Carboxyesterase mutant with improved stability and use thereof

Abstract

The application discloses an esterase mutant with improved thermal stability, and belongs to the technical field of enzyme engineering. Bacillus cereus The wild-type carboxylic acid esterase derived from the genus Pseudomonas is used as an evolution template, and through rational design and directional evolution means, multiple mutants are obtained, which are obviously superior to the wild type in catalytic activity, thermal stability, temperature tolerance and organic solvent tolerance. The mutant can adapt to more severe industrial reaction conditions, and has stronger application advantages in industrial production.

Description

Technical Field This invention belongs to the field of enzyme engineering technology, specifically relating to a carboxylesterase mutant, its preparation method, and its uses. Background Technology

[0001] Carboxylesterases (EC 3.1.1.1) belong to an important subclass of esterases. Their main function is to catalyze the hydrolysis of carboxylic acid ester bonds, breaking down ester compounds into the corresponding carboxylic acids and alcohols. The reaction formula is as follows: R-COO-R' + H2O → R-COOH + R'-OH. Carboxylesterases mainly act on small-molecule carboxylic acid ester substrates, such as drug esters, environmental toxin esters, and endogenous lipid esters. Due to their very broad substrate spectrum, carboxylesterases play a central role in several key physiological processes in organisms, including detoxification metabolism, drug activation / inactivation, lipid metabolism, and signal transduction.

[0002] With the increasing application of biocatalysis technology in industry, enzyme catalysis has attracted widespread attention due to its mild reaction conditions. However, many industrial applications (such as papermaking) require catalytic reactions at higher temperatures, which places higher demands on the thermal stability of enzymes. Although naturally derived carboxylesterases exhibit good catalytic performance under physiological conditions, their catalytic activity, stability, and applicability are often limited when faced with common non-natural substrates and harsh reaction conditions in industrial processes. Therefore, developing recombinant carboxylesterases with broad substrate adaptability, high catalytic efficiency, and excellent thermal and pH stability has become crucial to enhancing their application potential in fields such as medicine and environmental protection. To achieve this goal, we can start from the following two aspects: first, we can discover novel carboxylesterase genes with natural tolerance from microorganisms in extreme environments; second, we can use protein engineering techniques (such as rational design and directed evolution) to directionally modify existing enzymes to enhance their catalytic performance, thermal stability, and selectivity for non-natural substrates, thereby overcoming the technical bottlenecks in industrial applications. Summary of the Invention

[0003] This invention provides a carboxylesterase mutant, which is obtained by mutation based on a wild-type carboxylesterase. The wild-type carboxylesterase has at least 90% homology with the amino acid sequence shown in SEQ ID NO.1. The mutation site is at least one of the following sites: position 34, position 67, position 74, position 104, position 122, position 126, position 127, position 143, position 178, position 195, position 209, and position 238.

[0004] Furthermore, the wild-type carboxylesterase has 90%, 95%, 96%, 97%, 98%, or 99% or more homology with the amino acid sequence shown in SEQ ID NO.1.

[0005] Furthermore, the amino acid sequence of the wild-type carboxylesterase is shown in SEQ ID NO.1.

[0006] Further, the mutation includes at least one of the following mutation modes: the amino acid residue at position 34 is mutated to G, the amino acid residue at position 67 is mutated to E, the amino acid residue at position 74 is mutated to L, the amino acid residue at position 104 is mutated to A, the amino acid residue at position 122 is mutated to Q, the amino acid residue at position 126 is mutated to Q, the amino acid residue at position 127 is mutated to L, the amino acid residue at position 143 is mutated to G, the amino acid residue at position 178 is mutated to L, the amino acid residue at position 195 is mutated to L, the amino acid residue at position 209 is mutated to K, and the amino acid residue at position 238 is mutated to I.

[0007] Furthermore, the mutation includes at least one of the following mutation modes: .

[0008] The present invention also provides a gene encoding the above-mentioned carboxylesterase mutant.

[0009] The present invention also provides a recombinant expression vector containing the above-mentioned genes.

[0010] The expression vector may be selected from any vector known in the art suitable for expressing exogenous genes in host cells, including but not limited to at least one of the pET series vectors, pGEX series vectors, pUC series vectors, pBAD series vectors, and pMAL series vectors. Preferably, the recombinant expression vector is pET28a, pET21a, pBV220, or pGEX6P.

[0011] The present invention also provides a recombinant host cell comprising the above-described recombinant expression vector.

[0012] Further, the host cell is selected from at least one of Escherichia coli, Bacillus subtilis, Pichia pastoris, or Saccharomyces cerevisiae. Preferably, the host cell is Escherichia coli BL21(DE3).

[0013] The present invention also provides a method for preparing the above-mentioned carboxylesterase mutant, the method comprising the following steps: culturing the above-mentioned recombinant host cells, inducing expression, and collecting and purifying the carboxylesterase mutant.

[0014] This invention also provides the application of the above-mentioned carboxylesterase mutant in the biocatalytic synthesis of carboxylic acid or alcohol compounds from ester compounds.

[0015] The present invention also provides a method for synthesizing carboxylic acid or alcohol compounds, the method comprising the following steps: in the presence of the above-mentioned carboxylesterase mutant or the above-mentioned recombinant host cell, reacting with an ester compound as a substrate to generate a carboxylic acid or alcohol compound.

[0016] Furthermore, the substrate is p-nitrophenylbutyrate, p-nitrophenylacetic acid ester, or ethyl 2-phenylpropionate.

[0017] Furthermore, the reaction temperature is 25-70°C, preferably 25-60°C, 25-50°C or 25-40°C.

[0018] Furthermore, the pH value of the reaction is 7.5-8.5, preferably 8.0.

[0019] Furthermore, the solvent for the reaction is sodium phosphate buffer, or a mixture of sodium phosphate buffer and an organic solvent.

[0020] Furthermore, the organic solvent is DMSO.

[0021] Regarding the definition of terms used in this invention: Unless otherwise stated, the initial definitions provided herein apply to the term throughout the specification; for terms not specifically defined herein, the meanings that a person skilled in the art would give them should be given based on the disclosure and context.

[0022] In the nucleotide mutation code of this invention, the numbers are the nucleotide site numbers of the mutation, the letters before the numbers represent the nucleotides before the mutation, and the letters after the numbers represent the nucleotides after the mutation. For example, ATG100-102GGC means that the nucleotides at positions 100, 101, and 102 are mutated from ATG to GGC, and so on.

[0023] In the amino acid mutation code of this invention, the numbers are the amino acid site numbers of the mutation, the letters before the numbers represent the amino acid before the mutation, and the letters after the numbers represent the amino acid after the mutation. For example, M34G means that the 34th amino acid is mutated from M to G, and so on.

[0024] In the amino acid mutation codes of this invention, the letters before and after the numbers are amino acid abbreviations known in the art. For example, V represents Valine, A represents Alanine, M represents Methionine, L represents Leucine, Q represents Glutamine, C represents Cysteine, K represents Lysine, T represents Threonine, I represents Isoleucine, S represents Serine, H represents Histidine, E represents Glutamic acid, P represents Proline, F represents Phenylalanine, D represents Aspartic acid, Y represents Tyrosine, G represents Glycine, N represents Asparagine, W represents Tryptophan, and R represents Arginine.

[0025] In the amino acid combination mutation code of this invention, "+" indicates that a mutation has been performed before and after "+". For example, V178L+M34G means that the 178th amino acid is mutated from V to L and the 34th amino acid is mutated from M to G, and so on.

[0026] In the nucleotide combination mutation code of this invention, ";" indicates that mutations were performed before and after ";". For example, "GTG532-534CTG;ATG100-102GGC" means that nucleotides at positions 532, 533, and 534 were mutated from ACG to CAG and nucleotides at positions 100, 101, and 102 were mutated from TGG to TTT, and so on.

[0027] The present invention has achieved the following beneficial effects: This invention is derived from Bacillus cereus Using wild-type carboxylesterase as an evolutionary template, several mutants with significantly superior catalytic activity, thermal stability, temperature tolerance, and organic solvent tolerance were obtained through enzyme engineering. These mutants can adapt to more demanding industrial reaction conditions and have greater application advantages in industrial production.

[0028] Using the mutant of this invention to catalyze the synthesis of carboxylic acid compounds can reduce the amount of enzyme used and lower production costs.

[0029] The engineered Escherichia coli expressing the mutant of this invention has the advantages of short culture cycle, simple culture conditions, high yield of target protein, and simple purification.

[0030] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0031] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0032] Figure 1 The results show the temperature tolerance assessment of the BcEst mutant of the carboxylesterase of this invention.

[0033] Figure 2 The results of the organic reagent acceptability assessment of the BcEst mutant of the carboxylesterase of this invention are shown. Detailed Implementation

[0034] The raw materials and equipment used in the specific embodiments of this invention are all known products, obtained by purchasing commercially available products. The carboxylesterase BcEst involved in this invention was obtained by gene mining from the NCBI database using genome mining technology; this gene originates from... Bacillus cereus (UniprotKB:Q815L3). It has the amino acid sequence shown in SEQ ID No. 1 and the DNA sequence shown in SEQ ID No. 2.

[0035] In a specific embodiment of the present invention, the construction of the carboxylesterase BcEst expression vector involves the commonly used pET / 28a(+) vector in the art. Specifically, the carboxylesterase BcEst gene is constructed on the pET / 28a(+) vector with an N / terminal His tag for the preparation, purification, and activity assay of BcEst lyophilized powder.

[0036] Heterologous expression of the carboxylesterase BcEst was performed in engineered *E. coli* bacteria. Specifically, a vector carrying the carboxylesterase BcEst gene was transformed into *E. coli* BL21(DE3) to construct recombinant bacteria. Afterwards, extracted single clones were cultured and induced to express the target protein, resulting in bacterial cells containing the target protein. *E. coli* TOP10 was used for plasmid construction, and *E. coli* BL21(DE3) was used for protein expression.

[0037] The preparation of carboxylesterase lyophilized powder follows general methods in the art. Specifically, the recombinant bacteria induced and cultured are centrifuged for 10 min (6,000 rpm, 4°C), and the bacterial cells are collected. 1 g of bacterial cells is added to 7 mL of PBS phosphate buffer (pH 7.2 / 7.4, 0.01M). The bacterial cells are disrupted by sonication, and the supernatant after centrifugation (12,000 rpm, 4°C, 20 min) is the crude enzyme solution. The crude enzyme solution is rapidly frozen in liquid nitrogen and then freeze-dried to obtain lyophilized powder. This powder is used for evaluating protein activity.

[0038] This invention relates to protein purification technology. Specifically, the carboxylesterase BcEst containing an N / terminal histidine tag is purified using affinity chromatography, and the specific affinity chromatography purification method is as follows: 1. Centrifuge the broken bacterial culture at 18,000 rpm and 4°C for 20 min; 2. After centrifugation, the supernatant was equilibrated with 2 mL of Ni-Sepharose PBS phosphate buffer (pH 7.2-7.4, 0.01 M). TM 6. Fast Flow packing material was rotated and mixed for 1 hour. 3. Let the packing material stand still, elute the non-specifically bound proteins with the same buffer (30 mL) as in step 2, then elute the proteins with PBS phosphate buffer (pH 7.2-7.4, 0.01 M, 20-30 mL) containing 20 mM imidazole, and finally elute the target protein with 3-4 mL of buffer containing 250 mM imidazole and collect it. 4. Desalt the protein containing 250 mM imidazole collected in step 3 using a Cytiva PD-10 desalting column (follow the instructions for use). Collect the desalted protein.

[0039] The desalted protein is the purified esterase. The protein concentration of the purified esterase is determined using A280 nm, which is used to assess the protein melting temperature, activity, and temperature tolerance.

[0040] Example 1: Selection of BcEst mutant sites for carboxylesterase and acquisition of dominant mutants Through sequence conservation analysis, substrate binding pocket, active site, substrate channel analysis, structural and stability analysis, site-directed mutagenesis was performed at multiple sites. Further screening of more advantageous multi-site mutants was conducted using iterative strategies commonly used in this field. V0 represents wild-type. The advantageous mutants V1-V6 obtained after screening are shown in Table 1.

[0041] Table 1. Preferred mutants of carboxylesterase BcEst The following experimental examples demonstrate the beneficial effects of the present invention.

[0042] Experimental Example 1: Determination of the melting temperature (Tm) of the dominant BcEst mutant of carboxylesterase The method used in this experimental example is a common method in the art. Specifically, the melting temperature (Tm) of the protein was determined using a CFX96 Real / Time PCR Detection System, with a program of increasing the temperature from 25°C to 95°C in 0.5°C increments every 30 seconds. The specific reaction system used for the sample consisted of 25 μL of 25 μL containing 0.2 mg / mL of purified esterase, 10× fluorescent dye (5000X, SYPRO™ Orange dye), and 1xPBS buffer. The Tm values ​​of the mutants are shown in Table 2.

[0043] Table 2. Stability determination of the dominant mutant of carboxylesterase BcEst Experiments have demonstrated that the superior mutants of this invention exhibit significantly higher Tm values ​​and better thermal stability compared to the wild-type V0. Specifically, the melting temperature (Tm) of V1 and V2 is increased to 71-71.5 °C, an increase of 23-23.5 °C compared to the wild type; the Tm of the remaining mutants V3-V6 is increased to over 68 °C, an increase of over 20 °C compared to the wild type. The superior mutants of this invention demonstrate significantly better thermal stability than the wild type, enabling them to adapt to higher temperature reaction conditions and showing greater promise for industrial applications.

[0044] Experimental Example 2: Activity Assessment of the Dominant Mutant of Carboxylesterase BcEst 1. Experimental Methods 1.1 Enzyme activity assay In this embodiment, the activity assessment used two universal model substrates, p-PNB (p-nitrophenylbutyrate, C4) and p-PNA (p-nitrophenylacetate, C2), containing different acyl chain lengths. Esterase activity was determined spectrophotometrically at a wavelength of λ = 405 nm. The substrates were prepared fresh before use. A 96-well plate was used for activity detection. A 200 μL reaction mixture contained 1 mM of substrate and lyophilized esterase powder (0.02 mg / mL–0.04 mg / mL). The reaction was carried out at 25 °C in 10 mM sodium phosphate buffer at pH 8.0 for 2–3 min. All experiments were performed in triplicate, with a blank control containing no lyophilized esterase powder to subtract the background of spontaneous substrate hydrolysis. Specific enzyme activity was used to assess activity, and the activity values ​​are shown in Table 3. Specific enzyme activity (U / mg) = total enzyme activity (U) / total protein mass (mg).

[0045] Table 3. Specific activity determination of dominant BcEst mutants of carboxylesterase 1.2 Conversion Rate Test In addition to evaluating the mutant activity using the universal substrates p-PNB and p-PNA, this experiment also used ethyl 2-phenylpropionate (CAS: 2510-99-8) as an extended substrate for activity evaluation. This substrate is converted to 2-phenylpropionic acid under the catalysis of esterases. 2-phenylpropionic acid (α-phenylpropionic acid), also known as α-phenylpropionic acid, is the simplest aryl propionic acid and is an intermediate in the synthesis of aryl propionic acid anti-inflammatory and analgesic drugs and fine chemical products, and is widely used in the synthesis of pharmaceuticals, fragrances, and pesticides. By evaluating the hydrolytic activity of the mutant on this substrate, the potential application prospects of the mutant of this invention in the enzymatic resolution of chiral drug intermediates can be verified. The 500 μL reaction system contained 5 mM substrate and 0.15 mg / mL purified esterase. The reaction was carried out in a mixed solution of 100 mM sodium phosphate buffer and 10% DMSO at pH 8 and 30 °C for 18 h. All experiments were performed in triplicate, with the reaction solution without esterase as a blank control. The conversion rate of 2-phenylpropionic acid was calculated using the product standard curve, and the esterase activity was evaluated. The results are shown in Table 3.

[0046] 2. Experimental Results As shown in Table 3, when the activities of wild-type and mutant strains were measured using p-PNA and p-PNB as universal substrates, the activities of all mutant strains were higher than those of wild-type strains (1.06-2.36 times higher with p-NPB as substrate and 1.05-1.85 times higher with p-PNPA as substrate). Combined with the results of Experimental Example 1, it can be seen that the mutants of this invention exhibit enhanced catalytic activity for universal esterase substrates while improving thermal stability.

[0047] Further activity evaluation using ethyl 2-phenylpropionate as a substrate revealed that, except for V3, whose activity was lower than that of the wild type, the activities of the other mutants were 3%-13% higher than those of the wild type. This indicates that the mutants of this invention not only perform well in catalyzing universal lipase substrates, but also have excellent application potential in catalyzing the synthesis of chiral drug intermediates.

[0048] It should be noted that although the activity of mutant V3 in catalyzing ethyl 2-phenylpropionate is slightly reduced, its thermal stability, temperature tolerance and organic solvent tolerance are significantly improved compared with the wild type, and it can adapt to more demanding industrial reaction conditions (see Experimental Examples 1, 3 and 4 for details).

[0049] Those skilled in the art will understand that p-NPA and p-NPB are the most widely used standard substrates for esterase activity assessment. The mutants of this invention exhibit superior catalytic performance compared to the wild type when catalyzing substrates of different chain lengths (C2 and C4), sufficiently demonstrating that the catalytic performance of the mutants of this invention is significantly improved and possesses substrate universality. Ethyl 2-phenylpropionate, as an application substrate, is only used to illustrate the application of the mutant in a specific industrial scenario and is not considered a necessary parameter limiting the technical characteristics of the mutant.

[0050] Experimental Example 3: Assessment of the temperature tolerance of wild-type and mutant carboxylesterase BcEst This experiment assesses the temperature tolerance of the six dominant mutants (V1-V6) in Table 1 and the wild-type V0. The specific experimental methods are described below.

[0051] Temperature incubation settings: 0.2 mg / mL purified wild-type BcEst V0 and purified mutants V1, V2, V3, V4, V5, and V6 were placed in potassium phosphate buffer (pH 8.0, 10 mM) and incubated in water baths at 40°C, 50°C, 60°C, and 70°C. Samples were taken after 0 h, 0.5 h, 1 h, 2 h, 3 h, 4 h, and 5 h for residual activity determination. Residual activity was calculated as: (enzyme activity after temperature treatment / enzyme activity without temperature treatment) × 100%. The enzyme activity determination method in this experimental example is as follows: Enzyme activity was determined spectrophotometrically using p-nitrophenylbutyrate (p-NPB) as the substrate, which was prepared fresh before use. During the enzymatic hydrolysis of p-NPB, the absorbance of the released p-nitrophenol anion (which appears yellow at 22°C when the pH is above its pKa = 7.08) at a wavelength of λ = 410 nm was monitored to quantify enzyme activity. Activity was detected using 96-well plates. A 200 μL reaction mixture contained 1 mM of substrate and 2 μg / mL–4 μg / mL esterase (to minimize the influence of other conditions, the esterase concentration was the same for the same esterase treated at different temperatures). The reaction was carried out in 10 mM sodium phosphate buffer at pH 8.0. Temperature tolerance test results are shown below. Figure 1 .

[0052] Experimental results demonstrate that the wild-type (V0) of BcEst exhibits low temperature tolerance, losing its catalytic activity after 1 hour of incubation at temperatures above 50°C. In contrast, the six dominant mutants of this invention, after incubation at different temperatures (40°C-60°C), retained at least 75% of their residual activity compared to the control group (untreated). Two of the dominant mutants, V1 and V2, retained at least 80% of their residual activity even after incubation at 40°C-70°C for 5 hours. This indicates that the temperature tolerance of the mutants in this invention is significantly improved.

[0053] Experiment Example 4: Assessment of Organic Reagent Tolerance of Wild-Type and Mutant Carboxylesterase BcEst Since the chemical stability of the standard substrates p-PNB and p-PNA is affected by the concentration of organic reagents, in order to accurately evaluate the stability and catalytic ability of the mutants under different concentrations of organic solvents, this experimental example uses ethyl 2-phenylpropionate, a substrate directly related to industrial applications, to determine its tolerance to organic solvents.

[0054] In this experimental example, the tolerance of the six mutants V1-V6 of this invention to the commonly used co-solvent DMSO was evaluated. The enzyme activity test method is the same as that in Experiment 2, "1.2 Conversion Test", except that "10% DMSO" is replaced with the following DMSO systems: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, and 50% v / v DMSO. The relative activity in this experimental example is calculated as follows: conversion rate at different DMSO contents / conversion rate at 10% DMSO content.

[0055] The results of the tolerance assays of various carboxylesterases to the cosolvent DMSO are shown in the figure. Figure 2 .

[0056] Experiments have shown that the wild-type (V0) of BcEst exhibits relatively low tolerance to co-solvents. Using 10% DMSO as a co-solvent as a control, the activity remained at 80% of the control when the DMSO concentration was below 30%, but decreased sharply when the DMSO concentration exceeded 30%. In contrast, the dominant mutants V1-V6, even under high DMSO conditions (50% DMSO), still maintained 80% of the activity of the control, indicating that the mutants of this invention have significantly improved tolerance to high concentrations of organic solvents.

[0057] In summary, this invention is derived from... Bacillus cereus Using wild-type carboxylesterase as an evolutionary template, several mutants with significantly superior catalytic activity, thermal stability, temperature tolerance, and organic solvent tolerance were obtained through enzyme engineering technology. The mutants of this invention can adapt to more demanding industrial reaction conditions and have very high potential for industrial application.

[0058] The nucleotide and amino acid sequences involved in this invention are as follows: The amino acid sequence of SEQ ID No.1 BcEst Met Met Lys Leu Ala Ser Pro Lys Pro Phe Thr Phe Glu Gly Gly Asp 1 5 10 15 Arg Ala Val Leu Leu Leu His Gly Phe Thr Gly Asn Ser Ala Asp Val 20 25 30 Arg Met Leu Gly Arg Phe Leu Glu Lys Lys Lys Gly Tyr Thr Cys His Ala 35 40 45 Pro Ile Tyr Lys Gly His Gly Val Pro Pro Glu Glu Leu Val His Thr 50 55 60 Gly Pro Thr Asp Trp Trp Gln Asp Val Thr Glu Ala Tyr Gln Leu Leu 65 70 75 80 Lys Asp Lys Gly Phe Glu Lys Ile Ala Val Val Gly Leu Ser Leu Gly 85 90 95 Gly Val Phe Ser Leu Lys Leu Gly Tyr Thr Val Pro Val Leu Gly Val 100 105 110 Val Pro Met Cys Ala Pro Met Tyr Ile Lys Ser Glu Glu Thr Met Tyr 115 120 125 Gln Gly Ile Leu Ala Tyr Ala Arg Glu Tyr Lys Lys Arg Glu Gln Lys 130 135 140 Ser Pro Glu Gln Ile Glu Gln Glu Met Leu Glu Phe Gln Lys Thr Pro 145 150 155 160 Met Asn Thr Leu Lys Ala Leu Gln Gln Leu Ile Ala Asp Val Arg Asn 165 170 175 Asn Val Asp Met Ile Tyr Ala Pro Thr Phe Val Val Gln Ala Arg His 180 185 190 Asp Glu Met Ile Asn Thr Asp Ser Ala Asn Ile Ile Tyr Asn Gly Val 195 200 205 Glu Ser Thr Leu Lys Asp Ile Lys Trp Tyr Glu Asp Ser Thr His Val 210 215 220 Ile Thr Leu Asp Lys Gln Arg Asp Glu Leu His Glu Asp Val Tyr Asn 225 230 235 240 Phe Leu Glu Gln Leu Asp Trp 245 DNA Sequence of SEQ ID No.2 BcEst atgatgaaat tagcatctcc gaaaccattt acatttgagg gtggagaccg cgctgtttta 60 ttactacatg gattcacagg gaactcagct gatgtacgta tgttagggcg tttcttagaa 120 aagaaaggct atacttgtca tgcgccaatt tataaagggc acggtgtacc accagaagag 180 cttgttcata caggccctac agattggtgg caagatgtaa cggaggcata tcagctttta 240 aaagataaag gttttgagaa aattgctgtt gttggattgt cacttggcgg cgtattttct 300 ttaaaattag gttatacagt accggttcta ggtgtagtac caatgtgtgc accgatgtat 360 attaagagcg aagaaacgat gtaccaaggt atattggcat atgcccgcga atataaaaaa 420 cgtgagcaaa aatcaccaga acaaattgaa caagaaatgt tggaattcca aaagacaccg 480 atgaatacat taaaagcatt acaacaatta attgctgacg tacgtaacaa tgtggatatg 540 atttatgcac caacatttgt tgtacaagca cgtcatgacg aaatgattaa tacagatagt 600 gctaacatta tttataacgg tgtagaatct acgttaaaag acattaaatg gtatgaagac 660 tctacgcatg tcattacact tgataaacaa cgtgacgagc tacatgagga tgtatataac 720 ttcttggagc aactagattg g 741

Claims

1. A carboxylesterase mutant, characterized in that, The carboxylesterase mutant is obtained by mutation based on the wild-type carboxylesterase. The amino acid sequence of the wild-type carboxylesterase is shown in SEQ ID NO.

1. The mutation site is at least one of the following sites: position 34, position 67, position 74, position 104, position 122, position 126, position 127, position 143, position 178, position 195, position 209, and position 238.

2. The carboxylesterase mutant according to claim 1, characterized in that, The mutation includes at least one of the following mutation types: the amino acid residue at position 34 is mutated to G, the amino acid residue at position 67 is mutated to E, the amino acid residue at position 74 is mutated to L, the amino acid residue at position 104 is mutated to A, the amino acid residue at position 122 is mutated to Q, the amino acid residue at position 126 is mutated to Q, the amino acid residue at position 127 is mutated to L, the amino acid residue at position 143 is mutated to G, the amino acid residue at position 178 is mutated to L, the amino acid residue at position 195 is mutated to L, the amino acid residue at position 209 is mutated to K, and the amino acid residue at position 238 is mutated to I.

3. The carboxylesterase mutant according to claim 2, characterized in that, The mutation includes at least one of the following mutation modes: 。 4. A gene encoding a carboxylesterase mutant according to any one of claims 1-3.

5. A recombinant expression vector comprising the gene of claim 4.

6. A recombinant host cell comprising the recombinant expression vector of claim 5.

7. A method for preparing the carboxylesterase mutant according to any one of claims 1-3, characterized in that, The method includes the following steps: culturing the recombinant host cell as described in claim 6, inducing expression, and collecting and purifying the carboxylesterase mutant.

8. The use of the carboxylesterase mutant according to any one of claims 1-3 in the biocatalytic synthesis of carboxylic acids or alcohols from esters.

9. A method for synthesizing carboxylic acid or alcohol compounds, characterized in that, The method includes the following steps: in the presence of the carboxylesterase mutant according to any one of claims 1-3 or the recombinant host cell according to claim 6, a reaction is carried out using an ester compound as a substrate to generate a carboxylic acid or alcohol compound.

10. The method according to claim 9, characterized in that, The substrate is p-nitrophenylbutyrate, p-nitrophenylacetic acid ester, or ethyl 2-phenylpropionate.

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