CN115094048B - A method and application of mutating and modifying the 163th threonine residue to improve DcaE4 esterase activity - Google Patents

Abstract

The present disclosure relates to a method for increasing esterase activity, a protein having esterase activity, a gene encoding the protein, a recombinant vector into which the gene is inserted, a transformant transformed with the gene, a method for producing esterase, and an application of esterase in degrading p-nitrophenyl ester compounds. The method realizes amino acid optimization of key sites of the esterase and improves the use effect of the esterase in the fields of biopharmaceutical, bioremediation, low-temperature washing and the like.

Description

A method for improving the activity of DcaE4 esterase by mutating the 163th threonine residue and applications

related application

This application is a divisional application of a Chinese patent application with an application date of April 28, 2021 and an application number of 202110469012.9.

technical field

The present disclosure relates to the field of biotechnology, in particular, to a method for increasing esterase activity, a protein with esterase activity, a gene encoding the protein, a recombinant vector inserted with the gene, and a transformant transformed with the gene , the method for preparing esterase and the application of esterase in degrading p-nitrophenyl ester compounds.

Background technique

Esterase is a class of enzymes that can catalyze the cleavage and formation of various ester bonds, and its structure usually consists of an N-terminal lid domain and a C-terminal α/β hydrolase catalytic domain. The N-terminal lid domain is usually two α-helices located directly above the active center, and the C-terminal catalytic domain contains a Ser-Asp-His catalytic triad with multiple phosphorylation sites, and an oxyanion hole that stabilizes the tetrahedral intermediate state , these structures are crucial for the catalytic characteristics and engineering design of esterases. Most esterases have certain stereoselectivity, regioselectivity and broad substrate spectrum, and can be applied in various industrial fields, while cold-adapted esterases have the characteristics of stable products, energy saving and environmental protection due to their low temperature activity. Therefore, the application in the fields of biopharmaceuticals, bioremediation, and low-temperature washing has attracted extensive attention.

Extremophiles have adapted to extreme environments such as high temperature, low temperature, and high pressure during natural evolution, and are a high-quality source of extremophile gene resources. The variety of enzyme preparations currently produced is relatively single and cannot meet the needs of the industry. Therefore, it is still necessary to develop enzyme resources with special properties. The thermal stability of biological enzymes found so far is generally poor, which is not conducive to industrial applications, so the improvement of enzyme thermal stability has become particularly important.

Therefore, it is urgent to provide a method for optimizing esterase, improve the efficiency of the degrading enzyme, and enhance the thermal stability of the enzyme.

Contents of the invention

In order to further meet the needs of practical applications, the present disclosure provides a method for increasing esterase activity, a protein with esterase activity, a gene encoding the protein, a recombinant vector inserted with the gene, and a transformed gene transformed with the gene. Body, method for preparing esterase and application of esterase in degrading p-nitrophenyl esters.

The first aspect of the present disclosure provides a method for improving the activity of esterase, the method comprising mutating one or more of the amino acid residues to be mutated in the wild-type esterase, characterized in that the wild-type esterase The amino acid sequence is shown in SEQ ID NO.2, and the amino acid residues to be mutated include the 132nd serine residue, the 163rd threonine residue, the 238th serine residue and the 285th tyrosine residue residue; said mutation is engineered as a substitution of an amino acid residue.

The second aspect of the present disclosure provides a protein with esterase activity, which is a protein derived from the amino acid sequence of the wild-type esterase shown in SEQ ID NO.2 by substituting 1, 2, 3 or 4 amino acids and, the amino acid sequence of the protein is shown in SEQ ID NO.1.

The third aspect of the present disclosure provides a gene encoding the protein described in the second aspect, wherein the gene is a DNA molecule whose nucleotide sequence is shown in SEQ ID NO.3, 4, 5 or 6.

The fourth aspect of the present disclosure provides a recombinant vector, wherein the gene described in the second aspect is inserted into the recombinant vector.

The fifth aspect of the present disclosure provides a transformant, wherein, the host of the transformant is a genetically engineered bacterium; the gene introduced into the transformant includes the gene described in the second aspect, or, the gene introduced into the transformant The recombinant vector includes the recombinant vector described in the fourth aspect.

The sixth aspect of the present disclosure provides a method for preparing esterase, wherein the method comprises: inoculating the transformant described in the fifth aspect in a culture medium to obtain a cultured material.

The seventh aspect of the present disclosure provides an application of an esterase in degrading p-nitrophenyl esters, wherein the esterase contains the protein described in the second aspect.

Through the above technical solution, the present disclosure provides a method for improving esterase activity, a protein with esterase activity, a gene encoding the protein, a recombinant vector inserted with the gene, a transformant transformed with the gene, and preparation of esterase The method and the application of esterase in the degradation of p-nitrophenyl esters have important application value in the optimization and transformation of esterase.

Other features and advantages of the present disclosure will be described in detail in the detailed description that follows.

Description of drawings

The accompanying drawings are used to provide a further understanding of the present disclosure, and constitute a part of the description, together with the following specific embodiments, are used to explain the present disclosure, but do not constitute a limitation to the present disclosure. In the attached picture:

Figure 1 is the enzymatic activity of esterase DcaE4 and mutant S132.

Figure 2 is the enzymatic activity of esterase DcaE4 and mutant T163.

Figure 3 is the enzymatic activity of esterase DcaE4 and mutant S238.

Figure 4 is the enzymatic activity of esterase DcaE4 and mutant Y285.

Fig. 5 is the determination of the optimum temperature (A), thermal stability (B) and half-life at 40°C (C) and 50°C (D) of esterase DcaE4 and its mutants.

Detailed ways

Specific embodiments of the present disclosure will be described in detail below in conjunction with the accompanying drawings. It should be understood that the specific embodiments described here are only used to illustrate and explain the present disclosure, and are not intended to limit the present disclosure.

The first aspect of the present disclosure provides a method for improving the activity of esterase, the method comprising mutating one or more of the amino acid residues to be mutated in the wild-type esterase, characterized in that the wild-type esterase The amino acid sequence is shown in SEQ ID NO.2, and the amino acid residues to be mutated include the 132nd serine residue, the 163rd threonine residue, the 238th serine residue and the 285th tyrosine residue residue; said mutation is engineered as a substitution of an amino acid residue.

According to the present disclosure, wherein, optionally, the mutation modification includes at least one of the following (a)-(d):

(a) Mutate the 132nd ser amino acid residue into alanine residue, tryptophan residue, valine residue, proline residue, arginine residue, histidine residue, gluten residue one of aminoamide residues, glutamic acid residues and tyrosine residues;

(b) Mutation of the 163rd threonine residue to an alanine residue, a histidine residue, a leucine residue, an asparagine residue, an arginine residue, a lysine residue , a cysteine residue, a glutamic acid residue;

(c) mutating the 238th serine residue into an asparagine residue, an aspartic acid residue, an arginine residue, a histidine residue, a lysine residue, an asparagine residue, One of tyrosine residues, proline residues, valine residues, and tryptophan residues;

(d) Mutation of the 285th tyrosine residue to tryptophan residue, phenylalanine residue, valine residue, arginine residue, histidine residue, serine residue, gluten residue One of amino acid residues and cysteine residues.

Wherein, the mutation modification of the above (a) to (d) can be combined arbitrarily in the same esterase.

Preferably, the mutation modification includes any one of the following (i)-(iv):

(i) mutating the 132nd ser amino acid residue to an alanine residue;

(ii) mutating the threonine residue at position 163 to an alanine residue;

(iii) mutating the 238th serine residue to an asparagine residue;

(iv) Mutating the tyrosine residue at position 285 to a phenylalanine residue.

The second aspect of the present disclosure provides a protein with esterase activity, which is a protein derived from the amino acid sequence of the wild-type esterase shown in SEQ ID NO.2 by substituting 1, 2, 3 or 4 amino acids and, the amino acid sequence of the protein is shown in SEQ ID NO.1.

According to the present disclosure, wherein, the amino acid sequence of the protein is shown in SEQ ID NO.3, 4, 5 or 6.

The third aspect of the present disclosure provides a gene encoding the protein described in the second aspect, wherein the gene is a DNA molecule whose nucleotide sequence is shown in SEQ ID NO.7, 8, 9 or 10.

The fourth aspect of the present disclosure provides a recombinant vector, wherein the gene described in the second aspect is inserted into the recombinant vector.

According to the present disclosure, the recombinant vector may be a recombinant expression vector or a recombinant cloning vector, and the recombinant expression vector is a DNA molecule whose nucleotide sequence is shown in SEQ ID NO.11, 12, 13 or 14.

Wherein, the difference between SEQ ID NO.12 and SEQ ID NO.11 is: the 5659th base in the nucleotide sequence of SEQ ID NO.12 is G; the difference between SEQ ID NO.13 and SEQ ID NO.11 is : the 6026th base in the nucleotide sequence of SEQ ID NO.13 is T; the difference between SEQ ID NO.14 and SEQ ID NO.11 is: the 5885th base in the nucleotide sequence of SEQ ID NO.14 The base is A.

The fifth aspect of the present disclosure provides a transformant, wherein, the host of the transformant is a genetically engineered bacterium; the gene introduced into the transformant includes the gene described in the second aspect, or, the gene introduced into the transformant The recombinant vector includes the recombinant vector described in the fourth aspect.

According to the present disclosure, the genetically engineered bacteria can be wild-type genetically engineered bacteria or artificially modified genetically engineered bacteria, such as E. coli BL21 (DE3) competent cells, Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae and filamentous at least one of the fungi.

The sixth aspect of the present disclosure provides a method for preparing esterase, wherein the method comprises: inoculating the transformant described in the fifth aspect in a culture medium to obtain a cultured material.

Among them, the medium and culture conditions can be selected from various known suitable ones. The cultured material contains the protein with esterase activity provided by the first aspect of the present disclosure, so it has esterase enzyme activity, and can be directly used as an esterase composition according to needs, or can be purified according to needs. Proteins with esterase activity are then used.

The seventh aspect of the present disclosure provides an application of an esterase in degrading p-nitrophenyl esters, wherein the esterase contains the protein described in the second aspect.

The present disclosure is further illustrated by the following examples, but the present disclosure is not limited thereby.

Example 1

In this example, a recombinant coliform coli strain expressing the DcaE4 gene and mutants of Deinococcus radiodurans was constructed.

The expression plasmid pET28a in this disclosure is a commercially available product from Merck, Germany; Escherichia coli BL21 (DE3) is a commercially available product from Beijing Novozyme; the site-directed mutagenesis kit (Mut Express II Fast Mutagenesis Kit V2) was purchased from Nanjing Novozyme company.

1. Design PCR-specific primers according to the DcaE4 gene sequence in the Deinococcus radiodurans genome: DcaE4-F: 5' CCATGGCTGATATCGGATCCATGCCCGTAGACCCCCAACCT 3' (SEQ ID NO.12); DcaE4-R: 5' CTCGAGTGCGGCCGCAAGCTTTCAGCCGCGCAGTTGCTCG 3' (SEQ ID NO. 13); Sequence of site-directed saturation mutation primer: S132N-F: 5′-GCGCCGGTAGACGACGCCCTGGCGNNKGTGGTCTGGGC-3′ (SEQ ID NO.14); S132N-R: 5′-GGCGGCGTGCGCGGCGGCCCAGACCACMNNCGCCAGGG-3′ (SEQ ID NO.15); T163N-F : 5′-GACAGCGCGGGGGCCAACCTCGCCNNKGTCACGGCG-3′ (SEQ ID NO.16); T163N-R: 5′-CGTCACGCGACCGCAGCGCCGTGACMNNGGCGAGGT-3′ (SEQ ID NO.17); S238N-F: 5′-ACGCCTCGCCGCTCAACGCTGAGNNKCTCGCGGGGT T-3' (SEQ ID NO .18); S238N-R: 5'-ACCAGGGCCGGCGGCAACCCCGCGAGMNNCTCAGCG-3' (SEQ ID NO.19); Y285N-F: 5'-GCATGATTCACGGTNNKGCCAACATGACCGCGTTT-3' (SEQ ID NO.20); Y285N-R: 5'-GGCGAAACCGTMNNTCATGCC GGGGCCGGGGCGGTA- 3' (SEQ ID NO. 21); wherein, M=A/C; K=G/T; N=A/G/C/T.

2. Amplify the target gene sequence from the genomic DNA of Deinococcus radiodurans by PCR method.

3. After the PCR product was recovered by gel, it was connected to the pET-28a vector containing cohesive ends obtained by Bamh I/Hind III double enzyme digestion by recombinase to construct the E. coli expression vector pET28a-DcaE4.

4. The expression vector was transformed into Escherichia coli BL21, and the inserted sequence was verified to be correct by PCR, enzyme digestion and sequencing, and the strain was named BL21-pET28a-DcaE4.

5. Carry out site-directed saturation mutation according to the selected mutation sites Ser238, Ser132, Thr163 and Tyr285, design primers containing the mutation sites, and use site-directed mutagenesis kits to construct mutant expression vectors. The whole plasmid was amplified by inverse PCR using the wild-type plasmid (pET28a-DcaE4) as a template.

6. After the PCR reaction, take 2 μL for agarose gel electrophoresis detection, and purify if the band is correct. The purified DNA is digested with DpnI for demethylation, and then recombined.

The recombinant product was transformed into Escherichia coli BL21(DE3), and positive clones were identified by sequencing for subsequent experiments.

Experimental results of this example: the present disclosure successfully constructed recombinant E. coli engineering strains expressing DcaE4, and screened and obtained recombinant E. coli engineering strains of DcaE4 mutants with site-directed saturation mutations of Ser238, Ser132, Thr163 and Tyr285. Mutants mutated into four different types of amino acids were selected by gene sequencing, and the mutants selected at the Ser132 site were S132A, S132W, S132V, S132P, S132R, S132H, S132Q, S132E, and S132Y. The mutants selected for the Thr163 site were T163A, T163H, T163L, T163N, T163R, T163K, T163C, and T163E. The mutants selected for the Tyr285 site were Y285W, Y285F, Y285V, Y285R, Y285H, Y285S, Y285E, and Y285C. The mutants selected for the Ser238 site are S238D, S238R, S238H, S238K, S238N, S238Y, S238P, S238V, and S238W.

The specific sequence is as follows:

Example 2

This example is an experiment for detecting the enzyme activity of the esterase DcaE4 mutant.

The experimental materials of this embodiment include: recombinant engineering strains: the recombinant expression strains of esterase DcaE4 and DcaE4 mutants obtained in Example 1; enzyme activity assay reagents: substrate solution: 0.3% p-nitrophenol octanoate pNPC (C8)) was dissolved in isopropanol and stored at 4°C; buffer: 20mM Tris-HCl buffer (pH7.5, 0.11% gum arabic); substrate test solution: substrate solution and buffer respectively by 1: The ratio of 3 was mixed and used as the substrate test solution.

The experimental method of the present embodiment comprises:

1. Induced expression and purification of recombinant protein: Inoculate the bacteria into 20 mL of LB liquid medium with antibiotics at an inoculum of 1%, and cultivate overnight on a shaker at 37°C; the inoculum with an initial concentration of OD ₆₀₀ of 0.1 Transfer the bacterial solution to 500 mL of LB liquid medium added with kanamycin. Cultivate at 37°C until the concentration of the bacterial solution is 0.6-0.8, add IPTG (final concentration 0.1 μmol/L) to induce protein expression, the induction condition is 25°C, 6-8h. The induced bacterial liquid was centrifuged to collect the bacterial cells, and the bacterial cells were resuspended with NTA-0. The bacterial liquid was ultrasonically disrupted, and the ultrasonically disrupted samples were centrifuged for 30 minutes to collect the supernatant and precipitate of the crushed liquid respectively. The crushed supernatant obtained by centrifugation was the crude enzyme liquid, and the crushed samples were used for subsequent experiments.

2. Purification of recombinant esterase protein by affinity chromatography: Take out the nickel column, wash the nickel column twice with deionized water after the ethanol has run out, and then equilibrate the column with NTA-0, keeping the flow rate at 1mL/min. The crude enzyme solution was hung on the column and penetrated twice. Use the prepared NTA-10, NTA-30, NTA-50, NTA-80, NTA-100, NTA-150, NTA-200, NTA-250, NTA-300 gradient elution, use the protein detection solution to detect the protein, The eluted peaks were collected, and the size and purity of the obtained protein were detected by SDS-PAGE. The buffer of the protein solution is replaced by ultrafiltration centrifugation to remove the imidazole in the protein solution, and the obtained protein solution is the enzyme solution.

3. Determination of activity of esterase protein: Determination of esterase activity is carried out with a general colorimetric method and a general model substrate, p-nitrophenyl ester compounds. The detection principle is to generate p-nitrophenol (pNP) by hydrolyzing the p-nitrophenyl ester compound substrate, pNP is yellow and has an absorption peak at 410nm, and the enzyme activity of the esterase is determined by detecting the content of the product pNP. One enzyme activity unit (U) is defined as: the amount of enzyme required to release 1 μmol of product pNP per unit time. 600 μL of substrate test solution was added to a 1.5 mL centrifuge tube, and 25 μL of appropriately diluted enzyme solution was added; 25 μL of Tris-HCl buffer solution with pH=8 was added to the control group. After incubation at 30° C. for 5 min, 500 μL of 95% ethanol was added to terminate the reaction. The absorbance was measured at 410nm, and the enzyme activity was calculated.

Effect of temperature on enzyme activity and thermal stability of enzyme: add enzyme solution to Tris-HCl buffer solution at pH 8, respectively at different temperatures (5, 10, 20, 30, 40, 50, 60, 70°C) Measure the degradation activity of lipase/esterase, react for 5 minutes, and determine the optimum temperature of lipase. Further research on the thermal stability of lipase/esterase: heat the purified enzyme solution at 5, 10, 20, 30, 40, and 50°C for 6 hours, and measure the remaining enzyme activity at the optimum temperature every 1 hour to determine The highest enzyme activity is 100% to calculate the relative enzyme activity.

The experimental result of this embodiment:

The enzyme activities of DcaE4 wild-type and mutant proteins were measured, and the mutant proteins at the Ser238, Ser132, Thr163 and Tyr285 sites of DcaE4 were purified and diluted to the same concentration (40 μg/mL). Under the conditions, the enzyme activity was measured with the substrate pNPC8, and the results are shown in Figures 1-4. Compared with the wild-type DcaE4 esterase activity, only S132A and S132V at the Ser132 site had no significant changes compared with the wild type, and the enzyme activities of the other mutants all decreased to varying degrees. Among the mutations at the Thr163 site, except for T163A, T163L and T163C, the other mutations all resulted in different degrees of reduction in enzyme activity. Among the mutations at the Tyr285 site, only the enzyme activity of Y285F had no significant change compared with the wild type, and the other mutants decreased significantly. The enzymatic activity of the mutation of Ser238 site to S238N was significantly increased, and the enzymatic activity of the mutants S238P, S238V, S238W and S238K was significantly decreased compared with the wild type, and the enzymatic activities of the other mutants had no significant changes.

The mutants S132A, S132V, T163A, T163L, T163C, Y285F and S238N with no significant change in enzyme activity were selected to measure the temperature stability, and the optimum temperature of each mutant and wild-type DcaE4 was determined respectively (Fig. 5A), 20-60 The remaining enzyme activity after incubation at ℃ for 1 h (Fig. 5B) and the half-lives at 40°C and 50°C (Fig. 5C and Fig. 5D). As shown, the optimum temperature of each mutant and wild-type esterase is still 30°C. The half-life of each mutant and wild-type esterase DcaE4 was calculated by the first-order inactivation model, and the results are shown in Table 1. The half-lives of the mutants at 40 and 50°C were increased, among which the half-lives of S132A at 40 and 50°C were increased by 44.29% and 89.89%, and the half-lives of T163A at 40 and 50°C were increased by 89.59% and 119.11%, respectively. Y285F The half-lives at 40 and 50°C increased by 59.41% and 65.79%, respectively, and the half-lives of S238N at 40°C and 50°C increased by 47.15% and 70.10%, respectively. Among these mutants, T163A had the highest improvement in thermostability.

Table 1 The half-life of each mutant at 40℃ and 50℃

Example 3

This example is an analysis of the degradation ability of the esterase DcaE4 mutant to pesticides.

The experimental materials of this embodiment include: esterase protein: DcaE4 and mutants S132A, T163A, Y285F and S238N purified and obtained in Example 2; insecticide: carbaryl (CAR) was purchased from Aladdin Company; fenpropathrin (FEN), cis-cypermethrin (α-CYP), and deltamethrin (DEL) were purchased from Dr. Ehrenstorfer Company.

The experimental method of the present embodiment comprises:

1. Esterase hydrolysis system: In order to measure the degradation of insecticides by esterase DcaE4 and mutants, the purified enzyme protein was concentrated and then diluted with Tris-HCl (50mM, pH=8) to the same concentration of 40μg/mL , add carbaryl, fenpropathrin, cis-cypermethrin and deltamethrin (final concentration 50 μg/mL) to a certain amount of enzyme solution respectively, and the control group uses high temperature inactivated enzyme solution to add the same concentration of insecticidal reagent, the reaction system was mixed and incubated at 30°C for 8 hours to carry out the enzymatic hydrolysis reaction.

2. Sample preparation: Add an equal volume of ethyl acetate to the reaction solution, vortex and shake for 10 minutes, then let it stand for 1 hour, take a certain amount of upper organic phase nitrogen and blow it to dryness, add methanol to redissolve, and use an organic filter membrane to obtain the sample After filtration, it is used for chromatographic detection.

Chromatographic conditions: chromatographic column: Agilent Poroshell 120 SB-C18 (2.1mm×75mm, 2.7μm); mobile phase A: water (containing 10mM ammonium formate and 0.1% formic acid); mobile phase B: methanol (containing 10mM ammonium formate and 0.1 % formic acid); Gradient elution (0～2min, 10%B; 2～6min, 10%→40%B; 6～10min, 40%→80%B; 10～12min, 80%→95%B; 12 ～16min, 95%B; 16～18min 95%→5%B; 18～20min, 5%B); flow rate: 0.2mL/min; column temperature: 25℃; injection volume: 10μL; running time: 20min;

Mass spectrometry conditions: using Agilent jet stream electrospray ionization source (ESI), positive ion MRM mode acquisition; drying gas temperature: 350°C; drying gas flow rate: 12L/min; nebulizer pressure: 35psi; capillary voltage: 3500V;

The experimental result of this embodiment:

Degradation activity of esterase was measured, and the results showed that DcaE4 had a degradation function to the four insecticides tested, among which DcaE4 had the fastest degradation rate to carbaryl, and the degradation rate reached 100% in 8 hours, and fenpropathrin , cis-cypermethrin and deltamethrin decreased in turn, respectively 85.19%, 54.19%, 34.23%.

After 8 hours of incubating, DcaE4 and its mutants were resistant to carbaryl (CAR), fenpropathrin (FEN), cis-cypermethrin (α-CYP) and deltamethrin (DEL) at a concentration of 50 μg/mL, respectively. With degradation rates of 85.83% to 93.26%, 54.01% to 61.95%, 30.45% to 40.96%, and 26.64% to 35.37%, the overall degradation trend of the mutant is similar to that of the wild type, and the degradation of CAR, FEN, α-CYP and DEL However, the degradation rates of each mutant were different for different insecticides. Compared with the wild, the degradation rate of S238N for the four insecticides was significantly improved, and the degradation rate increased by 6.08% to 7.94%. (Table 2). In addition, other mutants did not have a significant impact on the degradation rate of pesticides.

Table 2 Degradation rate of DcaE4 and its mutants to insecticides and toxins

Enzymes CAR(%) FEN(%) α-CYP(%) DEL(%) WT 87.18±0.44 54.01±2.39 33.29±2.04 27.29±2.68 S132A 85.83±0.71 56.25±1.12 30.45±0.62 28.22±5.14 T163A 86.35±1.23 55.17±1.18 34.33±2.84 27.27±4.57 Y285F 88.42±0.52 58.84±1.25 34.61±4.08 26.64±2.32 S238N 93.26±0.33 61.95±0.98 40.96±1.30 34.05±1.23

The disclosure uses site-directed mutagenesis to optimize the amino acid at the key site of the low-temperature esterase DcaE4 in Deinococcus radiodurans; the mutation of the Ser238 site in the DcaE4 esterase protein can significantly improve the enzyme activity; the catalytic efficiency of the mutant S238N and stability were improved, and the degradation rate of the four insecticides was significantly improved; Ser132, Thr163 and Tyr285 were crucial to the structure of esterase, and mutants S132A, T163A and Y285F significantly improved the esterase stability.

The disclosure provides a method for improving esterase activity, a protein with lipase activity, a gene encoding the protein, a recombinant vector inserted with the gene, a transformant transformed with the gene, a method for preparing esterase, and esterase The application in the degradation of p-nitrophenyl ester compounds has laid the foundation for the application of esterase in the fields of biopharmaceuticals, bioremediation and low-temperature washing, and meets the needs of esterase in industry.

The preferred embodiments of the present disclosure have been described in detail above in conjunction with the accompanying drawings. However, the present disclosure is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present disclosure, various simple modifications can be made to the technical solutions of the present disclosure. These simple modifications all belong to the protection scope of the present disclosure.

In addition, it should be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable way if there is no contradiction. The combination method will not be described separately.

In addition, various implementations of the present disclosure can also be combined in any way, as long as they do not violate the idea of the present disclosure, they should also be regarded as the content disclosed in the present disclosure.

sequence listing

<110> Institute of Biotechnology, Chinese Academy of Agricultural Sciences

<120> A method and application of mutating and modifying the 163th threonine residue to improve DcaE4 esterase activity

<130> 20217CAAS-B-ZW

<160> 21

<170> SIPOSequenceListing 1.0

<210> 1

<211> 312

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> UNSURE

<222> (132)..(132)

<223> The 'Xaa' at location 132 stands for Ala, Trp, Val, Pro, Arg, His,Gln, Glu, or Tyr.

<220>

<221> UNSURE

<222> (163)..(163)

<223> The 'Xaa' at location 163 stands for Ala, His, Leu, Asn, Arg, Lys,Cys, or Glu.

<220>

<221> UNSURE

<222> (238)..(238)

<223> The 'Xaa' at location 238 stands for Asn, Asp, Arg, His, Lys, Asn, Tyr, Pro, Val, or Trp.

<220>

<221> UNSURE

<222> (285)..(285)

<223> The 'Xaa' at location 285 stands for Phe, Trp, Val, Arg, His, Ser, Glu, or Cys.

<400> 1

Met Pro Val Asp Pro Asn Leu Tyr Gln Leu Leu Leu Gln Leu Ser Gln

1 5 10 15

Ala Pro Glu Pro Ala Gly Leu Glu Glu Leu Arg Ala Gly Val Ile Ala

20 25 30

Asn Ala Ala Arg Ser Pro Lys Arg Pro Val Thr Ile Gly Glu Val Arg

35 40 45

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

50 55 60

His Pro Ala Gly Gln Ala Pro Ala Ser Gly Trp Pro Leu Thr Val Phe

65 70 75 80

Phe His Gly Gly Gly Phe Val Val Tyr Asp Leu Asp Thr His Asp Ala

85 90 95

Leu Cys Arg Glu Leu Cys Ala Thr Ser Gly Ala Ala Val Leu Ser Val

100 105 110

Ala Tyr Arg Leu Ala Pro Glu Ala Arg Phe Pro Ala Pro Val Asp Asp

115 120 125

Ala Leu Ala Xaa Val Val Trp Ala Ala Ala His Ala Ala Glu Leu Gly

130 135 140

Ala Asp Ala Gly Arg Leu Ala Val Ala Gly Asp Ser Ala Gly Ala Asn

145 150 155 160

Leu Ala Xaa Val Thr Ala Leu Arg Ser Arg Asp Glu Gly Gly Pro Ala

165 170 175

Leu Arg Ala Gln Leu Leu Ile Tyr Pro Ala Ala Asp Phe Glu His Pro

180 185 190

Glu Arg Tyr Pro Ser Arg Gln Glu Asn Gly Arg Gly Tyr Phe Leu Thr

195 200 205

Asp Glu Arg Met Arg Phe Phe Gly Gln Met Tyr Leu Ala Arg Pro Glu

210 215 220

Asp Ala Ala His Pro His Ala Ser Pro Leu Asn Ala Glu Xaa Leu Ala

225 230 235 240

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

245 250 255

Asp Glu Gly Ala Ala Tyr Ala Glu Ala Leu Lys Ala Ala Gly Val Ser

260 265 270

Ala Glu Tyr Arg Pro Gly Pro Gly Met Ile His Gly Xaa Ala Asn Met

275 280 285

Thr Ala Phe Ser Pro Val Ala Ala Gln Leu Ile Asp Glu Ala Gly Val

290 295 300

Trp Leu Gly Glu Gln Leu Arg Gly

305 310

<210> 2

<211> 312

<212> PRT

<213> Deinococcus radiodurans

<400> 2

Met Pro Val Asp Pro Asn Leu Tyr Gln Leu Leu Leu Gln Leu Ser Gln

1 5 10 15

Ala Pro Glu Pro Ala Gly Leu Glu Glu Leu Arg Ala Gly Val Ile Ala

20 25 30

Asn Ala Ala Arg Ser Pro Lys Arg Pro Val Thr Ile Gly Glu Val Arg

35 40 45

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

50 55 60

His Pro Ala Gly Gln Ala Pro Ala Ser Gly Trp Pro Leu Thr Val Phe

65 70 75 80

Phe His Gly Gly Gly Phe Val Val Tyr Asp Leu Asp Thr His Asp Ala

85 90 95

Leu Cys Arg Glu Leu Cys Ala Thr Ser Gly Ala Ala Val Leu Ser Val

100 105 110

Ala Tyr Arg Leu Ala Pro Glu Ala Arg Phe Pro Ala Pro Val Asp Asp

115 120 125

Ala Leu Ala Ser Val Val Trp Ala Ala Ala His Ala Ala Glu Leu Gly

130 135 140

Ala Asp Ala Gly Arg Leu Ala Val Ala Gly Asp Ser Ala Gly Ala Asn

145 150 155 160

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

165 170 175

Leu Arg Ala Gln Leu Leu Ile Tyr Pro Ala Ala Asp Phe Glu His Pro

180 185 190

Glu Arg Tyr Pro Ser Arg Gln Glu Asn Gly Arg Gly Tyr Phe Leu Thr

195 200 205

Asp Glu Arg Met Arg Phe Phe Gly Gln Met Tyr Leu Ala Arg Pro Glu

210 215 220

Asp Ala Ala His Pro His Ala Ser Pro Leu Asn Ala Glu Ser Leu Ala

225 230 235 240

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

245 250 255

Asp Glu Gly Ala Ala Tyr Ala Glu Ala Leu Lys Ala Ala Gly Val Ser

260 265 270

Ala Glu Tyr Arg Pro Gly Pro Gly Met Ile His Gly Tyr Ala Asn Met

275 280 285

Thr Ala Phe Ser Pro Val Ala Ala Gln Leu Ile Asp Glu Ala Gly Val

290 295 300

Trp Leu Gly Glu Gln Leu Arg Gly

305 310

<210> 3

<211> 312

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Pro Val Asp Pro Asn Leu Tyr Gln Leu Leu Leu Gln Leu Ser Gln

1 5 10 15

Ala Pro Glu Pro Ala Gly Leu Glu Glu Leu Arg Ala Gly Val Ile Ala

20 25 30

Asn Ala Ala Arg Ser Pro Lys Arg Pro Val Thr Ile Gly Glu Val Arg

35 40 45

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

50 55 60

His Pro Ala Gly Gln Ala Pro Ala Ser Gly Trp Pro Leu Thr Val Phe

65 70 75 80

Phe His Gly Gly Gly Phe Val Val Tyr Asp Leu Asp Thr His Asp Ala

85 90 95

Leu Cys Arg Glu Leu Cys Ala Thr Ser Gly Ala Ala Val Leu Ser Val

100 105 110

Ala Tyr Arg Leu Ala Pro Glu Ala Arg Phe Pro Ala Pro Val Asp Asp

115 120 125

Ala Leu Ala Ala Val Val Trp Ala Ala Ala His Ala Ala Glu Leu Gly

130 135 140

Ala Asp Ala Gly Arg Leu Ala Val Ala Gly Asp Ser Ala Gly Ala Asn

145 150 155 160

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

165 170 175

Leu Arg Ala Gln Leu Leu Ile Tyr Pro Ala Ala Asp Phe Glu His Pro

180 185 190

Glu Arg Tyr Pro Ser Arg Gln Glu Asn Gly Arg Gly Tyr Phe Leu Thr

195 200 205

Asp Glu Arg Met Arg Phe Phe Gly Gln Met Tyr Leu Ala Arg Pro Glu

210 215 220

Asp Ala Ala His Pro His Ala Ser Pro Leu Asn Ala Glu Ser Leu Ala

225 230 235 240

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

245 250 255

Asp Glu Gly Ala Ala Tyr Ala Glu Ala Leu Lys Ala Ala Gly Val Ser

260 265 270

Ala Glu Tyr Arg Pro Gly Pro Gly Met Ile His Gly Tyr Ala Asn Met

275 280 285

Thr Ala Phe Ser Pro Val Ala Ala Gln Leu Ile Asp Glu Ala Gly Val

290 295 300

Trp Leu Gly Glu Gln Leu Arg Gly

305 310

<210> 4

<211> 312

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Pro Val Asp Pro Asn Leu Tyr Gln Leu Leu Leu Gln Leu Ser Gln

1 5 10 15

Ala Pro Glu Pro Ala Gly Leu Glu Glu Leu Arg Ala Gly Val Ile Ala

20 25 30

Asn Ala Ala Arg Ser Pro Lys Arg Pro Val Thr Ile Gly Glu Val Arg

35 40 45

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

50 55 60

His Pro Ala Gly Gln Ala Pro Ala Ser Gly Trp Pro Leu Thr Val Phe

65 70 75 80

Phe His Gly Gly Gly Phe Val Val Tyr Asp Leu Asp Thr His Asp Ala

85 90 95

Leu Cys Arg Glu Leu Cys Ala Thr Ser Gly Ala Ala Val Leu Ser Val

100 105 110

Ala Tyr Arg Leu Ala Pro Glu Ala Arg Phe Pro Ala Pro Val Asp Asp

115 120 125

Ala Leu Ala Ser Val Val Trp Ala Ala Ala His Ala Ala Glu Leu Gly

130 135 140

Ala Asp Ala Gly Arg Leu Ala Val Ala Gly Asp Ser Ala Gly Ala Asn

145 150 155 160

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

165 170 175

Leu Arg Ala Gln Leu Leu Ile Tyr Pro Ala Ala Asp Phe Glu His Pro

180 185 190

Glu Arg Tyr Pro Ser Arg Gln Glu Asn Gly Arg Gly Tyr Phe Leu Thr

195 200 205

Asp Glu Arg Met Arg Phe Phe Gly Gln Met Tyr Leu Ala Arg Pro Glu

210 215 220

Asp Ala Ala His Pro His Ala Ser Pro Leu Asn Ala Glu Ser Leu Ala

225 230 235 240

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

245 250 255

Asp Glu Gly Ala Ala Tyr Ala Glu Ala Leu Lys Ala Ala Gly Val Ser

260 265 270

Ala Glu Tyr Arg Pro Gly Pro Gly Met Ile His Gly Tyr Ala Asn Met

275 280 285

Thr Ala Phe Ser Pro Val Ala Ala Gln Leu Ile Asp Glu Ala Gly Val

290 295 300

Trp Leu Gly Glu Gln Leu Arg Gly

305 310

<210> 5

<211> 312

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Met Pro Val Asp Pro Asn Leu Tyr Gln Leu Leu Leu Gln Leu Ser Gln

1 5 10 15

Ala Pro Glu Pro Ala Gly Leu Glu Glu Leu Arg Ala Gly Val Ile Ala

20 25 30

Asn Ala Ala Arg Ser Pro Lys Arg Pro Val Thr Ile Gly Glu Val Arg

35 40 45

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

50 55 60

His Pro Ala Gly Gln Ala Pro Ala Ser Gly Trp Pro Leu Thr Val Phe

65 70 75 80

Phe His Gly Gly Gly Phe Val Val Tyr Asp Leu Asp Thr His Asp Ala

85 90 95

Leu Cys Arg Glu Leu Cys Ala Thr Ser Gly Ala Ala Val Leu Ser Val

100 105 110

Ala Tyr Arg Leu Ala Pro Glu Ala Arg Phe Pro Ala Pro Val Asp Asp

115 120 125

Ala Leu Ala Ser Val Val Trp Ala Ala Ala His Ala Ala Glu Leu Gly

130 135 140

Ala Asp Ala Gly Arg Leu Ala Val Ala Gly Asp Ser Ala Gly Ala Asn

145 150 155 160

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

165 170 175

Leu Arg Ala Gln Leu Leu Ile Tyr Pro Ala Ala Asp Phe Glu His Pro

180 185 190

Glu Arg Tyr Pro Ser Arg Gln Glu Asn Gly Arg Gly Tyr Phe Leu Thr

195 200 205

Asp Glu Arg Met Arg Phe Phe Gly Gln Met Tyr Leu Ala Arg Pro Glu

210 215 220

Asp Ala Ala His Pro His Ala Ser Pro Leu Asn Ala Glu Asn Leu Ala

225 230 235 240

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

245 250 255

Asp Glu Gly Ala Ala Tyr Ala Glu Ala Leu Lys Ala Ala Gly Val Ser

260 265 270

Ala Glu Tyr Arg Pro Gly Pro Gly Met Ile His Gly Tyr Ala Asn Met

275 280 285

Thr Ala Phe Ser Pro Val Ala Ala Gln Leu Ile Asp Glu Ala Gly Val

290 295 300

Trp Leu Gly Glu Gln Leu Arg Gly

305 310

<210> 6

<211> 312

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Met Pro Val Asp Pro Asn Leu Tyr Gln Leu Leu Leu Gln Leu Ser Gln

1 5 10 15

Ala Pro Glu Pro Ala Gly Leu Glu Glu Leu Arg Ala Gly Val Ile Ala

20 25 30

Asn Ala Ala Arg Ser Pro Lys Arg Pro Val Thr Ile Gly Glu Val Arg

35 40 45

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

50 55 60

His Pro Ala Gly Gln Ala Pro Ala Ser Gly Trp Pro Leu Thr Val Phe

65 70 75 80

Phe His Gly Gly Gly Phe Val Val Tyr Asp Leu Asp Thr His Asp Ala

85 90 95

Leu Cys Arg Glu Leu Cys Ala Thr Ser Gly Ala Ala Val Leu Ser Val

100 105 110

Ala Tyr Arg Leu Ala Pro Glu Ala Arg Phe Pro Ala Pro Val Asp Asp

115 120 125

Ala Leu Ala Ser Val Val Trp Ala Ala Ala His Ala Ala Glu Leu Gly

130 135 140

Ala Asp Ala Gly Arg Leu Ala Val Ala Gly Asp Ser Ala Gly Ala Asn

145 150 155 160

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

165 170 175

Leu Arg Ala Gln Leu Leu Ile Tyr Pro Ala Ala Asp Phe Glu His Pro

180 185 190

Glu Arg Tyr Pro Ser Arg Gln Glu Asn Gly Arg Gly Tyr Phe Leu Thr

195 200 205

Asp Glu Arg Met Arg Phe Phe Gly Gln Met Tyr Leu Ala Arg Pro Glu

210 215 220

Asp Ala Ala His Pro His Ala Ser Pro Leu Asn Ala Glu Ser Leu Ala

225 230 235 240

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

245 250 255

Asp Glu Gly Ala Ala Tyr Ala Glu Ala Leu Lys Ala Ala Gly Val Ser

260 265 270

Ala Glu Tyr Arg Pro Gly Pro Gly Met Ile His Gly Phe Ala Asn Met

275 280 285

Thr Ala Phe Ser Pro Val Ala Ala Gln Leu Ile Asp Glu Ala Gly Val

290 295 300

Trp Leu Gly Glu Gln Leu Arg Gly

305 310

<210> 7

<211> 939

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atgcccgtag accccaacct gtaccaactt ctgctgcaac tctcgcaggc gcctgaaccc 60

gccggactgg aagaactgcg ggcgggcgtg atcgccaacg cggcgcgcag ccccaaacgt 120

ccggtgacta ttggcgaagt ccgtgacctg agcgtggcgg gcgcggaggg ctccctgccc 180

gcccgcctgt accacccccgc cgggcaggcc cccgcgtccg gctggccgct gacggtgttc 240

ttccacggtg gcggcttcgt ggtctacgac ctcgacaccc acgacgcgct gtgccgcgag 300

ctgtgcgcga cgtcgggcgc ggcggtgctg agcgtggcct accgcctcgc gcccgaagcc 360

cgctttcccg cgccggtaga cgacgccctg gcggctgtgg tctgggccgc cgcgcacgcc 420

gccgaactcg gcgcagacgc ggggcgactc gcggtggcgg gcgacagcgc gggggccaac 480

ctcgccaccg tcacggcgct gcggtcgcgt gacgagggcg gcccggcttt gcgggcgcag 540

cttctcattt accccgccgc cgatttcgag caccccgaac gctaccccag ccgccaggaa 600

aacggacgcg gctatttcct cactgacgag cggatgcgct ttttcggaca gatgtacctt 660

gctcgcccgg aagacgccgc gcatccccac gcctcgccgc tcaacgctga gagtctcgcg 720

gggttgccgc cggccctggt cctgaccgcc gaattcgacc ccctgcgcga tgaaggcgcc 780

gcttacgccg aagctctcaa ggccgctggc gtaagcgccg agtaccgccc cggccccggc 840

atgattcacg gttacgccaa catgaccgcg ttttcgcccg tcgccgcaca actgattgac 900

gaggcgggcg tatggctcgg cgagcaactg cgcggctga 939

<210> 8

<211> 939

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atgcccgtag accccaacct gtaccaactt ctgctgcaac tctcgcaggc gcctgaaccc 60

gccggactgg aagaactgcg ggcgggcgtg atcgccaacg cggcgcgcag ccccaaacgt 120

ccggtgacta ttggcgaagt ccgtgacctg agcgtggcgg gcgcggaggg ctccctgccc 180

gcccgcctgt accacccccgc cgggcaggcc cccgcgtccg gctggccgct gacggtgttc 240

ttccacggtg gcggcttcgt ggtctacgac ctcgacaccc acgacgcgct gtgccgcgag 300

ctgtgcgcga cgtcgggcgc ggcggtgctg agcgtggcct accgcctcgc gcccgaagcc 360

cgctttcccg cgccggtaga cgacgccctg gcgagtgtgg tctgggccgc cgcgcacgcc 420

gccgaactcg gcgcagacgc ggggcgactc gcggtggcgg gcgacagcgc gggggccaac 480

ctcgccgccg tcacggcgct gcggtcgcgt gacgagggcg gcccggcttt gcgggcgcag 540

cttctcattt accccgccgc cgatttcgag caccccgaac gctaccccag ccgccaggaa 600

aacggacgcg gctatttcct cactgacgag cggatgcgct ttttcggaca gatgtacctt 660

gctcgcccgg aagacgccgc gcatccccac gcctcgccgc tcaacgctga gagtctcgcg 720

gggttgccgc cggccctggt cctgaccgcc gaattcgacc ccctgcgcga tgaaggcgcc 780

gcttacgccg aagctctcaa ggccgctggc gtaagcgccg agtaccgccc cggccccggc 840

atgattcacg gttacgccaa catgaccgcg ttttcgcccg tcgccgcaca actgattgac 900

gaggcgggcg tatggctcgg cgagcaactg cgcggctga 939

<210> 9

<211> 939

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

atgcccgtag accccaacct gtaccaactt ctgctgcaac tctcgcaggc gcctgaaccc 60

gccggactgg aagaactgcg ggcgggcgtg atcgccaacg cggcgcgcag ccccaaacgt 120

ccggtgacta ttggcgaagt ccgtgacctg agcgtggcgg gcgcggaggg ctccctgccc 180

gcccgcctgt accacccccgc cgggcaggcc cccgcgtccg gctggccgct gacggtgttc 240

ttccacggtg gcggcttcgt ggtctacgac ctcgacaccc acgacgcgct gtgccgcgag 300

ctgtgcgcga cgtcgggcgc ggcggtgctg agcgtggcct accgcctcgc gcccgaagcc 360

cgctttcccg cgccggtaga cgacgccctg gcgagtgtgg tctgggccgc cgcgcacgcc 420

gccgaactcg gcgcagacgc ggggcgactc gcggtggcgg gcgacagcgc gggggccaac 480

ctcgccaccg tcacggcgct gcggtcgcgt gacgagggcg gcccggcttt gcgggcgcag 540

cttctcattt accccgccgc cgatttcgag caccccgaac gctaccccag ccgccaggaa 600

aacggacgcg gctatttcct cactgacgag cggatgcgct ttttcggaca gatgtacctt 660

gctcgcccgg aagacgccgc gcatccccac gcctcgccgc tcaacgctga gaatctcgcg 720

gggttgccgc cggccctggt cctgaccgcc gaattcgacc ccctgcgcga tgaaggcgcc 780

gcttacgccg aagctctcaa ggccgctggc gtaagcgccg agtaccgccc cggccccggc 840

atgattcacg gttacgccaa catgaccgcg ttttcgcccg tcgccgcaca actgattgac 900

gaggcgggcg tatggctcgg cgagcaactg cgcggctga 939

<210> 10

<211> 939

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

atgcccgtag accccaacct gtaccaactt ctgctgcaac tctcgcaggc gcctgaaccc 60

gccggactgg aagaactgcg ggcgggcgtg atcgccaacg cggcgcgcag ccccaaacgt 120

ccggtgacta ttggcgaagt ccgtgacctg agcgtggcgg gcgcggaggg ctccctgccc 180

gcccgcctgt accacccccgc cgggcaggcc cccgcgtccg gctggccgct gacggtgttc 240

ttccacggtg gcggcttcgt ggtctacgac ctcgacaccc acgacgcgct gtgccgcgag 300

ctgtgcgcga cgtcgggcgc ggcggtgctg agcgtggcct accgcctcgc gcccgaagcc 360

cgctttcccg cgccggtaga cgacgccctg gcgagtgtgg tctgggccgc cgcgcacgcc 420

gccgaactcg gcgcagacgc ggggcgactc gcggtggcgg gcgacagcgc gggggccaac 480

ctcgccaccg tcacggcgct gcggtcgcgt gacgagggcg gcccggcttt gcgggcgcag 540

cttctcattt accccgccgc cgatttcgag caccccgaac gctaccccag ccgccaggaa 600

aacggacgcg gctatttcct cactgacgag cggatgcgct ttttcggaca gatgtacctt 660

gctcgcccgg aagacgccgc gcatccccac gcctcgccgc tcaacgctga gagtctcgcg 720

gggttgccgc cggccctggt cctgaccgcc gaattcgacc ccctgcgcga tgaaggcgcc 780

gcttacgccg aagctctcaa ggccgctggc gtaagcgccg agtaccgccc cggccccggc 840

atgattcacg gtttcgccaa catgaccgcg ttttcgcccg tcgccgcaca actgattgac 900

gaggcgggcg tatggctcgg cgagcaactg cgcggctga 939

<210> 11

<211> 6289

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60

cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120

ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180

gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240

acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300

ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360

ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgatta 420

acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480

tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540

tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600

tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660

actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720

gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780

aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840

agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900

cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960

aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020

tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080

tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140

taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200

ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260

tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320

tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380

cccttgtatt actgtttatg taagcagaca gttttatgt tcatgaccaa aatcccttaa 1440

cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500

gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560

gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620

agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680

aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740

agtggcgata agtcgtgtct taccgggttg gactcaagac gtagttacc ggataaggcg 1800

cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860

accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920

aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980

ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040

cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100

gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160

tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220

agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280

tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340

caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400

ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460

gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520

gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580

gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640

aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700

ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760

acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820

ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880

tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940

tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000

cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060

gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120

ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180

catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240

ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300

gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360

gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420

ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480

atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540

cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600

tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660

ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720

aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780

atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840

cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900

gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960

tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020

agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080

gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140

ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acaagcaatgg 4200

catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260

tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320

tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380

gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440

ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500

tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560

catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620

cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680

tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740

ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800

ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860

cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920

gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980

aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040

ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac 5100

agcagcggcc tggtgccgcg cggcagccat atggctagca tgactggtgg acagcaaatg 5160

ggtcgcggat ccatgcccgt agaccccaac ctgtaccaac ttctgctgca actctcgcag 5220

gcgcctgaac ccgccggact ggaagaactg cgggcgggcg tgatcgccaa cgcggcgcgc 5280

agccccaaac gtccggtgac tattggcgaa gtccgtgacc tgagcgtggc gggcgcggag 5340

ggctccctgc ccgcccgcct gtaccacccc gccgggcagg cccccgcgtc cggctggccg 5400

ctgacggtgt tcttccacgg tggcggcttc gtggtctacg acctcgacac ccacgacgcg 5460

ctgtgccgcg agctgtgcgc gacgtcgggc gcggcggtgc tgagcgtggc ctaccgcctc 5520

gcgcccgaag cccgctttcc cgcgccggta gacgacgccc tggcggctgt ggtctgggcc 5580

gccgcgcacg ccgccgaact cggcgcagac gcggggcgac tcgcggtggc gggcgacagc 5640

gcgggggcca acctcgccac cgtcacggcg ctgcggtcgc gtgacgaggg cggcccggct 5700

ttgcgggcgc agcttctcat ttaccccgcc gccgatttcg agcaccccga acgctacccc 5760

agccgccagg aaaacggacg cggctatttc ctcactgacg agcggatgcg ctttttcgga 5820

cagatgtacc ttgctcgccc ggaagacgcc gcgcatcccc acgcctcgcc gctcaacgct 5880

gagagtctcg cggggttgcc gccggccctg gtcctgaccg ccgaattcga ccccctgcgc 5940

gatgaaggcg ccgcttacgc cgaagctctc aaggccgctg gcgtaagcgc cgagtaccgc 6000

cccggccccg gcatgattca cggttacgcc aacatgaccg cgttttcgcc cgtcgccgca 6060

caactgattg acgaggcggg cgtatggctc ggcgagcaac tgcgcggctg aaagcttgcg 6120

gccgcactcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag 6180

gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct 6240

aaacgggtct tgaggggttttttgctgaaa ggaggaacta tatccggat 6289

<210> 12

<211> 40

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ccatggctga tatcggatcc atgcccgtag accccaacct 40

<210> 13

<211> 40

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

ctcgagtgcg gccgcaagct ttcagccgcg cagttgctcg 40

<210> 14

<211> 38

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gcgccggtag acgacgccct ggcgnnkgtg gtctgggc 38

<210> 15

<211> 38

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

ggcggcgtgc gcggcggccc agaccacmnn cgccaggg 38

<210> 16

<211> 36

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

gacagcgcgg gggccaacct cgccnnkgtc acggcg 36

<210> 17

<211> 36

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

cgtcacgcga ccgcagcgcc gtgacmnngg cgaggt 36

<210> 18

<211> 37

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

acgcctcgcc gctcaacgct gagnnkctcg cggggtt 37

<210> 19

<211> 36

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

accagggccg gcggcaaccc cgcgagmnnc tcagcg 36

<210> 20

<211> 35

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

gcatgattca cggtnnkgcc aacatgaccg cgttt 35

<210> 21

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

ggcgaaaccg tmnntcatgc cggggccggg gcggta 36

Claims (7)

1. A method for improving esterase activity, the method comprising carrying out mutation transformation to one of the amino acid residues to be mutated in the wild-type esterase, characterized in that, the amino acid sequence of the wild-type esterase is as SEQ ID NO. 2, the amino acid residue to be mutated includes the 163th threonine residue; the mutation is transformed into an alanine residue at the 163rd threonine residue. 2. A protein with esterase activity, said protein is a protein derived from the amino acid sequence of wild-type esterase shown in SEQ ID NO.2 through substitution of 1 amino acid; and, the amino acid sequence of said protein is as shown in SEQ ID NO.2 Shown in ID NO.4. 3. The gene encoding the protein according to claim 2, wherein the gene is a DNA molecule whose nucleotide sequence is shown in SEQ ID NO.8. 4. A recombinant vector, wherein the gene of claim 3 is inserted into the recombinant vector. 5. A transformant, wherein, the host of the transformant is a genetically engineered bacterium; the gene introduced in the transformant comprises the gene of claim 3, or, the recombinant vector introduced in the transformant comprises the gene of claim 3 The recombinant vector described in claim 4. 6. A method for preparing esterase, wherein the method comprises: inoculating the transformant according to claim 5 in a culture medium to obtain cultured material. 7. An application of an esterase in degrading p-nitrophenyl esters, wherein the esterase contains the protein according to claim 2.