CN116656645A

CN116656645A - Group of thermostable GDSL family esterase mutants with improved catalytic efficiency and expanded substrate spectrum

Info

Publication number: CN116656645A
Application number: CN202310649844.8A
Authority: CN
Inventors: 张勇; 陈润莎; 谢文涛; 奥斯曼·阿里; 王玲; 吴金鸿; 李向红
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2023-06-02
Filing date: 2023-06-02
Publication date: 2023-08-29

Abstract

The application provides a group of preparation methods of thermostable esterase variants with improved catalytic efficiency and expanded substrate spectrum. Specifically, the application designs a group of variants of EstL5 by analyzing the crystal structure of esterase EstL5 and using the protein rationality and semi-rationality by taking wild gene EstL5 as a template based on the in-depth analysis of the protein structure of the enzyme. These activity improvements and substrate spectrum expansion EstL5 variants promote the practical application value of esterases.

Description

Group of thermostable GDSL family esterase mutants with improved catalytic efficiency and expanded substrate spectrum

Technical Field

The application relates to the field of genetic engineering, in particular to a group of thermostable esterase variants with improved catalytic efficiency and expanded substrate spectrum.

Background

Esterase (EC 3.1.1.1) can catalyze ester bond hydrolysis in acyl compounds to release fatty acid and corresponding alcohols, and the esterase can often generate transesterification and synthesis reaction in an organic phase, so that the esterase is widely applied to the fields of fine chemical industry, medicines, food processing, washing additives and the like. The normal temperature esterase is prevented from long-term storage and recycling in industrial application due to instability of the esterase. Therefore, it is particularly important to develop stable esterases suitable for severe industrial production environments.

The strain G.thermodetitricans is thermophile bacteria with an optimal growth temperature of 65 ℃, is used as an important source of thermophilic enzymes and has potential application value in the industrial and biotechnology fields. Because the g.thermodifitins strain NG80-2 whole genome has been sequenced and released in the NCBI database (Genbank Accession No CP 000557), it provides very useful information for screening new potential biocatalysts.

By screening thermophilic genome sequence data mining, researchers have noted a putative new GDSL family esterase gene. The esterase gene EstL5 is cloned, and EstL5 protein is expressed and purified, and the zymology is characterized by esterase with better cold activity and heat temperature qualitative property, and the esterase mainly acts on a C4 acyl substrate. However, the natural enzymes have relatively low activity and limited substrate spectrum, which restricts the practical application in the fields of food, chemistry, synthetic biology and the like.

In view of the foregoing, there is a need in the art to develop an esterase variant with good and stable low-temperature activity that has improved catalytic efficiency and expanded substrate spectrum.

Disclosure of Invention

The application aims to provide a group of thermostable GDSL family esterase mutants with improved catalytic efficiency and expanded substrate spectrum.

In a first aspect, the application provides a thermostable esterase mutant having a mutation in the amino acid sequence corresponding to the wild-type EstL5 protein, including a mutation in one or more core amino acid positions selected from the group consisting of:

(a)F237；

(b) loop142-153 missing;

(c) Y182 deletion, N183 deletion, S186 deletion, D187 deletion, and L188 deletion (i.e., Y182/N183/S186/D187/L188 deletion);

(d) P184 and F185;

(e) Y182; and/or

(f)L142；

Wherein, the amino acid sequence of the wild EstL5 protein is shown in SEQ ID NO: 1.

In another preferred embodiment, the thermostable esterase mutant has one or more mutations selected from the group consisting of:

(1)F237A；

(2) loop142-153 missing;

(3) Y182 deletion, N183 deletion, S186 deletion, D187 deletion, and L188 deletion (i.e., Y182/N183/S186/D187/L188 deletion);

(4) P184A and F185A;

(5) P184A, F185A, and loop142-153 deleted;

(6) P184A, F185A, Y deletion, N183 deletion, S186 deletion, D187 deletion, and L188 deletion;

(7) loop142-153, Y182, N183, S186, D187, and L188 deletions;

(8) Y182F; and

(9)L142A；

wherein the mutation is based on SEQ ID NO:1, and a sequence shown in 1.

In another preferred embodiment, the thermostable esterase mutant also includes an active fragment, variant or derivative protein thereof.

In another preferred embodiment, the active fragment, variant or derivative protein of the thermostable esterase mutant has a deletion of P184A/F185A, loop 142-153; and/or Y182/N183/S186/D187/L188 deletion mutation, and has a sequence identity of ≡85%,. Gtoreq.90%,. Gtoreq.91%,. Gtoreq.92%,. Gtoreq.93%,. Gtoreq.94%,. Gtoreq.95%,. Gtoreq.96%,. Gtoreq.97%,. Gtoreq.98%,. Gtoreq.99% as compared to the wild-type esterase.

In another preferred embodiment, the thermostable esterase mutant derived protein has, in addition to one or more amino acid mutations, deletions, insertions and/or substitutions of one or more (e.g., typically 1-30, preferably 1-10, more preferably 1-6, still more preferably 1-3, most preferably 1) amino acid residues, which still have the catalytic activity of the esterase mutant.

In another preferred embodiment, the wild type EstL5 protein is derived from thermophilic bacteria (G.thermodemitricens).

In another preferred embodiment, the thermostable esterase mutant has an amino acid sequence selected from any of SEQ ID NOs 2-11.

In another preferred embodiment, the ratio Q1/Q1 of the enzyme activity Q1 of the thermostable esterase mutant under the predetermined temperature condition to the enzyme activity Q0 of the wild type under the same condition is not less than 1.2, preferably not less than 1.5, more preferably not less than 2.0, and most preferably not less than 4.0. In another preferred embodiment, the predetermined temperature condition is 20-50deg.C, preferably 25-40deg.C, more preferably 28-32deg.C.

In another preferred embodiment, the ratio Q1/Q0 is 1.0-8.0, preferably 2.0-6.5.

In another preferred embodiment, the ratio Q1/Q1 of the enzyme activity Q1 of the thermostable esterase mutant under low temperature conditions to the enzyme activity Q0 of the wild type under the same conditions is not less than 1.2, preferably not less than 1.5, more preferably not less than 2.0, most preferably not less than 4.0. In another preferred example, the low temperature condition refers to-5 to 5 degrees celsius, more preferably-2 to 2 degrees celsius, or most preferably-1 to 1 degree celsius.

In another preferred example, the term "enzyme activity" refers to activity that catalyzes the following reaction:

in a second aspect, the application provides an isolated polynucleotide encoding a thermostable esterase mutant according to the first aspect of the application.

In a third aspect, the application provides a vector comprising a polynucleotide according to the second aspect of the application.

In another preferred embodiment, the vector comprises a recombinant vector, an expression vector and an integration vector.

In another preferred embodiment, the vector is an expression vector.

In another preferred embodiment, the vector is a pET plasmid.

In a fourth aspect the present application provides a genetically engineered host cell comprising a vector according to the third aspect of the application, or having incorporated into its genome an exogenous polynucleotide according to claim 2.

In another preferred embodiment, the host cell includes a prokaryotic cell and a eukaryotic cell.

In another preferred embodiment, the host cell comprises a cell derived from a microorganism selected from the group consisting of:

saccharomyces cerevisiae (Saccharomyces cerevisiae), pichia pastoris (Pichia pastoris), morganella salina (Saccharomyces monacensis), saccharomyces bayanus (Saccharomyces bayanus), pasteur yeast (Saccharomyces pastorianus), saccharomyces carlsbergensis (Saccharomyces carlsbergensis), schizosaccharomyces pombe (Saccharomyces pombe), kluyveromyces marxianus (Kluyveromyces marxiamus), kluyveromyces lactis (Kluyveromyces lactis), kluyveromyces fragilis (Kluyveromyces fragilis), pichia stipitis (Pichia stipitis), candida shehatae (Candida shehatae), candida tropicalis (Candida tropicalis), and Escherichia coli (Escherichia coli).

In another preferred embodiment, the host cell comprises Saccharomyces cerevisiae, pichia pastoris, or myceliophthora thermophila.

In another preferred embodiment, the host cell is E.coli.

In another preferred embodiment, the host cell expresses the thermostable esterase mutant of the first aspect of the application.

In a fifth aspect, the application provides a method of preparing a thermostable esterase mutant according to the first aspect, the method comprising:

(a) Culturing the host cell of claim 4 under conditions suitable for expression, thereby expressing the thermostable esterase mutant of the first aspect of the application; and

(b) Isolating the expression product, thereby obtaining the thermostable esterase mutant.

In a sixth aspect, the application provides the use of a thermostable esterase mutant according to the first aspect of the application for catalyzing the hydrolysis of an acyl substrate or for preparing a catalytic agent for hydrolyzing an acyl substrate to the corresponding acid and alcohol.

In another preferred embodiment, the acyl substrate comprises p-nitrophenol (pNP) -carboxylate.

In another preferred embodiment, the pNP-carboxylic acid esters include pNP-C2, pNP-C4, pNP-C8, pNP-C10, and pNP-C12.

In a seventh aspect, the application provides a method of catalyzing an acyl substrate comprising the steps of:

the esterase mutant according to the first aspect of the application is contacted with an acyl substrate, thereby obtaining the corresponding acid and alcohol.

In another preferred embodiment, the acyl substrate comprises: pNP-C2, pNP-C4, pNP-C8, pNP-C10, pNP-C12.

It is understood that within the scope of the present application, the above-described technical features of the present application and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

The following drawings are illustrative of particular embodiments of the application and are not intended to limit the scope of the application as defined by the claims.

Figure 1 shows that the EstL5 structure consists of a central 5 beta sheet and two side 6 alpha helices, where the alpha helices, beta sheet and the connecting loops between the two are represented by blue, magenta and coral colors, respectively.

FIG. 2 shows the results of the docking of different chain length pNP carboxylate with EstL5 binding molecules and the preference of EstL5 for different substrates.

FIG. 3 shows the distribution of the thermal parameters of E.coli homologous enzyme TAP (factor B) and EstL5 (factor B).

FIG. 4 shows the selectivity of EstL5 combinatorial mutants against different chain length pNP carboxylate substrates.

FIG. 5 shows the measurement of the protein thermostability parameters Tm of wild-type and mutant enzymes by differential scanning fluorescence.

FIG. 6 shows the low temperature activity of EstL 5.

Detailed Description

Through extensive and intensive research, the inventors have unexpectedly obtained a group of thermostable esterase mutants with improved catalytic efficiency and expanded substrate spectrum for the first time through a large number of screens. Specifically, the catalytic efficiency of the mutated esterase is obviously improved, and the esterase mutant disclosed by the application expands the preference of the esterase mutant for catalyzing a medium-long acyl chain substrate.

Specifically, the inventors designed a group of mutants of EstL5 protein, including EstL5-F237A, estL5-M1 (P184A/F185A), estL5-M2 (deletion loop 142-153), estL5-M4 (deletion Tyr182/Asn183/Ser186/Asp187/Leu 188) and M1 and M4 combined variants EstL5-M5, M2 and M4 combined variants EstL5-M6, by analyzing the crystal structure of the esterase EstL5 and based on in-depth analysis of the enzyme protein structure, using the wild-type gene EstL5 as a template, and using the protein rationality and semi-rationality.

The substrate preference test results show that the efficiency of the EstL5-F237A in the mutant enzymes for catalyzing pNP-C2 and pNP-C4 is obviously improved; the EstL5-M2/M6 variant deleted of loop142-153 has significantly improved activity of catalyzing long-chain substrates pNP-C10 and pNP-C12; the variant EstL5-M1 on loop182-198 preferentially catalyzes both short-chain pNP-C4 and long-chain pNP-C10 substrates, with altered substrate specificity.

EstL5 enzyme has more than 40% activity at 0 ℃, and a series of EstL5 enzyme mutants are analyzed, designed, synthesized and tested through a structure in order to further improve the low-temperature activity of the EstL5 enzyme and enable the EstL5 enzyme to be better applied under different conditions. Compared with the wild type EstL5 enzyme, the activities of mutant enzymes EstL5-Y182F and EstL5-L142A are obviously improved at the low temperature of 0 ℃.

The EstL5 mutant with improved low-temperature activity and expanded substrate spectrum has excellent practical application value.

Terminology

In order that the present disclosure may be more readily understood, certain terms are first defined. As used in the present application, each of the following terms shall have the meanings given below, unless explicitly specified otherwise herein. Other definitions are set forth throughout the application.

As used herein, the term "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

"transduction," "transfection," "transformation," or the terms used herein refer to the process of transferring an exogenous polynucleotide into a host cell, and transcription and translation to produce a polypeptide product, including the use of plasmid molecules to introduce the exogenous polynucleotide into the host cell (e.g., E.coli).

"Gene expression" or "expression" refers to the process by which a gene is transcribed, translated, and post-translationally modified to produce an RNA or protein product of the gene.

"Polynucleotide" refers to polymeric forms of nucleotides of any length, including Deoxynucleotides (DNA), ribonucleotides (RNA), hybrid sequences and the like. Polynucleotides may include modified nucleotides, such as methylated or capped nucleotides or nucleotide analogs. The term polynucleotide as used herein refers to single-and double-stranded molecules that are interchangeable. Unless otherwise indicated, polynucleotides in any of the embodiments described herein include a double stranded form and two complementary single strands that are known or predicted to constitute the double stranded form.

Conservative amino acid substitutions are known in the art. In some embodiments, the potential substituted amino acids are within one or more of the following groups: glycine, alanine; and valine, isoleucine, leucine and proline; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine lysine, arginine and histidine; and/or phenylalanine, tryptophan and tyrosine; methionine and cysteine. Furthermore, the application provides non-conservative amino acid substitutions that allow amino acid substitutions from different groups.

The meaning of all parameters, dimensions, materials and configurations described herein will be readily understood by those skilled in the art. The actual parameters, dimensions, materials, and/or configurations may depend upon the specific application for which the application is used. It will be appreciated by those skilled in the art that the examples or claims are given by way of example only and that the scope of the application which can be covered by the embodiments of the application is not limited to the specifically described and claimed scope within the scope of the equivalents or claims.

All definitions and uses herein should be understood to exceed dictionary definitions or definitions in documents incorporated by reference.

All references, patents and patent applications cited herein are incorporated by reference with respect to the subject matter in which they are cited, and in some cases may contain the entire document.

Wild type esterase EstL5 protein

Wild EstL5 is GDSL family esterase from thermophilic bacteria G.thermodenicin, the optimal catalytic temperature of the enzyme is 30 ℃, the residual enzyme activity after incubation for 12h at 55 ℃ is 100%, and 40% of the enzyme activity is still reserved at 0 ℃, so that the enzyme has better low-temperature activity and thermal stability.

Wild type EstL5 catalyzes the following reaction:

esterase variants

As used herein, the terms "esterase variant of the application", "thermostable esterase variant of the application", "mutant of the application", "esterase mutant of the application" or "thermostable mutant of the application" are used interchangeably and refer to an esterase variant of the first aspect of the application.

The application designs a group of variants of EstL5 by analyzing the crystal structure of esterase EstL5 and using wild gene EstL5 as a template and utilizing the protein rationality and semi-rationality through analyzing the protein structure based on the enzyme, and the variants comprise EstL5-F237A, estL5-M1 (P184A/F185A), estL5-M2 (delete loop 142-153), estL5-M3 (P184A/F185A/delete loop 142-153), estL5-M4 (delete Tyr182/Asn183/Ser186/Asp187/Leu 188) and

EstL5-M5 (P184A/F185A/delete Tyr182/Asn183/Ser186/Asp187/Leu 188), estL5-M6 (delete Loop 142-153/delete Tyr182/Asn183/Ser186/Asp187/Leu 188), estL5-M7 (P184A/F185A/delete Loop 142-153/delete Tyr182/Asn183/Ser186/Asp187/Leu 188). The substrate preference test result shows that the efficiency of catalyzing pNP-C2 and pNP-C4 of EstL5-F237A in the mutant enzymes is obviously improved; while

EstL5-M1 (P184A/F185A), estL5-M2 (delete Loop 142-153) and EstL5-M6 (delete Loop 142-153/delete Tyr182/Asn183/Ser186/Asp187/Leu 188) enhanced their catalytic activity on long-chain substrates pNP-C10. In addition, estL5 enzyme has more than 40% of activity at 0 ℃, and in order to further improve the low-temperature activity of the EstL5 enzyme so that the EstL5 enzyme can be better applied under different conditions, a series of mutants are designed through structural analysis and calculation, wherein the activities of mutant enzymes EstL5-Y182F and EstL5-L142A are obviously improved at the low temperature of 0 ℃. These activity improvements and substrate spectrum expansion EstL5 variants promote the practical application value of esterases.

As used herein, an "isolated polypeptide" means that the polypeptide is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. The person skilled in the art is able to purify the polypeptides using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the polypeptide can also be further analyzed by amino acid sequence.

In the application, the wild type esterase is subjected to mutation at a specific site, and the corresponding mutant with remarkably improved activity is obtained. As used herein, the terms "mutant protein", "mutant" are used interchangeably to refer to mutants of esterases.

As used herein, the term "P184A/F185A" is taken as an example when describing mutations, and refers to mutant proteins derived from wild-type esterases, with P at position 184 mutated to a and F at position 185 mutated to a. Other mutations are described in a similar manner.

In view of the teachings of the present application and the prior art, those skilled in the art will appreciate that the muteins of the present application shall also include active fragments, modified or unmodified variant forms of the muteins or derived proteins thereof.

Specifically, the active fragments, variant forms or derived proteins of the esterase mutants include: the amino acid sequences formed by one or more site-directed mutations in the sequences shown, further having deletions, insertions and/or substitutions of one or more (e.g.typically 1 to 30, preferably 1 to 10, more preferably 1 to 6, still more preferably 1 to 3, most preferably 1) amino acid residues, still have the catalytic activity of the esterase mutant.

One skilled in the art can generate mutants with conservative variations based on, for example, conservative amino acid substitutions as shown in Table A below.

Table A

Initial residues	Representative substitution	Preferred substitution
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala	Leu

As used herein, modified (typically not altering the primary structure) forms of proteins include: chemically derivatized forms of proteins such as acetylated or carboxylated in vivo or in vitro. Modifications also include glycosylation. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Proteins modified to increase their proteolytic resistance or to optimize their solubility properties are also included. These techniques are known to those skilled in the art.

In addition, the mutant protein derivative protein of the application also includes the mutant protein or its active fragments and other proteins or markers formed fusion protein or conjugates.

The muteins of the application may also contain one or more additional mutations, thereby further enhancing the catalytic activity of the esterase mutant.

Coding nucleic acids and combinations thereof

On the basis of the esterase mutants of the application, the application also provides isolated polynucleotides encoding the esterase mutants or simple variants thereof. The polynucleotides of the application may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be identical to the nucleotide sequence encoding the esterase mutant in the examples of the application or it may be a degenerate pathogen.

As used herein, "degenerate variant" refers in the present application to sequences encoding esterase mutants described in the first aspect of the application, but which differ from the nucleotide sequences encoding esterase mutants in the examples of the application.

The application also relates to variants of the above polynucleotides which encode polypeptides having the same amino acid sequence as the application or fragments, analogs and derivatives of the polypeptides. Variants of the polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. Such nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the encoded polypeptide.

The polypeptides and polynucleotides of the application are preferably provided in isolated form, and more preferably purified to homogeneity.

The full-length nucleotide sequence of the esterase mutant or a fragment thereof of the application can be obtained usually by a PCR amplification method, a recombinant method or an artificial synthesis method. For the PCR amplification method, primers can be designed according to the nucleotide sequences disclosed in the present application, particularly the open reading frame sequences, and amplified to obtain the relevant sequences using a commercially available cDNA library or a cDNA library prepared according to a conventional method known to those skilled in the art as a template. When the sequence is longer, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

At present, it is already possible to obtain the DNA sequences encoding the proteins of the application (or fragments or derivatives thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the protein sequences of the application by chemical synthesis.

Methods of amplifying DNA/RNA using PCR techniques are preferred for obtaining the genes of the present application. In particular, when it is difficult to obtain full-length cDNA from a library, it is preferable to use RACE method (RACE-cDNA end rapid amplification method), and primers for PCR can be appropriately selected according to the sequence information of the present application disclosed herein and synthesized by a conventional method. The amplified DNA/RNA fragments can be isolated and purified by conventional methods, such as by gel electrophoresis.

Carrier body

The application also relates to vectors comprising the polynucleotides of the application, host cells genetically engineered with the vectors or thermostable esterase mutant protein coding sequences of the application, and methods of producing the polypeptides of the application by recombinant techniques.

The polynucleotide sequences of the present application may be used to express or produce recombinant thermostable esterase mutants by conventional recombinant DNA techniques. Generally, there are the following steps:

(1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a thermostable esterase mutant of the application, or with a recombinant expression vector containing the polynucleotide;

(2) A host cell cultured in a suitable medium;

(3) Separating and purifying the protein from the culture medium or the cells.

In the present application, the polynucleotide sequence of the thermostable esterase mutant may be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. Any plasmid or vector may be used as long as it is replicable and stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translational control elements.

Methods well known to those skilled in the art can be used to construct expression vectors containing thermostable esterase mutant-encoding DNA sequences and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E.coli; a lambda phage PL promoter; eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, LTRs from retroviruses, and other known promoters that control the expression of genes in prokaryotic or eukaryotic cells or viruses thereof. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

In addition, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and green fluorescent protein for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli.

Host cells

Vectors comprising the appropriate DNA sequences as described above, as well as appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples: bacteria such as Escherichia coli, bacillus subtilis, etc.; fungal cells such as yeast; a plant cell; insect cells Sf9, etc.; mammalian cells CHO, COS, 293, and the like.

When expressed in eukaryotic cells, the polynucleotides of the application will have enhanced transcription if an enhancer sequence is inserted into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs, that act on a promoter to increase the transcription of a gene. Examples include the SV40 enhancer 100 to 270 base pairs late in the replication origin, and adenovirus enhancers.

It will be clear to a person of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells, which can take up DNA, can be obtained after the exponential growth phase and then treated with CaCl ₂ Or other chemical treatment of the transformation, using procedures well known in the art. Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present application. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods.

Preparation of esterase mutants

The application also provides a method for preparing an esterase mutant, comprising culturing a host cell of the application under conditions suitable for expression, thereby expressing the esterase mutant; and isolating the esterase mutant.

The muteins obtained may optionally also be purified, whereby a purer mutein product is obtained.

Preferably, the conditions suitable for expression include techniques conventional in the art, and purification techniques include nickel column purification, ion exchange chromatography, and the like.

Application of

The application also provides application of the esterase mutant as a catalytic reagent in catalyzing hydrolysis of acyl substrate to generate corresponding acid and alcohol or in catalyzing transesterification or synthesis of esters.

Substrate(s)

pNP-C2: the p-nitrophenol reacts with acetic acid to produce p-nitrophenol acetate and water. The molecular weight is 195.17200.

pNP-C4: the p-nitrophenol reacts with butyric acid to produce p-nitrophenol butyrate and water. The molecular weight is 209.199.

pNP-C8: the p-nitrophenol reacts with octanoic acid to produce p-nitrophenol octanoate and water. The molecular weight is 265.31.

pNP-C10: the reaction of p-nitrophenol with decanoic acid produces p-nitrophenol decanoate and produces water. The molecular weight is 293.36.

pNP-C12: the p-nitrophenol reacts with lauric acid to produce p-nitrophenol laurate and water. The molecular weight is 321.41.

The application has the main advantages that:

1. compared with wild esterase, the mutant obtained by screening of the application has obviously improved enzyme catalysis efficiency.

2. The mutant of the application expands the preference of the mutant for catalyzing the medium-long acyl chain substrate.

3. The application demonstrates the importance of conformational changes in the flexible region for substrate selectivity, providing a new direction for the directed engineering of other esterases of this family.

The application will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer.

Primer synthesis:

the primers used in the present application were prepared by synthesis by commercial companies.

The enzyme preparation such as DpnI enzyme used in the experiment is purchased from Thermo Scientific company; primeSTAR Max high-fidelity polymerase is available from TakaRa; restriction enzymes were purchased from Fermentas; the DNA gel recovery kit and the plasmid miniprep kit used were all purchased from Axygen.

E.coli BL21 (DE 3), E.coli DH 5. Alpha. Strain and pET-28a (+) plasmids referred to in the examples below were all from the laboratory (E.coli and plasmids as described above are also commercially available and do not require preservation for the proprietary program).

The following examples relate to the following media:

LB liquid medium: 5.0g/L yeast powder, 10.0g/L, naCl 10.0.0 g/L tryptone and 100 mu g/L kanamycin.

LB solid medium: 5.0g/L yeast powder, 10.0g/L, naCl 10.0.0 g/L tryptone, 15g/L agar powder and 100 mu g/L kanamycin.

The sequences of the application

EstL5 wild type amino acid sequence SEQ ID NO 1

EstL5 single point mutant M0 amino acid sequence SEQ ID NO. 2

EstL5 double point combined mutant M1 amino acid sequence SEQ ID NO 3

EstL5 mutant M2 amino acid sequence SEQ ID NO. 4

EstL5 mutant M3 amino acid sequence SEQ ID NO. 5

EstL5 mutant M4 amino acid sequence SEQ ID NO. 6

EstL5 mutant M5 amino acid sequence SEQ ID NO. 7

EstL5 mutant M6 amino acid sequence SEQ ID NO. 8

EstL5 mutant M7 amino acid sequence SEQ ID NO 9

EstL5 mutant M8 amino acid sequence SEQ ID NO 10

EstL5 mutant M9 amino acid sequence SEQ ID NO. 11

Example 1

Purifying the recombinant expressed wild esterase EstL5 protein, and screening protein crystals. Protein crystal optimization by hanging drop method, finally in (0.01M CoCl ₂ ·6H ₂ O, ph 5.2.1M sodium acetate and 1M 1, 6-hexanediol) improved crystals were obtained for diffraction. Data were collected on an open sea light source (SSPF) BL17U line station X-ray diffraction. Diffraction data processing on-line station HKL3000 software is integrated to obtain scale suffix file, and resolution is up toThe esterase EstL5 crystal structure was resolved by molecular replacement.

Esterase EstL5 has a typical hydrolase structure of the GDSL family, with a structure center of 5 parallel β -strands surrounded by 6 major α -helices (as shown in FIG. 1). Wherein the 5 parallel beta strands are arranged in parallel in a manner similar to the beta sheets in alpha/beta hydrolase.

Based on multiple sequence alignment analysis, the conserved amino acid residues of the GDSL hydrolase family are present in the esterase EstL5 amino acid sequence. To analyze the function of these highly conserved amino acid residues in the EstL5 catalytic reaction, 10 related mutants were constructed by alanine mutation of the conserved amino acid residues in EstL 5.

Table 1 primers used for mutants

Mutant	Content
		M0	F237A
M1	P184A/F185A
		M2	Deletion of amino acid residues from position 142-153
M3	Combine M1 and M2
		M4	Deletion of amino acid residues Y182, N183, S186, D187 and L188
M5	Combination of M1 and M4
		M6	Combining M2 and M4
M7	Combination of M1 and M6
		M8	Y18AF
M9	L142A

The primer is used to carry out full plasmid PCR by taking the nucleotide sequence of the parent esterase as a template, and the corresponding mutation is introduced at a specific position of the gene. The total reaction volume was 50. Mu.l, and the components and amounts of the reaction system were as shown in Table 2, and after centrifugation, they were mixed and amplified by an Eppendorf PCR amplification apparatus. Whole plasmid PCR site-directed mutagenesis reaction procedure set up: pre-denaturation at 98℃for 2min,30 cycles of amplification (denaturation at 98℃for 15s, annealing at 50-65℃for 15s, extension at 72℃for 2 min); extending at 72 ℃ for 5min; the PCR product was buffered at 4 ℃. The annealing temperature depends on the Tm of the upstream and downstream primers, and the extension time depends on the whole plasmid gene length (generally 30 s/kb).

After gel purification of the obtained PCR product, dpnI enzyme is treated for 2 hours at 37 ℃, E.coli DH5 alpha chemical competence is transformed, the transformed product is coated on LB solid culture medium, the culture is carried out for 12 hours at 37 ℃, transformant is picked up on LB solid culture medium, LB liquid culture medium is inoculated for culture, plasmid is extracted after culture is carried out for 10 hours at 37 ℃, and the plasmid is subjected to sequence determination, thus obtaining mutant with correct sequencing.

TABLE 2 PCR site-directed mutagenesis reaction System

EXAMPLE 2 heterologous expression and purification of esterase EstL5 mutant

The method comprises the following specific steps:

transformation of the already constructed mutant expression plasmid pET28b-EstL5 into E.coli BL21 (DE 3)

Chemically competent cells. Positive monoclonal is selected and inoculated in 5mL of LB liquid medium supplemented with 50 mg.L-1 kanamycin, cultured overnight, inoculated in 1L of LB liquid medium according to the proportion of 1 percent, and cultured in shaking mode at 37 ℃ and 200 r/min. Cell proliferation to exponential growth phase (OD) ₆₀₀ IPTG (0.8 mM. L-1) is added when the concentration is 0.6-0.8%, shake culture is carried out for 18-20h at 20 ℃ to induce the heterologous expression of the protein. Centrifugation to collect the cells and re-suspension in the knotThe buffer (50 mM Tris-HCl pH 8.0, 300mM NaCl,20mM imidazole) was combined, and after disruption by a high pressure homogenizer, 13,000Xg was centrifuged at high speed for 1 hour, and the crude enzyme solution was purified using a 5mL Ni-NTA pre-packed gravity column. Finally, the protein of interest was eluted with elution buffer (250 mM imidazole, 50mM Tris-HCl pH 8.0, 300mM NaCl). Protein concentration was checked with BCA protein quantification kit using bovine serum albumin as standard. The molecular weight and purity of the target protein were analyzed by 12% SDS-PAGE. Gel filtration chromatography uses an AKTA protein purification system, superdex 200 inch 10/300G column.

Example 3 investigation and engineering of the substrate-specific mechanism of esterase EstL5

The molecular docking structure of the esterase EstL5 with 5 different chain length pNP-carboxylates (pNP-C2, pNP-C4, pNP-C8, pNP-C10, pNP-C12) was predicted using an AutoDock Vina, wherein pNP is p-nitrophenyl.

The molecular docking results show that 5 pNP-carboxylic esters with different chain lengths are combined in a similar way in the EstL5 active pocket, the hydrophobic fatty chain is positioned between the hydrophobic carboxyl combining pocket and the externally accessible solvent, and the carboxylic esters can be combined with a catalytic centerWithin the scope are Tyr182, phe237, asn133 and Ser63 residues that form hydrogen bonding interactions (as shown in FIGS. 2A-E).

EstL5 catalytic pocket is relatively small and in a distorted state, and the predicted 5 docking structures (pNP-C2, pNP-C4, pNP-C8, pNP-C10, pNP-C12) have lowest binding free energies of-5.54 kcal. Mol-1, -7.83 kcal. Mol-1, -6.39 kcal. Mol-1, -5.26 kcal. Mol-1, and-5.61 kcal. Mol-1, respectively. The EstL5 substrate binding affinity, pNP-C4 > pNP-C8 > pNP-C12 > pNP-C2 > pNP-C10, i.e., pNP-C4, was shown to be the optimal binding substrate, as shown in FIG. 2F.

The key loop of the enzyme catalytic binding region is subjected to conformational change or substrate specificity can be changed, so that the novel functional esterase is obtained. The single crystal structure (PDB code:1 IVN) from E.coli thioesterase I/protease I/lysophospholipase L1 (TAP) has been resolved by the Taiwan Lo et al team (PDB code:1U 8U). TAP preferentially catalyzes the hydrolysis of acyl chain length (C10) thioesters of 10 carbon atoms and medium short-chain pNP-carboxylate substrates (C8 most active). "Switch loop" (loop 75-80) and flexible region loops 110-120 interacting therewith were found based on factor B flexible region analysis. Octanoic acid (OCA) molecules bind to the substrate site of TAP, and the continuous hydrophobic surface causes a significant change in loop75-80 conformation.

By aligning EstL5 with TAP sequences and structures, it was found that the EstL5 loop136-154 flexible region was far away from the substrate binding pocket of the TAP "Switch loop" and the loop182-198 region was significantly less flexible (as shown in FIGS. 3A-B). Thus, mutants EstL5-M1 (P184A/F185A), estL5-M2 (loop 142-153 deleted), estL5-M4 (Tyr 182/Asn183/Ser186/Asp187/Leu188 deleted), and M2 and M4 combined mutants EstL5-M6 were designed according to the application.

The substrate preference test result shows that after loop142-153 is deleted, the activity of EstL5-M2/M6 mutant for catalyzing long-chain substrates pNP-C10 and pNP-C12 is obviously improved, and the flexibility and conformational change of the switch loop in the catalysis process can be restored. The mutant EstL5-M1 on the loop182-198 preferentially catalyzes the short-chain pNP-C4 and long-chain pNP-C10 substrates (shown in figure 4), and can be related to the flexible change of the loop182-198 after the mutation of EstL5-M1, thereby providing a new direction for the directional modification of other esterases in the family.

Example 4 substrate Spectrum expansion and stability analysis of variant enzymes

The specificity of the mutants for substrates of different chain lengths was determined using the p-nitrophenol method. The specificity of the EstL5 variant enzyme for substrates of different chain lengths is shown in FIG. 4. Compared with the wild type, the relative activity of EstL5-F237A on pNP-C2 and pNP-C4 is obviously improved; estL5-M1 (P184A/F185A) and the variant EstL5-M2/M6 (both containing deleted loop 142-153) catalyze the activity of long-chain substrate pNP-C10 to be obviously improved, which indicates that the site mutations can widen the substrate spectrum and catalyze the reaction of the long-chain substrate.

The protein thermostability parameters Tm of the wild-type and mutant enzymes were determined by differential scanning fluorescence. The results are shown in FIG. 5. The thermal stability Tm of wild type EstL5 WT is 62.5deg.C, the variants EstL5-F237A, estL5-M1 (P184A/F185A), estL5-M2 (loop 142-153 deleted) and EstL5-M6 (loop 142-153 deleted/Tyr 182/Asn183/Ser186/Asp187/Leu 188) are 62.6deg.C, 61.5deg.C, 58.2C and 54.7deg.C respectively, which indicate that these mutations do not have a major effect on the stability of the protein, thus improving the catalytic activity of the esterase and widening its substrate spectrum while ensuring that the stability is substantially unchanged.

Example 6 Activity enhancement of mutant enzyme at Low temperature

During the course of the study, estL5 was found to have low temperature activity, and retained 40% of the enzyme activity at 0deg.C for 1.0h in ice bath. In order to further improve the low-temperature activity, a series of mutants are designed based on the enzyme protein structure and molecular dynamics simulation, wherein the enzyme activities of the variants EstL5-Y182F and EstL5-L142A are obviously improved at the temperature of 0 ℃.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims

1. A thermostable esterase mutant characterized in that the mutant has a mutation in the amino acid sequence corresponding to the wild-type EstL5 protein, said mutation comprising a mutation in one or more core amino acid positions selected from the group consisting of:

(a)F237；

(b) loop142-153 missing;

(d) P184 and F185;

(e) Y182; and/or

(f)L142；

2. The thermostable esterase mutant according to claim 1, wherein the mutant has one or more mutations selected from the group consisting of:

(1)F237A；

(2) loop142-153 missing;

(4) P184A and F185A;

(5) P184A, F185A, and loop142-153 deleted;

(7) loop142-153, Y182, N183, S186, D187, and L188 deletions;

(8) Y182F; and

(9)L142A；

wherein the mutation is based on SEQ ID NO:1, and a sequence shown in 1.

3. The thermostable esterase mutant according to claim 1, wherein the mutant has an amino acid sequence selected from any of SEQ ID NO. 2-11.

4. The thermostable esterase mutant according to claim 1, wherein the ratio Q1/Q1 of the enzyme activity Q1 of the thermostable esterase mutant under predetermined temperature conditions to the enzyme activity Q0 of the wild-type under the same conditions is not less than 1.2, preferably not less than 1.5, more preferably not less than 2.0, most preferably not less than 4.0. In another preferred embodiment, the predetermined temperature condition is 20-50deg.C, preferably 25-40deg.C, more preferably 28-32deg.C.

5. An isolated polynucleotide encoding the thermostable esterase mutant of claim 1.

6. A vector comprising the polynucleotide of claim 5.

7. A genetically engineered host cell comprising the vector of claim 6, or having incorporated into its genome an exogenous polynucleotide of claim 5.

8. A method of making the thermostable esterase mutant of claim 1, comprising:

(a) Culturing the host cell of claim 7 under conditions suitable for expression, thereby expressing the thermostable esterase mutant of claim 1; and

9. Use of a thermostable esterase mutant according to claim 1, for catalyzing the hydrolysis of an acyl substrate or for preparing a catalytic agent for hydrolyzing an acyl substrate to the corresponding acid and alcohol.

10. A method of catalyzing an acyl substrate comprising the steps of:

contacting the esterase mutant of claim 1 with an acyl substrate to obtain the corresponding acid and alcohol.