CN112301014A

CN112301014A - Esterase mutant with improved thermal stability and application thereof

Info

Publication number: CN112301014A
Application number: CN202011216111.8A
Authority: CN
Inventors: 杨广宇; 马富强; 秦朕龙; 郭天杰; 李长龙
Original assignee: Shanghai Shendao Biotechnology Co ltd
Current assignee: Shanghai Hannover Biotechnology Co Ltd; Shanghai Shendao Biotechnology Co ltd
Priority date: 2020-11-04
Filing date: 2020-11-04
Publication date: 2021-02-02
Anticipated expiration: 2040-11-04
Also published as: CN112301014B

Abstract

The invention discloses an Escherichia coli esterase (EstWY enzyme) mutant with improved thermal stability, belonging to the technical field of enzyme engineering. The mutant enzyme with remarkably improved thermal stability is obtained by carrying out mutation and codon optimization on the EstWY enzyme gene derived from an Escherichia coli strain. Compared with the wild type, the half-life period of the mutant at 45 ℃ is about 5 times that of the wild type, which shows that the mutant is compared with the wild type, and the constructed high-efficiency expression genetic engineering bacteria have the advantages of short culture period, simple culture condition, high target protein yield and simple purification.

Description

Esterase mutant with improved thermal stability and application thereof

Technical Field

The invention relates to the field of bioengineering, and particularly relates to an esterase mutant with improved thermal stability and application thereof.

Background

Esterases (esterases) are enzyme systems that catalyze the hydrolysis of ester compounds, which function to hydrolyze both aliphatic and aromatic esters. The esters are hydrolyzed into acids and alcohols by hydrolysis in the presence of water molecules. The reaction formula is as follows: R-COOR/(ester) + H₂O (water) ═ RCOOH (fatty acid) + R/OH (alcohol). It is widely found in animals, plants and microorganisms. Among them, animal pancreatic esterase and microbial esterase are the main sources of esterase. Because of rich microbial resources and the advantages of convenient industrial production and the like of microbial fermentation enzyme production, the esterase is widely applied to the fields of agriculture, food brewing, medicinal chemistry, sewage treatment, bioremediation and the like, and because the enzymatic reaction of the esterase has higher substrate specificity, regioselectivity/enantioselectivity, the esterase is a high-efficiency biocatalyst for synthesizing chiral compounds. However, since natural enzymes all function in a relatively mild environment in vivo, and industrial application processes require enzymes to function in a relatively harsh environment (such as high temperature, extreme pH value, organic solvent, non-natural substrate, product inhibition, etc.), natural enzymes often suffer from poor stability in application. For this purpose, enzymes with good stability must be selected to meet the requirements of industrial production. The key to solve the problem is to improve the thermal stability of EstWY enzyme by means of protein engineering.

Commonly used protein engineering methods are rational design (rational design) and irrational design (irrational design), based on structural function-relationship and high-throughput screening, respectively. The rational design has the defect of accuracy, mainly the structure-function relationship of the protein is too numerous and complicated, and the prior art still lacks sufficient knowledge. To optimize the thermal stability of proteins efficiently, Markus Wys et al, 2001, proposed a Consensus theory (Consensus). Unlike rational design based on the precise structure-function relationship of proteins, the Consensus Concept is based on the amino acid sequence information of homologous proteins and analyzes the information capable of improving the thermal stability of enzymes from the evolutionary point of view.

The original strain (such as Fervidobacterium nodosum) is used for fermentation, the problems of harsh culture conditions and low enzyme production level exist, and the target enzyme is difficult to obtain. Meanwhile, the original strain enzyme system is complex and cannot be directly used, the purification steps are complicated, the purified enzyme cannot be directly used, and the problem of poor stability exists.

Natural evolution limits the function of the original EstWY enzyme and often cannot be applied in an industrial approach in an optimal state. By using a protein engineering method, the original gene can be subjected to molecular modification, the limit of natural evolution is broken, and a new enzyme gene which is suitable for industrial application and has excellent properties is obtained. Therefore, the requirement of EstWY for efficient expression, purification and application is always the focus and difficulty of the research of the technicians in the field.

Disclosure of Invention

The invention takes Consensus theory (Consensus) as a guiding idea, integrates and analyzes the sequence of an esterase family, and combines bioinformatics and crystallography assistance to obtain a novel esterase mutant with high stability.

In order to solve the technical problems, the invention obtains the EstWY enzyme mutant with improved thermal stability by using a consensus design method, screens 5 amino acid mutation sites by using a consensus method which is improved based on a traditional consensus method and has no systematic development bias, performs site-specific mutation on the amino acid mutation sites to obtain the mutant enzyme with obviously improved thermal stability, overcomes the defects that the existing EstWY enzyme has poor thermal stability and cannot meet the requirements of reagents, and lays a foundation for widening the industrial application of the EstWY enzyme.

The first purpose of the invention is to provide an EstWY enzyme, wherein the EstWY enzyme is derived from Escherichia coli and is named as ENND-TOP protein, the nucleotide sequence of the encoded ENND-TOP protein is shown as SEQ ID NO.1, and the amino acid sequence of the EstWY enzyme is shown as SEQ ID NO. 2.

Furthermore, the EstWY enzyme is a derived protein which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO.2 and has the same function with the protein shown in SEQ ID NO. 2.

Further, the EstWY enzyme is an EstWY enzyme mutant obtained by substituting one or more amino acid residues for an amino acid residue at one or more positions in the amino acid sequence shown in SEQ ID No. 2.

Further, the EstWY enzyme is an EstWY enzyme mutant obtained by substituting one or more amino acid residues for an amino acid residue at one or more positions of the amino acid sequence of the EstWY enzyme exhibiting an amino acid sequence showing at least 90% homology among the amino acid sequences shown in SEQ ID No. 2.

Further, the mutation substitution site of the amino acid sequence of the amine dehydrogenase represented by SEQ ID NO.2 includes at least one of: 276, 279, 289, 322, 358 or corresponding positions thereof.

Further, the EstWY enzyme mutant comprises a single-point mutant of any one single-point mutation site of S276V, L279F, T289R, R322G and N358D in an amino acid sequence shown in SEQ ID NO. 2.

Further, the EstWY enzyme mutant comprises a combined mutant on the amino acid sequence shown in SEQ ID NO. 2: L279F/T289R, L279F/R322G, L279F/N358D, T289R/R322G, T289R/N358D, R322G/N358D, L279F/T289R/R322G, L279F/T289R/N358D, L279F/R322G/N358D, T289R/R322G/N358D, L279F/T289R/R322G/N358D.

In a second aspect of the invention, a gene encoding an EstWY enzyme mutant is disclosed.

In the third aspect of the invention, a recombinant plasmid containing EstWY enzyme mutant gene is disclosed.

The fourth aspect of the invention discloses soluble protein, immobilized enzyme or engineering bacteria for expressing EstWY enzyme mutant.

Further, the engineering bacteria are gram-positive bacteria, gram-negative bacteria, yeast and fungi.

Further, the engineering bacteria are escherichia coli BL21(DE3) engineering bacteria.

The fifth aspect of the invention discloses a construction method of an EstWY enzyme mutant, which comprises the following steps:

a1, searching ENND-TOP amino acid sequences in a Pfam database and an NCBI database, removing repeated identical sequences, selecting a protein sequence with the consistency of more than 40% with a target protein sequence, then performing multi-sequence comparison through Clusalx1.83 software to generate a fasta file, uploading the fasta file to a Consensus Maker v2.0.0 server, modifying setting parameters, generating an Consensus sequence which can be edited in a later period through the Clusalx1.83 software, and performing comparison and comparison on the amino acid sequence of the ENND-TOP protein with the family Consensus sequence and an amino acid abundance map of each site;

a2, obtaining an ENND-TOP structural model through an online software Swiss-model;

a3, observing the crystal structure by PyMOL, rechecking the mutation site and the mutation form to be selected according to the structure information, setting the screening condition, and screening the ENND-TOP mutant with thermal stability;

a4, analyzing the mutant obtained in A3 according to an ENND-TOP structural model, and screening out mutants S276V, L279F, T289R, R322G and N358D which improve the heat stability of ENND-TOP protein according to a judgment criterion;

further, the judgment criteria in step a4 are: firstly, the mutation eliminates the original acting force form which is not beneficial to thermal stability, such as electrostatic repulsion and charge accumulation; secondly, the mutation does not damage the existing acting force form which is beneficial to thermal stability and the stable protein structure; ③ the mutation should introduce a new form of action favorable to thermal stability, such as hydrogen bond, salt bridge, hydrophobic interaction.

Further, the setting of the screening conditions in step a3 includes:

b1, judging whether a certain locus is a candidate locus standard is as follows: the amino acid abundance of most proteins in the family at the position is high overall; ② the amino acid at the site is conserved; the amino acid with higher frequency of occurrence at the site has larger physical and chemical property difference with the amino acid at the ENND-TOP site, such as charge difference, polarity intensity, steric hindrance and the like;

b2, removal of active sites in the vicinity, i.e.from the catalytic residues

Amino acid residues within the range, excluding amino acid residues in the embedded or semi-embedded state.

In a sixth aspect of the invention, an EstWY enzyme mutant is disclosed for use in hydrolyzing aliphatic and aromatic esters, by hydrolysis, the esters are hydrolyzed to acids or alcohols.

The technical scheme of the invention has the following advantages:

1. by taking the consensus theory as a guiding idea, database retrieval, multi-sequence screening and comparison are established, and the efficiency is high. .

2. The EstWY enzyme mutant with improved thermal stability is obtained, and the half-life period at 45 ℃ can reach 5 times of that of a wild type.

3. The constructed high-efficiency expression genetic engineering bacteria have the advantages of short culture period, simple culture condition, high target protein yield and simple purification, and are beneficial to the application of the EstWY enzyme mutant in hydrolysis of aliphatic ester and aromatic ester.

Drawings

FIG. 1 shows the crystal structure and mutation site of ENND-TOP protein.

Detailed Description

The present invention is described in further detail below with reference to specific examples and with reference to the data. It will be understood that these examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.

General description of the sources of the biological materials described in the present invention:

1. primer synthesis: the primers used in the present invention were all prepared by synthesis from Shanghai bioengineering, Inc.

2. The KOD high-fidelity enzyme used in the experiment was purchased from Toyo Boseki; restriction enzymes were purchased from NEB; t4 DNA ligase, DNA gel recovery kit and plasmid mini kit were purchased from Takara.

EXAMPLE 1 cloning of the wild EstWY enzyme ENND-TOP Gene

The wild-type EstWY enzyme gene is codon-optimized by using Escherichia coli as a host, synthesized by Jinwei Zhi, Suzhou, and amplified by an upstream primer 5' -ACTGCTCATATGACCGATCCGACCTTTGATGCA-3 '(underlined bases are recognition sites for restriction enzyme NdeI) and downstream primer 5' -TCAGCTCTCGAGTTAATGGCCCAGTGCTTTA-3' (underlined bases are recognition sites of restriction enzyme XhoI), the target gene is amplified by PCR amplification using KOD high fidelity polymerase from Toyobo under the following conditions: 95 ℃ for 2min, then 55 ℃ for 20sec, 72 ℃ for 100sec for 30 cycles, and finally 72 ℃ for 10 min.

After the reaction, the PCR product was detected by 1.5% agarose gel electrophoresis to obtain a 1.0kb band, the length of which was consistent with the expected result. The desired fragment was recovered and purified by the standard procedures of the kit, the recovered fragment and pET28a plasmid were digested simultaneously with restriction endonucleases XhoI and NdeI, T4 DNA ligase was ligated, the ligation product was transformed into competent cells of Escherichia coli BL21(DE3), and the competent cells were plated on LB plate containing kanamycin (50ug/ml) to extract a positive cloning plasmid, which was sequenced, and the cloned EstWY enzyme ENND-TOP gene was correctly sequenced and ligated into pET28a plasmid, named pET28 a-ENND-TOP.

Example 2 expression, purification and Activity assay of ENND-TOP

The engineering bacteria in the glycerin pipe are inoculated into a 5mL LB culture medium test tube containing 100ug/mL Kan according to the volume ratio of 1 percent, and cultured for 12h at 37 ℃ and 220 rpm. Transferring the 5mL of the bacterial liquid into a 1L LB culture medium shake flask containing 50ug/mL Kan, culturing at 37 ℃ and 220rpm for about 2h to enable OD600 to reach about 0.8, adding 0.1mM IPTG inducer, and performing induction culture at 25 ℃ and 220rpm for 12-18 h. And ultrasonically crushing the escherichia coli thallus suspension obtained after fermentation, and performing Ni-NTA affinity chromatography treatment in one step to obtain the target protein with the purity of more than 95%.

The method for determining the EstWY enzyme comprises the following steps: the enzyme activity was measured in real time using p-nitrobenzoate ester at a concentration of 20mM as a reaction substrate and a buffer system of 50mM Tris-HCl buffer (pH 9.0). Measuring enzyme activity at 30-60 deg.C every 5 deg.C, increasing temperature density at the position close to optimum temperature, and analyzing optimum reaction temperature of esterase. And (3) respectively preserving the purified enzyme solution at 10, 20, 30, 40, 50 and 60 ℃ for 15min, measuring the enzyme activity, and analyzing the thermal stability of the enzyme.

Example 3 multiple sequence alignment and Consensus analysis of ENND-TOP homologous proteins

The specific operation is as follows:

1. entering the Pfam database homepage (http:// Pfam. xfam. org /), the amino acid SEQUENCE of FPOX-E was entered in the SEQUENCE SEARCH tool for searching. The server directly feeds back the amino acid sequence comparison result of the whole family of the protein, and displays the abundance of various amino acids of each site in a bar graph mode. The website may also automatically generate consensus sequences for the family of proteins.

2. The amino acid sequence of ENND-TOP was imported into NCBI protein database or Pfam database and all protein sequences with identity (identity) greater than 40% to the target protein sequence were found using Blast tool. Deleting the repeated identical sequence, sorting the rest sequence into fasta format, and inputting into Clustalx1.83 software for multi-sequence alignment. The results of the alignment are output in the format of aln, dnd, and fasta. The dnd file is used for constructing an evolutionary tree file, and the aln and fasta files are sequence files in different forms. Uploading the fasta. file to a Weblogo 3(http:// Weblogo. threeplusone. com /) server, and after setting parameters are modified according to needs, displaying the amino acid abundance of each site of a protein sequence in a multi-sequence alignment result by the online software in a form of a histogram. Uploading the fasta. file to a Consensus Maker v2.0.0(http:// www.hiv.lanl.gov/content/sequence/CONSENSUS/Consensus. html) server, and after setting parameters are modified as required, generating a Consensus sequence which can be edited later by the online software.

3. The amino acid sequence of the target protein ENND-TOP was compared with the consensus sequence of the family and the amino acid abundance map at each site.

Example 4 homology modeling and selection of mutation sites

1. We obtained a structural model of ENND-TOP using the online software Swiss-model.

2. The crystal structure is observed by PyMOL, the mutant site to be selected and the mutation form are rechecked according to the structural information, and the mutant which is most likely to improve the ENND-TOP thermal stability is screened out, wherein the screening conditions are as follows:

(1) the standard for judging a certain locus as a candidate locus is as follows: the amino acid abundance of most proteins in the family at the position is high overall; ② the amino acid at the site is conserved; and the amino acid with higher occurrence frequency at the site has larger physical and chemical property difference with the amino acid at the ENND-TOP site, such as charge difference, polarity strength, steric hindrance and the like.

(2) Removal of active sites in the vicinity, i.e. from catalytic residues

After two-step screening, a total of 28 differential sites remained, most of which were located on the surface of the protein molecules.

(3) According to the ENND-TOP structural model, the 28 mutation forms are analyzed in detail one by one, and mutants which can improve the ENND-TOP thermal stability are screened out.

The main judgment criteria are: firstly, the mutation eliminates the original acting force form which is not beneficial to thermal stability, such as electrostatic repulsion, charge aggregation and the like; secondly, the mutation does not damage the existing acting force form which is beneficial to thermal stability and the stable protein structure; and thirdly, new acting force forms which are beneficial to thermal stability, such as hydrogen bonds, salt bridges, hydrophobic interaction and the like, are introduced into the mutation.

A single-point mutant is designed in total, 5 mutants are designed: S276V, L279F, T289R, R322G, N358D.

Example 5 construction, expression, purification and characterization of the mutants

5.1 construction of ENND-TOP site-directed mutants

The recombinant plasmid pET28a-ENND-TOP was used as a template, a pair of complementary oligonucleotides having a mutation site was used as primers, and whole plasmid PCR amplification was performed using KOD high fidelity enzyme (Takara) to obtain a recombinant plasmid having a specific mutation site. The primer sequences are shown in table 1:

TABLE 1 primer sequence Listing

The KOD high-fidelity polymerase of Toyobo is used for PCR amplification, and the amplification conditions are as follows: 95 ℃ for 2min, then 55 ℃ for 20sec, 72 ℃ for 100sec for 30 cycles, and finally 72 ℃ for 10 min. The PCR product was recovered from the gel, and the gel-recovered product was digested with DpnI enzyme (Fermentas Corp.) at 37 ℃ for 2h to degrade the original template. The digestion products were transformed into BL21(DE3), spread on LB agar plates containing 50. mu.g/mL kanamycin, cultured overnight at 37 ℃, screened for positive clones, and verified by sequencing. Obtaining the recombinant strain of the EstWY enzyme mutant.

5.2 characterization of the Properties of the mutants

Pure enzyme solutions of site-directed mutants of EstWY were obtained according to the method of example 2 and the 5 single-point mutants were characterized. The results are shown in table 2, and among the 5 single-point mutants, the thermal stability of 4 mutants is obviously improved, namely L279F, T289R, R322G and N358D. Table 2 summarizes the enzymatic properties of the mutants with improved thermostability.

Mutants with improved stability were additively combined: using a similar construction method to the single-point mutant, 11 combination mutants were successfully constructed: L279F/T289R, L279F/R322G, L279F/N358D, T289R/R322G, T289R/N358D, R322G/N358D, L279F/T289R/R322G, L279F/T289R/N358D, L279F/R322G/N358D, T289R/R322G/N358D, L279F/T289R/R322G/N358D, and are expressed, purified and characterized one by one. The expression quantity and soluble protein quantity of the combined mutants are obviously improved compared with the wild ENND-TOP. All combination mutants showed additive effects of thermostabilization compared to single point mutants. The detailed table summarizes the enzymological properties of ENND-TOP wild-type enzyme and thermostable mutants, and the half-life at 45 ℃ of the most stable mutant is about 5 times that of the wild-type.

TABLE 2 characterization of enzymatic Properties of ENND-TOP wild type and Single-Point mutants

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Sequence listing

<110> Shanghai Shendao Biotechnology Co., Ltd

<120> esterase mutant with improved thermal stability and application thereof

<130> 2020

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1182

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

catatgaccg atccgacctt tgatgcactg catagcgcaa tgcgtgcaca ggttgatcag 60

cagtttctgc ctggtgttac caccgcactg ctgcgtggtc gtaaagttgt tgatcgtttt 120

tgttatggtc atgcagatcg tgaagcaggt attgcactgc gtgaagatca cctgtttcgt 180

atgtttagca gcaccaaact gattaccagc tgtgcagtta tgctgctggt tgaagaaggt 240

cgcgttcgtc tgagcgatcc ggttgatgca tatattccgg aactggcaaa tcgtcaggtt 300

ctgcgtgcag atgcaaaaac cctggcagat accgaaccgg cacgtagccc gattacactg 360

cagcatctga tgacccatac cagcggtctg agctatggtg tttttgatcc gggtagcctg 420

ctgtatcgtg catataatga agccggtgtt ctgaatccgc tgcaggatct ggcaggtatg 480

acccgtgttc tggcaaccct gccgctggca tttcatccgg gtacacagtg ggaatatagc 540

gttgcaaccg atgttctggg tcgtgttgtt gaagttgcaa gcggtgaaac ctttggtaat 600

tttctggcac gtcgtatttt tggtccgctg gaaatggttg ataccgattt ttgggttccg 660

cctgcaaaac aggatcgtct gtgtgcactg tatgttggtg ttgatctgct ggatccgacc 720

aaaccgggtc tgttacgtgc agataataaa ccgtttccgg gtgcatatcg tagtaaattt 780

gcacgtgaaa gcggtggtgg tggtctggtt agcaccctgg atgatagcat tcgtctgatt 840

cagagcctga ttcctggtgg tccgacactg ctgaaaccgg caacactgga acacatgttt 900

gcaaatcatc tgcctgcagg tatgcatgtt cgttttccga atgttccggc acagcctggt 960

tggcgttttg gtctgggtag ctcagttcgt gaaagtgcag gtctgggtga accgagcgaa 1020

gttgttggtg aagcaagctg gggtggcctg gcaggcaccc tgtggtggat caatccgcgt 1080

ctgggtattg cagcagttct gctgacccag cgttattttg gttttggtaa tccgtatgcc 1140

gtgcacttta aaaaccatgc atataaagca ctgggccatt aa 1182

<210> 2

<211> 393

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

His Met Thr Asp Pro Thr Phe Asp Ala Leu His Ser Ala Met Arg Ala

1 5 10 15

Gln Val Asp Gln Gln Phe Leu Pro Gly Val Thr Thr Ala Leu Leu Arg

20 25 30

Gly Arg Lys Val Val Asp Arg Phe Cys Tyr Gly His Ala Asp Arg Glu

35 40 45

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

50 55 60

Thr Lys Leu Ile Thr Ser Cys Ala Val Met Leu Leu Val Glu Glu Gly

65 70 75 80

Arg Val Arg Leu Ser Asp Pro Val Asp Ala Tyr Ile Pro Glu Leu Ala

85 90 95

Asn Arg Gln Val Leu Arg Ala Asp Ala Lys Thr Leu Ala Asp Thr Glu

100 105 110

Pro Ala Arg Ser Pro Ile Thr Leu Gln His Leu Met Thr His Thr Ser

115 120 125

Gly Leu Ser Tyr Gly Val Phe Asp Pro Gly Ser Leu Leu Tyr Arg Ala

130 135 140

Tyr Asn Glu Ala Gly Val Leu Asn Pro Leu Gln Asp Leu Ala Gly Met

145 150 155 160

Thr Arg Val Leu Ala Thr Leu Pro Leu Ala Phe His Pro Gly Thr Gln

165 170 175

Trp Glu Tyr Ser Val Ala Thr Asp Val Leu Gly Arg Val Val Glu Val

180 185 190

Ala Ser Gly Glu Thr Phe Gly Asn Phe Leu Ala Arg Arg Ile Phe Gly

195 200 205

Pro Leu Glu Met Val Asp Thr Asp Phe Trp Val Pro Pro Ala Lys Gln

210 215 220

Asp Arg Leu Cys Ala Leu Tyr Val Gly Val Asp Leu Leu Asp Pro Thr

225 230 235 240

Lys Pro Gly Leu Leu Arg Ala Asp Asn Lys Pro Phe Pro Gly Ala Tyr

245 250 255

Arg Ser Lys Phe Ala Arg Glu Ser Gly Gly Gly Gly Leu Val Ser Thr

260 265 270

Leu Asp Asp Ser Ile Arg Leu Ile Gln Ser Leu Ile Pro Gly Gly Pro

275 280 285

Thr Leu Leu Lys Pro Ala Thr Leu Glu His Met Phe Ala Asn His Leu

290 295 300

Pro Ala Gly Met His Val Arg Phe Pro Asn Val Pro Ala Gln Pro Gly

305 310 315 320

Trp Arg Phe Gly Leu Gly Ser Ser Val Arg Glu Ser Ala Gly Leu Gly

325 330 335

Glu Pro Ser Glu Val Val Gly Glu Ala Ser Trp Gly Gly Leu Ala Gly

340 345 350

Thr Leu Trp Trp Ile Asn Pro Arg Leu Gly Ile Ala Ala Val Leu Leu

355 360 365

Thr Gln Arg Tyr Phe Gly Phe Gly Asn Pro Tyr Ala Val His Phe Lys

370 375 380

Asn His Ala Tyr Lys Ala Leu Gly His

385 390

<210> 3

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tggatgatta tattcgtctg attcagagc 29

<210> 4

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

agacgaatat aatcatccag ggtgctaac 29

<210> 5

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gcattcgttt tattcagagc ctgattcct 29

<210> 6

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ctctgaataa aacgaatgct atcatccag 29

<210> 7

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gtggtccgcg tctgctgaaa ccggcaaca 29

<210> 8

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ttcagcagag ccggaccacc aggaatcag 29

<210> 9

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ctggttgggg ttttggtctg ggtagctca 29

<210> 10

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

agaccaaaac cccaaccagg ctgtgccgg 29

<210> 11

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ggtggatcga tccgcgtctg ggtattgca 29

<210> 12

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

agacgcggat cgatccacca cagggtgcc 29

Claims

1. An EstWY enzyme, which is an EstWY enzyme derived from Escherichia coli and is named as ENND-TOP protein, wherein the nucleotide sequence for coding the ENND-TOP protein is shown as SEQ ID NO.1, and the amino acid sequence of the EstWY enzyme is shown as SEQ ID NO. 2.

2. The EstWY enzyme according to claim 1, which is a derived protein having one or more amino acids substituted, deleted or added to the amino acid sequence shown in SEQ ID No.2 and having the same function as the protein shown in SEQ ID No. 2.

3. The EstWY enzyme according to claim 1, which is an EstWY enzyme mutant obtained by substituting one or more amino acid residues for an amino acid residue at one or more positions in the amino acid sequence shown in SEQ ID No. 2.

4. The EstWY enzyme according to claim 1, which is an EstWY enzyme mutant obtained by substituting one or more amino acid residues for an amino acid residue at one or more positions of the amino acid sequence of the EstWY enzyme exhibiting an amino acid sequence showing at least 90% homology among the amino acid sequences shown in SEQ ID No. 2.

5. A gene encoding the EstWY enzyme mutant of claim 3 or 4.

6. A recombinant plasmid comprising the gene of claim 4.

7. A soluble protein, immobilized enzyme or engineered bacterium comprising the EstWY enzyme mutant of any of claims 1-4.

8. A method for constructing the EstWY enzyme mutant as claimed in any one of claims 1 to 4, comprising the steps of:

wherein, the judgment criterion in the step a4 is: firstly, the mutation eliminates the original acting force form which is not beneficial to thermal stability, such as electrostatic repulsion and charge accumulation; secondly, the mutation does not damage the existing acting force form which is beneficial to thermal stability and the stable protein structure; ③ the mutation should introduce a new form of action favorable to thermal stability, such as hydrogen bond, salt bridge, hydrophobic interaction.

9. The method for constructing an EstWY enzyme mutant according to claim 8, wherein the setting of the screening conditions in step A3 includes:

b2, removal of active sites in the vicinity, i.e.from the catalytic residues

10. Use of an EstWY enzyme mutant according to claim 3 or 4 for the hydrolysis of aliphatic and aromatic esters to hydrolyze the esters to acids or alcohols.