CN112301014B

CN112301014B - Esterase mutant with improved thermal stability and application thereof

Info

Publication number: CN112301014B
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 Hannover Biotechnology Co ltd; 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: 2022-07-22
Anticipated expiration: 2040-11-04
Also published as: CN112301014A

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 usedIt is widely found in animals, plants and microorganisms. Among them, animal pancreatic esterase and microbial esterase are major 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 effectively optimize the thermal stability of proteins, Markus Wys et al, 2001, proposed 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 optimal state in an industrial approach. 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 one or more amino acid residues 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 one or more amino acid residues in the amino acid sequence of 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 on the 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.

The third aspect of the invention discloses a recombinant plasmid containing EstWY enzyme mutant gene.

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

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 structural information, setting the screening condition, and screening the ENND-TOP heat-stable mutant;

a4, analyzing the mutants obtained in A3 according to an ENND-TOP structure 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 should eliminate 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, the standard for judging a certain locus as a candidate locus is as follows: the majority of proteins in the family have higher total amino acid abundance at the position; the amino acid at the position 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 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 synthetically by Shanghai bioengineering, Inc.

2. The KOD Hi-Fi enzyme used in the experiments 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 subjected to codon optimization by taking escherichia coli as a host, is synthesized by Jinwei Zhi company of Suzhou, and is subjected to 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 E.coli BL21(DE3), and plated on LB plate containing kanamycin (50ug/ml) to extract a positive cloning plasmid, which was sequenced, and it was found that the cloned EstWY enzyme ENND-TOP gene had the correct sequence and correctly ligated into pET28a plasmid, named pET28 a-ENND-TOP.

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

The engineering bacteria in the glycerinum tube 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 12 hours 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 obtained escherichia coli thallus suspension after fermentation, and performing one-step Ni-NTA affinity chromatography treatment to obtain the target protein with the purity of more than 95%.

The EstWY enzyme determination method comprises the following steps: the enzyme activity was measured in real time using p-nitrobenzoate as a reaction substrate at a concentration of 20mM and Tris-HCl buffer (pH9.0) at a concentration of 50mM as a buffer system. 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 keeping the purified enzyme solution at the temperature of 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 can also automatically generate the consensus sequence of the protein family.

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. And uploading the fasta. files to a Consensus Maker v2.0.0(http:// www.hiv.lanl.gov/content/sequence/CONSENSUS/Consensus. html) server, and modifying the setting parameters as required to generate 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, 28 differential sites are remained, and most of the differential sites are 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, introducing new acting force forms which are beneficial to thermal stability, such as hydrogen bonds, salt bridges, hydrophobic interaction and the like 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 and the 5 single-point mutants were characterized as in example 2. The results are shown in table 2, and among the 5 single point mutants, the thermal stability of 4 mutants was significantly improved, which are 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 the compounds 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 table details the enzymatic properties of ENND-TOP wild-type enzyme, thermostable mutants, the half-life at 45 ℃ of the most stable mutants is about 5 times that of the wild-type.

TABLE 2 characterization of enzymatic Properties of ENND-TOP wild type and Single site 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 one skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.

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Claims

1. An EstWY enzyme mutant is characterized in that the EstWY enzyme mutant is derived from a wild-type EstWY enzyme of Escherichia coli, the wild-type EstWY enzyme is named as ENND-TOP protein, the amino acid sequence of the ENND-TOP is shown in SEQ ID No.2, and the EstWY enzyme mutant is shown in SEQ ID No.2

One of the following mutants on the amino acid sequence shown in SEQ ID NO. 2: L279F, T289R, R322G, N358D, 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.

2. A gene encoding the EstWY enzyme mutant of claim 1.

3. A recombinant plasmid comprising the gene of claim 2.

4. A soluble protein, immobilized enzyme or engineered bacterium comprising the EstWY enzyme mutant of claim 1.

5. Use of the EstWY enzyme mutant of claim 1 to hydrolyze aliphatic and aromatic esters by hydrolysis to convert the esters to acids or alcohols.