CN107893060A - A kind of marine bacteria source thermostabilization salt tolerant SGNH family's hydrolases and application - Google Patents

Abstract

The invention discloses a kind of marine bacteria source Novel thermostable salt tolerant organic solvent-resistant SGNH family hydrolase A linE4 and its encoding gene and application.The present invention relates to hydrolase gene aline4 to come from marine bacteria Altererythrobacter indicus DSM 18604, and nucleotide sequence is as shown in SEQ ID NO.1.For hydrolase gene of the present invention after heterogenous expression, the hydrolase has esterase active, catalytic activity highest when substrate is p-nitrophenol butyrate (C4), enzyme activity 25.8U/mg.AlinE4 catalyzing hydrolysis optimal pH and temperature are 7.5 and 40 DEG C；After being incubated 60min in 40,50 and 60 DEG C, remain to keep more than 50% activity；Under organic solvent and metal ion existence condition, the enzyme activity is higher.The hydrolase has heat endurance and basic character, can be applied to the field such as detergent, wastewater treatment, fine chemistry industry, pharmacy and environment remediation high temperature, saliferous and containing the industrial production under the conditions of organic solvent.

Description

A kind of marine bacteria source thermostabilization salt tolerant SGNH family's hydrolases and application

Technical field

The invention belongs to genetic engineering field, and in particular to a kind of marine bacteria source Novel thermostable salt tolerant SGNH families Hydrolase, its encoding gene and its application.

Background technology

SGNH families hydrolase be a kind of catalytic residue serine containing 4 strict conservations in 4 conserved sequence areas, The hydrolase of glycine, asparagine and histidine.SGNH families hydrolase is widely present in eucaryon and prokaryotes, is had The various actives such as lipase, protease, thioesterase, Arylesterase activity, lysophospholipase, carbohydrate esterase, acyltransferase, It is a kind of hydrolase with extensive substrate spectrum, may participate in bacterial virulence and growth and development of plants, form generation and defence etc. Different physiological roles.Extensive substrate spectrum and functional diversity make family's hydrolase have extensive potential using value, turn into Domestic and international study hotspot.

The SGNH families hydrolase multi-source reported is in eucaryote at present, family's hydrolase of bacterial origin compared with Few, function is also indefinite.Ocean is a huge microbial resources treasure-house, and the hydrolase of marine source generally has and ocean The advantageous property of environmental correclation, such as temperature stability, salt tolerance, alkali resistance, low temperature resistant and excellent chiral selectivity etc. Deng therefore, the hydrolase to show unique characteristics being filtered out from marine microorganism just into weight of exploitation infant industry enzyme preparation Want direction.

The present invention screens a kind of new SGNH families hydrolase gene from a kind of marine bacteria, and the gene is carried out Recombination expression, recombinase have the characteristics that thermostabilization, salt tolerant and organic solvent-resistant, available for fine chemistry industry, pharmacy, washing, The industrial circle such as wastewater treatment and environment remediation.

The content of the invention

It is an object of the invention to provide a kind of new marine bacteria source hydrolase, its encoding gene and preparation method thereof, The hydrolase can be used for esters degraded and living things catalysis and the conversion of other ester type compounds under the conditions of high temperature and high salt.

The present invention relates to the polypeptide of the separation with hydrolytic enzyme activities, and it is selected from the group：

(a) polypeptide, itself and SEQ ID NO:Sequence shown in 2 polypeptide is consistent；

(b) polypeptide, it is SEQ ID NO:The remote catalytic center position of polypeptide shown in 2 carry out various substitutions, addition and/ Or the mutant that one or several amino acid of missing obtain, the mutant have and SEQ ID NO:Protein sequence shown in 2 is extremely Few more than 90% homology and at least more than 90% enzymatic activity.

Polypeptide of the present invention with hydrolytic enzyme activities, its bacterium from mangrove wild rice root nodule soil Altererythrobacter indicus。

The present invention bacterium Altererythrobacter indicus native for being isolated from mangrove wild rice root nodule DSM 18604, bacterial strain are bought from Germany Microbiological Culture Collection Center DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen).Based on its whole genome sequence, screening obtains hydrolase gene Aline4, its nucleotide sequence is as shown in SEQ ID No.1.Gene aline4 sizes are 573bp, base composition 139A (24.26%), 115T (20.07%), 149C (26.00%) and 170G (29.67%), encoding proteins size are 190 amino Sour residue, its amino acid sequence is as shown in SEQ ID No.2.By hydrolase A linE4 amino acid sequences in GenBank nr numbers According to Homology search is carried out in storehouse, uniformity highest is grand genomic source arylesterase therewith, uniformity be 71% (its Number of registration in GenBank databases is OJW68931.1).Amino acid sequence analysis shows that active site serine is attached Nearly sequence is the conserved region (amino acid position is 11 to 14) with Gly-Asp-serine-leucine composition, 13 Position serine and 162 aspartic acids and 165 hyte propylhomoserins collectively form serine hydrolase catalytic triads.In addition, 13 Serine, 50 glycine and 81 asparagines collectively form negative oxygen ion hole.Its amino acid sequence feature meets SGNH water Solve enzyme family feature.In summary, AlinE4 should be a newcomer in SGNH hydrolase families.

, can be to SEQ ID NO under the premise of AlinE4 protein actives are not influenceed:Remote catalytic center amino acid shown in 2 The amino acid sequence of position (preferably away from the amino acid position of 11-14,162 and 165) carries out various substitutions, additions and/or deletions One or several amino acid obtain the derived protein with AlinE4 activity.According to the common knowledge of art technology, albumen The biological activity of matter is closely related with its functional domain.In general, only occur in the site of functional domain Mutation may have an impact to the 2 and 3 dimensional organization of protein, so as to influence its biological activity.And for occurring remote From the amino acid sites of functional domain (amino acid position of preferably 11-14,162 and 165), because this region is not involved in albumen Functional conformation, indivedual point mutation of amino acid will not produce substantial effect to the biological activity of protein, so as to base The biological function of this reservation crude protein.Preferable hydrolase A linE4 mutant have at least with SEQ ID NO:Shown in 2 Amino acid sequence more than 90% homology, more preferably have at least more than 95% homology, most preferably have at least More than 99% homology.Described mutant can retain hydrolase A linE4 biological function, the preferably mutation substantially Body has and SEQ ID NO:The enzymatic activity of the hydrolase of amino acid sequence shown in 2 at least more than 90%, more preferably have at least More than 95% enzymatic activity, most preferably at least more than 99% enzymatic activity.

The present invention relates to SEQ ID NO:2 mature polypeptide or including for its homologous sequence substitute, lack and/or inserted one The artificial variants of individual or multiple amino acid, mutated site are preferably smaller than 5, more preferably less than 3, most preferably only 1 position The mutation of amino acid.The example of conservative replacement is within the following group：Basic amino acid group (arginine, lysine and group ammonia Acid), acidic amino acid group (glutamic acid and aspartic acid), polar amino acid group (glutamine and asparagine), hydrophobic amino Sour group (leucine, isoleucine and valine), aromatic amino acid group (phenylalanine, tryptophan and tyrosine) and p1 amino acid Group (glycine, alanine, serine, threonine and methionine).The 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor for not changing specific activity generally is ability Known to domain, and by such as Η .Neurath and R.L.Hill, 1979 in TheProteins, Academic Press, New Described in York.The exchange most generally occurred be Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn、Ala/Val、Ser/Gly、Tyr/Phe、Ala/Pro、Lys/Arg、Asp/Asn、Leu/Ile、Leu/Val、Ala/ Glu and Asp/Gly etc..

Known mutagenesis, restructuring and/or Shuffling Method can be used, then carry out the screening process of correlation, such as by Reidhaar-Olson and Sauer, 1988, Science, 241：53-57；Bowie and Sauer, 1989, Proc.Natl.Acad.Sci.USA86:2152-2156；Those disclosed in WO95/17413 or WO 95/22625, carry out One or more 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, missing and/or insertion are simultaneously tested.Other workable methods include fallibility PCR, phagocytosis Body display (such as Lowman etc., 1991, Biochemistry30:10832-10837；

U.S. Patent number 5,223,409；) and regiondirected mutagenesis (region-directed WO92/06204 Mutagenesis) (Derbyshire etc., 1986, Gene46:145 and 1988, DNA7:127).

The invention further relates to the polynucleotides of separation, and it includes the nucleotide sequence for encoding hydrolase A linE4 of the present invention, Or the nucleotide sequence by the coding present invention with the mutant of hydrolase A linE4 activity forms.

The present invention relates to the polynucleotides of separation of the coding with hydrolase A linE4 active peptides, it is selected from the group：

(a) polynucleotides, itself and SEQ ID NO:Sequence shown in 1 nucleotides is consistent；

(b) polynucleotides, it is coding SEQ ID NO:The amino acid sequence of remote catalytic center amino acid position shown in 1 Row carry out the polynucleotides for the mutant that various one or several amino acid of substitutions, additions and/or deletions obtain, the polynucleotides With with SEQ ID NO:The homology of nucleotide sequence at least more than 90% shown in 1.

The invention provides encoding hydrolytic enzymes AlinE4 gene aline4, its nucleotides shown in SEQ ID NO.1 Sequence is consistent；Gene aline4 sizes are 573bp, and base composition is 139A (24.26%), 115T (20.07%), 149C (26.00%) and 170G (29.67%), encoding proteins size is 190 amino acid residues, its amino acid sequence such as SEQ ID Shown in No.2.The present invention is also provided to removing 31-42,484-186 and 493-495 in the nucleotide sequence shown in SEQ ID NO.1 Other nucleotides outside the nucleotides of position are replaced, add and/or lacked one or several nucleotides can base so as to obtain coding The mutant gene of this reservation AlinE4 biological activity of albumen.Preferable hydrolase A linE4 mutant genes have at least with SEQ ID NO:The homology of nucleotide sequence more than 90% shown in 1, more preferably there is at least more than 95% homology, most It is preferred that with least more than 99% homology.

The invention further relates to the nucleic acid construct of the polynucleotides of the separation comprising the present invention, can use multi-mode operation perhaps The polynucleotides of the separation of hydrolase of the present invention are encoded to provide the expression of hydrolase.The polynucleotides of the separation and one or Multiple regulating and controlling sequences are operably connected, and the regulating and controlling sequence is in suitable host cell in the bar compatible with the regulating and controlling sequence The expression of coded sequence is instructed under part.Regulating and controlling sequence can be appropriate promoter sequence, and it is by for expressing code book hair The nucleotide sequence of the host cell identification of the polynucleotides of bright polypeptide.Promoter sequence contains the transcription of the expression of direct polypeptide Regulating and controlling sequence.Promoter can be that any nucleotide sequence of transcriptional activity is shown in selected host cell, including mutation , promoter truncate and heterozygosis, and can be from coding with the homologous or heterologous extracellular or intracellular polypeptide of host cell Gene obtains.

Using gene clone technology, the hydrolase A linE4 genes being cloned into can be connected on suitable carrier, and turned Change or be transfected into prokaryotes or eucaryote host expresses Prepare restructuring hydrolase A linE4.Suitable prokaryotes host bag Various bacteriums such as E.coli etc. is included, suitable eucaryote host includes yeast (such as methanol yeast) and mammalian cell (such as Chinese hamster ovary cell) etc., it is preferred to use prokaryotic expression system E.coli.

The protokaryon or eukaryotic expression for the various commercially viable purchases that suitable carrier is well known to those skilled in the art carry Body, prokaryotic expression carrier such as pET serial carriers, pQE serial carriers；Yeast expression carrier pPICZ- α-A, pHIL-D2, pPIC9, pHIL-S1(Invitrogen Corp.San Diego.California.USA)；Animal cell expression vectors pSVK3, pMSG (Amersham Pharmacia Biotech Inc.USA) etc..

The invention further relates to recombinant host cell, and it includes the polynucleotides of the separation of the present invention, it may be advantageous to for water In the recombinant production for solving enzyme AlinE4.By the vector introduction host cell of the polynucleotides comprising the present invention, the choosing of host cell Select the gene for being largely dependent upon coded polypeptide and its source.Host cell can be the hydrolase in the present invention AlinE4 restructuring any cell useful in producing, for example, protokaryon or eukaryotic., can general gram using gene clone technology It is grand to hydrolase A linE4 genes be connected on suitable carrier, and convert or be transfected into prokaryotes or eucaryote host Express Prepare restructuring hydrolase A linE4.Suitable prokaryotes host includes various bacteriums such as E.coli etc., can be by as follows Protoplast transformation or or electroporation carrier is transformed into prokaryotic.Suitable eucaryote host includes yeast (such as Methanol yeast) and mammalian cell (such as Chinese hamster ovary cell).Present invention preferably employs prokaryotic expression system E.coli Expression product hydrolase As linE4.One preferable example is that the hydrolase gene aline4 for screening the present invention connects It is connected on coli expression carrier pET28a, and is transformed into e. coli bl21 (DE3), goes out high activity through induced expression Recombinase.

The invention further relates to the method for producing hydrolase A linE4 of the present invention, it includes：

(a) recombinant host cell is cultivated under conditions of hydrolase A linE4 is helped to create, wherein the host cell Include SEQ ID NO:The nucleotides of nucleotides shown in 1 or its at least one mutational site；

(b) polypeptide is reclaimed.

In the production method of the present invention, it is being suitable for producing the hydrolase A linE4 using methods known in the art Nutrient medium in cultivate cell.For example, can be by suitable culture medium and allowing to express and/or separating the hydrolysis Small-scale or large scale fermentation (bag in the Shaking culture carried out under conditions of enzyme AlinE4, and laboratory or industrial fermentation tank Include it is continuous, in batches, fed-batch or solid state fermentation) cultivate cell.Trained using methods known in the art in suitable nutrition Support and cultivated in base, the nutrient medium includes carbon source and nitrogen source and inorganic salts.Suitable culture medium can supply from business Business is answered to obtain or can be prepared according to disclosed composition.If polypeptide is secreted into nutrient medium, the polypeptide can be from described Directly reclaimed in culture medium.If polypeptide is not secreted, it can be reclaimed from cell lysate.

Gained hydrolase A linE4 can be reclaimed using methods known in the art.For example, can by conventional method from Reclaimed in nutrient medium, the conventional method includes but is not limited to centrifugation, filtering, extraction, spray drying, evaporation or precipitated. Can be purified by a variety of methods known in the art, methods described include but is not limited to chromatograph (for example, ion exchange, it is affine, Hydrophobic, chromatofocusing and size exclusion) or the methods of differential solubility (such as ammonium sulfate precipitation).

Present invention also offers hydrolase A linE4 or the application of hydrolase A linE4 Host Strains industrially can be expressed, Such as available for being catalyzed ester-type hydrolysis.Show that hydrolase A linE4 has esterase active by esterase activity measure.AlinE4 or The above-mentioned Host Strains that can express AlinE4 can be used for hydrolyzing short-chain fatty acid ester, such as C2-C8 short carbon chain fatty acid esters, while right C10-C14 Long carbon chain fatty acid ester also has certain degradation.Preferable short chain fatty acids fat is with C2-C8 short carbon chains P-nitrophenyl phenolic ester, such as p-nitrophenol acetic acid esters, p-nitrophenol butyrate, p-nitrophenol capronate and to nitro Catalytic activity highest when phenol caprylate etc., wherein substrate are p-nitrophenol butyrate (C4), enzyme activity 25.8U/mg.

AlinE4 catalyzing hydrolysis temperature range is 15~60 DEG C, preferably 40 DEG C；The pH value of the hydrolysis be 6.0~ 10.5, preferably 7.5.It is incubated in 10~60 DEG C under the conditions of 1h and 4h, remains to keep more than 80% activity；It is incubated in 90 DEG C 0.5h and 1h, AlinE4 can keep more than 45% activity；0.5mol/L and 1mol/L NaCl conditions can REINFORCED Al inE4 activity, More than 95% activity can be retained under the conditions of 2mol/L and 1mol/L NaCl；In Ba²⁺、Ca²⁺、Mg²⁺And Sr²⁺In the presence of can retain More than 85% activity；Ethanol, DMSO, glycerine, isopropanol and methanol can strengthen its activity.

The present invention sieves from mangrove wild rice root nodule soil bacterium Altererythrobacter indicus DSM18604 Choosing obtains new thermostabilization salt tolerant organic solvent-resistant hydrolase gene, it was found that the gene coded protein has excellent zymetology special Property, it can be applied in catalysis solution ester and enzymatic clarification ester process of producing product.The hydrolase gene of acquisition can be cloned into suitably Heterogenous expression is realized in host, realizes industrialized production thermostabilization salt tolerant organic solvent-resistant hydrolase, is follow-up commercial Application There is provided cost cheap thermostabilization salt tolerant organic solvent-resistant hydrolase parent material.The production of the enzyme can be at detergent, waste water Shown in the high temperature such as reason, fine chemistry industry, pharmacy and environment remediation, saliferous and production technology containing organic solvent important economy and Social value.

Brief description of the drawings

Fig. 1 is purifying hydrolase AlinE4 policapram gel electrophoresis analysis figure.

Fig. 2 is hydrolase A linE4 substrate specificity figure.C2：P-nitrophenol acetic acid esters；C4：P-nitrophenol butyric acid Ester, C6：P-nitrophenol capronate；C8：P-nitrophenol caprylate；C10：P-nitrophenol decylate；C12：P-nitrophenyl Phenol dodecanoate；C14：P-nitrophenol myristinate；C16：P-nitrophenol Palmitate；Define measured value when substrate is C4 For 100%.

Fig. 3 schemes for hydrolase A linE4 optimal reactions pH.

Fig. 4 is hydrolase A linE4 optimal reactive temperature figures.

Fig. 5 is heat endurance figure under hydrolase A linE4 different temperatures.

Fig. 6 is different time heat endurance figure under hydrolase A linE4 high temperature.

Fig. 7 is NaCl to hydrolase A linE4 activity images figures.

Fig. 8 is bivalent cation to hydrolase A linE4 activity influence figures.

Fig. 9 is organic solvent to hydrolase A linE4 activity influence figures.

Embodiment

The hydrolase gene aline4 of embodiment 1 acquisition

Based on the bacterium Altererythrobacter indicus DSM for being isolated from mangrove wild rice root nodule soil 18604 full-length genomes, ORFs prediction and gene annotation result, screen lipid hydrolyzing enzyme related gene.Pass through Blastp (http://blast.ncbi.nlm.nih.gov/) homology of known hydrolase gene sequence in aligned sequences and database. Analysis is compared through database and obtains aline4 genes, and size 573bp, base composition is 139A (24.26%), 115T (20.07%), 149C (26.00%) and 170G (29.67%), its nucleotide sequence such as SEQ ID No:Shown in 1.Encoding proteins Size is 190 amino acid residues, and its amino acid sequence is as shown in SEQ ID No.2.The AlinE4 protein sequences are existed Homology search is carried out in GenBank, Amino acid sequence identity highest is non-culture source esterase therewith, and uniformity is 71%, its number of registration in GenBank databases is OJW68931.1.

Phylogenetic Analysis shows that hydrolase A linE4 belongs to esterase II family, also falls within SGNH hydrolase families.Amino Sequence analysis result shows that nearby sequence is with Gly-Asp-serine-leucine group to active site serine Into conserved region (amino acid position be 11 to 14), 13 serines collectively form with 162 aspartic acids and 165 hyte propylhomoserins Serine hydrolase catalytic center.13 serines, 50 glycine and 81 asparagines collectively form negative oxygen ion hole.Its Amino acid sequence feature meets SGNH hydrolase family features.

In summary, AlinE4 should be a newcomer in esterase family and SGNH hydrolase families.

The gene aline4 of embodiment 2 recombinant expression plasmid and the structure of recombinant bacterial strain

The gene aline4 that the present invention obtains is cloned on expression vector, builds recombinant strains.Based on NCBI The gene open reading frame sequence that ORF Finder ORF analyses obtain, the sense primer aline4F of design amplification full genome (5’-TCGCGGATCCATGGGCGAATCGCGC-3 ', BamHI) and anti-sense primer aline4R (5 '- TCCGCTCGAGTCACTTCTTCGCAGGCAGCGCC-3 ', XhoI), PCR amplifications confirm full length gene sequence.Using digestion gram Grand method construction expression plasmid, i.e., with BamHI and XhoI double digestion PCR primers, fragment after purification with through BamHI and XhoI The plasmid pSMT3 connections of double digestion, using CaCl₂Conversion method is converted into E.coli DH5 α, and kalamycin resistance screening is positive Clone.Using the plasmid of plasmid extraction kit (Omega, the U.S.) extraction positive colony, reflected through BamHI and XhoI double digestions It is fixed, 600bp or so DNA fragmentation is obtained, is gene aline4 through sequencing identification.Recombinant expression plasmid is transformed into E.coli In Rosetta (DE3) expression bacterial strains, construction expression recombinant bacterial strain.

Embodiment 3 utilizes recombinant strains expression recombination aline4

The 3ml recombinant strains built are transferred to 100ml contains 20 μ g/ml kanamycins and 34 μ g/ml chlorine are mould In the LB fluid nutrient mediums of element, 37 DEG C of shaken cultivations to OD₆₀₀Reach 0.6, the IPTG for adding final concentration of 0.5mM is induced Expression, is transferred to 20 DEG C with 150r/min shaken cultivations 16h.Low-temperature centrifugation collects thalline, is resuspended in NTA-10 solution (500mM chlorine Change sodium, 10mM imidazoles, 20mM Tris hydrochloric acid, pH 8.0) in, ultrasonic disruption processing is being carried out on ice.On low-temperature centrifugation is collected Clearly, using NTA-Ni²⁺Affinity column chromatography purifies expressing protein.Expressed recombinant protein contains the 6 × His tag of N-terminal, affable Inhaled with layer is adsorbed onto on post, by the imidazole solution gradient elution of various concentrations, collect eluent.Through SDS-PAGE testing goals Distribution situation of the albumen in eluent.The ubiquitin-like SUMO at recombinant protein N end is cut off in bag filter using ULP1 enzymes, and is adopted Use NTA-Ni²⁺Affinity column chromatography removes SUMO albumen, collects sample and carries out SDS-PAGE detections.Obtain electrophoretically pure recombinant protein AlinE4, molecular weight about 20kDa (Fig. 1).Protein concentration is determined with Brandford methods.

The recombination aline4 of embodiment 4 Activity determination

Utilize the restructuring hydrolase A linE4 activity of p-nitrophenol butyric acid ester process measure purifying.Concrete operations：1ml reacts System includes 1mM p-nitrophenol butyrates, 100mM Tris-HCl buffer solutions (pH 7.5) and 185ng pure enzyme proteins, adopts With ultra-violet and visible spectrophotometer (Beckman DU800 types, the U.S.) the METHOD FOR CONTINUOUS DETERMINATION light absorption value A under the conditions of 40 DEG C₄₀₅ 2min, the enzyme liquid using inactivation are used to return to zero as control.One enzyme activity unit is defined as per minute from p-nitrophenyl phenolic ester Catalysis produces the required enzyme amount of l μm of ol p-nitrophenol.The esterase active measured is 25.8U/mg.

The hydrolase A linE4 substrate specificities of embodiment 5 are analyzed

Hydrolase A linE4 substrate specificity analysis uses system (1ml)：100mM Tris-HCl buffer solutions (pH 7.5), 1mM substrates, 185ng pure enzyme proteins, the METHOD FOR CONTINUOUS DETERMINATION light absorption value A at 25 DEG C are added₄₀₅2min.Determine the substrate used For：P-nitrophenol acetic acid esters (C2), p-nitrophenol butyrate (C4), p-nitrophenol capronate (C6), p-nitrophenol Caprylate (C8), p-nitrophenol decylate (C10), p-nitrophenol dodecanoate (C12), p-nitrophenol myristinate (C14), p-nitrophenol Palmitate (C16).Show after measured, p-nitrophenyl phenolic ester shorter to acyl group carbochain AlinE4 (C4, C6 and C8) has higher catalytic activity, catalytic activity highest when wherein substrate is p-nitrophenol butyrate (C4), to acyl The longer p-nitrophenyl phenolic ester (C10, C12 and C14) of base carbochain also has certain catalytic activity (Fig. 2).As a result show, hydrolyze Enzyme AlinE4 has preferable catalytic activity to the shorter lipid material of acyl group carbochain, and the hydrolysis vigor for short-chain lipid is better than long-chain Lipid.

The hydrolase A linE4 optimum reaction conditionses of embodiment 6 are analyzed

Hydrolase A linE4 optimal reactions pH is determined in the range of 3.0~10.5.Concrete operations are：In different pH buffer solutions Middle addition 1mM p-nitrophenols butyrate and 185ng pure enzyme proteins, the METHOD FOR CONTINUOUS DETERMINATION light absorption value A at 40 DEG C₃₄₈2min.Measure The buffer solution used is：100mM citric acid-sodium citrate buffer solutions (pH 3.0~6.0), 100mM potassium dihydrogen phosphates-hydroxide Sodium buffer solution (pH 6.0~7.5), 100mM Tris hydrochloride buffers (pH 7.5~9.0) and 100mM 2- cyclohexylamino second sulphurs Acid-sodium hydrate buffer solution (pH 9.0~10.5).Measurement result shows, AlinE4 optimal reactions pH is 7.5, pH 6.0~ (Fig. 3) active in the range of 10.5.

Hydrolase A linE4 optimal reactive temperatures determine in the range of 15~60 DEG C.Concrete operations are：1ml reaction systems In, 1mM p-nitrophenol butyrates, 100mM Tris-HCl buffer solutions (pH 7.5) and 185ng pure enzyme proteins are added, is existed respectively 15th, METHOD FOR CONTINUOUS DETERMINATION light absorption value A under the conditions of 20,25,30,35,40,45,50,55 and 60 DEG C₄₀₅2min.Measurement result shows AlinE4 range of reaction temperature is 15~60 DEG C, and optimal reactive temperature is 40 DEG C (Fig. 3).

The hydrolase A linE4 zymetology stability analyses of embodiment 7

Hydrolase A linE4 thermal stability analysis concrete operations are：(1) by enzyme liquid respectively 10,20,30,40,50, 60th, 1h and 4h is incubated under the conditions of 70,80,90 and 100 DEG C, determines the activity of enzyme；(2) by enzyme liquid respectively in 90,95 and 100 DEG C of bars 0.5,1,1.5,2 and 2.5h is incubated under part, determines the activity of enzyme.Live body system is：In 1ml reaction systems, 1mM is added to nitro Phenol butyrate, 100mM Tris-HCl buffer solutions (pH 7.5) and 185ng pure enzyme proteins, the METHOD FOR CONTINUOUS DETERMINATION light absorption value at 40 DEG C A₄₀₅2min.As a result show, 4h is incubated in 10~60 DEG C and is incubated in 10~70 DEG C under the conditions of 1h, AlinE4 remains to keep More than 80% active (Fig. 5)；0.5h and 1h, AlinE4 are incubated in 90 DEG C can keep 50% and more than 45% active (Fig. 6)； 0.5h and 1h, AlinE4 are incubated in 100 DEG C can keep 30% and more than 20% active (Fig. 6), and it is good to illustrate that AlinE4 has Heat endurance.

Measure concrete operations of the NaCl to hydrolase A linE4 activity influences are：Be separately added into 0 in reaction system, 0.5, 1st, 2,3,4 and 5mol/L NaCl, the activity of enzyme is determined.Live body system is：In 1ml reaction systems, 1mM p-nitrophenols are added Butyrate, 100mM Tris-HCl buffer solutions (pH 7.5) and 185ng pure enzyme proteins, the METHOD FOR CONTINUOUS DETERMINATION light absorption value A at 40 DEG C₄₀₅ 2min.As a result show, under the conditions of 0.5~3mol/L NaCl, NaCl (retains 95% without influence substantially on AlinE4 activity Above activity), wherein 0.5mol/L and 1mol/L NaCl conditions can REINFORCED Al inE4 activity；When NaCl concentration reaches 5mol/L, AlinE4 remains to retain more than 40% activity, illustrates that AlinE4 has good salt-tolerant trait (Fig. 7).

Measure concrete operations of the bivalent cation to hydrolase A linE4 activity influences are：It is separately added into reaction system 10mM Ba²⁺、Ca²⁺、Cd²⁺、Co²⁺、Cu²⁺、Mg²⁺、Mn²⁺、Ni²⁺、Sr²⁺、Zn²⁺With ethylenediamine tetra-acetic acid (EDTA), enzyme activity is determined Property.Surveying enzyme activity system is：In 1ml reaction systems, 1mM p-nitrophenol butyrates, 100mM Tris-HCl buffer solutions (pH are added 7.5) with 185ng pure enzyme proteins, the METHOD FOR CONTINUOUS DETERMINATION light absorption value A at 40 DEG C₄₀₅2min.Measurement result shows that AlinE4 activity can quilt Cd²⁺、Cu²⁺、Ni²⁺And Zn²⁺Ion substantially suppresses, in Ba²⁺、Ca²⁺、Mg²⁺And Sr²⁺In the presence of on enzyme activity influence less (retain More than 85% activity), EDTA can strengthen its active (Fig. 8).

Measure concrete operations of the organic solvent to hydrolase A linE4 activity influences are：It is separately added into reaction system 15% (v/v) organic solvent：Acetone (Acetone), acetonitrile (Acetonitrile), ethanol (Ethanol), dimethylformamide (DMF), dimethyl sulfoxide (DMSO) (DMSO), glycerine (Glycerol), isopropanol (Isopropanol) and methanol (Methanol), survey Determine the activity of enzyme.Live body system is：In 1ml reaction systems, 1mM p-nitrophenol butyrates are added, 100mM Tris-HCl delay Fliud flushing (pH 7.5) and 185ng pure enzyme proteins, the METHOD FOR CONTINUOUS DETERMINATION light absorption value A at 40 DEG C₄₀₅2min.Measurement result shows, AlinE4 activity can be completely inhibited by acetonitrile, and ethanol, DMSO, glycerine, isopropanol and methanol can strengthen its activity, particularly in second AlinE4 activity doubles the above (Fig. 9) in the presence of alcohol and glycerine.

Sequence table

<110>The Second Institute of Oceanograghy,SOA

<120>A kind of marine bacteria source thermostabilization salt tolerant SGNH family's hydrolases and application

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 573

<212> DNA

<213> Altererythrobacter indicus

<400> 1

atgggcgaat cgcgcgtgat tctcgccttc ggagacagcc tgtttgcagg ctatggcctt 60

gataaggggg agagctatcc ggcaaagctg gaaactgcgc tgcgcagcca tggcatcaat 120

gccagaatca ttaatgccgg cgtttcgggt gacaccactg cggcagggct gcagcgaatc 180

aaattcgtgc tggatagcca gccggacaag ccggaattgg ccatagtgga actgggcggg 240

aatgaccttt tacgcggcct ctcaccagcc gaagcgcggc agaacctcag cggaatcctc 300

gaagaattgc agaggcggaa aattccaatc ctgttgatgg gaatgcgagc gccgcccaat 360

ctaggggcaa aatatcagcg cgaatttgat gggatttatc cctatctggc cgaaaaatat 420

gacgccaagc tggtaccttt cttccttgag gccgtggcag atagacctga cctcattcag 480

aaggatcacg ttcaccccac tgcgcgcggt gtggaggaac tcgtgtctgc aacatcgaat 540

gcagttgcca aggcgctgcc tgcgaagaag tga 573

<210> 2

<211> 190

<212> PRT

<213> Altererythrobacter indicus

<400> 2

Met Gly Glu Ser Arg Val Ile Leu Ala Phe Gly Asp Ser Leu Phe Ala

1 5 10 15

Gly Tyr Gly Leu Asp Lys Gly Glu Ser Tyr Pro Ala Lys Leu Glu Thr

20 25 30

Ala Leu Arg Ser His Gly Ile Asn Ala Arg Ile Ile Asn Ala Gly Val

35 40 45

Ser Gly Asp Thr Thr Ala Ala Gly Leu Gln Arg Ile Lys Phe Val Leu

50 55 60

Asp Ser Gln Pro Asp Lys Pro Glu Leu Ala Ile Val Glu Leu Gly Gly

65 70 75 80

Asn Asp Leu Leu Arg Gly Leu Ser Pro Ala Glu Ala Arg Gln Asn Leu

85 90 95

Ser Gly Ile Leu Glu Glu Leu Gln Arg Arg Lys Ile Pro Ile Leu Leu

100 105 110

Met Gly Met Arg Ala Pro Pro Asn Leu Gly Ala Lys Tyr Gln Arg Glu

115 120 125

Phe Asp Gly Ile Tyr Pro Tyr Leu Ala Glu Lys Tyr Asp Ala Lys Leu

130 135 140

Val Pro Phe Phe Leu Glu Ala Val Ala Asp Arg Pro Asp Leu Ile Gln

145 150 155 160

Lys Asp His Val His Pro Thr Ala Arg Gly Val Glu Glu Leu Val Ser

165 170 175

Ala Thr Ser Asn Ala Val Ala Lys Ala Leu Pro Ala Lys Lys

180 185 190

Claims (10)

1. a kind of polypeptide of the separation with hydrolytic enzyme activities, it is selected from the group：

(a) polypeptide, itself and SEQ ID NO:Sequence shown in 2 polypeptide is consistent；

(b) polypeptide, it is SEQ ID NO:The remote catalytic center position of polypeptide shown in 2 carries out various substitutions, addition and/or lacked The mutant that one or several amino acid obtain is lost, the mutant has and SEQ ID NO:Protein sequence shown in 2 is at least More than 90% homology and at least more than 90% hydrolytic enzyme activities.

2. polypeptide according to claim 1, it is characterised in that：The catalytic center of the hydrolase is SEQ ID NO:2 institutes 11-14,162 and No. 165 amino acid positions shown.

3. polypeptide according to claim 1, it is characterised in that：Described mutant is SEQ ID NO:Polypeptide shown in 2 Various substitutions, additions and/or deletions are carried out less than the mutant that 5 amino acid obtains away from catalytic center position.

4. a kind of encode the polynucleotides with polypeptide described in claim 1, it is selected from the group：

(a) polynucleotides, itself and SEQ ID NO:Sequence shown in 1 nucleotides is consistent；

(b) polynucleotides, it is to removing 31-42,484-186 and 493-495 position in the nucleotide sequence shown in SEQ ID NO.1 Other nucleotides outside nucleotides are replaced, add and/or lacked the mutant gene that one or several nucleotides obtain, should Polynucleotides have and SEQ ID NO:The homology of nucleotide sequence at least more than 90% shown in 1.

5. a kind of nucleic acid construct, it includes the multinuclear for the claim 4 being operably connected with one or more regulating and controlling sequences Thuja acid, the regulating and controlling sequence instruct the generation of the polypeptide in suitable expressive host.

6. a kind of recombinant expression carrier, it includes the nucleic acid construct of claim 5.

7. a kind of host, it is inverted or transfected prokaryotic is biological or eucaryote host obtains as the carrier described in claim 6.

8. host according to claim 7, it is bacterium, yeast or mammalian cell.

9. a kind of method for producing any one of the claim 1-3 polypeptide, it includes：

(a) recombinant host cell described in claim 7, is cultivated under conditions of hydrolase is helped to create, wherein the place Chief cell includes SEQ ID N0:The nucleotides of nucleotides shown in 1 or its at least one mutational site；

(b) polypeptide, is reclaimed.

10. the Host Strains that can express hydrolase described in hydrolase or claim 7 described in claim 1 are in catalysis esters water Application in solution.