

The present invention relates to methods for producing improved protein variants, comprising: a) testing a plurality of protein variants with single substitutions in the first test of the first property and the second property in the second test, where the property of the parent protein is a predetermined value of 1.0 in each test , a favorable first or second property has a value in excess of 1.0, and an excessively unfavorable first or second property has a value of less than approximately 0.80; b) identification of a substitution in at least one variant protein with single substitutions that is associated with a favorable first property and which is not associated with an excessively unfavorable second property; c) identifying a substitution in at least one variant protein with single substitutions that is associated with a favorable second property and which is not associated with an overly unfavorable first property; and d) introducing a substitution from step b) and a substitution from step c) into a protein to obtain a variant protein with multiple substitutions. In some embodiments, the methods further comprise the step of e) testing a protein variant with multiple substitutions in the first test and second test, where the improved protein variant reaches a value in excess of 1.0 in both the first and second tests, or in excess of 1 , 0 in the first test, and values from 0.80 to 1.0 in the second test.

In some embodiments, the protein is an enzyme selected from the group consisting of protease, amylase, cellulase, polyesterase, esterase, lipase, cutinase, pectinase, oxidase, transferase, catalase and alkalase. In some preferred embodiments, the enzyme is a protease or amylase. In some embodiments, the first and second properties of interest comprise two or more properties selected from the group consisting of substrate binding, enzyme inhibition, expression, detergent stability, thermal stability; reaction rates; degree of completion of the reaction; thermal activity; starch thinners; decomposition of biomass, saccharification, hydrolysis of ether, enzymatic bleaching, washing efficiency and textile modification. In some particularly preferred embodiments, the methods further comprise producing an improved variant of the protein. In some embodiments, the first and second properties are negatively correlated. In some embodiments, the beneficial first or second property has a value of approximately greater than 1.2, and / or the excessively unfavorable first or second property has a value of less than approximately 0.60. In some preferred embodiments, the first property is stability, and the second property is washing efficiency. In a subset of these embodiments, stability comprises stability in the detergent, and washing efficiency comprises the efficiency of washing off blood-milk-ink (CMP) in the detergent. In some embodiments, the first property is protein expression, and the second property is enzymatic activity. In a subset of these embodiments, the enzymatic activity comprises the washing efficiency of rice starch in the detergent. In some preferred embodiments, the protease is selected from the group consisting of neutral metalloprotease, serine protease, and subtilisin. In a subset of these embodiments, the neutral metalloprotease is a neutral metalloprotease of the Bacillaceae family. In certain exemplary embodiments, a neutral metalloprotease from the genus Bacillus (e.g., B. subtilis NprE). In further embodiments, the amylase is α-amylase of the Bacillaceae family. In exemplary embodiments, α-amylase from the genus Bacillus (e.g., AmyS B. stearothermophilus ). In some preferred embodiments, the washing efficiency is tested in a powder or liquid detergent composition having a pH between 5 and 12.0 and / or cold water with a liquid detergent having a basic pH. In some embodiments, the implementation of at least one of the substitutions comprises a change in the total charge equal to 0, -1, or -2 with respect to the parent enzyme. In some embodiments, the implementation of at least one of the substitutions comprises a change in the total charge equal to +1 or +2 with respect to the parent enzyme. In some preferred embodiments, the implementation of at least one of the substitutions comprises at least two substitutions, the first with a change in the total charge equal to 0, -1, or -2 with respect to the parent enzyme; and the second with a change in the total charge equal to +1 or +2 with respect to the parent enzyme. In some embodiments, the implementation of at least one replacement contains from one to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19 or 20) substitutions. In some embodiments, an improved variant of the enzyme has a change in total charge of -2, -1, 0, +1, or +2 with respect to the parent enzyme. In some embodiments, the substitutions are at positions in the parent enzyme having a solvent accessible surface (DRP) of about more than 25%, about more than 50%, or about more than 65%. The present invention also provides isolated polynucleotides encoding the enzyme variants set forth herein. In further embodiments, the present invention relates to an expression vector comprising a polynucleotide in functional combination with a promoter. In some embodiments, host cells comprising an expression vector are provided. In further embodiments, cleaning compositions are provided that comprise enzyme variants produced using the methods of the present invention.

The present invention relates to methods for producing improved protein variants, comprising, in functional order: a) testing the first property of a plurality of protein variants with single substitutions in the first test and the second property in the second test, where the property of the parent protein is a predetermined value of 1.0, in each test, a favorable first or second property has a value in excess of 1.0, and an excessively unfavorable first or second property has a value of less than approximately 0.80; b) identification of a substitution in at least one variant protein with single substitutions that is associated with a favorable first property and which is not associated with an excessively unfavorable second property; c) identification of a substitution in at least one variant protein with single substitutions that is associated with a favorable second property and which is not associated with an overly unfavorable first property; and d) introducing a substitution from step b) and a substitution from step c) into a protein to obtain a multiple-substitution protein variant, wherein the multiple-substitution protein variant is an improved protein variant. In some preferred embodiments, the methods further comprise: step e) testing a protein variant with multiple substitutions in the first test and second test, wherein the improved protein variant reaches a value greater than 1.0 in both the first and second tests, or exceeding 1.0 in the first test, and values from 0.80 to 1.0 in the second test. In some further embodiments, the parent protein is an enzyme, wherein the improved protein variants are enzymes. In some further embodiments, the enzyme is selected from proteases, amylases, cellulases, polyesterases, esterases, lipases, cutinases, pectinases, oxidases, transferases and catalases. In some particularly preferred embodiments, the enzyme is a protease or amylase. In some additional embodiments, the first and second properties of interest comprise two or more of the group consisting of substrate binding, enzyme inhibition, expression, detergent stability, thermal stability, reaction rate, degree of completion of the reaction, thermal activity, liquefaction of starch, decomposition of biomass , saccharification, ether hydrolysis, enzymatic bleaching, washing efficiency and textile modification. In some other further embodiments, the methods further comprise the step of producing an improved variant of the protein. In some further embodiments, the first and second properties are negatively correlated. In some particularly preferred embodiments, the favorable first or second property has a value of approximately greater than 1.2. In some additional preferred embodiments, the overly unfavorable first or second property has a value of approximately less than 0.60. In some other additional embodiments, the overly unfavorable first or second property has a value of approximately less than 0.40. In some preferred embodiments, the first property is stability, and the second property is washing efficiency. In some particularly preferred embodiments, the stability comprises stability in detergent formulations and the washing efficiency comprises the efficiency of washing off blood-milk-ink (CMP). In some other additional embodiments, the washing efficiency is tested in a powder or liquid detergent composition having a pH between about 5 and about 12. In some additional preferred embodiments, the washing efficiency is tested in cold water with a liquid detergent having a basic pH. In some further embodiments, the first property is protein expression, and the second property is enzymatic activity. In some further embodiments, the protease is selected from neutral metalloproteases and serine proteases. In some particularly preferred embodiments, the serine protease is subtilisin. In some further embodiments, the neutral metalloprotease is a neutral metalloprotease derived from a member of the Bacillaceae family. In some alternative embodiments, the amylase is an α-amylase derived from a member of the Bacillaceae family. In some additional embodiments, the implementation of at least one of the substitutions comprises a change in the total charge equal to 0, -1, or -2 relative to the parent enzyme. In some alternative embodiments, the implementation of at least one of the substitutions comprises a change in the total charge equal to +1 or +2 with respect to the parent enzyme. In some other additional embodiments, the implementation of at least one of the substitutions comprises a change in the total charge equal to 0, -1, or -2 relative to the parent enzyme. In some additional alternative embodiments, the implementation of at least one of the substitutions comprises a change in the total charge equal to +1 or +2 relative to the parent enzyme. In some other additional embodiments, the implementation, an improved version of the enzyme has a change in the total charge equal to +1 or +2 relative to the parent enzyme. In some alternative embodiments, the substitutions are at positions in the parent enzyme having a solvent accessible surface (DRP) of greater than about 25%. In some preferred embodiments, the substitutions are at positions in the parent enzyme having a solvent accessible surface (DRP) of more than about 50%, about more than 65%. In some particularly preferred embodiments, the parent enzyme is a wild-type enzyme.

The present invention also relates to cleaning compositions containing an improved version of the protein produced in accordance with the methods described herein.

The present invention also relates to isolated neutral metalloprotease variants having an amino acid sequence containing at least one amino acid substitution made at a position equivalent to that of a neutral metalloprotease containing the amino acid sequence set forth in SEQ ID NO: 3. In some preferred embodiments, the implementation of at least one substitution is made at a position equivalent to position 83 of the amino acid sequence set forth in SEQ ID NO: 3. In some particularly preferred embodiments, the substitution is L83K.

Brief Description of the Drawings

On figa shows the relative specific hydrolytic activity for BODIPY-starch with respect to the expression of the combinatorial charge library (KZB) AmyS-S242Q in shake tubes.

FIG. 1B shows the relative purification activity on rice starch microsamples in TIDE 2 × with respect to the expression of AmyS-S242Q KZB in shake tubes.

FIG. 2A shows the relative expression in shake tubes relative to the relative change in total charge for the AmyS-S242Q KZB.

2B shows the relative hydrolytic activity for BODIPY starch with respect to the relative change in total charge for the AmyS-S242Q KZB.

FIG. 3A shows the relative expression in shake tubes relative to the relative change in total charge for the AmyS-S242Q KZB.

FIG. 3B shows the purification activity of micro starch with rice starch with respect to the relative change in total charge for the AmyS-S242Q KZB.

General Description of the Invention

The present invention relates to methods for constructing proteins in order to optimize their effectiveness under specific environmental conditions of interest. In some embodiments, the present invention relates to methods for constructing enzymes to optimize their catalytic activity under specific environmental conditions. In some preferred embodiments, the present invention relates to methods for constructing enzymes to optimize their catalytic activity and / or stability under adverse environmental conditions. In some preferred embodiments, the present invention relates to methods for constructing enzymes to optimize their storage stability, particularly under adverse environmental conditions. In some preferred embodiments, the present invention relates to methods for changing the total surface charge and / or distribution of the surface charges of enzymes (e.g. metalloproteases) in order to obtain enzyme variants that exhibit improved efficiency and / or stability in detergent compositions compared to the initial or parent an enzyme.

Protease subtilisin is the main enzyme used in laundry detergents and possibly the most widely used enzyme in the world. It has been noted that surface electrostatic effects can modulate the catalytic activity of subtilisin (see, for example, Russell and Fersht, Nature 328: 496-500 [1987]). More recently, mutations have been discovered that are involved in changing the total charge of subtilisin and have a strong influence on the washing efficiency in detergents (see, for example, EP Patent No. 0479870 B1). It was believed that this positive effect is the result of a shift in pI (isoelectric point) of subtilisin towards the pH of the washing solution. However, recent work has shown that this conclusion is not always applicable (see, for example, US patent No. 6673590 B1). As indicated in this patent, the effect of charge mutations in subtilisin is highly dependent on the concentration of detergent, with mutations that reduce the pI of the parent subtilisin provide an enzyme that is more effective at a low concentration of detergent, and mutations that increase the pI provide an enzyme that is more effective at a high detergent concentration. This has significant benefits, as the concentration of detergent in detergent solutions varies significantly on a global scale. Thus, it will be apparent to those skilled in the art that there is an optimal pI of subtilisin for effective washing, which depends on pH and the concentration of detergent in the washing solution. Further attempts to increase subtilisin activity in laundry detergents have been described (see US Patent Application No. 2005/0221461). Surprisingly, it was found that subtilisin variants having the same total electrostatic charge as the parent subtilisin have increased washing efficiency under washing conditions with both high and low detergent concentrations.

Unless otherwise indicated, practicing the present invention involves conventional methods that are commonly used in the construction of proteins, molecular biology, microbiology, and recombinant DNA, which are known to those skilled in the art. Such methods are known to those skilled in the art and are described in a variety of texts and reference works well known to those skilled in the art. All patents, patent applications, articles, and publications noted herein above and below are hereby expressly incorporated herein by reference because they contain relevant information regarding the methods and compositions provided by the present invention.

Unless otherwise stated herein, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to the methods and materials described herein find use in practicing the present invention, some preferred methods and materials are described herein. Thus, the terms that are defined immediately below are described more fully in relation to the description as a whole.

As used herein, the singular includes the plural until the contrary is clearly indicated in the context. Numeric ranges include numbers that define this range. Unless otherwise indicated, nucleic acids are written from left to right in the direction from 5 ′ to 3 ′; amino acid sequences are written from left to right in the direction from the amino to the carboxy terminus, respectively. It should be understood that this invention is not limited to the specific methods, protocols, and reagents described, as they may vary depending on the context in which they are used by those skilled in the art.

It is intended that each maximum numerical limitation set throughout this description includes each lower numerical limitation, as if such lower numerical limitations were expressly written herein. Each minimum numerical limitation set throughout this description will include each upper numerical limitation, as if such upper numerical limitations were expressly written herein. Each numerical range defined throughout this description will include each narrower numerical range that falls within such a wider numerical range, as if all such narrower numerical ranges were expressly written herein.

In addition, the headers provided herein are not limiting of the various aspects or embodiments of the invention that may occur in relation to the entire description. Thus, the terms defined directly below are more fully defined in relation to the description as a whole. However, in order to facilitate understanding of the invention, a certain number of terms are defined below.

Definitions

As used herein, the terms “protease” and “proteolytic activity” refer to a protein or peptide exhibiting the ability to hydrolyze peptides or substrates having peptide bonds. There are many well-known procedures for measuring proteolytic activity (see, for example, Kalisz, Microbial Proteases, In: Fiechter (ed.), Advances in Biochemical Engineering / Biotechnology [1988]). For example, proteolytic activity can be established using comparative assays that analyze the ability of the corresponding protease to hydrolyze a commercial substrate. Exemplary substrates suitable for such a protease or proteolytic activity assay include, but are not limited to, dimethyl casein (Sigma C-9801), bovine collagen (Sigma C-9879), bovine elastin (Sigma E-1625), and bovine keratin (ICN Biomedical 902111) . Colorimetric tests using these substrates are well known in the art (see, for example, WO 99/3401 1; and US Patent No. 6376450). The pNA assay (see, for example, Del Mar et al, Anal. Biochem, 99: 316-320 [1979]) also find use in determining the active concentration of an enzyme for fractions collected during gradient elution. This assay measures the rate at which p-nitroaniline is released by enzyme hydrolysis of a soluble synthetic substrate, succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide (sAAPF-pNA). The rate of development of yellow color as a result of the hydrolysis reaction is measured at 410 nm on a spectrophotometer and it is proportional to the active concentration of the enzyme. In addition, the measurement of optical density at 280 nm can be used to determine the total protein concentration. The ratio of active enzyme to total protein gives the purity of the enzyme.

As used herein, the terms “ASP protease”, “ASP protease” and “ASP” refer to the serine proteases described herein and described in US Patent Serial No. 10/576331). In some preferred embodiments, the ASP protease is a protease designated herein as 69B4 protease derived from Cellulomonas strain 69B4 . Thus, in preferred embodiments, the term “protease 69B4” refers to a naturally occurring mature protease derived from Cellulomonas strain 69B4 (DSM 16035). In alternative embodiments, the present invention provides ASP protease fragments.

The term “Cellulomonas protease homologs” refers to naturally occurring proteases having amino acid sequences substantially identical to the mature protease derived from Cellulomonas strain 69B4 , or the polynucleotide sequence that encodes such naturally occurring proteases, and these proteases retain the functional characteristics of the serine protease encoded by such nucleic acids. In some embodiments, these protease homologues are referred to as "cellulomonadins."

As used herein, the terms “ASP variant”, “ASP protease variant” and “Protease 69B variant” are used with respect to proteases that are similar to wild-type ASPs, in particular their function, but have mutations in their amino acid sequence that distinguish their sequence from wild-type protease.

As used herein, “ Cellulomonas ssp.” Refers to all species of the “ Cellulomonas ” genus, which are gram-positive bacteria systematized as members of the Cellulomonadaceae family, Micrococcineae suborder, Actinomycetales order, Actinobacteria class. It was established that the genus Cellulomonas continues to undergo taxonomic reorganization. Thus, it is assumed that the genus includes species that have been re-systematized.

As used herein, «Streptomyces ssp.» Refers to all species of the genus «Streptomyces», which are Gram-positive bacteria, systematized as members of the family Streptomycetaceae, suborder Streptomycineae, the order Actinomycetales, class Actinobacteria. It was established that the genus Streptomyces continues to undergo taxonomic reorganization. Thus, it is assumed that the genus includes species that have been re-systematized.

As used herein, the "genus Bacillus " includes all species of the genus " Bacillus " known to those skilled in the art, including but not limited to B. subtilis , B. licheniformis , B. lentus , B. brevis , B. stearothermophilus , B . alkalophilus , B. amyloliquefaciens, B. clausii , B. halodurans , B. megaterium , B. coagulans , B. circulans , B. lautus and B. thuringiensis . It has been established that the genus Bacillus continues to undergo taxonomic reorganization. Thus, it is assumed that the genus includes species that have been re-systematized, including but not limited to organisms such as B. stearothermophilus , now called “Geo bacillus stearothermophilus ”. Sustainable production of endospores in the presence of oxygen considered the defining feature of the genus Bacillus, although this characteristic also applies to the recently named Alley clobacillus, Amphibacillus, Aneurinibacillus, Anoxybacillus , Brevibacillus, Filobacillus, Gracilibacillus, Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus and Virgibacillus.

The terms “polynucleotide” and “nucleic acid” used interchangeably herein refer to the polymeric form of nucleotides of any length, or ribonucleotides, or deoxyribonucleotides. These terms include, but are not limited to, single, double, or triple stranded DNA, genomic DNA, cDNA, RNA, DNA-RNA hybrids or a polymer containing purine and pyrimidine bases, or other natural, chemically, biochemically modified, non-natural nucleotide bases or their derivatives. Non-limiting examples of polynucleotides are provided: genes, gene fragments, chromosome fragments, EST, exons, introns, mRNAs, tRNAs, rRNAs, ribozymes, cDNAs, recombinant polynucleotides, branched polynucleotides, plasmids, vectors isolated with any DNA sequence, isolated with any RNA sequence from , nucleic acid probes and primers. In some embodiments, polynucleotides comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracil, other sugars and linking groups, such as fluorribose and thioate, and branching nucleotides. In alternative embodiments, the nucleotide sequence is interrupted by non-nucleotide components.

As used herein, the terms “DNA construct” and “transforming DNA” are used interchangeably with respect to DNA used to introduce sequences into a host cell or organism. DNA can be created in vitro by PCR or any other suitable method (s) known for this purpose in this field. In particularly preferred embodiments, the DNA construct contains the sequence of interest (for example, as an input sequence). In some embodiments, the sequence is operably linked to additional elements, such as control elements (e.g., promoters, etc.). The DNA construct may further comprise a selective marker. It may further comprise an input sequence flanked by homologous fragments. In a further embodiment, the transforming DNA contains other non-homologous sequences added at the ends (e.g., spacer sequences or flanking sequences). In some embodiments, the ends of the introduced sequence are closed so that the transforming DNA forms a closed ring. Transforming sequences may be wild-type, mutant, or modified. In some embodiments, the DNA construct contains sequences homologous to the chromosome of the host cell. In other embodiments, the DNA construct comprises non-homologous sequences. When the DNA construct is assembled in vitro , it can be used to: 1) insert heterologous sequences into the desired target sequence of the host cell; and / or 2) mutating the region of the chromosome of the host cell (i.e., replacing the endogenous sequence with a heterologous sequence) and / or 3) removing the targeted genes; and / or introducing a replicating plasmid into the host organism.

As used herein, the terms “expression cassette” and “expression vector” refer to a nucleic acid construct created recombinantly or synthetically, with a number of specific nucleic acid elements that allow transcription of a particular nucleic acid in a target cell. A recombinant expression cassette can be inserted into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus or nucleic acid fragment. Typically, a portion of an expression vector with a recombinant expression cassette contains, among other sequences, a nucleic acid sequence to be transcribed and promoters. In preferred embodiments, expression vectors are capable of incorporating and expressing heterologous DNA fragments in a host cell. Many prokaryotic and eukaryotic expression vectors are commercially available. The selection of suitable expression vectors is the responsibility of specialists in this field. The term “expression cassette” is used interchangeably herein with a “DNA construct” and its grammatical equivalents. The selection of suitable expression vectors is the responsibility of specialists in this field.

As used herein, the term “vector” refers to a polynucleotide construct designed to introduce nucleic acids into one or more types of cells. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, cassettes, and the like. In some embodiments, the polynucleotide construct comprises a DNA sequence encoding a protease (e.g., a precursor or mature protease) that is operably linked to a suitable pro-sequence (e.g., secretory, etc.) that allows for the expression of DNA in a suitable host organism.

As used herein, the term "plasmid" refers to a circular double-stranded (dc) DNA construct used as a cloning vector that forms an extrachromosomal self-replicating genetic element in some eukaryotes and prokaryotes, or integrates into the host chromosome.

As used herein in the context of introducing a nucleic acid sequence into a cell, the term “introduced” refers to any method suitable for transferring a nucleic acid sequence to a cell. Such methods for administration include, but are not limited to, protoplast fusion, transfection, transformation, conjugation, and transduction (see, for example, Ferrari et al., Genetics, in Hardwood et al., (Eds.), Bacillus . Plenum Publishing Corp. ., pp. 57-72 [1989]).

As used herein, the terms “transformed” and “stably transformed” refer to a cell that has a negative (heterologous) polynucleotide sequence integrated into its genome or as an episomal plasmid that lasts for at least two generations.

As used herein, the term “nucleotide sequence encoding a selectable marker” refers to a nucleotide sequence that allows expression in host cells and where the expression of the selectable marker gives cells containing the expressed gene the ability to grow in the presence of an appropriate selective agent or when lack of essential nutrient.

As used herein, the terms “selective marker” and “permanent selective marker” refer to a nucleic acid (eg, a gene) that allows expression in a host cell, which makes it easy to select those host organisms that contain the vector. Examples of such selective markers include, but are not limited to, antimicrobial agents. Thus, the term "selective marker" refers to genes that provide an indication that the host cell has captured the incoming DNA of interest, or the onset of some other reaction. Typically, selective markers are genes that confer antimicrobial resistance or a metabolic advantage to the host cell in order to distinguish cells containing exogenous DNA from cells that did not accept any exogenous sequence during the transformation. A “permanent selectable marker” is a marker that is located on the chromosome of a microorganism to be transformed. A constant selectable marker encodes a gene that is different from a selectable marker in a DNA transform construct. Selective markers are well known to those skilled in the art. As noted above, preferably, the marker is an antimicrobial resistance marker (e.g., amp ^R ; phleo ^R ; spec ^R ; kan ^R ; ery ^R ; tet ^R ; cmp ^R and neo ^R (see, for example, Guerot-Fleury, Gene, 167: 335-337 [1995]; Palmeros et al, Gene 247: 255-264 [2000]; and Trieu-Cuot et al., Gene, 23: 331-341 [1983])). Other suitable markers of the invention include, but are not limited to, auxotrophic markers, such as tryptophan; and detection markers, such as β-galactosidase.

As used herein, the term “promoter” refers to a nucleic acid sequence that directs the transcription of a gene located in the 5′-3 ′ direction. In preferred embodiments, the promoter corresponds to a host cell in which the target gene is to be expressed. A promoter, together with other transcriptional and translational regulatory nucleic acid sequences (also called “control sequences”), is necessary for the expression of a given gene. Basically, transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences, ribosome binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.

A nucleic acid is “operably linked” when it is located in a functional relationship with another nucleic acid sequence. For example, DNA encoding a secretory leader sequence (i.e., a signal peptide) is operably linked to DNA for a polypeptide if it is expressed as a precursor protein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to facilitate translation. Generally, “functionally linked” means that the DNA sequences are connected adjacent, and, in the case of a secretory leader sequence, are adjacent and are in the same reading phase. However, enhancers should not be contiguous. Binding is accomplished by ligation at suitable restriction sites. If such sites are absent, synthetic oligonucleotide adapters or linkers are used in accordance with the state of the art.

As used herein, the term “gene” refers to a polynucleotide (eg, a DNA segment) that encodes a polypeptide and contains regions that come before and after the coding regions, as well as intermediate sequences (introns) between the individual coding segments (exons).

As used herein, “homologous genes” refers to a pair of genes from different, but usually related species that correspond to each other and which are identical or very similar to each other. The term encompasses genes that are separated by speciation (i.e., the development of new species) (e.g., orthologous genes), as well as genes that are separated by genetic doubling (e.g., paralogous genes).

As used herein, “ortholog” and “orthologous genes” refer to genes in various species that have evolved from a common ancestral gene (ie, a homologous gene) through speciation. Typically, orthologs retain one function during evolution. Identification of orthologs finds application in reliable prediction of gene function in newly sequenced genomes.

As used herein, “paralog” and “paralogous genes” refer to genes that are linked through duplication within the genome. While orthologs retain one function during evolution, paralogs develop new functions, even if some functions are often associated with the original function. Examples of paralogous genes include, but are not limited to, genes encoding trypsin, chymotrypsin, elastase, and thrombin, all of which are serine proteases and occur together in the same species.

As used herein, “homology” refers to the similarity or identity of a sequence, with identity being preferred. This homology is established using methods known in the art (see, for example, Smith and Waterman, Adv. Appl. Math., 2: 482 [1981]; Needleman and Wunsch, J. Mol. Biol., 48: 443 [1970 ]; Pearson and Lipman, Proc. Natl. Acad.Sci. USA, 85: 2444 [1988]; programs such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics, Genetics Computer Group, Madison, WI; software package; and Devereux et ah, Nucl. Acid Res. 12: 387-395 [1984]).

As used herein, a “similar sequence" is a sequence in which the function of a gene is basically the same since the gene is based on a parent gene (for example, the protease of Cellulomonas strain 69B4 ). Additionally, similar genes include at least about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, approximately 95%, approximately 97%, approximately 98%, approximately 99%, or approximately 100% sequence identity with the sequence of the parent gene. Alternatively, similar sequences align with 70-100% of the genes found in the region of the parent gene (for example, the protease of 69B4 Cellulomonas strain) and / or have at least 5-10 genes found in the region aligned with the genes on the chromosome, containing the parent gene (for example, the chromosome of strain 69B4 Cellulomonas ). In further embodiments, more than one of the above properties relates to a sequence. Similar sequences are established by known methods for sequence alignment. A common alignment method is BLAST, although, as described above and below, there are other methods that also find use in sequence alignment.

One suitable example is the PILEUP algorithm. PILEUP creates multiple sequence alignment from a group of related sequences using progressive, paired alignments. He can also build a tree that shows the family relationships used to create the alignment. PILEUP uses a simplified method of progressive alignment by Feng and Doolittle (Feng and Doolittle, J. Mol. Evol., 35: 351-360 [1987]). The method is similar to the method described by Higgins and Sharp (Higgins and Sharp, CABIOS 5: 151-153 [1989]). Useful PILEUP options include a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.

Another example of a suitable algorithm is the BLAST algorithm described by Altschul et al. (Altschul et al., J. Mol. Biol., 215: 403-410 [1990]; and Karlin et al., Proc. Natl. Acad. Sci., USA, 90: 5873-5787 [1993]). In particular, a useful BLAST software implementation is the WU-BLAST-2 program (see Altschul et al., Meth. Enzymol., 266: 460-480 [1996]). WU-BLAST-2 uses several search options, most of which are set to default values. The following values of adjustable parameters were set: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11. The parameters HSP S and HSP S2 are dynamic values and are set by the program itself depending on the composition of a specific sequence and the composition of a specific database against which the sequence of interest is searched. However, values can be adjusted to increase sensitivity. The value of% amino acid sequence identity is established by dividing the number of identical identical residues by the total number of residues in the "longer" sequence in the aligned region. A “longer” sequence is a sequence that contains more true residues in the aligned region (gaps introduced by WU-Blast-2 to maximize alignment scores are ignored).

Thus, “percent identity (%) of the nucleic acid sequence” is defined as the percentage of nucleotide residues in the test sequence that are identical to the nucleotide residues of the start sequence (ie, the sequence of interest). The preferred method uses the BLASTN module of the WU-BLAST-2 program, in which the parameters are set by default, with the overlap span and overlap fraction set to 1 and 0.125, respectively.

As used herein, the term “hybridization” refers to a process by which a nucleic acid strand is coupled to a complementary strand via base pairing, which is known in the art.

A nucleic acid sequence is considered to be “selectively hybridizable” with a reference nucleic acid sequence if the two sequences specifically hybridize to each other under medium to extremely stringent hybridization and washing conditions. Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex or probe. For example, “maximum stringency” typically occurs at about Tm-5 ° C (5 ° C below the Tm probe); "Extreme stringency" at about 5-10 ° C below Tm; "Medium stringency" at about 10-20 ° C below the Tm probe; and “low stringency” at about 20-25 ° C. below Tm. Functionally, the most stringent conditions can be used to identify a sequence that has a strict identity or an almost strict identity with a hybridization probe; whereas hybridization under medium or low stringency conditions can be used to detect or establish homologues of a polynucleotide sequence.

Moderate and extremely stringent hybridization conditions are well known in the art. An example of conditions with high stringency includes hybridization at approximately 42 ° C in 50% formamide, 5 × SSC, 5 × Denhardt's solution, 0.5% SDS and 100 μg / ml denatured DNA carrier, followed by two washing in 2 × SSC and 0 , 5% SDS at room temperature and two additional times in 0.1 × SSC and 0.5% SDS at 42 ° C. An example of moderately stringent conditions includes overnight incubation at 37 ° C in a solution containing 20% formamide, 5 × SSC (150 mm NaCl, 15 mm sodium tricitrate), 50 mm sodium phosphate (pH 7.6), 5 × Denhardt's solution , 10% dextran sulfate and 20 mg / ml denatured cut salmon sperm DNA, followed by washing the filters in 1 × SSC at approximately 37-50 ° C. Specialists in this field know how to select the temperature, ionic strength, etc. as necessary, to adapt to factors such as probe length, etc.

As used herein, “recombinant” includes a reference to a cell or vector that has been modified by introducing a heterologous nucleic acid sequence, or a cell obtained from a cell so modified. Thus, for example, recombinant cells express genes that are not found in identical form in the natural (non-recombinant) form of the cell, or express native genes that are otherwise abnormally expressed, insufficiently expressed, or not expressed at all as a result of intentional human intervention. "Recombination", "recombination" and the creation of a "recombined" nucleic acid, as a rule, is an assembly of two or more nucleic acid fragments, where the assembly gives rise to a chimeric gene.

In a preferred embodiment, mutant DNA sequences are generated by site-saturating mutagenesis in at least one codon. In another preferred embodiment, site-saturating mutagenesis is performed for two or more codons. In an additional embodiment, the mutated DNA sequences have more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95% or more than 98% homology with the wild-type sequence. In alternative embodiments, the mutant DNA is created in vivo using any known mutagenesis procedure, for example, such as irradiation, nitrosoguanidine, and the like. The desired DNA sequence is then isolated and used in the methods provided herein.

As used herein, the term “target sequence” refers to a DNA sequence in a host cell that encodes a sequence where an inserted sequence is to be inserted into the genome of the host cell. In some embodiments, the target sequence encodes a wild-type functional gene or operon, while in other embodiments, the target sequence encodes a functionally mutant gene or operon, or a non-functional gene or operon.

As used herein, a “flanking sequence” refers to any sequence that is located either in the 3′-5 ′ direction or 5′-3 ′ direction relative to the sequence in question (for example, for ABC genes, gene B is flanked by gene sequences A and C) In a preferred embodiment, the input sequence is flanked by homologous fragments on each side. In another embodiment, the introduced sequence and homologous fragments comprise a block that is flanked by spacer sequences on each side. In some embodiments, the flanking sequence is present on only one side (either 3 ′ or 5 ′), but in preferred embodiments, it is on each side of the flanking sequence. In some embodiments, the flanking sequence is present on only one side (either 3 ′ or 5 ′), while in preferred embodiments, it is present on each side of the flanking sequence.

As used herein, the term “spacer sequence” refers to any additional DNA that flanks homologous fragments (typically vector sequences). However, the term covers any non-homologous DNA sequence. Not limited to any theory, the spacer sequence provides a non-critical target for the cell to initiate DNA uptake.

As used herein, the terms “amplification” and “gene amplification” refer to a process by which a specific DNA sequence is disproportionately replicated so that the amplified gene becomes present in an increased number of copies than was originally present in the genome. In some embodiments, selection of cells by growth in the presence of a drug (e.g., an inhibitor for an inhibitory enzyme) results in amplification of either an endogenous gene encoding the gene product necessary for growth in the presence of a drug or in amplification of exogenous (i.e., administered ) the sequence encoding this gene product, or both.

Amplification is a special case of nucleic acid replication using matrix specificity. It differs from nonspecific matrix replication (i.e., replication that depends on the matrix but does not depend on the specific matrix). Here, the specificity of the matrix differs in the accuracy of replication (i.e., the synthesis of the correct polynucleotide sequence) and nucleotide (ribo or deoxyribo) specificity. The specificity of the matrix is often described in terms of the specificity of the “target”. Target sequences are “targets” in the sense that they search and sort the “targets” from other nucleic acids. Amplification methods were originally created for this sorting.

As used herein, the term “co-amplification” refers to the introduction into one cell of an amplifiable marker in combination with other gene sequences (ie, containing one or more non-selectable genes, such as those contained in an expression vector) and the use of an appropriate selection pressure so that the cells amplify both the amplifiable marker and other gene sequences that are not to be selected. An amplifiable marker can be physically linked to other gene sequences, or alternatively two separate DNA fragments can be introduced into one cell, one contains an amplifiable marker and the other contains a non-selectable marker.

As used herein, the terms “amplifiable marker”, “amplifiable gene” and “amplifiable vector” refer to a gene or vector encoding a gene that allows amplification of this gene under appropriate growth conditions.

In most amplification methods, “matrix specificity” is achieved by selecting an enzyme. Amplification enzymes are enzymes that, under the conditions of their use, will process only specific nucleic acid sequences in a heterologous mixture of nucleic acids. For example, in the case of Qβ replicase, MDV-1 RNA is a specific matrix for replicase (see, for example, Kacian et al., Proc. Natl. Acad. Sci. USA 69: 3038 [1972]), and other nucleic acids do not are replicated by this amplification enzyme. Similarly, in the case of T7 RNA polymerase, this amplification enzyme has strict specificity for its own promoters (see, Chamberlin et al., Nature 228: 227 [1970]). In the case of T4 ligase DNA, the enzyme will not ligate two oligonucleotides or polynucleotides if there is a mismatch between the oligonucleotide or polynucleotide substrate and the matrix at the ligation point (see, Wu and Wallace, Genomics 4: 560 [1989]). Ultimately, it was found that Taq and Pfu polymerases, due to their ability to work at high temperatures, exhibit high specificity for the sequences linked and, thus, indicated by primers; high temperature leads to thermodynamic conditions that promote hybridization of primers with target sequences, but not hybridization with non-target sequences.

As used herein, the term “amplifiable nucleic acid” refers to nucleic acids that can be amplified by any amplification method. It is contemplated that an “amplifiable nucleic acid” would typically contain a “sample matrix”.

As used herein, the term “sample matrix” refers to a nucleic acid that originates from a sample that is analyzed for the presence of a “target” (defined below). In contrast, a “background matrix” is used for a nucleic acid that is different from the template matrix that may or may not be present in the sample. The background matrix is most often arbitrary. This may be the result of a transfer, or it may be due to the presence of contaminants from nucleic acids that must be found and removed from the sample. For example, nucleic acids from organisms other than those to be detected may be present as a background in the test sample.

As used herein, the term “primer” refers to an oligonucleotide that either occurs in nature as a purified restriction enzyme cleavage product, or is synthetically engineered to act as a point of synthesis initiation when placed under conditions in which product synthesis is induced a primer extension segment that is complementary to the nucleic acid chain (i.e. in the presence of nucleotides and an inducing agent such as DNA polymerase and at suitable temperature and pH). For maximum amplification efficiency, the primer is preferably single stranded, but can alternatively be double stranded. If the primer is double-stranded, then it is first processed to separate its chains before use for the manufacture of elongation products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be long enough to direct the synthesis of elongation products in the presence of an inducing agent. The exact length of the primers will depend on many factors, including temperature, source of primer, and the method used.

As used herein, the term “probe” refers to an oligonucleotide (i.e., a nucleotide sequence) that either occurs in nature as a purified restriction enzyme digest product, or is synthetically, recombinantly generated, or by PCR amplification that can be hybridized with another oligonucleotide of interest. The probe may be single-stranded or double-stranded. Probes are suitable for detecting, identifying and isolating specific genetic sequences. It is contemplated that any probe used in the present invention will be labeled with any “reporter molecule” so that it can be detected in any detection system, including but not limited to enzymatic (eg, ELISA, as well as enzyme-based histochemical analyzes) ), fluorescent, radioactive and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label.

As used herein, the term “target,” as used in relation to a polymerase chain reaction, refers to a nucleic acid region linked by primers used in the polymerase chain reaction. Thus, the search and sorting of the “target” from other nucleic acid sequences occurs. A “segment” is defined as a region of a nucleic acid within a target sequence.

As used herein, the term "polymerase chain reaction" ("PCR") refers to methods from US patents Nos. 4,683,195, 4,683,202 and 4,965,188, which include methods for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification, which are known to specialists in this field. Since the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be “PCR amplified”.

As used herein, the term “amplification reagents” refers to those reagents (deoxyribonucleotide triphosphates, buffer, etc.) that are necessary for amplification, with the exception of primers, a nucleic acid template, and an amplification enzyme. Typically, amplification reagents, along with other reaction components, are placed and contained in a reaction vessel (test tube, microwell, etc.).

Using PCR, one copy of a specific target sequence in genomic DNA can be amplified to a level that can be determined using several different methods (for example, hybridization with a labeled probe; insertion of biotinylated primers followed by detection of an enzyme-avidin conjugate; insertion of deoxynucleotide triphosphates labeled ³² P, such as dCTP or dATP, in the amplified segment). In addition to genomic DNA, any oligonucleotide or polynucleotide sequence can be amplified using an appropriate set of primer molecules. In particular, the amplified segments created by the PCR process itself are themselves effective matrices for subsequent PCR amplifications.

As used herein, the terms “PCR product”, “PCR fragment” and “amplification product” refer to the resulting mixture of compounds after completion of two or more PCR cycles from the steps of denaturation, annealing and elongation. These terms encompass a case in which amplification of one or more segments of one or more target sequences occurs.

As used herein, the term “RT-PCR” refers to the replication and amplification of RNA sequences. In this method, in which reverse transcription is combined with PCR, the most common is a single enzyme procedure that uses thermostable polymerase as described in US Pat. No. 5,327,770. In RT-PCR, the RNA matrix is converted to cDNA due to reverse transcriptase activity of the polymerase and then amplified using the polymerase activity of the polymerase (i.e., as in other PCR methods).

As used herein, the terms "restriction endonucleases" and "restriction enzymes" refer to bacterial enzymes, each of which cuts double-stranded DNA directly in or adjacent to a particular nucleotide sequence.

A "restriction site" refers to a nucleotide sequence recognized and cut using a given restriction endonuclease and often represents a site for embedding DNA fragments. In certain embodiments, restriction sites are created within the selectable marker and at the 5 ′ and 3 ′ ends of the construct DNA.

As used herein, the term “chromosome integration” refers to a process by which an introduced sequence is introduced into the chromosome of a host cell. Homologous regions of transforming DNA align with homologous regions of the chromosome. Subsequently, the sequence between homologous fragments is replaced by the introduced sequence through double crossing over (i.e., homologous recombination). In some embodiments of the present invention, the homologous regions of the inactivating chromosomal DNA segment of the construct align with the flanking homologous regions of the natural chromosome region of the Bacillus chromosome. Subsequently, the natural chromosome region in double crossing over (i.e., homologous recombination) is deleted using the DNA construct.

"Homologous recombination" means the exchange of DNA fragments between two DNA molecules or paired chromosomes in a site with identical or almost identical nucleotide sequences. In a preferred embodiment, the chromosomal integration is homologous recombination. “Homologous sequences”, as used herein, mean a nucleic acid or polypeptide sequence having 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90 %, 88%, 85%, 80%, 75% or 70% sequence identity with a different nucleic acid or polypeptide sequence with optimal alignment for comparison. In some embodiments, homologous sequences have from 85% to 100% sequence identity, while in other embodiments, from 90% to 100% sequence identity, and in more preferred embodiments, 95% and 100% sequence identity occurs.

As used herein, “amino acid” refers to peptide or protein sequences or fragments thereof. The terms “protein,” “peptide,” and “polypeptide” are used interchangeably.

As used herein, “protein of interest” and “polypeptide of interest” refer to the desired and / or evaluated protein / polypeptide. In some embodiments, the “protein of interest” is a “parent protein” (ie, a starting protein). In some embodiments, the parent protein is a wild-type enzyme that is used as a starting point in the construction / design of the protein. In some embodiments, the protein of interest is expressed intracellularly, while in other embodiments, it is a secreted polypeptide. In particularly preferred embodiments, these enzymes include serine proteases and metalloproteases described herein. In some embodiments, the protein of interest is a secreted polypeptide fused to a signal peptide (i.e., an amino terminal extension segment on a protein to be secreted). Nearly all secreted proteins use the amino-terminal protein extension segment, which plays a key role in directing and moving precursor proteins across the membrane. This extension segment is proteolytically removed by signal peptidase during or immediately after passing through the membrane.

As used herein, the term “heterologous protein” refers to a protein or polypeptide that does not naturally occur in the host cell. Examples of heterologous proteins include enzymes such as hydrolases, including proteases. In some embodiments, the implementation of the genes encoding the proteins are naturally occurring genes, while in other embodiments, mutated and / or synthetic genes are used.

As used herein, “homologous protein” refers to a protein or polypeptide that is native or found in a cell in nature. In preferred embodiments, the cell is a gram-positive cell, while in particularly preferred embodiments, the cell is a Bacillus host cell. In alternative embodiments, the homologous protein is a native protein synthesized by other organisms, including but not limited to E. coli , Cellulomonas , Bacillus , Streptomyces , Trichoderma, and Aspergillus . This invention relates to host cells that produce a homologous protein through recombinant DNA technology.

As used herein, an “operon region” contains a group of contiguous genes that are transcribed as a single transcriptional unit from a common promoter and thus undergo coregulation. In some embodiments, the operon comprises a regulatory gene. In the most preferred embodiments, operons are used that have increased expression, judging by measurements of RNA levels, but have unknown or excessive function.

As used herein, an “antimicrobial region” is an region containing at least one gene that encodes an antimicrobial protein.

It is said that a polynucleotide “encodes” an RNA or polypeptide if, in its native state or by manipulation by methods known to those skilled in the art, it can be transcribed and / or translated to synthesize an RNA, polypeptide or fragment thereof. The antisense strand of such a nucleic acid is also said to encode sequences.

As is known in the art, DNA can be translated using RNA polymerase to form RNA, but RNA can be reverse transcribed using reverse transcriptase to form DNA. Thus, DNA can encode RNA and vice versa.

The terms “regulatory segment” or “regulatory sequence” or “expression control sequence” refer to a polynucleotide DNA sequence that is operably linked to a polynucleotide DNA sequence that encodes an amino acid sequence of a polypeptide chain to affect the expression of the encoded amino acid sequence. A regulatory sequence can inhibit, inhibit, or facilitate the expression of a functionally linked polynucleotide sequence encoding an amino acid.

"Host strain" or "host cell" refers to a host organism suitable for expression of a vector containing the DNA of the present invention.

An enzyme is “overexpressed” in a host cell if the enzyme is expressed in the cell at a level higher than the level at which it is expressed in the corresponding wild-type cell.

As used herein, the terms “protein” and “polypeptide” are used interchangeably. Throughout this disclosure, a three-letter code for amino acids is used as determined in accordance with the IUPAC-IUB of the Joint Commission on Biochemical Nomenclature (JCBN). It should also be understood that the polypeptide can be encoded by more than one nucleotide sequence due to the degeneracy of the genetic code.

A "sequence" is an amino acid sequence between a signal sequence and a mature protease, which is necessary for secretion of the protease. Cleavage of the prosequence will result in the formation of a mature active protease.

The terms “signal sequence” or “signal peptide” refer to any sequence of nucleotides and / or amino acids that are involved in the secretion of a mature or precursor form of a protein. This determination of the signal sequence is functional, which means that all those amino acid sequences encoded by the N-terminal part of the protein gene that are involved in the secretion of the protein are included. They are often, but not always, linked to the N-terminal portion of the protein or to the N-terminal portion of the precursor protein. The signal sequence may be endogenous or exogenous. The signal sequence may be naturally associated with a protein (eg, a protease) or may be from another gene encoding another secreted protein. One exemplary exogenous signal sequence contains the first seven amino acid residues of the signal sequence from B. subtilis subtilisin fused to the residue of the subtilisin signal sequence from B. lentus (ATCC 21536).

The term "hybrid signal sequence" refers to signal sequences in which part of the sequence obtained from the expressing host organism is fused to the signal sequence of the gene to be expressed. In some embodiments, synthetic sequences are used.

The term “substantially the same signaling activity” refers to signaling activity, which is indicated to a large extent by the same protease secretion into the fermentation medium, for example, the protease level in the fermentation medium is at least 50%, at least 60%, at least at least 70%, at least 80%, at least 90%, at least 95%, at least 98% of the levels of secreted protease in the fermented medium, which is provided with a signal sequence.

The term “mature” form of a protein or peptide refers to the final functional form of a protein or peptide. As an example, the mature form of the NprE protease of the present invention at least contains the amino acid sequence of SEQ ID NO: 3.

The term “precursor” form of a protein or peptide refers to a mature form of a protein that has a sequence that is operably linked to the amino or carboxy terminus of the protein. The precursor may also have a “signal” sequence functionally linked to the amino terminus of the pro sequence. The precursor may also contain additional polynucleotides that are involved in post-translational activity (for example, polynucleotides are cleaved from there to leave the mature form of the protein or peptide).

“Naturally occurring enzyme” and “naturally occurring protein” indicate an enzyme or protein having an unmodified amino acid sequence identical to that found in nature. Naturally occurring enzymes include native enzymes, such enzymes are naturally expressed or found in a particular microorganism.

The terms “isolated from” and “obtained from” refer not only to an enzyme (eg, a protease) synthesized or synthesized by a strain of the organism in question, but also to an enzyme encoded by a DNA sequence isolated from such a strain and produced in a host organism containing such DNA sequence. Additionally, the term refers to an enzyme that is encoded by a DNA sequence of synthetic and / or cDNA origin and which has the identification characteristics of the enzyme in question.

A “derivative” as part of this definition, as a rule, retains the characteristic proteolytic activity observed in the wild type, native or parent form, if the derivative is suitable for purposes similar to the purpose of using the wild type, native or parent form. Functional derivatives of the enzyme encompass naturally occurring, synthetically or recombinantly generated peptides or fragments of peptides that possess the basic characteristics of the parent enzyme.

The term “functional derivative” refers to a nucleic acid derivative having the functional characteristics of a nucleic acid encoding an enzyme. Functional nucleic acid derivatives that encode the enzymes provided herein encompass naturally occurring, synthetically or recombinantly generated nucleic acids or fragments. The wild-type nucleic acid encoding the enzymes of the present invention includes naturally occurring alleles and homologues based on the degeneracy of the genetic code known in the art.

The term "identical" in the context of two sequences of nucleic acids or polypeptides refers to residues in two sequences that are the same when aligned with the maximum match, which is measured using one of the following sequence comparison or analysis algorithms.

The term “optimal alignment” refers to alignment giving the highest percentage of identity.

“Percentage of sequence identity”, “Percentage of amino acid sequence identity”, “Percentage of gene sequence identity” and / or “Percentage of nucleic acid / polynucleotide sequence identity” with respect to two amino acid, polynucleotide and / or gene sequences (as appropriate) refer to the percentage of residues that are identical in two sequences when these sequences are optimally aligned. Thus, 80% amino acid sequence identity means that 80% of the amino acids in the two optimally aligned polypeptide sequences are identical.

Thus, the phrase “substantially identical” in the context of two nucleic acids or polypeptides refers to a polynucleotide or polypeptide that has at least 70% sequence identity, preferably at least 75%, preferably at least 80% preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97%, preferably at least 98% and preferably at least 99% sequence identity spine as compared to a reference sequence using the programs or algorithms (e.g., BLAST, ALIGN, CLUSTAL) using standard settings. One sign that the two polypeptides are substantially identical is that the first polypeptide has immunological cross-reactivity with the second polypeptide. Typically, polypeptides that differ in conservative amino acid substitutions are immunologically cross-reactive. Thus, the polypeptide is substantially identical to the second polypeptide, for example, when two peptides differ only by conservative substitution. Another indication that the two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions (for example, in the range from medium to extremely stringent).

The term “isolated” or “purified” refers to material that is removed from its original environment (for example, the natural environment, if it occurs in nature). For example, it is said about a material that it is “purified” when it is present in a particular composition in a higher or lower concentration than that found in an organism existing in nature, or in a wild-type organism, or in combination with components that are not usually present during expression in a naturally occurring organism or in a wild-type organism. For example, a naturally occurring polynucleotide or polypeptide present in a living organism is not isolated, but the same polynucleotide or polypeptide separated from some or all of the materials coexisting with it in the natural system is isolated. In some embodiments, the implementation of such polynucleotides are part of the vector and / or such polynucleotides or polypeptides are part of the composition, and yet isolated in such a vector or composition that are not part of their natural environment. In some preferred embodiments, it is said that the nucleic acid or protein is purified, for example, if they are the source of substantially the same band on an electrophoretic gel or in blotting.

The term "isolated" when used in relation to a DNA sequence refers to a DNA sequence that has been removed from its natural genetic environment and thus free from other extraneous or undesirable coding sequences, and exists in a form suitable for use in genetically engineered production systems squirrel. Such isolated molecules are molecules that are separated from their natural environment and include cDNA and genomic clones. The isolated DNA molecules of the present invention are free from other genes with which they are usually associated, but may contain naturally occurring 5'- and 3'-untranslated regions, such as promoters and terminators. Identification of related areas will be apparent to those skilled in the art (see, for example, Dynan and Tijan, Nature 316: 774-78, 1985). The term "isolated DNA sequence" is alternatively referred to as "cloned DNA sequence".

The term "isolated", when used in relation to a protein, refers to a protein that is found in a state different from the natural environment. In a preferred form, the isolated protein is substantially free of other proteins, particularly other homologous proteins. The isolated protein has more than 10% purity, preferably more than 20% purity and even more preferably more than 30% purity, which is determined using SDS-PAGE. Additional aspects of the invention encompass protein in a highly purified form (i.e., more than 40% pure, more than 60% pure, more than 80% pure, more than 90% pure, more than 95% pure, more than 97% purity and even more than 99% purity), which is determined using SDS-PAGE.

As used herein, the term “combinatorial mutagenesis” refers to methods in which libraries of variants of starting sequences are created. In these libraries, variants contain one or more mutations selected from a predefined set of mutations. In addition, the methods provide means for introducing random mutations that are not members of a predefined set of mutations. In some embodiments, the methods comprise the methods set forth in US Application No. 09/699250, filed October 26, 2000. In alternative embodiments, combinatorial mutagenesis methods encompass commercially available kits (e.g. QUIKCHANGE® Multisite, Stratagene, La Jolla, CA).

As used herein, the term “variant” refers to a protein that has been derived from a precursor protein (eg, a “parent” protein) by the addition, substitution or deletion of one or more amino acids. In some embodiments, the embodiment comprises at least one modification that comprises charge changes compared to the precursor protein. In some preferred embodiments, the precursor protein is a parent protein that is a wild-type protein.

As used herein, the term “mutant library” refers to a population of cells that are identical in most of their genomes but include different homologues of one or more genes. Such libraries can be used, for example, to identify genes or operons with improved characteristics.

As used herein, the term “start gene” refers to a gene of interest that encodes a protein of interest that needs to be improved and / or modified using the present invention.

As used herein, the terms “multiple sequence alignment” and “MVP” refer to sequences of a plurality of start gene homologues that are aligned using an algorithm (eg, Clustal W).

As used herein, the terms "consensus sequence" and "canonical sequence" refer to the original amino acid sequence with which all variants of a particular protein or sequence of interest are compared. The terms also refer to the sequence in which the nucleotides that are most often present in the DNA sequence of interest are listed. For each position in the gene, the consensus sequence gives the amino acid that is most often found at this position in the MEP.

As used herein, the term “consensus mutation” refers to differences in the start gene sequence and the consensus sequence. Consensus mutations are established by comparing the start gene sequence and the consensus sequence obtained from the MEP. In some embodiments, consensus mutations are introduced into the starting gene so that it becomes more like a consensus sequence. Consensus mutations also include amino acid substitutions that change the amino acid in the starting gene to the amino acid that is more often found in the MEP at this position relative to the frequency of this amino acid in the starting gene. Thus, the term consensus mutation includes all single amino acid substitutions that replace the amino acid of the starting gene with an amino acid that is more common than the amino acid in the MEP.

As used herein, the term “parental variant with favorable properties” refers to a variant that has been identified by screening a library of combinatorial consensus mutagenesis. In preferred embodiments, the initial variants with favorable properties have improved performance compared to the starting gene.

As used herein, the term “improved variant with favorable properties” refers to a variant that has been established by screening an expanded library of combinatorial consensus mutagenesis.

As used herein, the terms “enhancing mutation” and “mutation enhancing efficacy” refer to a mutation that leads to improved efficacy when introduced into the starting gene. In some preferred embodiments, these mutations are identified by sequencing variants with favorable properties established during the screening step of this method. In most embodiments, it is likely that mutations, which are more often found in variants with favorable properties, are improving mutations compared to an unscreened library of combinatorial consensus mutagenesis.

As used herein, the term “enriched library of combinatorial consensus mutagenesis” refers to a KKM library that is designed and constructed based on the results of screening and / or sequencing from an early cycle of KKM mutagenesis and screening. In some embodiments, the enriched CMC library is based on the sequence of an initial variant with favorable properties obtained from an early CMC cycle. In further embodiments, the enriched CMC is designed so that mutations that are often observed in the parental variants with favorable properties from the early mutagenesis and screening cycles are preferred. In some preferred embodiments, this is accomplished by skipping primers that code for mutations that decrease efficiency, or by increasing the concentration of primers that code for mutations that increase efficiency with respect to other primers that were used in early KKM libraries.

As used herein, the term “efficacy-reducing mutations” refers to mutations in the library of combinatorial consensus mutagenesis that are less likely to be found in variants with favorable properties as a result of screening compared to an unscreened library of combinatorial consensus mutagenesis. In preferred embodiments, the screening process removes and / or reduces the relative content of variants that contain "mutations that reduce effectiveness."

As used herein, the term “functional assay” refers to an assay that provides a measure of protein activity. In particularly preferred embodiments, the term refers to assay systems in which a protein is analyzed for its ability to function under its normal load. For example, in the case of enzymes, functional analysis concerns the determination of the effectiveness of an enzyme in catalysing a reaction.

As used herein, the term “intended property” refers to a property of a start gene that needs to be changed. It is not intended that the present invention be limited to any particular intended property. However, in some preferred embodiments, the intended property is the stability of the gene product (for example, resistance to denaturation, proteolysis or other destructive factors), while in other embodiments, the level of production in the producing host is changed. In fact, it is contemplated that any property of the starting gene will find use in the present invention.

The term “property” or its grammatical equivalents in the context of a nucleic acid, as used herein, refers to any characteristic or property of a nucleic acid that can be selected or detected. These properties include, but are not limited to, an effect on binding to a polypeptide, a property conferred on a cell containing a particular nucleic acid, a property on transcription of a gene (e.g., promoter strength, recognition of a promoter, regulation of a promoter, enhancer function), a property on RNA processing (for example, RNA splicing, RNA stability, RNA conformation and post-transcriptional modification), the ability to affect translation (for example, level, regulation , Binding of mRNA to ribosomal proteins, post-translational modification). For example, in nucleic acid, the binding site of a transcription factor, polymerase, regulatory factor, etc. can be modified to create the desired characteristics or to identify undesirable characteristics.

The term “property” or its grammatical equivalents in the context of a polypeptide, as used herein, refers to any characteristic or property of a polypeptide that can be selected or detected. These properties include, but are not limited to, oxidative stability, substrate specificity, catalytic activity, thermal stability, alkali stability, pH activity profile, resistance to proteolytic degradation, K _M , k _cat , k _cat / K _M ratio, protein folding, immune stimulation response, the ability to bind to the ligand, the ability to bind to the receptor, the ability to secrete, the ability to exhibit on the surface of the cell, the ability to oligomerize, the ability to transmit signals, ability to stimulate cell proliferation, ability to inhibit cell proliferation, ability to induce apoptosis, ability to be modified by phosphorylation or glycosylation, ability to treat a disease.

As used herein, the term "screening" has its usual meaning in this field and, basically, is a multi-stage process. In a first step, a mutant nucleic acid or variant polypeptide thereof is provided. In a second step, the property of a mutant nucleic acid or polypeptide variant is determined. In a third step, the property that has been determined is compared with the property of the corresponding parent nucleic acid, with the property of the corresponding naturally occurring polypeptide, or with the property of the starting material (e.g., the initial sequence) to create the mutant nucleic acid.

It will be clear to a person skilled in the art that the screening procedure for obtaining a nucleic acid or protein with a modified property depends on the property of the starting material, the modification of which is intended to facilitate the creation of mutant nucleic acid. Therefore, a professional will appreciate that the invention is not limited to any particular property to be screened, and that the following description of properties lists only illustrative examples. Methods for screening a particular property are generally described in the art. For example, you can measure binding, pH, specificity, etc. before and after the mutation, where a change indicates a change. Preferably, the screening is carried out in a high throughput manner, including at the same time a plurality of samples to be screened, including, but not limited to, analysis using chips, phage display and a plurality of substrates and / or indicators.

As used herein, in some embodiments, screening encompasses screening steps at which options of interest are enriched from the totality of options. Examples of these embodiments include selecting options that give the host a growth advantage, as well as a phage display or any other detection method, where the options can be captured from the totality of options based on their binding or catalytic properties. In a preferred embodiment, the library of variants is exposed to external influences (heating, protease, denaturation) and subsequently variants that remain intact are identified by screening or enriched by selection. The term is intended to encompass any suitable means for selection. In fact, it is not intended that the present invention be limited to any particular screening method.

As used herein, the term “targeted random selection” refers to a process in which multiple sequences are created in which one or more positions have been randomized. In some embodiments, the randomization is complete (i.e., all four nucleotides, A, T, G, and C, may occur in a randomized position). In alternative embodiments, nucleotide randomization is limited to a subset of four nucleotides. Targeted random selection can be applied to one or more codons of a sequence encoding one or more proteins of interest. After expression from the obtained libraries, protein populations are created in which one or more amino acid positions may contain a mixture of all 20 amino acids or a subset of amino acids, which is determined by the randomization scheme of a randomized codon. In some embodiments, individual members of a targeted random selection population differ in the number of amino acids due to targeted or random insertion or deletion of codons. In further embodiments, synthetic amino acids are included in the generated protein populations. In some preferred embodiments, a large portion of the population from targeted random selection exhibits higher sequence homology with respect to the consensus sequence than the start gene. In some embodiments, the sequence encodes one or more proteins of interest. In alternative embodiments, the proteins have various biological functions. In some further preferred embodiments, the input sequence comprises at least one selective marker. This sequence can encode one or more proteins of interest. They may have other biological function (s). In many cases, the introduced sequence will contain a selectable marker, such as a gene, that provides antibiotic resistance.

The terms “modified sequence” and “modified genes” as used herein interchangeably refer to a sequence that contains a deletion, insertion, or violation of a naturally occurring nucleic acid sequence. In some preferred embodiments, the expression product of the modified sequence is a truncated protein (for example, if the modification is a deletion or violation of the sequence). In some particularly preferred embodiments, the truncated protein retains biological activity. In alternative embodiments, the expression product of the modified sequence is an elongated protein (eg, modifications containing an insert in a nucleic acid sequence). In some embodiments, the implementation leads to a truncated protein (for example, when the insert leads to the formation of a stop codon). Thus, the insert can result in either a truncated protein or an elongated protein as an expression product.

As used herein, the terms “mutant sequence” and “mutant gene” are used interchangeably and refer to a sequence that has a change in at least one codon found in a wild-type host cell sequence. The expression product of the mutant sequence is a protein with a modified amino acid sequence in relation to the wild type. The expression product may have altered functional capabilities (for example, improved enzymatic activity).

The terms “mutagenic primer” or “mutagenic oligonucleotide” (used interchangeably herein) are intended to refer to oligonucleotide structures that correspond to a portion of the template sequence and which hybridize to it. Regarding mutagenic primers, the primer will not exactly match the template nucleic acid; mismatches or mismatches in the primer are used to introduce the desired mutation into the nucleic acid library. As used herein, "non-mutagenic primer" or "non-mutagenic oligonucleotide" refers to oligonucleotide structures that exactly match the template nucleic acid. In one embodiment, only mutagenic primers are used. In another preferred embodiment, the primers are designed so that, in at least one region in which the mutagenic primer has been incorporated, a non-mutagenic primer included in the oligonucleotide mixture also takes place. Adding a mixture of mutagenic primers and non-mutagenic primers corresponding to at least one of the mutagenic primers, as a result, you can create a library of nucleic acids, which presents many combinatorial mutational patterns. For example, if you want some members of the mutant nucleic acid library to maintain their parental sequence at certain positions, while other members are mutants at such sites, non-mutagenic primers provide the ability to obtain a specific level of non-mutant members in the nucleic acid library for a given residue. The methods of the invention utilize mutagenic and non-mutagenic oligonucleotides, which typically have a length between 10-50 bases, more preferably about 15-45 bases long. However, it may be necessary to use primers that are either shorter than 10 bases or longer than 50 bases to obtain the desired mutagenesis result. With respect to the respective mutagenic and non-mutagenic primers, it is not necessary that the corresponding oligonucleotides are the same length, but only that overlap occurs in the region corresponding to the introduced mutation.

In some embodiments, the primers are added in a predetermined ratio. For example, if you want the resulting library to have a significant level of a specific specific mutation and fewer different mutations at the same or different site, you can create the desired offset library by selecting the amount of primer to be added. Alternatively, by adding larger or smaller amounts of non-mutagenic primers, it is possible to control the frequency with which the corresponding mutation (s) are generated in the library of mutant nucleic acids.

As used herein, the phrase “adjacent mutations” refers to mutations that are present in a single oligonucleotide primer. For example, adjacent mutations may be adjacent or close to each other, however, they will be introduced into the final mutant template nucleic acids using a single primer.

As used herein, the phrase “non-contiguous mutations” refers to mutations that are represented by individual oligonucleotide primers. For example, non-contiguous mutations will be introduced into the final mutant maternal nucleic acids using separately prepared oligonucleotide primers.

The terms “wild-type sequence”, “wild-type nucleic acid sequence” and “wild-type gene” used interchangeably herein refer to a sequence that is native or naturally occurring in the host cell. In some embodiments, the wild-type sequence refers to the sequence of interest, which is the starting point of a protein construct project. The wild-type sequence can encode either a homologous or heterologous protein. A homologous protein is a protein that a host cell will produce without interference. A heterologous protein is a protein that the host cell will not produce without intervention.

The term “oxidatively stable” refers to the proteases of the present invention that retain a certain amount of enzymatic activity for a given period of time under conditions prevailing in the process of proteolysis, hydrolysis, purification or other process of the invention, for example when they are exposed to or brought into contact with bleaches or oxidizing agents. In some embodiments, proteases retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% proteolytic activity after contact with a bleaching or oxidizing agent for a predetermined period of time, for example, at least 1 minute, 3 minutes, 5 minutes, 8 minutes, 12 minutes, 16 minutes, 20 minutes, etc.

The term "chelator-stable" refers to the proteases of the present invention, which retain a certain amount of enzymatic activity for a given period of time under conditions prevailing during the proteolysis, hydrolysis, purification or other process of the invention, for example, when they are exposed to or brought into contact with chelating agents. In some embodiments, proteases retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% proteolytic activity after contact with a chelating agent for a predetermined period of time, for example, at least 10 minutes, 20 minutes, 40 minutes, 60 minutes, 100 minutes, etc.

The terms “thermally stable” and “thermostable” refer to the enzymes of the present invention that retain a certain amount of enzymatic activity after exposure to set temperatures for a predetermined period of time under conditions prevailing during the enzymatic treatment, hydrolysis, purification, or other process of the invention, for example when they are exposed to altered temperatures. Altered temperatures include elevated or decreased temperatures. In some embodiments, proteases retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% proteolytic activity after exposure to altered temperatures for a given period of time, for example, at least 60 minutes, 120 minutes, 180 minutes, 240 minutes, 300 minutes, etc.

The terms “increased stability” or “reduced stability” in the context of oxidation, chelation, thermally and / or pH-stable enzyme refer to increased stored enzymatic activity over time compared to other serine proteases (eg, subtilisin proteases) and / or wild-type enzymes .

The terms "increased stability" or "reduced stability" in the context of oxidation, chelation, thermally and / or pH-stable enzyme refers to less persistent enzymatic activity over time compared with other serine proteases (e.g. subtilisin proteases) and / or wild-type enzymes .

As used herein, the term “cleaning composition” includes, unless otherwise indicated, granular or powder detergents for general use or for harsh conditions, in particular cleaning detergents; general purpose liquid, gel or paste-like detergents, in particular so-called liquids for harsh conditions; liquid detergents for delicate matter; detergents for washing dishes or lightweight detergents for washing dishes, in particular, foaming agents; automatic dishwashing detergents, including various tableted, granular, liquid detergents and dishwashing rinses for home and institutional use; liquid cleaners and disinfectants, including antibacterials for hand washing, cleaning tiles, mouthwashes, cleaning products for teeth and oral cavity, shampoos for cars and carpets, cleaning products for bathrooms; shampoos and hair rinses; shower gels and bath foams and metal cleaners; as well as auxiliary cleaners, such as bleach additives and stain-sticks or pre-treatments.

Unless otherwise specified, the concentrations of all components or compositions are in accordance with the active concentration of that component or composition, with the exception of impurities, for example, residual solvents or by-products that may be present in commercially available sources.

Masses of enzymatic components are based on a common active protein. All percentages and ratios are calculated by weight unless otherwise indicated. All percentages and ratios are calculated based on the whole composition, unless otherwise indicated.

It should be understood that each maximum numerical limitation given throughout this description includes each lower numerical limitation, as if such lower numerical limitations were expressly indicated herein. Each minimum numerical limitation given throughout this description will include each upper numerical limitation, as if such upper numerical limitations were expressly indicated herein. Each numerical range indicated throughout this description will include every numerical range that falls within such a wide numerical range, as if the narrower numerical ranges were expressly indicated herein.

The term "purification activity" refers to the purification efficiency achieved by the enzyme under conditions prevailing during the enzymatic treatment, hydrolysis, purification, or other process of the invention. In some embodiments, the cleaning efficiency is determined by applying various cleaning tests related to enzyme sensitive paints, for example grass, blood, milk or egg protein, which is determined by various chromatographic, spectrophotometric or other quantitative methods after the paints are subjected to standard washing conditions. Exemplary assays include, but are not limited to, the assays described in WO 99/34011 and US Pat. No. 6,605,458, as well as those methods that are included in the examples.

The term “cleaning amount effective” of a protease refers to the amount of protease described herein before which the desired level of enzymatic activity is achieved in a particular cleaning composition. Such effective amounts are readily determined by a person skilled in the art and are based on many factors, such as the particular protease used, the cleaning application, the specific composition of the cleaning composition, and whether a liquid or dry (e.g., granular, lumpy) composition is required, etc.

The term “auxiliary cleaning agents”, as used herein, means any liquid, solid or gaseous substance selected for the specific type of cleaning composition and product form desired (eg, liquid, granules, powder, lump, paste, spray, tablet, gel ; or foam composition), these substances are also preferably compatible with the proteolytic enzyme used in the composition. In some embodiments, the granular compositions are in a “compact” form, while in other embodiments, the liquid compositions are in a “concentrated” form.

The term "increased effectiveness" in the context of cleaning activity refers to increased or increased cleaning activity of certain enzyme-sensitive paints, such as eggs, milk, grass or blood, which is determined by a routine evaluation after a standard washing cycle and / or multiple washing cycles.

The term "reduced effectiveness" in the context of cleaning activity refers to reduced or less cleaning activity of specific enzyme-sensitive paints, such as eggs, milk, grass or blood, which is determined by a routine evaluation after a standard washing cycle.

The term "comparative effectiveness" in the context of purification activity refers to at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the purification activity a comparable protease (e.g., commercially available proteases). The cleaning efficiency can be determined by comparing the proteases of the present invention with other proteases in various cleaning tests for enzyme sensitive paints, such as blood, milk and / or ink (CMC), which is determined using conventional spectrophotometric or analytical methods after the wash cycle under standard conditions.

As used herein, a “low detergent concentration” system contains detergents in which approximately less than 800 ppm. detergent components are present in wash water. Japanese detergents are typically considered systems with a low concentration of detergent, since they typically contain approximately 667 ppm. detergents present in washing water.

As used herein, “medium detergent concentration” systems contain detergents where between about 800 ppm. and approximately 2000 ppm detergent components are present in the wash water. As a rule, North American detergents are considered as systems with an average concentration of detergent, since they usually contain approximately 975 ppm. detergents present in washing water. Brazilian detergents typically contain approximately 1,500 ppm. detergents present in washing water.

As used herein, “high detergent concentration” systems contain detergents, where approximately more than 2000 ppm. Detergent components present in washing water. European detergents are generally regarded as systems with a high concentration of detergent, as they contain approximately 3000-8000 ppm. detergent components in water for washing.

As used herein, “fabric cleaning compositions” include hand and machine wash detergents, including additional laundry compositions and compositions suitable for use in soaking and / or pretreating dyed fabrics (eg, clothes, linens, and other textile materials).

As used herein, “non-material cleaning compositions” include cleaning compositions for non-textile (ie non-fabric) surfaces, including, but not limited to, dishwashing detergents, oral cleaning compositions, dental cleaning compositions, and personalized cleaning compositions.

The "compact" form of cleaning compositions herein is best reflected in density and, in terms of composition, in the amount of fillers from inorganic salts. Inorganic salt fillers are the traditional ingredients of detergent compositions in powder form. In traditional detergent formulations, salt fillers are present in substantial amounts, typically 17-35% by weight of the total composition. In contrast, in compact compositions, salt fillers are present in amounts not exceeding 15% of the total composition. In some embodiments, salt fillers are present in amounts that do not exceed 10% or more preferably 5% by weight of the composition. In some embodiments, inorganic salt fillers are selected from sulfates and chlorides of alkali and alkaline earth metals. A preferred salt filler is sodium sulfate.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for constructing proteins in order to optimize their effectiveness under specific environmental conditions of interest. In some embodiments, the present invention relates to methods for constructing enzymes to optimize their catalytic activity under specific environmental conditions. In some preferred embodiments, the present invention relates to methods for constructing enzymes to optimize their catalytic activity and / or stability under adverse environmental conditions. In some preferred embodiments, the present invention relates to methods for constructing enzymes to optimize their storage stability, particularly under adverse environmental conditions. In some preferred embodiments, the present invention relates to methods for changing the total surface charge and / or distribution of surface charges of enzymes (e.g. metalloproteases) to produce enzyme variants that exhibit improved efficiency and / or stability in detergent compositions compared to the initial or parent enzyme .

In some embodiments, the present invention provides methods for constructing an enzyme to simultaneously optimize its catalytic activity and stability under adverse environmental conditions, even when these two properties are negatively correlated when analyzing the effects of individual mutations. In particular, the present invention relates to methods for changing the total surface charge and / or distribution of surface charges of a metalloprotease in order to obtain enzyme variants exhibiting improved efficiency in detergent compositions.

The present invention relates to methods and compositions containing at least one version of a neutral metalloprotease, which has improved washing efficiency and / or stability in the detergent composition (s). In some particularly preferred embodiments, the present invention relates to neutral metalloprotease variants of Bacillus amyloliquefaciens . The present invention finds particular use in applications, including but not limited to cleaning, bleaching, and disinfection. Additionally, the present invention relates to methods for constructing an enzyme to optimize its catalytic activity under adverse environmental conditions. In particular, the present invention relates to methods for changing the total surface charge and / or distribution of surface charges of a metalloprotease in order to obtain enzyme variants exhibiting improved efficiency and / or stability in detergent compositions.

Many proteins and enzymes are extremely susceptible to denaturation and are subject to irreversible denaturation when stored in detergents for washing. It is known that detergents for washing contain anionic, cationic and nonionic surfactants, where a surfactant is classified based on its ionic (electric charge) properties in water. These ingredients interact with the surface charge of the protein molecule, which leads to protein denaturation (for example, loss of structure and function). NprE (neutral metalloprotease) has been shown to be unstable when stored in detergent formulations containing a surfactant such as LAS. LAS is an anionic surfactant in which the total negative charge enhances the interaction with the positively charged side chains of amino acids located on the surface of the protein. Such electrostatic interactions affect important protein stability by weakening or disrupting stabilizing electrostatic interactions. Then the styling of the destabilized protein is disrupted and it becomes inactive. During the development of the present invention, it was found that the surface charge of the enzyme seriously affects the washing efficiency and / or stability in the detergent. Additionally, it was found that the distribution of charged residues on the surface of the protease greatly affects the washing efficiency and / or stability. The methods for constructing the proteins of the present invention efficiently optimize proteases to increase the effectiveness of one or more properties in detergent compositions by optimizing the total surface charge and / or distribution of surface charges.

Briefly, in some embodiments of the present invention, the methods involve creating libraries for evaluating sites involving multiple amino acid residues of an enzyme of interest and testing enzyme variants for properties of interest. This makes it possible to identify beneficial, neutral and harmful mutations, as well as the optimal charge change (relative to the parent enzyme) for the property of interest (c). In some alternative embodiments, the charge scans all residues to create mutation variants that change charge at each site (for example, mutates neutral residues to positive and / or negative charges, and mutates charged residues to oppositely charged and / or neutral residues). In some additional preferred embodiments, the methods relate to creating combinatorial “charge balanced” libraries of variants that contain useful mutations that change the charge of the enzyme in the desired direction and useful or neutral mutations that change the charge in the opposite direction, and then test the charge balanced libraries for the property of interest (c). Thus, the surface charge of the enzyme and the distribution of surface charge are optimized at the same time, and enzyme variants possessing improvements in many properties can be identified.

The methods of the present invention find application in improving the efficiency of various classes of enzymes as well as proteases (for example, amylases, cellulases, oxidases, kutinases, mannanases, pectinases, amylases, lipases, etc.). In fact, it is not intended that the present invention be limited by any particular enzyme or class of enzymes. In addition, the present invention finds application in optimizing the non-enzymatic properties of a protein that require a specific surface charge and charge distribution (for example, expression, binding to the cell surface, compliance to form a composition, etc.).

I. Production of protease variants with improved properties

A large number of site assessment libraries have been designed for NprE, in which each mature protein amino acid has been replaced by most other amino acids (see US Patent Application Serial No. 10/576331 and WO 2005/052146). These libraries were screened for stability in detergent and the effectiveness of purification from CMC. Then, the screening data was analyzed in relation to the effect of a change in charge introduced by mutations. When designing NprE variants, both increased stability and good cleaning efficiency from CMC (blood-milk-ink), which from the very beginning seemed mutually exclusive, are desirable. The present invention relates to means for the production of a more stable variant, which exhibits good cleaning efficiency from CMC.

Thus, the present invention relates to methods for establishing mutations that increase the stability or efficiency of purification from CMC without unduly reducing another parameter. As used herein, the phrase "excessively unfavorable" refers to properties of a protein having less than the required values. This term covers some low-potency proteins with neutral mutations (less than 80% of the potency of the parent protein or wild-type protein); low-efficiency proteins with non-harmful mutations (less than 50%); and mostly inactive proteins with harmful mutations (less than 5%). In some embodiments, the relative effectiveness values are expressed as an efficiency indicator (PE), which is the ratio of the effectiveness of a protein variant to the effectiveness of the parent protein. Then the charge mutations are balanced so that the final variant is from +1 to +3 with respect to the wild-type enzyme. In addition, the present invention relates to means for selecting amino acid residues that do not appear to interact in the three-dimensional structure of the enzyme, thereby minimizing the non-summability between multiple mutations.

As described herein, four NprE variants were constructed using the methods of the present invention. These variants contained from ten to eighteen mutations. As described in fine detail in the examples, these options showed increased stability and cleaning efficiency from CMC, similar to the wild-type enzyme.

II. The main methods for producing useful enzyme variants

As described herein, the relationship between the washing efficiency in the analysis with CMC micro-samples and the total charge on the surface of the enzyme was determined. The methods of the present invention find application in increasing the efficiency of various enzymes and proteins (for example, amylases, cellulases, oxidases, cutinases, mannanases, pectinases, lipases, proteases and other enzymes). Briefly, amino acid residues located on the surface of the wild-type enzyme that are approximately greater than 25% available for the solvent, approximately greater than 50% available for the solvent, or approximately greater than 65% available for the solvent, have been identified, and libraries were created to evaluate sites in which each wild-type residue was replaced by many other naturally occurring amino acids. In some embodiments, the construction of proteins on the surface of a molecule involves the substitution of neutral amino acid side chains with acidic or basic side chains and / or the replacement of positively charged side chains with neutral or negatively charged side chains, or vice versa. In addition, a change in the total charge of enzyme variants that showed improved cleansing efficiency from CMC was noted in order to determine this structural-functional relationship. In additional embodiments, when the optimal charge value was set for a given enzyme, natural isolates were screened to identify enzyme variants with the optimal charge value / charge distribution.

III. Production of improved amylase variants

A combinatorial charge library for AmyS-S242Q was constructed by introducing combinations of substitutions at four positions in a mature enzyme. This library was screened for the hydrolysis of BODIPY starch, the purification efficiency of micro samples with rice starch, and the expression of the enzyme. Then, the screening data was analyzed in relation to the effect of a change in charge introduced by mutations. When designing AmyS-S242Q variants, both increased protein expression and good enzyme efficiency are desirable, although it has been found that these properties are negatively correlated. The present invention relates to means for producing a variant with a higher level of expression, which exhibits good cleaning efficiency from rice starch. Thus, the present invention relates to methods for identifying mutations that increase expression or enzymatic activity without unduly reducing another parameter.

experimental part

The following examples are provided to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and should not be construed as limiting its scope.

In the following disclosure of experiments, the following abbreviations were used: ° C (degrees Celsius); rpm (revolutions per minute); H ₂ O (water); HCl (hydrochloric acid); A.K. and A.K. (amino acid); by. (couple of reasons); tbp (one thousand base pairs); kDa (kilodaltons); g (grams); mcg (micrograms); mg (milligram); ng (nanograms); μl (microliter); ml (milliliter); mm (millimeter); nm (nanometer); μm (micrometer); M (molar); mM (millimolar); μM (micromolar); ED (unit); V (volt); MM (molecular weight); sec (second); min (s) (minute / minutes); hour (s) (hour / hours); MgCl ₂ (magnesium chloride); NaCl (sodium chloride); OD ₂₈₀ (optical density at 280 nm); OD ₄₀₅ (optical density at 405 nm); OD ₆₀₀ (optical density at 600 nm); PAGE (polyacrylamide gel electrophoresis); EtOH (ethanol); PBS (phosphate buffered saline [150 mM NaCl, 10 mM sodium phosphate buffer, pH 7.2]); LAS (sodium lauryl sulfonate); SDS (sodium dodecyl sulfate); Tris (Tris (hydroxymethyl) aminomethane); TAED (N, N, N'N'-tetraacetylethylenediamine); BES (polyethersulfone); MES (2-morpholinoethanesulfonic acid, monohydrate; total weight 195.24; Sigma # M-3671); CaCl ₂ (calcium chloride, anhydrous; total weight 110.99; Sigma # C-4901); DMF (N, N-dimethylformamide, total weight 73.09, d = 0.95); Abz-AGLA-Nba (2-aminobenzoyl-L-alanylglycyl-L-leucyl-L-alanino-4-nitrobenzylamide, total weight 583.65; Bachem # H-6675, VWR catalog H 100040-598); SBG 1% ("Super broth with glucose"; 6 g of soyton [Difco], 3 g of yeast extract, 6 g of NaCl, 6 g of glucose); the pH was adjusted to 7.1 with NaOH before sterilization using methods known in the art; mass./about. (mass to volume); about / about (volume to volume); Npr and npr (neutral metalloprotease); SEQUEST® (SEQUEST database search engine, University of Washington); Npr and npr (neutral metalloprotease gene); nprE and NprE (neutral metalloprotease B. amyloliquefaciens ); PMN (purified metalloprotease MULTIFECT®); PMT (microtiter plate); MS (mass spectroscopy); IAA (paint removal index); TIGR (The Institute for Genomic Research, Rockville, MD); AATCC (American Association of Textile and Coloring Chemists); Procter & Gamble (Procter & Gamble, Inc., Cincinnati, OH); Beckman (Beckman Coulter, Inc., Fullerton, CA); Amersham (Amersham Life Science, Inc. Arlington Heights, IL); ICN (ICN Pharmaceuticals, Inc., Costa Mesa, CA); Pierce (Pierce Biotechnology, Rockford, IL); EMPA (Eidgenossische Material Prufungs und Versuch Anstalt, St. Gallen, Switzerland); CFT (Center for Test Materials, Vlaardingen, The Netherlands); Amicon (Amicon, Inc., Beverly, MA); ATCC (American Type Culture Collection, Manassas, VA); Becton Dickinson (Becton Dickinson Labware, Lincoln Park, NJ); Perkin-Elmer (Perkin-Elmer, Wellesley, MA); Rainin (Rainin Instrument, LLC, Woburn, MA); Eppendorf (Eppendorf AG, Hamburg, Germany); Waters (Waters, Inc., Milford, MA); Geneart (Geneart GmbH, Regensburg, Germany); Perseptive Biosystems (Perseptive Biosystems, Ramsey, MN); Molecular Probes (Molecular Probes, Eugene, OR); BioRad (BioRad, Richmond, CA); Clontech (CLONTECH Laboratories, Palo Alto, CA); Difco (Difco Laboratories, Detroit, MI); GIBCO BRL or Gibco BRL (Life Technologies, Inc., Gaithersburg, MD); Epicentre (Epicentre Biotechnologies, Madison, WI); Zymo Research (Zymo Research Corp., Orange, CA); Integrated DNA Technologies (Integrated DNA Technologies, Inc., Coralville, IA): New Brunswick (New Brunswick Scientific Company, Inc., Edison, NJ); Thermoelectron (Thermoelectron Corp., Waltham, MA); BMG (BMG Labtech, GmbH, Offenburg, Germany); Greiner (Greiner Bio-One, Kremsmuenster, Austria); Novex (Novex, San Diego, CA); Finnzymes (Finnzymes OY, Finland) Qiagen (Qiagen, Inc., Valencia, CA); Invitrogen (Invitrogen Corp., Carlsbad, CA); Sigma (Sigma Chemical Co., St. Louis, MO); DuPont Instruments (Asheville, NY); Global Medical Instrumentation or GMI (Global Medical Instrumentation; Ramsey, MN); MJ Research (MJ Research, Waltham, MA); Infors (Infors AG, Bottmingen, Switzerland); Stratagene (Stratagene Cloning Systems, La Jolla, CA); Roche (Hoffmann La Roche, Inc., Nutley, NJ); Ion Beam Analysis Laboratory (Ion Bean Analysis Laboratory, The University of Surrey Ion Beam Center (Guildford, UK); TOM (Terg-o-Meter); CMC (blood-milk-ink); BaChem (BaChem AG, Bubendorf, Switzerland) ; Molecular Devices (Molecular Devices, Inc., Sunnyvale, CA); MicroCal (Microcal, Inc., Northhampton, MA); Chemical Computing (Chemical Computing Corp., Montreal, Canada); NCBI (National Center for Biotechnology Information); GE Healthcare (GE Healthcare, UK).

Example 1

Analyzes

The following assays were used in the examples described below. Any deviation from the following protocols is noted in the examples. In these experiments, a spectrophotometer was used to measure the optical density of the products formed after completion of the reactions. A refractometer was used to measure the reflection coefficient of the samples.

A. Determination of protein

1. Analysis with BHC (bicinchoninic acid) to determine the protein content

In these assays, an analysis with BHC (Pierce) was used to determine protein concentration in protease samples on a microtiter plate (PMT) scale. The following chemical and reagent solutions were used in this analytical system: a protein reagent with BCC and Pierce dilution buffer (50 mM MES, pH 6.5, 2 mM CaCl ₂ , 0.005% TWEEN®-80). Used equipment PMT reader SpectraMAX (type 340). PMT were obtained from Costar (type 9017).

In the test, 200 μl of BHC reagent was instilled into each well, and then 20 μl of diluted protein was added. After thorough mixing, the PMT was incubated for 30 minutes at 37 ° C. Air bubbles were removed and the absorbance (OD) of the solution in the wells was read at 562 nm. To determine the protein concentration, the background readings were subtracted from the readings for the samples. Using OD ₅₆₂ values for protein standards (purified protease), a calibration curve was constructed. Protein concentrations in the samples were extrapolated from the calibration curve.

2. Bradford analysis to determine protein content

In these assays, a Bradford staining reagent analysis (Quick Start) was used to determine protein concentration in protease samples at the PMT scale.

The following chemical and reagent solutions were used in this analytical system: the Bradford stain quick start reagent (BIO-RAD Cat. No. 500-0205), dilution buffer (10 mM NaCl, 0.1 mM CaCl ₂ , 0.005% TWEEN®-80 ) We used Biomek FX Robot equipment (Beckman) and a SpectraMAX PMT reader (type 340; Molecular Devices). PMT were from Costar (type 9017).

In the test, 200 μl of Bradford staining reagent was instilled into each well, and then 15 μl of dilution buffer was added. Finally, 10 μl of filtered culture broth was added to the wells.

After thorough mixing, the PMTs were incubated for at least 10 minutes at room temperature. Air bubbles were removed and the OD wells were read at 595 nm. To determine the protein concentration, the background reading of the device (i.e., from uninoculated wells) was subtracted from the readings of the device for samples. The obtained OD ₅₉₅ values gave a relative measure of the protein content in the samples.

B. Analysis with microsamples to test the effectiveness of the protease

Equipment used included the Eppendorf Thermomixer and the SpectraMAX PMT reader (type 340). PMT were obtained from Costar (type 9017). Detergent preparations (liquid detergent for washing TIDE® 2 × Ultra Clean Breeze (Procter &Gamble); US washing conditions).

Milli-Q water was adjusted to a water hardness of 6 gpg (Ca / Mg = 3/1) and 0.78 g / L of TIDE® 2 × Ultra Clean Breeze detergent was added. The detergent was preliminarily subjected to heat treatment at 95 ° C for one hour to inactivate all the enzymes present in the composition. The washing solution was shaken for 15 minutes. Then 5 mM HEPES (free acid) was added and the pH was adjusted to 8.2.

Microsamples

Round 0.25 inch diameter microsamples were obtained from CFT Vlaardingen. Before cutting samples, matter (EMPA 116) was washed with water. One microtiter was placed in each well of a 96-well microtiter plate.

Testing method

The required cleaning solution was prepared as described above. After equilibrating the Thermomixer at 25 ° C, 190 μl of the washing solution was added to each well of the PMT containing the microsample. To this mixture, 10 μl of a diluted enzyme solution was added so that the final concentration of the enzyme was 1 μg / ml (established in the analysis with BChK). The PMT was sealed with tape and placed in an incubator for 30 minutes with shaking at 1400 rpm. After incubation under appropriate conditions, 100 μl of the solution from each well was transferred to fresh PMT. A new PMT containing 100 μl of solution per well was read at 405 nm using a SpectraMax PMT reader. Empty controls were also included, as well as a control containing a microsample and detergent, but not an enzyme.

Calculation of the effectiveness of CMC

The obtained optical density value was corrected for an empty value (i.e., obtained after incubation of the microsamples in the absence of the enzyme). The resulting optical density gave a measure of the hydrolytic activity of the test enzyme.

C. Stability Analysis with TIDE®

The stability of the wild-type and protease variants was measured after the incubation step in the presence of 25% heat-treated liquid detergent for washing TIDE® 2 × Ultra Clean Breeze. Initial and residual activity was determined using the AGLA assay described below, a fluorescent 96-well plate reader, an incubator / shaker (iEMS; Thermoelectron) and an incubator / shaker (Innova; New Brunswick (type 4230). PMT were from Costar (type 9017) and from Greiner (black tablets, type 655076).

Chemicals and reagents

The following chemical solutions and reagent solutions were used in this analytical system:

TIDE® 2 × Ultra Clean Breeze

125 g of TIDE® 2 × Ultra Clean Breeze (heat treated at 95 ° C as described above) dissolved in a mixture of 50 g of 50 mM HEPES pH 8.2 and 275 ml of water; TIDE® concentration was 27.7%, after dilution with 25% supernatant (hereinafter referred to as “TIDE®”)

MES dilution buffer

52.6 mm MES / NaOH, 2.6 mm CaCl ₂ , 0.005% TWEEN®-80, pH 6.5

Substrate AGLA, BaChem, cat. No. H-6675 or American Peptide Co., cat. No. 81-0-31

AGLA Substrate Solution

451 mg of AGLA was dissolved in 16 ml of N, N-dimethylformamide; this solution was poured into 304 ml of MES buffer (52.6 mm MES / NaOH, 2.6 mm CaCl ₂ , 0.005% TWEEN®-80, pH 6.5) with shaking.

Testing Methods

Stress free conditions

First, 20 μl of filtered culture broth was diluted in 180 μl of MES dilution buffer. Then 20 μl of this diluted broth was diluted in 180 μl of MES dilution buffer. Then, 10 μl of this dilution was diluted in 190 μl of a solution of AGLA substrate in a plate, previously heated to 25 ° C. All vials present were removed and the plate was measured according to the protease assay protocol with AGLA.

Stress conditions

First, 20 μl of the filtered culture broth was diluted in 180 μl of TIDE® washing solution and, after being pre-mixed in an iEMS shaker for 5 minutes, further incubated in an Innova shaker. The plate was incubated for a total of 60 minutes at 32 ° C, at 200 rpm. In addition, 20 μl of the filtered culture broth was diluted in 180 μl of TIDE® washing solution and, after being pre-mixed in an iEMS shaker for 5 minutes, further incubated in an Innova shaker. The plate was incubated for a total of 40 minutes at 20 ° C, at 200 rpm. Then, 20 μl of any of these solutions was diluted in 180 μl of MES dilution buffer, and 10 μl of this dilution was diluted in 190 μl of a solution of AGLA substrate in a plate preheated to 25 ° C. All air bubbles present were removed and the plate was measured according to the protease assay protocol with AGLA.

Calculations

Fluorescence measurements were carried out with excitation at 350 nm and emission at 415 nm. The spectrophotometer software calculated the reaction fluorescence increase rates for each well for the MEOF / min linear regression line:

Percentage of residual activity: (Slope under stressful conditions) * 100 / (Slope under stress-free conditions)

D. Protease assay with 2-aminobenzoyl-L-alanylglycyl-L-leucyl-L-alanino-4-nitrobenzylamide (Abz-AGLA-Nba)

The method provided below provides a certain degree of technical detail that makes reproducible protease assay data independent of time and place. Although the analysis is adaptable to the given laboratory conditions, any data obtained using the modified procedure should be given in accordance with the results obtained by the original method.

Neutral metalloproteases cleave the peptide bond between glycine and leucine in 2-aminobenzoyl-L-alanylglycyl-L-leucyl-L-alanino-4-nitrobenzylamide (Abz-AGLA-Nba). Free 2-aminobenzoyl-L-alanylglycine (Abz-AG) in solution has fluorescence with a maximum emission at 415 nm and a maximum excitation at 340 nm. Abz-AG fluorescence was quenched by nitrobenzylamide in the intact Abz-AGLA-Nba molecule.

In these experiments, the release of Abz-AG by proteolytic cleavage of Abz-AGLA-Nba was monitored by fluorescence spectroscopy (Ex. 340 / Isp. 415). The rate of manifestation of Abz-AG was a measure of proteolytic activity. Assays were performed without limiting the initial substrate speed.

A temperature controlled microplate agitator (e.g. Eppendorf Thermomixer) was required for reproducible assay results. Analyzed solutions were incubated to the desired temperature (e.g. 25 ° C) in a microplate mixer before adding the enzyme. Enzyme solutions were added to the plate in a stirrer, vigorously mixed and quickly transferred to a plate reader.

A spectrophotometer was required with the possibility of continuous data recording and linear regression analysis, along with temperature control (for example, SpectraMax M5, Gemini EM, Molecular Devices). The reader always maintained the required temperature (e.g. 25 ° C). The reader was tuned to detect top-read fluorescence and the excitation was set at 350 nm and emission at 415 nm without the use of a filter with a limited passband. PMT was set to medium sensitivity and 5 readings for each well. Autocalibration was turned on, but only for calibration before the first read. The analysis was measured for 3 minutes with a minimum reading interval in accordance with the number of wells selected for tracking. The reader was set to calculate the speed of MEOF / min (thousandths of a unit of relative fluorescence per minute). The number of readings used to calculate the speed (Vmax points) was set to the amount equivalent to 2 minutes, which was determined using the read interval (for example, when reading every 10 seconds, 12 points will be used to calculate the speed). The maximum EOF was set at 50,000.

All pipetting of the main enzyme and substrate solutions was performed with piston pipettes (Rainin Microman). Working solutions of the buffer, enzyme, and working solution for analysis were pipetted with single or multi-channel air-displaced pipettes (Rainin LTS) from test tubes, reagent containers, or microplates with stock solutions. To minimize reagent losses, a repeater (Eppendorf) is convenient when transferring the solution for analysis to the wells of a microplate when only a few wells are used. Automated pipetters such as Beckman FX or Cybio Cybi-well are also convenient when transferring enzyme solutions from a microplate with stock solution to a microplate for analysis in order to inoculate an entire microplate at the same time.

Reagents and solutions

52.6 mm MES / NaOH, 2.6 mm CaCl ₂ , pH 6.5 - MES buffer

MES acid (10.28 g) and 292 mg of anhydrous CaCl _{2 were} dissolved in approximately 900 ml of purified water. The solution was titrated with NaOH to a pH of 6.5 (at 25 ° C or with a temperature-sensitive pH probe). The pH adjusted buffer was adjusted to a total volume of 1 liter. The final solution was filtered through a 0.22 μm sterile filter and stored at room temperature.

48 mM Abz-AGLA-Nba in DMF - Abz-AGLA-Nba stock solution

Approximately 28 mg of Abz-AGLA-Nba was placed in a small tube. It was dissolved in DMF (volume varies depending on the weight of Abz-AGLA-Nba) and shaken for several minutes. The solution was stored at room temperature in a dark place.

50 mM MES, 2.5 mM CaCl ₂ , 5% DMF, 2.4 mM Abz-AGLA-Nba pH 6.5 - solution for analysis

One ml of Abz-AGLA-Nba stock solution was added to 19 ml of MES buffer and shaken. The solution was stored at room temperature in a dark place.

50 mm MES, 2.5 mm CaCl ₂ , pH 6.5 - buffer for dilution of the enzyme

This buffer was prepared by adding 5 ml of purified water to 95 ml of MES buffer.

50 mM MES, 2.5 mM CaCl ₂ , 5% DMF, pH 6.5 - substrate dilution buffer

5 ml of pure DMF was added to 95 ml of MES buffer. This buffer was used to determine kinetic parameters.

Enzyme Solutions

The enzyme stock solutions were diluted with the enzyme dilution buffer to a concentration of approximately 1 ppm. (1 μg / ml). MULTIFECT® Neutral Protease (wild-type NprE) was diluted to a concentration below 6 ppm. (6 μg / ml). Serial dilutions were preferred. At room temperature, the solutions were stable for 1 hour, but for longer periods of storage, the solutions were stored in ice.

Procedure

First, all buffer, basic and working solutions were made. Each dilution of the enzyme was analyzed three times, unless otherwise indicated. If the filling is incomplete, the wells in the stock microplate are filled with the working solution of the enzyme so that complete vertical columns are formed, starting from the left side of the tablet (to adjust to the tablet reader). An appropriate assay plate was filled in a similar manner. The microplate spectrophotometer was set up as previously described.

First, a 200 μl aliquot of the assay solution was placed in the wells of a 96-well microplate. The plate was incubated for 10 min at 25 ° C in a microplate mixer with temperature control, protected from light. The analysis was started by transferring 10 μl of the working solutions of the enzyme from the microplate with the stock solution to the microplate for analysis in a stirrer. Optimally, a 96-well pipette head was used, or in some experiments, an 8-well multichannel pipette was used to transfer first from the leftmost column. The solutions were vigorously mixed for 15 seconds (900 rpm in Eppendorf Thermomixer). The microplate for analysis was immediately transferred to a microplate spectrophotometer and the recording of fluorescence measurements was started upon excitation at 350 nm and emission at 415 nm. The spectrophotometer software recalculated the reaction rates for increasing fluorescence for each well into direct linear regression in MEOF / min. In some experiments, to equalize the temperature, a second plate was placed in the microplate mixer while the first plate was read.

The initial velocity levels linearly depended on the concentration of the product (i.e., fluorescence of the released 2-aminobenzoyl) up to 0.3 mM of the product, which corresponds to approximately 50,000 EOF in solution with an initial 2.3 mM Abz-AGLA-Nba with background fluorescence of approximately 22,000 EOF . Abz-AGLA-Nba was dissolved in DMF and used on the day of its manufacture.

EXAMPLE 2

NprE protease production in B. subtilis

This example describes experiments conducted to produce the NprE protease in B. subtilis . In particular, methods are provided that are used to transform the plasmid pUBnprE into B. subtilis . Transformation was performed as is known in the art (see, for example, WO 02/14490, and US patent application series No. 11 / 581.102). The DNA sequence provided below (the nprE leader sequence, the nprE sequence and the mature nprE DNA sequence from B. amyloliquefaciens ) encodes the NprE precursor protein.

In the above sequence, bold is the DNA that encodes the mature NprE protease, the standard font denotes the leader sequence (nprE leader sequence), and the underscore denotes pro-sequences (nprE sequences). The amino acid sequence provided below (NprE leader sequence, NprE pro sequence and mature NprE DNA sequence) (SEQ ID NO: 2) corresponds to a full-sized NprE protein. In this sequence, underscore indicates pro-sequence and bold indicates mature NprE protease.

The mature NprE sequence is shown in SEQ ID NO: 3. This sequence was used as the basis for the manufacture of the libraries of the variants described herein.

The expression vector pUBnprE was constructed by amplification of the nprE gene from B. amyloliquefaciens chromosomal DNA using PCR using two specific primers:

Oligo AB 1740: CTGCAGGAATTCAGATCTTAACATTTTTCCCCTATCATTTTTCCCG (SEQ ID NO: 4); and

Oligo AB 1741: GGATCCAAGCTTCCCGGGAAAAGACATATATGATCATGGTGAAGCC (SEQ ID NO: 5)

PCR was performed in a thermal cycler with high-precision DNA polymerase Phusion (Finnzymes). The PCR mixture contained 10 μl of 5 × buffer (Finnzymes Phusion), 1 μl of 10 mM dNTP, 1.5 μl of DMSO, 1 μl of each primer, 1 μl of Phusion Finnzymes DNA polymerase, 1 μl of a 50ng / μl chromosomal DNA solution, 34.5 μl of MilliQ water. The following PCR protocol was used: 1) 30 sec at 98 ° C; 2) 10 sec at 98 ° C; 3) 20 sec at 55 ° C; 4) 1 min at 72 ° C; 5) 25 cycles from stages 2 to 4; and 6) 5 min at 72 ° C.

As a result, a 1.9 kb DNA fragment was obtained, which was digested with restriction enzymes Bgl II and BclI. Multicopy vector pUB110 Bacillus (. See, e.g., Gryczan, J Bacteriol, 134: 318-329 [1978]) was digested with restriction enzyme Bam HI, then PCR fragment × Bgl II × BclI was ligated to the vector pUB110 × Bam HI to form the expression vector pUBnprE.

pUBnprE was transformed into a B. subtilis strain (Δ aprE , Δ nprE , oppA, Δ spoIIE , degUHy32 , Δ amyE : :( xylR, pxylA-comK ). Transformation into B. subtilis was performed as described in WO 02/14490. Selective growth B. subtilis transformants carrying the pUBnprE vector were carried out in shake flasks containing 25 ml of MBD medium (medium of a specific composition based on MOPS) with 20 mg / L neomycin. The MBD medium was prepared mainly as is known in the art (see , Neidhardt et al., J Bacteriol, 119: 736-747 [1974]), with the exception that NH ₄ Cl ₂ , FeSO ₄ and CaCl _{2 were} excluded from the stock medium, 3 mM K ₂ HPO _{4 was used} and the stock medium added 60 mM urea, 75 g / l glucose and 1% soyton. Also, micronutrients were prepared as a 100 × basic solution containing 400 mg FeSO ₄ ∙ 7H ₂ O, 100 mg MnSO ₄ ∙ H ₂ O, 100 mg ZnSO ₄ in one liter ∙ 7H ₂ O, 50 mg CuCl ₂ ∙ 2H ₂ O, 100 mg CoCl ₂ ∙ 6H ₂ O, 100 mg NaMoO ₄ ∙ 2H ₂ O, 100 mg Na ₂ B ₄ O ₇ ∙ 10H ₂ O, 10 ml 1 M CaCl ₂ and 10 ml of 0.5 M sodium citrate. The culture was incubated for three days at 37 ° C in an incubator / shaker (Infors). This cultivation led to the formation of secreted NprE protease with proteolytic activity, which was demonstrated by protease assays. The gel analysis was performed using NuPage Novex 10% Bis-Tris gels (Invitrogen, Cat. No. NP0301BOX). To prepare samples for analysis, 2 volumes of the supernatant were mixed with 1 volume of 1 M HCl, 1 volume of 4 × LDS sample buffer (Invitrogen, cat. No. NP0007) and 1% PMSF (20 mg / ml) and subsequently heated for 10 minutes at 70 ° C. Then, 25 μl of each sample was loaded into the gel along with 10 μl of SeeBlue Plus2 pre-stained protein standards (Invitrogen, Cat. No. LC5925). The results clearly showed that the nprE cloning strategy described in this example is suitable for the production of active NprE in B. subtilis .

Example 3

Creation of libraries for the assessment of sites (BOS)

This example describes the methods used to create the nprE BOS.

The pUBnprE vector containing the nprE expression cassette described above served as a template DNA. This vector contained a unique Bgl II restriction site, which was used to create libraries for site evaluation. Briefly, to create a library for evaluating nprE sites, three PCR reactions were performed, including two PCR mutagenesis for introducing the mutated codon of interest into the mature DNA sequence nprE, and a third PCR for combining the two PCR mutagenesis in order to construct the expression vector pUBnprE, containing the desired mutated codon in the mature nprE sequence.

The mutagenesis method was based on a codon-specific mutation approach, in which all possible mutations were simultaneously created in a specific triple DNA using direct and reverse oligonucleotide primers from 25 to 45 nucleotides in length, including a specially designed triplet NNS DNA sequence (N = A, C , T or G; and S = C or G), which corresponded to the sequence of the codon to be mutated, and guaranteed random insertion of nucleotides into the specific codon of mature nprE. The number indicated in the name of the primer corresponds to the position of a particular codon of mature nprE. Evaluated sites included: 4, 12, 13, 14, 23, 24, 33, 45, 46, 47, 49, 50, 54, 58, 59, 60, 65, 66, 87, 90, 96, 97, 100, 186, 196, 211, 214, 228, and 280. An exemplary list of primer sequences is described in US Patent Application Serial No. 11 / 581.102.

To create libraries used to assess sites, two additional primers containing the Bgl II restriction site together with a part pUBnprE DNA sequence flanking the restriction site Bgl II. These primers were produced in Invitrogen (scale 50 nanomoles, desalted):

pUB-BglII-FW GTCAGTCAGATCTTCCTTCAGGTTATGACC (SEQ ID NO: 6); and

pUB-BglII-RV GTCTCGAAGATCTGATTGCTTAACTGCTTC (SEQ ID NO: 7).

The creation of each BOS began with two primary PCR amplifications using the primer pUB-BglII-FW and the specific reverse primer nprE for mutagenesis. For the second PCR, the primer pUB-BglII-RV and the specific forward primer nprE for mutagenesis were used (primers for direct and reverse mutagenesis corresponding to the positions of the mature nprE codon).

Mutations were introduced into the mature nprE sequence using high-precision Phusion DNA polymerase (Finnzymes; Cat. No. F-530L). All PCR was performed according to the Finnzymes protocol attached to the polymerase. PCR conditions for primary PCR were:

for primary PCR 1:

pUB-BglII-FW primer and NPRE specific reverse primer for mutagenesis - both 1 μl (10 μM);

for primary PCR 2:

pUB-BglII-RV primer and NPRE specific direct primer for mutagenesis - both 1 μl (10 μM); together with

5 × Phusion HF Buffer 10 μl 10 mm dNTP mixture 1 μl DNA polymerase Phusion 0.75 μl (2 units / μl) DMSO, 100% 1 μl DNA matrix pUBnprE 1 μl (0.1-1 ng / μl) Distilled, autoclaved water up to 50 μl

PCR program: 30 seconds at 98 ° C, 30 × (10 seconds at 98 ° C, 20 seconds at 55 ° C, 1.5 minutes at 72 ° C) and 5 min at 72 ° C were performed in a Peltier PTC-200 thermal cycler (MJ Research). PCR experiments led to the formation of two fragments of approximately 2 to 3 kb, which had an overlapping region of approximately 30 nucleotides in the region of the mature NprE codon of interest. Fragments were combined in a third PCR reaction using the two above fragments and the forward and reverse Bgl II primers. The PCR coupling reaction was carried out in the following solution: pUB-BglII-FW primer and pUB-BglII-RV primer, both 1 μl (10 μM) each

5 × Phusion HF Buffer 10 μl 10 mm dNTP mixture 1 μl DNA polymerase Phusion 0.75 μl (2 units / μl) DMSO, 100% 1 μl Mixture for primary PCR reaction 1 1 μl Mixture for primary PCR reaction 2 1 μl Distilled, autoclaved water up to 50 μl

The program for connecting PCR was as follows: 30 seconds at 98 ° C, 30 × (10 seconds at 98 ° C, 20 seconds at 55 ° C, 2:40 minutes at 72 ° C) and 5 minutes at 72 ° C in a Peltier thermal cycler PTC-200 (MJ Research).

Amplified linear fragment 6.5 kb purified using a QIAQUICK® PCR purification kit (Qiagen, cat. No. 28106) and digested with restriction enzyme Bgl II to form sticky ends at both ends of the connecting fragment:

- 35 μl of purified linear DNA fragment

- 4 μl REACT® 3 buffer (Invitrogen)

- 1 μl Bgl II, 10 units / ml (Invitrogen)

Reaction conditions: 1 hour, 30 ° C.

Ligation of the fragment cleaved with Bgl II and purified using the QIAQUICK® PCR purification kit (Qiagen, cat. No. 28106), led to the formation of ring and multimeric DNA containing the desired mutation:

- 30 μl of purified, cleaved using Bgl II DNA fragment

- 8 μl of T4 DNA ligase buffer (Invitrogen Cat. No. 46300-018)

- 1 μl T4 DNA ligase, 1 unit / μl (Invitrogen Cat. No. 15224-017)

Reaction conditions: 16-20 hours, at 16 ° C.

Subsequently, the dopant mixture was transformed into B. subtilis strain (Δ aprE , Δ nprE , oppA , Δ spoIIE , degUHy32 , Δ amyE : :( xylR, pxylA-comK ). Transformation into B. subtilis was performed as described in WO 02/14490. For each library, 96 individual colonies were collected and grown in MOPS with neomycin and 1.25 g / L yeast extract for sequence analysis (BaseClear) and screening purposes. Each library included no more than 19 site-specific nprE variants.

Variants were created by growing transformants from BF B. subtilis in 96-well PMT at 37 ° C for 68 hours in MBD medium with 20 mg / L neomycin and 1.25 g / L yeast extract.

Example 4

Creating protease variants using QUICKHANGE® mutagenesis

This example describes alternative methods for creating biofeedback nprE, although the methods provided herein are suitable for producing biofeedback of other enzymes of interest (e.g. Asp ). As in Example 3 above, the pUBnprE vector containing the nprE expression cassette served as the source of the template DNA to generate the biofeedback nprE and NprE variants. The main difference between the two methods is that this method requires amplification of the whole vector using complementary primers for site-directed mutagenesis.

Materials

Bacillus strain containing the vector pUBnprE

Qiagen Plasmid Midi Kit (Qiagen Cat. No. 12143)

Ready-Lyse Lysozyme (Epicentre Cat. No. R 1802M)

dam Methylase Kit (New England Biolabs Cat. No M0222L)

Zymoclean Gel DNA Recovery Kit (Zymo Research Cat. No D4001)

Site-specific nprE primers for mutagenesis, scale 100 nmol, 5 'phosphorylated, purified by PAGE (Integrated DNA Technologies)

QUIKCHANGE® Multi Site-Directed Mutagenesis Kit (Stratagene Cat. No. 200514)

MJ Research PTC-200 Peltier Thermal Cycler (Bio-Rad Laboratories)

1.2% Agarose E-gels (Invitrogen Cat. No. G5018-01)

TempliPhi Amplification Kit (GE Healthcare Cat. No. 25-6400-10)

Competent B. subtilis cells (∆ aprE , ∆ nprE , oppA , ∆ spoIIE , degUHy32 , ∆ amyE : :( xylR, pxylA-comK )

Ways

To obtain pUBnprE plasmids containing one mutation (identified by biofeedback screening of nprE as described above in Example 4 and US Patent Application Serial No. 11 / 581.102), a separate colony of each Bacillus strain of interest was used to inoculate a 5 ml LB + 10 tube ppm neomycin (e.g. starting culture). The culture was grown at 37 ° C, with shaking at 225 rpm for 6 hours. Then 100 ml of fresh LB + 10 ppm. neomycin was inoculated with 1 ml starter culture. This culture was grown overnight at 37 ° C, with shaking at 225 rpm. After this incubation, the cell pellet was collected using sufficient centrifugation to provide a cell pellet. The cell pellet was resuspended in 10 ml P1 buffer (Qiagen Plasmid Midi Kit). Then 10 μl of Ready-Lyse Lysozyme was added to the resuspended cell pellet and incubated at 37 ° C for 30 minutes. The Qiagen Plasmid Midi Kit protocol was continued using 10 ml of P2 and P3 buffers to allow for increased cell culture volume. After isolation of each plasmid pUBnprE containing a separate mutation in nprE from Bacillus , the concentration of each plasmid was determined. The dam plasmids were then methylated using the dam Methylase Kit (New England Biolabs) according to the manufacturer's instructions to methylate approximately 2 μg of each pUBnprE plasmid per tube. Zymoclean Gel DNA recovery kit was used to purify and concentrate dam- methylated pUBnprE plasmids. Then, the amount of dam- methylated pUBnprE plasmids was determined and diluted to a working concentration of 50 ng / μl each. A mixture of site-specific mutagenesis primers was prepared separately for each reaction. For example, when using the pUBnprE T14R plasmid as the template source, a tube with mixed site-specific mutagenesis primers will contain 10 μl nprE-S23R, 10 μl nprE-G24R, 10 μl nprE-N46K and 10 μl nprE-T54R (all primers are 10 μM each). The PCR reaction using the QuikChange Multi Site-Directed Mutagenesis Kit (Stratagene) was performed according to the manufacturer's instructions, for example, 1 μl dam- methylated plasmid pUBnprE containing one mutation (50 ng / μl), 2 μl nprE site-specific primers for mutagenesis (10 μM), 2.5 μl 10 × QuikChange Multi Reaction buffer, 1 μl dNTP mixture, 1 μl QuikChange Multi enzyme mixture (2.5 U / μl), and 17.5 μl distilled, autoclaved water to provide the final reaction volume 25 μl NprE variant libraries were amplified using the following conditions: 95 ° C for 1 min (1st cycle only), then 95 ° C for 1 min, 55 ° C for 1 min, 65 ° C for 13.5 min and 29 repetitions of the cycle. The reaction product was stored at 4 ° C. overnight. The reaction mixture was then digested with restriction enzyme Dpn I (supplied with QUIKCHANGE® Multi Site-Directed Mutagenesis Kit) to digest the parent plasmid pUB-nprE using the manufacturer's protocol, i.e. 1.5 μl of the restriction enzyme Dpn I was added to each tube and incubated at 37 ° C for 3 hours; then 2 μl of the Dpn I- digested PCR reaction product was analyzed for 1.2% E-gel to ensure that the PCR reaction was complete and that the parent matrix was destroyed. Then, to create a large amount of DNA to increase the size of the nprE multivariate library, TempliPhi was used as rolling ring amplification using the manufacturer's protocol, i.e. 1 μl PCR-processed in Dpn I QuikChange Multi Site-Directed Mutagenesis, 5 μl TempliPhi sample buffer, 5 μl TempliPhi reaction buffer and 0.2 μl TempliPhi enzyme mixture, to form a final reaction volume of ~ 11 μl; incubated at 30 ° C for 3 hours; the TempliPhi reaction was diluted by adding 200 μl of distilled, autoclaved water and, in summary, was vigorously mixed. Then, 1.5 μl of the diluted TempliPhi material was transformed into competent B. subtilis cells, and nprE multivariates were selected for use with LA + 10 ppm plates. neomycin + 1.6% skim milk. Colonies were selected and then sequenced to identify various combinations of NprE library variants.

Table 4-1 shows the names of the primers and the sequences used in these experiments. Integrated DNA Technologies synthesized all primers (100 nmol scale, 5'-phosphorylated and purified in PAGE). Additional primers for mutagenesis are described in US patent application Ser. No. 11/581102). The sites evaluated included: 4, 12, 13, 23, 45, 49, 50, 54, 59, 60, 65, 82, 90, 110, 119, 128, 129, 130, 135, 136, 137, 138, 139, 140, 151, 152, 155, 179, 190, 197, 198, 199, 204, 205, 214, 216, 217, 218, 219, 220, 221, 222, 224, 243, 244, 260, 261, 263, 265, 269, 273, 282, 285, 286, 289, 293, 296, 297 and 299.

Example 5

Expression, fermentation, purification and characterization of protease variants

This example describes the methods used for expression, fermentation and purification of proteases of transformed B. subtilis from the previous examples.

Recombinant Bacillus subtilis was cultured using traditional periodic fermentation in a nutrient medium. One glycerol vessel of a B. subtilis culture containing neutral metalloprotease B. amyloliquefaciens was used to inoculate 600 ml of 1% SBG medium containing 200 mg / l chloramphenicol. Cultures were grown for 36-48 hours at 37 ° C, after which time the culture fluid was collected by centrifugation at 12,000 rpm, as is known in the art. This procedure was performed twice. Final enzyme concentrations obtained for 48 hour cultures ranged between approximately 1.4 and 2 g / L. After 36 hours of incubation at 37 ° C, the fermentation broth was collected and centrifuged at 12,000 rpm (SORVALL® centrifuge, model RC5B). Secreted neutral metalloproteases were isolated from the culture fluid and concentrated approximately 10-fold using an Amicon filter system 8400 with BES (polyethersulfone) with a threshold of 10 kDa.

The concentrated supernatant was dialyzed overnight at 4 ° C. against 25 mM MES buffer, pH 5.4, containing 10 mM NaCl. The dialysate was then loaded onto a Poros HS20 cation exchange column (total volume ~ 83 ml; binding capacity ~ 4.5 g protein / ml column; water) as described below. The column was pre-equilibrated using 25 mM MES buffer, pH 5.4, containing 10 mM NaCl. Then, approximately 200-300 ml of the sample was loaded onto the column. Bound protein was eluted using a pH gradient of 5.4 to 6.2 through a 10 column volume of MES buffer. Protein elution was between pH 5.8 and 6.0, and was evaluated using proteolytic activity as described herein and 10% (w / v) NUPAGE® SDS-PAGE (Novex). Then the fractions containing the neutral protease were combined. 3: 1 calcium and zinc chlorides were added before adjusting the pH to 5.8. For protein purification, Perceptive Biosystems BIOCAD® Vision (GMI) was used.

The homogeneity of the purified protein was determined, evaluated using 10% (w / v) NUPAGE® SDS-PAGE, with a purity exceeding 95%. Typically, purified preparations showed negligible serine protease activity when evaluated using standard protease analysis with substrate N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (Bachem). This analysis was performed in the format of a microtiter plate (PMT) (96 wells) using 100 mM Tris-HCl buffer, pH 8.5, containing 10 mM CaCl ₂ and 0.005% TWEEN®-80. The substrate (p-AAPF NA) was prepared by preparing a stock solution of 160 mM in DMSO (dimethyl sulfoxide) (100 mg / ml) and diluting this stock solution 100 times with Tris-HCl buffer containing CaCl ₂ and 0.005% TWEEN®-80. Then, 10 μl of a dilute protease solution (dilutions were made using 100 mM Tris-HCl buffer, pH 8.5, containing 10 mM CaCl ₂ and 0.005% TWEEN®-80) were added to 190 μl of a 1 mg / ml p-AAPF NA solution. The sample was mixed for 5 minutes and kinetic changes were read at 410 nm for 2 to 5 minutes. The angular response coefficient was measured and used as an indicator of the amount of serine protease activity. A protein storage composition was prepared using 25 mM MES buffer, pH 5.8, containing 1 mM zinc chloride, 4 mM calcium chloride and 40% propylene glycol.

Example 6

Compensation of the effect of mutations on the activity and stability of proteases

This example describes multiple substitution protease variants designed to optimize two conflicting enzyme properties. Table 6-1 shows how the change in charge was calculated:

Table 6-1
Charge change calculation Amino Acid in the Wild Mutant amino acid Charge change Neutral Neutral 0 Positive Neutral -one Negative Neutral +1 Neutral Positive +1 Positive Positive 0 Negative Positive +2 Neutral Negative -one Positive Negative -2 Negative Negative 0

As established in the development process of the present invention, the average stability decreased with increasing positive charge. However, the efficiency of cleaning from CMC increased with increasing positive charge. The optimal value of the cleaning efficiency from CMC under testing conditions was achieved when the charge changed by approximately +1.

Increased stability and cleaning efficiency from CMC are desirable in the engineered version of NprE. However, these properties appear to be conflicting properties. As established in the development process of the present invention, when applying the methods of the present invention, it is possible to create a more stable version without significant compromise with the efficiency of purification from CMC by selective combining of single mutations. The strategy described herein has been successfully applied to produce multiple-substitution NprE variants that have improvements to the first property (e.g., stability as a primary property), while improving or not degrading the second property (e.g., cleaning efficiency from CMC as a secondary property ) In particular, the following criteria were used to select the substitutions of interest. Mutations were selected that provide increased stability in the detergent or the efficiency of purification from CMC without excessive reduction of another parameter. In addition, charge mutations were compensated, so that the final version had a total charge of +1 to +3 with respect to the wild-type enzyme. In addition, amino acid residues that did not appear to be involved in the three-dimensional structure to minimize the non-summability of multiple mutations.

In the process of developing the present invention, four variants were constructed, each containing from ten to eighteen substitutions. These options are shown in table 6-3. It is important that these multiple-substitution variants had increased detergent stability and similar cleaning efficiency compared to the wild-type enzyme. This was accomplished by introducing negative and neutral charge-stabilizing mutations that were not too harmful for the purification of MSCs, while compensating for positive charge mutations that affect efficiency, which did not affect stability too much. An additional set of pairs of options was constructed. The first of each pair had a stabilizing negative charge mutation, which reduced the purification of MSCs, and the second of each pair had a compensating positive charge mutation, which restored the cleaning of MSCs and at the same time maintained stability at the level of the above wild type. The cleaning performance and stability values for these options are also shown in Table 6-4.

Example 7

Compensation of the effect of mutations on the activity and expression of amylase

This example illustrates that two conflicting enzyme properties can be optimized simultaneously by introducing multiple amino acid substitutions.

This example describes how experiments were conducted to produce α-amylase of Bacillus stearothermophilus (also referred to herein as AmyS), a mutated truncated form of AmyS amylase (S242Q having a 29 amino acid deletion, also referred to as S242Q in this document), its variants in B . subtilis . Transformation was performed as is known in the art (see, for example, WO 02/14490). Briefly, a gene encoding parental amylases was cloned into a pHPLT expression vector that contains a LAT promoter sequence (PLAT) encoding a LAT signal peptide (preLAT) followed by Pstl and Hpal restriction sites for cloning.

Creation of KZB AmyS-S242Q B. stearothermophilus

AmyS-S242Q plasmid DNA was isolated from the transformed B. subtilis strain (genotype: Δ aprE , Δ nprE , amyE :: xylRPxylAcomK-phleo ) and sent to DNA2.0 Inc. as a matrix for the design of KZB. Fulfilled DNA2.0 Inc. request (Mountain View, CA) to create a positional library in each of the four AmyS-S242Q amylase sites (S242Q). Options were supplied as glycerol stock solutions in 96-well plates. The AmyS S242Q combinatorial charge library was designed by identifying the following four residues: Gln-97, Gln-319, Gln-358, and Gln-443. For four sites, a KZB of 81 members was created, performing all combinations of the three possibilities in each site: wild-type, arginine, or aspartic acid.

The amino acid sequence of mature truncated S242Q substituted amino acid amylase, shown in italics, was used as the basis for the production of the libraries of variants described herein:

Amylase expression - scale 2 ml

B. subtilis clones containing AmyS, S242Q, or AmyTS23t expression vectors were replicated using a 96-well steel replicator from glycerol stock solutions to 96-well culture plates (BD, 353075) containing 150 μl LB medium + 10 μg / ml neomycin, which were grown overnight at 37 ° C, 220 rpm in a humidified chamber. An aliquot of 100 μl from the overnight culture was used to inoculate 2000 μl of a medium of a specific composition + 10 μg / ml neomycin in 5 ml plastic culture tubes. The culture medium was enriched with a semi-determined medium based on MOPS buffer, with urea as the main source of nitrogen, glucose as the main source of carbon and the addition of 1% soyton and 5 mM calcium for active cell growth. Culture tubes were incubated at 37 ° C, 250 rpm, for 72 hours. After this incubation, the culture broths were centrifuged for 10 minutes at 3000 × g. The supernatant solution was decanted into 15 ml polypropylene conical tubes and 80 μl aliquots of each sample were taken into 96-well plates for protein quantification.

Determination of Amylase Concentration by Antibody Titration

As described herein, the concentration and specific activity of α-amylase was determined by titration with an inhibitory polyclonal antibody. It was found that polyclonal antibodies induced against Bacillus stearothermophilus α-amylase (AmyS) strongly inhibit AmyS and α-amylase from TS23 Bacillus sp. (e.g., binding is strong enough to give a linear loss of activity during titration). Therefore, this antibody can be used to measure the concentration of the enzyme, which in turn will be used to calculate specific activity. In summary, the degree of inhibition of an enzyme caused by several known antibody concentrations is measured. Based on this information, the antibody concentration necessary for complete inhibition is extrapolated, which is equivalent to the concentration of the enzyme in the sample. Α-amylase activity and inhibition were measured using fluorogenic BODIPY-starch assay. The buffer was 50 mM MOPS, pH 7.0, containing 0.005% Tween-80.

A polyclonal antibody directed against purified AmyS was induced in the rabbit and purified by standard methods. The empirical value of the "apparent concentration" of the antibody stock solution was determined by measuring the inhibition of an AmyS sample with known specific activity. An antibody sample was then used to determine the concentration and specific activity of the AmyS and TS23t variants. These values were used to create normalized 96-well main enzyme plates in which all variants were diluted to a total concentration.

BODIPY Starch Assays for Determining Amylase Activity

BODIPY starch analysis was performed using the EnzChek® Ultra Amylase Assay Kit (E33651, Invitrogen). A stock solution of starch substrate DQ 1 mg / ml was prepared by dissolving the contents of a vessel containing a lyophilized substrate in 100 μl of 50 mM sodium acetate buffer at pH 4.0. The vessel was vigorously stirred for approximately 20 seconds and left at room temperature, in the dark, with periodic stirring until dissolved. 900 μl of assay buffer (50 mM sodium acetate with 2.6 mM CaCl ₂ pH 5.8) was added and the vessel was vigorously stirred for 20 seconds. The substrate solution was stored at room temperature, in the dark, until it was ready for use, or at 4 ° C. For analysis, 100 μg / ml of the working substrate solution DQ was prepared from 1 mg / ml of the substrate solution in the analysis buffer. 190 μl of a 100 μg / ml substrate solution was added to each well in a 96-well flat-bottom microtiter plate. 10 μl of enzyme samples were added to the wells, mixed for 30 seconds using a stirrer with a thermostat at 800 rpm. A tangled sample that contained only buffer and substrate (no enzyme) was included in the analysis. The rate of change of fluorescence intensity was measured (excitation: 485 nm, emission: 520 nm) in a fluorescent microtiter plate reader at 25 ° C for 5 minutes.

Α-amylase binding

Amylase variants were incubated with microsamples with or without CS-28 rice starch under standard washing conditions for 30 minutes. The amount of free enzyme was measured by analysis with BODIPY-starch. The fraction of the enzyme associated with microsamples was calculated as follows:

Bound fraction = (Enzyme activity in the absence of a sample - Enzyme activity in the presence of a sample) / (Enzyme activity in the absence of a sample)

results

As established during the development of the present invention, the average expression value of AmyS-242Q decreased with increasing positive charge. However, the specific hydrolysis of BODIPY starch increased with increasing positive charge. Increased recombinant amylase expression and starch hydrolysis are desirable in the engineered version of AmyS-242Q, suitable for liquefying starch in the production of fuel ethanol or, for example, for cleaning in washing applications. However, these properties appear to be conflicting properties. As stated in the development process of the present invention, using the methods of the present invention, it is possible to create a variant of amylase with higher expression without a significant compromise with hydrolysis of starch by selective combining of individual mutations. The strategy described herein has been successfully used to create and select multiple AmyS-242Q variants with multiple substitutions that have improvements to the first property (e.g., expression as a primary property), while improving or not degrading the second property (e.g., starch hydrolysis as secondary property).

In addition, in contrast to the average expression of the AmyS-242Q variants, the purification of rice starch microsamples increased with increasing positive charge. Increased expression and purification efficiency of recombinant amylase are desirable in the construction of the AmyS-242Q variant. However, apparently, these properties are also conflicting properties. As established during the development of the present invention, when applying the methods of the present invention, it is possible to create a variant of amylase with higher expression without significant compromise with the efficiency of purification by selective combining of individual mutations. The strategy described in this document has been successfully used to create and select multiple-choice AmyS-242Q variants that have improvements to the first property (e.g., expression as a primary property), while improving or not degrading the second property (e.g., cleaning micro-samples with rice starch in as a secondary property).

In particular, eighteen members of the AmyS-S242Q combinatorial charge library (KZB), containing variants having combinations of one to four substitutions of charged residues, were tested for expression in shake tubes, hydrolysis of BODIPY starch, and rice starch decontamination activity. AmyS-S242Q winners are shown in Tables 7-1 and 7-2. It is important that the multiple substitution options from Table 7-1 have equal or improved expression and equal or improved hydrolysis of BODIPY starch compared to the parent enzyme. Similarly, multiple substitution variants from Table 7-2 have equal or improved expression and equal or improved rice starch decontamination activity compared to the parent enzyme.

In general, since enzyme activity and enzyme synthesis have different charge dependencies (see FIGS. 2A, 2B, 3A and 3B), they have a negative correlation (see FIGS. 1A and 1B). However, there are many options that have both improved expression and improved activity, and analysis of the library, thus, makes it possible to identify them.

Although the method was demonstrated by the example of amylase, it is applicable to other classes of enzymes, such as proteases, lipases, cellulases, transferases and pectinases. In addition, any combination of two or more properties can be analyzed simultaneously, for example, expression, activity, binding, thermal stability, stability in the presence of a detergent and / or chelator.

All patents and publications noted in the description show the level of specialists in the field to which the invention belongs. Specialists in this field will easily appreciate the fact that the present invention is well suited to perform the tasks and obtain the specified results and advantages, as well as what is associated with them. The compositions and methods described herein show exemplary preferred embodiments and are not intended to limit the scope of the invention. Professionals in this field it is obvious that the variation of substitutions and modifications can be made in the invention described in this document without departing from the scope and essence of the invention.

Illustratively described herein, the invention may be practiced appropriately in the absence of any element or elements, limitations or restrictions that are not specifically described herein. The terms and expressions that were used are used as description terms, but not for limitation, and there is no intention when using such terms and expressions to exclude equivalents of the properties shown and described or their fragments, but it has been established that various modifications are possible within the scope of the claimed scope of invention. Thus, it should be understood that although the present invention has been specifically disclosed using preferred embodiments and optional properties, those skilled in the art may resort to modifying and modifying the ideas disclosed herein, and that such modifications and variations are deemed to be encompassing. within the scope of this invention as defined herein.

In the present document, the invention has been described broadly and generally. Increasingly narrower varieties and less general grouping fall within the general disclosure, also forming part of the invention. This includes a general description of the invention with the exception or negative restriction that removes any object from the genus, regardless of whether the excluded object is specifically indicated in this document or not.

Claims (13)

1. A method for identifying improved protein variants, including in functional order:
a) testing multiple protein variants with single substitutions in the first test of the first property and in the second test of the second property, where the parent protein property is assigned a value of 1.0 in each test, a favorable first or second property has a value greater than 1.0, and excessively unfavorable the first or second property has a value of less than about 0.80, wherein said parent protein is a protease, said first property is stability, and said second property is pre sets washing efficiency;
b) identification of a substitution, in at least one variant protein with single substitutions, which is associated with a favorable first property and which is not associated with an excessively unfavorable second property;
c) identifying a substitution in at least one variant protein with single substitutions that is associated with a favorable second property and which is not associated with an overly unfavorable first property; and
d) introducing a substitution from step b) and a substitution from step c) into a protein to produce a multi-substitution protein variant, where
(i) the substitution from step b) and the substitution from step c) do not interact in the three-dimensional structure of said parent protein, and
(ii) at least one of the substitutions contains a change in the total charge equal to 0, -1 or -2 relative to the parent enzyme, and at least one of the substitutions contains a change in the total charge equal to +1 or +2 in relation to the parent enzyme;
e) testing the specified variant of the protein with multiple substitutions in the specified first test and in the specified second test; and
f) identification of the indicated variant of the protein with multiple substitutions as a variant of the protein with improved stability and washing efficiency, if it reaches a value exceeding 1.0 in both the first and second specified tests, or a value exceeding 1.0 in the first test, and values from 0.80 to 1.0 in the second test. 2. The method according to claim 1, where the specified favorable first or second property has a value of more than approximately 1.2. 3. The method according to claim 1, where the specified excessively unfavorable first or second property has a value of less than approximately 0.60. 4. The method according to claim 3, where the specified excessively unfavorable first or second property has a value of less than approximately 0.40. 5. The method according to claim 1, where the specified stability includes stability in detergent compositions and washing efficiency includes the effectiveness of washing the blood-milk-ink (CMC). 6. The method according to claim 1, where the specified washing efficiency is tested in a powder or liquid detergent composition having a pH between about 5 and about 12. 7. The method according to claim 1, where the specified washing efficiency is tested in cold water in a liquid detergent having a basic pH. 8. The method according to claim 1, where the specified protease is selected from a neutral metalloprotease and serine protease. 9. The method of claim 8, wherein said serine protease is subtilisin. 10. The method of claim 8, wherein said neutral metalloprotease is a neutral metalloprotease derived from a member of the Bacillaceae family. 11. The method according to claim 1, where the substitutions are in the parent enzyme at positions having a solvent accessible surface (DRP) of greater than about 25%. 12. The method according to claim 1, where the substitutions are in the parent enzyme at positions having a solvent accessible surface (DRP) of more than about 50% or more than about 65%. 13. The method according to claim 1, where the specified parent enzyme is a wild-type enzyme.

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