An ec hydrolase mutant with improved enzyme activity and tolerance and applications thereof

Abstract

This invention discloses an EC hydrolase mutant with enhanced enzyme activity and tolerance, and its applications, belonging to the field of enzyme engineering technology. First, this invention identifies the disulfide bond site Q149C-F157C in the flexible region, constructing disulfide bonds between the α-helical structures on the protein surface, successfully significantly improving the stability of multiple aspects of the ES8 mutant V129S / I229M. Experimental data show that the mutant exhibits improved stability under different environments; the stability at 80℃ half-life, pH 4.0, and 20% ethanol is 8.14 times, 1.49 times, and 2.72 times that of Est8-CE, respectively. Molecular dynamics simulation analysis of protein structural changes shows that the introduction of disulfide bonds significantly reduces the RMSD and RMSF of Est8-CE, demonstrating enhanced structural stability and promising suitability for use in extreme environments, thus possessing greater industrial application value.

Description

Technical Field

[0001] This invention relates to an EC hydrolase mutant with enhanced enzyme activity and tolerance and its application, belonging to the field of enzyme engineering technology. Background Technology

[0002] Ethyl carbamate (EC) is a trace harmful byproduct that is naturally formed in fermented foods and alcoholic beverages (such as rice wine, wine, soy sauce, etc.). It has potential carcinogenicity, so its content control has received widespread attention in the field of food safety.

[0003] EC (extracorporeal membrane oxygenation) is orally toxic, and high doses can cause cancer. Long-term consumption of foods with high EC content poses safety risks. Therefore, the World Health Organization and many countries around the world have established strict limits on EC content in traditional fermented foods. Currently, there are two main research methods for reducing EC content in traditional fermented foods: one is to reduce EC formation by eliminating precursors (such as urea, citrulline, carbamoyl phosphate, etc.) (indirect reduction method); the other is to directly degrade EC through EC hydrolases (EC.3.5.1.75) or EC-producing microorganisms (direct reduction method). Because EC molecules are stable and difficult to decompose once formed, the direct reduction method, which eliminates EC from all pathways at the end of the chain, is theoretically one of the best methods for controlling EC content in traditional fermented foods.

[0004] Previously, we molecularly modified wild-type EC hydrolases derived from Acinetobacter calcitoninus to obtain a hydrolases mutant Est8-CE (V129S / I229M) with improved acid tolerance and catalytic rate. However, its stability under extreme conditions was poor, and it could not perform EC degradation function well. Therefore, further modification is still needed to meet actual needs. Summary of the Invention

[0005] To address the limitation of the EC hydrolase mutant Est8-CE in extreme environments, this invention introduces paired cysteine ​​residues to form disulfide bonds on the surface of the high-catalytic-rate esterase mutant V129S / I229M (Est8 catalytic enhanced mutant, Est8-CE). Using Discovery Studio 2023 software and the Disulfide bydesign 2.0 server, combined with RMSF results, potential disulfide bonds are predicted to identify potential mutation sites located in more flexible regions of the molecular surface that are more prone to disulfide bond formation. These sites are then mutated to cysteine ​​residues, thereby increasing intermolecular forces between surface proteins and improving enzyme stability. Homology modeling of the protein structure is performed using trRosetta and AlphaFold3 servers. After structural alignment and molecular docking, disulfide bond prediction is performed on the enzyme-substrate complex, ultimately yielding a mutant Est8-CES1 with significantly improved tolerance to temperature, acidic environments, and ethanol.

[0006] The first objective of this invention is to provide an EC hydrolase mutant, wherein the amino acid sequence of the EC hydrolase mutant is as shown in SEQ ID NO.3 or has at least 90% homology with it. More preferably, it is 95%, 98%, or even 99%.

[0007] A second objective of this invention is to provide a nucleic acid molecule encoding the EC hydrolase mutant.

[0008] A third objective of this invention is to provide a gene integration expression cassette or recombinant plasmid carrying the nucleic acid molecule.

[0009] Furthermore, the recombinant plasmid can use any expression vector as its backbone, selected according to the host type, and can be a pET series vector.

[0010] A fourth object of the present invention is to provide recombinant cells containing the EC hydrolase mutant.

[0011] Furthermore, the host cell is a microorganism, such as bacteria or fungi.

[0012] Furthermore, the bacteria are Escherichia coli or Bacillus subtilis, preferably Escherichia coli BL21(DE3).

[0013] A fifth objective of this invention is to provide the use of the EC hydrolase mutant, nucleic acid molecule, gene expression cassette, recombinant plasmid, or recombinant cell in the hydrolysis of ethyl carbamate or the preparation of ethyl carbamate degradation products.

[0014] A sixth object of the present invention is to provide an enzyme composition comprising the EC hydrolase mutant.

[0015] A seventh objective of the present invention is to provide a recombinant Escherichia coli in which the EC hydrolase mutant is overexpressed.

[0016] An eighth object of the present invention is to provide the use of the recombinant Escherichia coli in the preparation of EC hydrolase mutants.

[0017] A ninth object of the present invention is to provide a method for producing an EC hydrolase mutant, comprising the step of fermentation production using the recombinant Escherichia coli.

[0018] Further fermentation was carried out at 28-32℃ and 180-280 rpm.

[0019] The beneficial effects of this invention are:

[0020] This invention characterizes the enzymatic properties of Est8-CE and its mutants through experiments, and elucidates the mechanism of enhanced stability of the mutants by combining substrate binding conformation analysis and molecular dynamics simulations. Experimental data show that the mutant Est8-CES1 (Q149C-F157C) exhibits significantly improved stability under extreme conditions. In terms of thermal stability, the half-life of Est8-CES1 at 80 °C is 8.14 times that of Est8-CE. After being placed at 4 °C for 6 h in an acidic environment (pH 4.0), the residual enzyme activity of Est8-CES1 is 1.49 times that of Est8-CE, and under 20% ethanol conditions, the residual enzyme activity of Est8-CES1 is as high as 2.72 times that of Est8-CE. Molecular dynamics trajectory analysis results indicate that the introduction of disulfide bonds significantly reduces the RMSD and RMSF of Est8-CE, demonstrating enhanced structural stability of the mutant and its potential for use in extreme environments. Attached Figure Description

[0021] Figure 1 The structural positions of the flexible regions within the protein were selected. Among them, (A) the molecular dynamics RMSF results of Est8 and Est8-CE; (B) the structural positions of 131-179 and 236-244.

[0022] Figure 2 The mutation sites and modified regions are shown. Among them, Helix1 (P139-K152); Helix2 (R154-P165); Helix3 (T209-K214); Helix4 (I232-L242).

[0023] Figure 3Gel images of Es8-CE and its mutants, along with measurements of free thiol groups and specific enzyme activity. (A) Gel images of cell lysis supernatant and precipitate, M: standard molecular weight protein, S: lysis supernatant, P: lysis precipitate; (B) Gel image of purified enzyme; (C) Measurement of free thiol groups; (D) Measurement of specific enzyme activity.

[0024] Figure 4 The effect of temperature on enzyme activity. Among them, (A) the optimal reaction temperature; (B) the half-life at 80 °C.

[0025] Figure 5 The effect of pH on enzyme activity. Among them, (A) optimal reaction pH; (B) pH stability.

[0026] Figure 6 The effect of ethanol on esterase activity. Among them, (A) relative enzyme activity at different ethanol concentrations; (B) residual enzyme activity at different ethanol concentrations. Detailed Implementation

[0027] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0028] The culture media involved in the following examples are as follows:

[0029] LB medium: yeast extract 5 g / L, tryptone 10 g / L, NaCl 10 g / L.

[0030] TB medium: yeast extract 24 g / L, tryptone 12 g / L, 4 mL glycerol, 2.313 g / L KH2PO4, 12.54 g / L K2HPO4. For solid medium, add 15 g / L agar powder.

[0031] The detection methods involved in this invention (such as enzyme activity determination, optimal reaction temperature of enzyme, optimal reaction pH, pH stability, ethanol tolerance, ethanol stability and enzyme kinetic parameter determination methods, etc.) are the same as those in the invention application with publication number CN120118877A.

[0032] Identification of introduced disulfide bonds: Free thiol groups were detected using Ellman's reagent 5,5'-dithio-bis-[2-nitrobenzoic acid] (DTNB). When free thiol groups from cysteine ​​are present, they react with DTNB to form 2-nitro-5-thiobenzoic acid (TNB). This reaction produces a maximum absorption peak at 412 nm. Therefore, absorbance at 412 nm can be used as a reliable indicator of the presence of free thiol groups in proteins. To assess disulfide bond formation in mutants, the levels of free thiol groups in Est8-CE and mutants were determined at the same concentration. If the mutant showed the same level of free thiol groups as Est8-CE, this indicated successful disulfide bond formation with the introduced cysteine ​​pair. Conversely, an increased level of free thiol groups in the mutant indicated the inability to form disulfide bonds. For disulfide bond detection, 1 mg / mL of purified enzyme solution was mixed with 5 mL of 0.1 mmol / L DTNB analytical solution. Incubate at 37 °C for 10 min, and measure the absorbance of the reaction mixture at 412 nm.

[0033] To assess the thermal stability of the Est8-CE mutant, the half-life T of Est8-CE and its mutant at 70 °C was determined. 1 / 2 The purified enzyme sample was incubated at high temperature at different time intervals, and the remaining enzyme activity was measured. The unheated enzyme reaction system served as a control (100%). The time required for the enzyme activity to decrease by half was recorded as T. 1 /

[0034] The sequence involved in this invention is as follows:

[0035] (1) Wild-type Est8 amino acid sequence (SEQ ID NO.1):

[0036] MGYVTTKDNVEIFYKDWGPKDAPVIFFHHGWPLSSDDWDAQLLFFLKEGFRVVAHDRRGHGRSSQIWDGHDMDHYASDVAAVVDHLGVHGAVHVGHSTGGGEVAHYVANYQNDPVAKAVLISAVPPLMVKTENNPNGL PKEVFDDLQNQLFKNRSQFYYDVPAGPFYGYNRPGAKVSEPVILNWWRQGMMGGAKAHYDGIVAFSQTDFTEDLKKIKIPVLVMHGEDDQIVPIEISGKLSAELVQNGKLITYPGFPHGMPTTEAETINKDLLEFIRS

[0037] (2) Amino acid sequence of mutant Est8-CE (es8-V129S / I229M) (SEQ ID NO.2):

[0038] MGYVTTKDNVEIFYKDWGPKDAPVIFFHHGWPLSSDDWDAQLLFFLKEGFRVVAHDRRGHGRSSQIWDGHDMDHYASDVAAVVDHLGVHGAVHVGHSTGGGEVAHYVANYQNDPVAKAVLISAVPPLMSKTENNPNGL PKEVFDDLQNQLFKNRSQFYYDVPAGPFYGYNRPGAKVSEPVILNWWRQGMMGGAKAHYDGIVAFSQTDFTEDLKKIKIPVLVMHGEDDQMVPIEISGKLSAELVQNGKLITYPGFPHGMPTTEAETINKDLLEFIRS

[0039] (3) Amino acid sequence of mutant Est8-CES1 (SEQ ID NO.3):

[0040] MGYVTTKDNVEIFYKDWGPKDAPVIFFHHGWPLSSDDWDAQLLFFLKEGFRVVAHDRRGHGRSSQIWDGHDMDHYASDVAAVVDHLGVHGAVHVGHSTGGGEVAHYVANYQNDPVAKAVLISAVPPLMSKTENNPNGL PKEVFDDLQNCLFKNRSQCYYDVPAGPFYGYNRPGAKVSEPVILNWWRQGMMGGAKAHYDGIVAFSQTDFTEDLKKIKIPVLVMHGEDDQMVPIEISGKLSAELVQNGKLITYPGFPHGMPTTEAETINKDLLEFIRS

[0041] The strains and plasmids involved in this invention are:

[0042] E. coli BL21 / pET30a-est8-ce: an engineered strain containing the mutant plasmid pET30a-es8-V129S / I229M;

[0043] Plasmid pET30a-es8-V129S / I229M: pET30a plasmid for overexpressing the 8 mutant V129S / I229M.

[0044] The methods for preparing, culturing, expressing, and purifying the enzyme mutant are the same as those in invention application CN120118877A. The primer sequences involved are as follows:

[0045] Table 1 PCR primer sequences

[0046]

[0047] Note: Mutation sites are underlined.

[0048] Example 1: Selection of mutation sites

[0049] Based on the protein sequence of the mutant Est8-CE, protein homology modeling and prediction were performed using the AlphaFold3 server to simulate its crystal structure. This was then optimized using Discovery Studio 2023, and the resulting structure was validated using Ramachandran plots. Molecular dynamics simulations were performed on the obtained Est8-CE, and the root mean square fluctuations were calculated after analyzing the simulation trajectory results to determine the modified flexible region to improve the structural rigidity of Est8-CE under acidic and ethanol environments.

[0050] The structure of the Est8-CE protein obtained from homology modeling was predicted based on the principles of disulfide bond formation using both the Disulfide by design 2.0 server and Discovery Studio 2023 software. The Disulfide by design 2.0 server predicted potentially stable disulfide bonds by combining multi-dimensional conditions such as geometric constraints, energy functions, B-factor analysis, and residue depth. Discovery Studio 2023 software predicted disulfide bonds based on the following parameters and provided scores: energy variation, temperature factor variation, amino acid spacing, amino acid embedding depth, volume variation, and changes in the angle between Coil-Coil pairs and Chi3. Using these two disulfide bond prediction methods, two sets of predicted structures were found to be pseudo-disulfide bonds that could form in the protein structure.

[0051] Among the predicted pseudo-disulfide bond sites, the results from the Disulfide by design 2.0 server were sorted by energy value. Based on the bond energy calculation of 1505 disulfide bonds from different proteins by the Disulfide by design 2.0 server, 90% of the disulfide bond energies were less than 2.176 kcal / mol, so pseudo-disulfide bond sites with bond energies greater than 2.176 kcal / mol were screened out. For the prediction results from Discovery Studio 2023 software, the comprehensive score was used as the standard, and pseudo-disulfide bond sites with scores greater than 90 were selected as references.

[0052] In enzyme stability enhancement strategies, a crucial aspect is the "trade-off effect" between enzyme stability and catalytic rate. To minimize the impact on enzyme catalytic activity, the selection of mutation sites should avoid those with high conservation, especially since some residues at these sites are key components of the enzyme's structure, serving both catalytic function and structural support. To prevent significant reductions or even inactivation of enzyme activity, the ConSurf server is used to predict the conservation of residue sites in the protein, and highly conserved sites containing structural or functional residues in the resulting pseudo-disulfide bonds are then screened out.

[0053] Accurate prediction of flexible regions is key to improving protein stability through disulfide engineering. Flexible regions of the WT protein were identified through 100 ns of disulfide engineering at 310.15 K, and then these regions were directionally fixed by introducing disulfide bonds. To determine the insertion sites of disulfide bonds within the flexible regions, the Disulfide by design 2.0 server and Discovery Studio 2023 software were used for comprehensive identification and selection of appropriate disulfide bonds for construction, based on the flexible regions. The Disulfide by design 2.0 server is available at http: / / cptweb.cpt.wayne.edu / DbD2 / index.

[0054] The three-dimensional structure of Est8-CE was constructed using AlphaFold3 and subsequently optimized using Discovery Studio 2023. Ramachandran plot data of the model structure showed that 99.8% of the residues were located in favorable and allowed regions, demonstrating the rationality and credibility of the Est8-CE structure.

[0055] Identifying flexible regions in enzyme structures can provide a basis for selecting sites for disulfide bond construction. The RMSF (Reactive Molecular Strength Spectrum) obtained from trajectory analysis in molecular dynamics simulations is an effective parameter for quantifying the flexibility of individual amino acids, and a higher RMSF value indicates poorer rigidity in this region. Based on the molecular dynamics results of the esterase Est8 and the high-catalytic-rate mutant Est8-CE (V129S / I229M), regions with significantly increased RMSF were identified as regions of enhanced flexibility. These regions were designated for further analysis and potential evolutionary improvements. Previous studies analyzing the catalytic rate enhancement mechanism have shown that increasing the flexibility of the region near the catalytic triplet can effectively increase the enzyme's catalytic rate. Figure 1Figure A shows five regions with significantly increased RMSF between Est8 and Est8-CE: 91-116, 131-179, 197-231, 236-244, and 249-263. Regions 91-116, 197-231, and 249-263 each contain one of the catalytic triplet components; to avoid reducing enzyme activity, these three regions are unsuitable for disulfide bond modification. Regions 131-179 and 236-244 are designated as flexible regions requiring improvement. Figure 1 (B)

[0056] To improve the structural rigidity of regions 131-179 and 236-244 and to screen for protomutation sites within these regions that better match the characteristics and structure of disulfide bonds, we used Disulfide by Design 2.0 and Discovery Studio 2023 software to predict potential disulfide bonds in the Est8-CE esterase structural model obtained from homology modeling on the trRosetta and AlphaFold3 servers, respectively. The two prediction results were sorted by energy and score, and disulfide bonds with energies below 2.176 kcal / mol in the Disulfide by Design 2.0 results and scores greater than 90 in the Discovery Studio 2023 results were selected. Highly conserved sites containing catalytic residues and structural residues were excluded. The prediction results after disulfide bond screening are shown in the table below. Of the nine predicted disulfide bonds, two pairs were repeated: Q149-F157 and L212-L242. This indicates that these two disulfide bonds are more likely to form in Est8-CE. Considering the high flexibility of Est8-CE in the 131-179 and 236-244 regions, disulfide bond mutants can be constructed by selecting the Q149-F157 and L212-L242 sites. This invention further analyzes the data of the mutant Es8-CES1 constructed at the Q149-F157 site.

[0057] Table 2. Predicted disulfide bond energies and scores

[0058]

[0059] Depend on Figure 2It is known that the pseudo-disulfide bond site Q149-F157 is located in the α-helix structure that constitutes the main conformational support in Est8-CE. The figure shows that disulfide bond Q149C-F157C is located between two long α-helix structures formed by the P139-K152 (Helix1) and R154-P165 (Helix2) site regions, and the α-helix structure connected by disulfide bond Q149C-F157C is close to the substrate channel, which may play an important role in maintaining the conformational stability of the enzyme under acidic or ethanolic conditions. Virtual amino acid mutation and stability assessments using Discovery Studio 2023 showed that the Es8-CES1 mutant exhibited excellent stability, consolidating the possibility of further improving its stabilizing effect.

[0060] Table 3. Virtual amino acid mutation results from Discovery Studio 2023

[0061]

[0062] Example 2: Characterization of the enzymatic properties of mutants

[0063] (1) Compared with enzyme activity

[0064] The Est8-CES1 mutant was constructed using site-directed mutagenesis with mutant primers. Subsequently, SDS-PAGE analysis confirmed the successful expression and purification of these mutant enzymes in *E. coli* BL21. Figure 3 The A-band showed that the protein was basically expressed in a soluble manner, with a small amount in the precipitate. The purified bands were uniform and free of other contaminants, indicating good purification. Figure 3 (B) The formation of the introduced cysteine ​​pair in the mutant was verified using Ellman's reagent, such as... Figure 3 As shown in Figure C, both EST8-CE and the mutant exhibited comparable levels of free thiol concentrations, indicating successful disulfide bond formation between the introduced cysteine ​​pairs. The enzyme activity assay results showed ( Figure 3 (D), the mutant Est8-CES1 has a 21% higher specific enzyme activity than Est8-CE.

[0065] (2) Temperature stability

[0066] To determine the optimal temperature for Est8-CE and the mutant, relative enzyme activity was measured within the range of 20–80 °C. Figure 4The optimal reaction temperatures for EST8-CE and Est8-CES1 were 55 ℃ and 65 ℃, respectively. At 65 ℃, the relative enzyme activity of EST8-CE decreased significantly to 49%, while the relative enzyme activity of Est8-CES1 remained at 100% under the same conditions. The relative enzyme activity of Est8-CES1 increased almost linearly before 65 ℃, but decreased to 47% after the reaction temperature was increased to 70 ℃, indicating that its catalytic conformation was significantly disrupted at 70 ℃. Temperature half-life measurements showed (…). Figure 4 (B) At 80 °C, the half-lives of Est8-CE and Est8-CES1 were 7 min and 57 min, respectively. The mutants not only showed higher activity at higher temperatures, but also better stability at high temperatures. The half-life of Est8-CES1 was 8.14 times that of Est8-CE. The results show that the construction of disulfide bonds effectively enhances the stability of the enzyme at high temperatures.

[0067] (3) pH stability

[0068] To investigate the optimal pH for Est8-CE and the mutant, enzyme activity was measured within a pH range of 3.0–8.0. Figure 5 As shown in Figure A, the optimal pH for EST8-CE is 7.0, while the optimal pH for Est8-CES1 decreases to 5.5. Both EST8-CE and Est8-CES1 exhibit 70% relative enzyme activity over a relatively wide pH range of 4.5-8.0, demonstrating good adaptability to various acidic and alkaline environments. Notably, compared to EST8-CE, Est8-CES1 maintains almost maximum relative enzyme activity within the weakly acidic range of 4.0-5.5, exhibiting better acid tolerance. pH stability results show ( Figure 5 Of the enzymes tested (B), Est8-CES1 showed the best stability at pH 7.0, while EST8-CE showed the best stability at pH 8.0. Furthermore, Est8-CES1 exhibited optimal stability in an acidic environment ranging from pH 3.0 to 7.0, retaining 73% and 83% residual enzyme activity after 6 hours at 4°C and pH 4.0, respectively. In contrast, Est8-CE retained only 49% and 67% residual enzyme activity at the same pH. These experimental data indicate that Est8-CES1 maintains good stability under acidic conditions.

[0069] (4) Ethanol tolerance

[0070] like Figure 6As shown in Figure A, at a 10% ethanol concentration, both enzymes retained over 60% relative enzyme activity. At ethanol concentrations of 10%–40% (v / v), the mutants showed significantly increased relative enzyme activity compared to Est8-CE. For example, at a 20% ethanol concentration, Est8-CES1 retained 49% of its relative enzyme activity, which is 2.72 times that of Est8-CE. Ethanol stability results are shown below. Figure 6 As shown in Figure B, Est8-CES1 maintains more than 50% residual enzyme activity in an environment of 0%-40% ethanol, while Est8-CE has only 36% residual enzyme activity in an environment of 40% ethanol. Figure 6 This indicates that Est8-CES1 has good ethanol tolerance and has the potential for direct application in low-alcohol beverages. In addition, the experimental results also show that constructing disulfide bonds can effectively increase the enzyme's resistance to extreme environments.

[0071] (5) Dynamic parameters

[0072] Kinetic parameters of Est8-CE and the mutant were determined at EC concentrations ranging from 10 to 1000 mmol / L. All data were fitted using nonlinear regression. Table 4 shows the Vt values ​​for the mutant and EST8-CE. max There are significant differences, V of Est8-CES1 max The concentration was 386.78 ± 9.41 μmol / (min·mg), an increase of 157.22% compared to EST8-CE's 246.03 ± 4.67 μmol / (min·mg). Est8-CES1 showed a significant increase in V... max The improvement indicates that the introduction of disulfide bonds into Est8-CE increases its catalytic rate under acidic conditions, which is presumably related to its enhanced acid tolerance.

[0073] Table 4 Kinetic parameters of Est8-CE and its mutants

[0074]

[0075] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. An EC hydrolase mutant, characterized in that, The amino acid sequence of the EC hydrolase mutant is shown in SEQ ID NO.

3.

2. A nucleic acid molecule encoding the EC hydrolase mutant of claim 1.

3. A gene integration expression cassette carrying the nucleic acid molecule of claim 2.

4. A recombinant plasmid carrying the nucleic acid molecule of claim 2.

5. A recombinant cell containing the gene integration expression cassette of claim 3 or the recombinant plasmid of claim 4, characterized in that, The recombinant cells include microorganisms.

6. The use of the EC hydrolase mutant of claim 1, the nucleic acid molecule of claim 2, the gene integration expression cassette of claim 3, the recombinant plasmid of claim 4, or the recombinant cell of claim 5 in the hydrolysis of ethyl carbamate or the preparation of ethyl carbamate degradation products.

7. An enzyme composition, characterized in that, The enzyme composition includes the EC hydrolase mutant of claim 1.

8. A recombinant Escherichia coli, characterized in that, The recombinant Escherichia coli overexpressed the EC hydrolase mutant of claim 1.

9. The use of the recombinant Escherichia coli according to claim 8 in the preparation of EC hydrolase mutants.

10. A method for producing an EC hydrolase mutant, characterized in that, It includes the step of fermentation using the recombinant Escherichia coli as described in claim 8.

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