CN113637653A

CN113637653A - Esterase mutant Est8-XL with improved activity and application thereof

Info

Publication number: CN113637653A
Application number: CN202110898705.XA
Authority: CN
Inventors: 丁俊美; 王晓亮; 主虎杰; 黄遵锡; 刘艳; 周峻沛; 韩楠玉
Original assignee: Yunnan Normal University
Current assignee: Yunnan Normal University
Priority date: 2021-08-05
Filing date: 2021-08-05
Publication date: 2021-11-12
Anticipated expiration: 2041-08-05
Also published as: CN113637653B

Abstract

The invention discloses an esterase mutant Est8-XL with improved activity and application thereof, wherein the amino acid sequence of the esterase mutant Est8-XL is shown as SEQ ID NO. 1. The mutant esterase Est8-XL with improved activity is obtained by three rounds of screening by error-prone PCR and a high-throughput screening method based on color change of a Bromothymol blue (BTB) indicator. Compared with wild esterase Est8, the deacetylation activity of the mutant esterase Est8-XL on 7-aminocephalosporanic acid is improved, and the deacetylation activity is between pH9.5 and pH510.0 Activity in buffer and in 10mM NaCl, KCl, LiCl, CuSO₄、NiSO₄And ZnSO₄The activity in the solution is improved. The mutant esterase Est8-XL can be used in the technical field of biological medicines.

Description

Esterase mutant Est8-XL with improved activity and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering and enzyme engineering, and particularly relates to an esterase mutant Est8-XL with improved activity and application thereof.

Background

China is a large country for using antibiotics and also a large country for producing antibiotics. Cephalosporin antibiotics are the largest family of domestic and foreign antibiotic drugs, and have important effects in the medical field due to the advantages of high efficiency, low toxicity, broad spectrum and beta-lactamase resistance. However, due to the excessive use of antibiotics, serious problems of spreading of drug-resistant bacteria are faced, and therefore the development of new antibiotics is in great tendency.

Deacetyl-7-aminocephalosporanic acid (D-7-ACA), an intermediate for synthesizing the novel antibiotic, can remove acetyl at the 3-position under the action of deacetylase to generate. D-7-ACA (Deacetyl-7-amino-cephalosporic a cid, D-7-ACA) is used as an intermediate for synthesizing the antibiotic, so that not only can a novel antibiotic be synthesized, but also the economic benefit of a production enterprise can be improved compared with 7-ACA. The traditional method for synthesizing the D-7-ACA is a chemical cracking method, but has the problems of high cost, complicated process flow, environmental pollution and the like. The novel enzyme method has the advantages of substrate specificity, simple and convenient operation, environmental friendliness and the like, and has good development prospect for producing the D-7-ACA.

Esterases are widely present in animals, plants and microorganisms and are a class of enzymes that catalyze ester hydrolysis, ester formation and transesterification. The GDSL family esterase of microbial origin does not have a G-x-S-x-G motif close to the middle part of a protein sequence but has a GDSL motif, has more flexible catalytic sites and can change the conformation of the esterase along with the existence and combination of different substrates. Much of the research on GDSL family esterases is now focused on plants and animals, and less on microbial sources, and the mechanism of catalytic action is less complete.

The applicant previously obtained esterase Est8 (belonging to GDSL family) derived from microorganisms (Bacillus sp.K91), and the generated D-7-ACA can provide a novel intermediate for the synthesis of cephalosporin antibiotics, but the enzyme catalyzes the 7-ACA to generate the D-7-ACA with lower activity.

Disclosure of Invention

Aiming at the problem that a novel cephalosporin antibiotic intermediate is urgently needed, the invention aims to provide an esterase mutant Est8-XL with improved GDSL family deacetylation activity and application thereof.

In order to achieve the technical purpose, the invention specifically adopts the following technical means:

an esterase mutant Est8-XL with improved activity, wherein the esterase mutant Est8-XL is obtained by directional mutation of Bacillus thermophilus K91(Bacillus sp.K91) esterase Est8, and the amino acid sequence of the esterase mutant Est8-XL is shown as SEQ ID No. 1.

Compared with the wild esterase Est8, the deacetylation activity of the esterase mutant Est8-XL on the functional substrate 7-aminocephalosporanic acid is improved from 2.71U/mg to 4.47U/mg.

Wherein the amino acid sequence and the nucleotide sequence of the wild esterase Est8 are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4.

In another aspect of the invention, the coding gene of the esterase mutant Est8-XL is provided, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2.

In another aspect of the invention, the esterase mutant Est8-XL was prepared by the following scheme:

1) connecting the gene shown as SEQ ID NO.2 with an expression vector pEASY-E2, and transforming Escherichia coli BL21(DE3) with the recombinant plasmid to obtain a recombinant bacterium;

2) fermenting and culturing the recombinant strain, and inducing the expression of a recombinant esterase mutant Est 8-XL;

3) recovering and purifying the expressed esterase mutant Est 8-XL;

4) determination of deacetylation Activity.

In another aspect of the invention, a recombinant plasmid and a recombinant bacterium are provided, wherein the recombinant plasmid and the recombinant bacterium contain a coding gene of the mutant Est 8-XL.

In another aspect of the invention, the esterase mutant Est8-XL, the coding gene, the recombinant plasmid and the recombinant bacterium are applied to preparation of cephalosporin antibiotics.

In particular to application in synthesizing a novel cephalosporin antibiotic intermediate deacetylated 7-aminocephalosporanic acid.

The beneficial technical effects of the invention are as follows:

compared with the wild esterase Est8, the obtained mutant esterase Est8-XL has changed enzyme activity and resistance to certain metal ions. The amino acid sequence and the nucleotide sequence of the wild esterase Est8 are shown as SEQ ID NO.1 and SEQ ID NO. 2. At pH9.5, the wild-type esterase Est8 and the mutant esterase Est8-XL had 74.8% and 86.4% enzyme activity, respectively, and at pH 10.0, the wild-type esterase Est8 and the mutant esterase Est8-XL had 13.9% and 23.6% enzyme activity, respectively. At 10mM NaCl, KCl, LiCl, CuSO₄、NiSO₄And ZnSO₄The enzyme activities of the medium mutant esterase Est8-XL are respectively 17.5%, 19.6%, 18.2%, 7.2%, 13.4% and 10.7% higher than that of the wild esterase Est 8. The mutant esterase Est8-XL had 1.6 times higher deacetylation activity on 7-ACA than the wild-type esterase Est 8.

Drawings

FIG. 1 is a screen of highly deacetylated active mutants of the invention dependent on color change of BTB indicator;

fig. 2 is a SDS-PAGE analysis of the recombinant esterase Est8 and mutant Est8-XL of the invention expressed in e.coli BL21(DE3), wherein M: a protein Marker; 1: post-expression supernatant of pEASY-E2/Est8 in E.coli BL21(DE3) cells; 2 and 3: purified recombinant esterase Est8 and mutant esterase Est 8-XL;

FIG. 3 shows the pH activity of the recombinant esterase Est8 and its mutant Est8-XL of the present invention;

FIG. 4 shows the pH stability of the recombinant esterase Est8 and its mutant Est8-XL of the present invention;

FIG. 5 shows the thermal activity of the recombinant esterase Est8 and its mutant Est8-XL of the present invention;

FIG. 6 shows the thermostability of the recombinant esterase Est8 and its mutant Est8-XL of the present invention.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The experimental materials and reagents in the experimental examples of the invention are as follows:

LB liquid medium: ddH for 4g NaCl, 4g Tryptone and 2g Yeast extract₂And O is metered to 400 mL.

Amp-LB solid Medium: 2g of Tryptone, 1.5g of Yeast extract, 2g of NaCl and 2% (w/v) of agar in ddH₂O to 200mL, sterilized, added ampicillin at a final concentration of 1mM (0.22 μm filter sterilized) at about 55 deg.C, and mixed well for use.

Vectors and strains: escherichia coli BL21(DE3) expression strain (available from Beijing Quanjin Biotechnology Co., Ltd.), Bacillus thermophilus K91(Bacillus sp.K91) (available from Yunnan university), pEASY-E2 expression vector (available from Beijing Quanjin Biotechnology Co., Ltd.).

Example 1 construction of a library of mutations

And (3) amplifying an esterase gene est8 in a Bacillus thermophilus K91(Bacillus sp.K91) strain by utilizing error-prone PCR (polymerase chain reaction) to obtain a mutant sequence. Error-prone PCR was performed according to the GeneMorph II EZCone Domain Mutagenesis Kit random Mutagenesis Kit (available from Agilent technologies, Inc.) using the instruction-based protocol.

1) Mutant big primer PCR of Est8

Using a recombinant plasmid containing a gene est8 extracted from an E.coli BL21(DE3) expression recombinant strain as a PCR template, designing primers:

upstream primer est 8F: 5'-GCAAATCATATTTATCTTGC-3', respectively;

downstream primer est 8R: 5'-CCTTTCTTTGATGATCGATTC-3' are provided.

The PCR reaction system is as follows:

PCR reaction parameters: pre-denaturation at 95 ℃ for 5 min; then denaturation at 95 ℃ for 30sec, annealing at 42 ℃ for 30sec, extension at 72 ℃ for 1min, and heat preservation at 72 ℃ for 10min after 30 cycles.

2) EZCone of Est8 (mutant amplified with the product of a mutant Large primer PCR as primer)

The PCR reaction system is as follows:

PCR reaction parameters: pre-denaturation at 95 ℃ for 1 min; then denaturation at 95 ℃ for 50sec, annealing at 60 ℃ for 50sec, extension at 68 ℃ for 12min, and heat preservation at 37 ℃ for 10min after 30 cycles.

The PCR product was treated with Dpn I restriction enzyme and the Est8 plasmid template was digested.

3) Transformation of mutant plasmids: adding 5 mu L of plasmid with random mutation of pEASY-E2/est8 into 100 mu L of E.coli BL21(DE3) competent cells just thawed, and gently shaking and uniformly mixing; performing ice bath for 30min, and performing heat shock for 45sec in a water bath kettle at 42 ℃; ice-cooling for 7min, and adding 890 μ L of nonreactive LB liquid medium on a clean bench for culture (37 deg.C, 180rpm, 1 h); centrifuging (7000rpm, 4min) to collect 100 μ L LB suspension thallus, and sucking and spreading on LB solid culture medium containing 1 ‰ Amp; the cells were cultured in an inverted state (37 ℃ C., 16 hours).

4) Screening and identifying: randomly selecting 5 single colonies from the inverted LB solid medium to culture in 500. mu.L LB liquid medium containing Amp antibiotics (37 ℃, 180rmp, 3-4 h); taking the bacterial liquid as a template, performing PCR identification by using a universal primer T7, verifying a PCR positive screening son, taking 200 mu L of the identified positive bacterial liquid, sending the positive bacterial liquid to a company Limited in Biotechnology engineering (Shanghai) for sequencing, and further verifying.

Primer T7 was as follows:

upstream primer T7F: 5'-TAATACGACTCACTATAGGG-3', respectively;

downstream primer T7R: 5'-TGCTAGTTATTGCTCAGCGG-3' are provided.

The PCR reaction system is as follows:

PCR reaction parameters: pre-denaturation at 94 ℃ for 5 min; then denaturation at 94 ℃ for 30sec, annealing at 55 ℃ for 30sec, extension at 72 ℃ for 1.5min, and heat preservation at 72 ℃ for 10min after 30 cycles.

5) Construction of a mutation library: randomly selecting a single colony of the esterase Est8 mutant, inoculating the single colony to a 96-well cell culture plate containing 150 mu L of liquid Amp-LB, and culturing for 16h at 37 ℃ and 200 rpm; add 50. mu.L of 40% glycerol to each well, mix well and store at-70 ℃. Wells a1 of each cell culture plate were blank against e.coli BL21(DE3) containing only empty vector of pEASY-E2, and wells a2 were wild-type against.

6) Induced expression of mutant libraries: transferring the bacterial liquid to a 96 deep-hole plate containing 200 mu L of liquid Amp-LB, and carrying out shake culture at 37 ℃ and 190rpm for 10 h; adding 200 μ L liquid Amp-LB (containing 1.4mM isopropyl thiogalactoside), inducing expression (20 ℃, 160 rpm); after inducing for 10h, centrifuging (3000rpm, 12min), washing the thalli with deionized water twice, centrifuging and then suspending again; add 20. mu.L/well PopCulture Reagent cell lysate (Merck) and 5. mu.L lysozyme to 96-well plate, mix well and shake the cell (25 ℃, 10 min); centrifuging (4 deg.C, 3000rpm, 10min), and storing in 4 deg.C refrigerator.

Example 2 screening of mutants having improved deacetylation Activity

1) Primary screening: 480 mutants were screened.

100 μ L of substrate/well (substrate 50mM 7-ACA, 0.02% BTB and 50mM phosphate buffer pH 7.3) was added to a 96-well microplate; adding 50 mu L of cell lysis product/hole into a 96 enzyme-labeled plate, uniformly mixing, reacting at 25 ℃ for 10min, and detecting the OD value under a 616nm absorbance enzyme-labeled instrument. The mutants were screened by colorimetric methods: since esterase catalyzes the production of D-7-ACA and acetic acid from 7-ACA, the pH of the solution decreases, and the solution changes from blue to green to yellow in the presence of 0.02% BTB indicator, with a more yellow solution indicating a higher enzyme activity.

2) Twice re-screening: screening 40 mutants screened in the initial screening

The screened 40 mutants with high deacetylation activity of 7-ACA were selected on a 96-well plate, each mutant was designed with two replicates, and wild type was set as control. Finally obtaining 1 mutant with 7-ACA deacetylation activity higher than that of wild type, and obtaining the sequence of the mutated site through sequencing. Designing a color development plate for color development reaction and further verifying: the control group and each experimental group were set up in 3 replicates and blank was supplemented with 50. mu.L ddH₂O, 1.1U of the corresponding protein (wherein the experimental groups Est8-G45A and Est8-K196N are mutants rationally designed for mutant Est8-XL) was added to each well of the experimental group and left to react at 25 ℃ for 7 min.

The results show that: the mutant esterase Est8-XL had a higher deacetylation activity towards 7-ACA than the wild esterase Est8 (FIG. 1).

Example 3 preparation of the wild-type enzyme Est8 and of the mutant Est8-XL

Adding the recombinant strain containing the wild enzyme Est8 and the mutant Est8-XL into a test tube containing 5mL of Amp-LB liquid culture medium by 1 per thousand of inoculation amount respectively for culture (37 ℃, 180 rpm); adding 4mL of the mixed solution into 400mL of Amp-LB liquid culture medium after 15h, and culturing for 2.5 h; adding Isopropyl Thiogalactoside (Isopropypyl beta-D-Thiogalactoside, IPTG) with a final concentration of 1.4mM, and inducing at 20 deg.C and 150rpm for 21 h; collecting thallus with refrigerated centrifuge (4 deg.C, 5000rpm, 10 min); resuspending the thalli by using Tris-HCl 8.0 buffer solution, and then crushing cells by using an ultrasonic cell crusher; centrifuging at 12000rpm and 4 deg.C for 20min to collect supernatant protein solution; 2mL of the collected supernatant protein solution was applied to a Nickel-NTAAgarose purification column and the objective protein was purified by elution with a gradient eluent containing 0 to 500mM imidazole.

SDS-PAGE showed that both the mutant enzyme Est8-XL and the wild-type enzyme Est8 were purified, and the products were single bands (

lanes

2 and 3 are the bands of the purified products of the wild-type enzyme Est8 and the mutant enzyme Est8-XL, respectively) (FIG. 2).

Example 4 determination of the deacetylation Activity of the wild enzyme Est8 and its mutant Est8-XL

The procedures were performed according to the BCA protein quantification kit instructions and the acetic acid assay kit instructions.

1) Carrying out ultrafiltration concentration on the purified wild esterase Est8 and mutant esterase Est8-XL by using 50mM phosphate buffer solution with the pH value of 7.0; the concentrated protein was quantified using BCA protein quantification kit (Shanghai Yazyme Biotech Co., Ltd.); the deacetylation activity of 7-ACA was determined by the wild-type esterase Est8 and the mutant esterase Est8-XL using an acetic acid assay kit (Megazyme).

2) Sucking 300 mu L of 7-ACA substrate solution with the concentration of 10mM and 100 mu L of diluted enzyme solution, and uniformly mixing in a water bath kettle at 25 ℃ for enzymatic reaction; after 10min, sucking 200. mu.L of the reaction solution, mixing with the corresponding reagent in the acetic acid assay kit, reading at 340nm with an ultraviolet spectrophotometer, and taking the reaction solution without enzyme solution as a control. The amount of enzyme required to produce 1. mu.M of substrate per minute was defined as one enzyme activity unit (1U/mg). The acetic acid content in the system is determined by using the kit, namely the product yield, and the deacetylation activities of the wild esterase Est8 and the mutant Est8-XL on the 7-ACA are respectively 2.71U/mg and 4.47U/mg according to the known reaction time and enzyme amount through an enzyme activity calculation formula.

TABLE 1 deacetylation activity of recombinant esterase Est8 and its mutant Est8-XL on functional substrate 7-ACA

EXAMPLE 5 determination of the Properties of the purified enzymes of the mutant esterase Est8-XL and of the wild-type esterase Est8

1) Determination of pH activity and stability of wild esterase Est8 and mutant Est8-XL

Determination of optimum pH: mixing the diluted enzyme solution with pre-heated 5min buffer solution with pH of 6.5-10.0 and ethanol under 37 deg.C water bath condition4-Nitrophenyl acid (p-NPC)₂) The reaction was carried out for 2min in the mixture. And (3) measuring the pH stability: the enzyme solution was diluted with a buffer solution of pH 2.4-12.0 and treated at 37 ℃ for 1h, and then the wild-type recombinant esterase Est8 and its mutant Est8-XL were subjected to enzymatic reactions at pH 8.5 and 40 ℃ and 35 ℃ respectively, with untreated enzyme solution as a control. The buffer solution is as follows: 50mM of Citrate-phosphate buffer (pH 2.4-6.0), 50mM of Tris-HCl (pH 7.0-9.5) and 50mM of Boric acid-NaOH (pH 10.0-12.0).

The results show that: both Est8 and Est8-XL had an optimum reaction pH of 8.5 (FIG. 3), and at pH 7.5, the relative enzymatic activity of Est8 was 16.2% higher than that of Est8-XL (FIG. 3); at pH9.5, the relative enzyme activity of Est8-XL was 11.6% higher than that of Est8, and at pH 10.0, the relative enzyme activity of Est8-XL was 9.7% higher than that of Est8 (FIG. 3). After the wild recombinant esterase Est8 and the mutant esterase Est8-XL are tolerant in a buffer solution with the pH value of 5.0-10.0 for 1 hour, the enzyme activities of the wild recombinant esterase Est8 and the mutant esterase Est8-XL are both kept above 50 percent; after the enzyme activity of Est8 and Est8-XL is stable after the enzyme activity is endured for 1 hour in a buffer solution with the pH value of 7.0-9.0, the enzyme activity of Est8 and Est8-XL is maintained to be more than 80 percent, and the enzyme activity of Est8 and the enzyme activity of Est8-XL are below 40 percent after the enzyme activity is processed for 1 hour in the buffer solution with the pH value of 2.4-4.0 and the pH value of 11.0-12.0 (figure 4). The above results illustrate that: both have better pH stability.

2) Determination of thermal activity and thermal stability of purified enzyme of wild esterase Est8 and mutant Est8-XL

And (3) measuring thermal activity: the enzymatic reaction is carried out in a buffer at pH 8.5 at 0-70 ℃. And (3) measuring the thermal stability: the diluted wild esterase Est8 and mutant Est8-XL enzyme solutions were respectively treated at 25 ℃, 50 ℃ and 55 ℃ for 1h (enzyme activity was measured every 10min), and enzymatic reactions were carried out at pH 8.5, 40 ℃ and 35 ℃ with the untreated enzyme solution as a control.

The results show that: the mutant esterase Est8-XL had a lower thermal activity between 40 ℃ and 70 ℃ than the wild esterase Est8 (FIG. 5), and both the wild esterase Est8 and the mutant Est8-XL were stable at 25 ℃ but had half-lives of less than 10min at 55 ℃ for both the mutant Est8-XL and the wild esterase Est8 (FIG. 6).

3) Influence of different metal ions and chemical reagents on activity of purified enzyme of wild enzyme Est8 and mutant Est8-XL

At 35 ℃ and 40 ℃ and pH 8.5 with p-NPC₂The enzyme activity was determined for the substrate. The results show (Table 2) that the sample was tested in the presence of 10mM NaCl, KCl, LiCl, and CuSO₄、NiSO₄And ZnSO₄The enzyme activity of the mutant esterase Est8-XL in the buffer solution is 17.5 percent, 19.6 percent, 18.2 percent, 7.2 percent, 13.4 percent and 10.7 percent higher than that of the wild esterase Est8 respectively. However, 10mM FeCl₃The enzyme activity of mutant esterase Est8-XL can be completely inhibited; FeCl₂But also greatly inhibits the activity of Est 8-XL. Wherein, chemical reagents SDS, DTT and CTAB have great inhibition effect on the enzyme activity of wild esterase Est8 and mutant esterase Est 8-XL.

TABLE 2 Effect of Metal ions and chemical reagents on Activity of mutant Est8-XL and wild enzyme Est8

Example 6 sequence analysis of mutants with improved deacetylation Activity

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Sequence listing

<110> university of Yunnan Master

<120> esterase mutant Est8-XL with improved activity and application thereof

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 217

<212> PRT

<213> esterase mutant (Est8-XL)

<400> 1

Met Ala Asn His Ile Tyr Leu Ala Gly Asp Ser Thr Val Gln Thr Tyr

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Gly Asp Ser Thr Asn Gln Gly Gly Trp Gly Gln Phe Leu Gly Ser His

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Leu Pro Glu His Ile Gln Val Ile Asn Arg Ala Ile Ala Gly Arg Ser

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Ser Lys Thr Phe Val Glu Glu Gly Arg Leu Gln Ala Ile Leu Asp Val

50 55 60

Ile Glu Pro Asp Asp Trp Leu Phe Val Gln Met Gly His Asn Asp Ala

65 70 75 80

Ser Lys Asn Lys Pro Glu Arg Tyr Thr Glu Pro Tyr Thr Thr Tyr Lys

85 90 95

Gln Tyr Leu Lys Gln Tyr Ile Ala Gly Ala Arg Glu Lys Gly Ala His

100 105 110

Pro Leu Leu Ile Thr Pro Val Ala Arg Phe His Tyr Glu Asn Gly Val

115 120 125

Phe Leu Asn Asp Phe Pro Asp Tyr Cys Ile Ala Met Lys Gln Thr Ala

130 135 140

Glu Glu Glu Asn Val Gln Leu Ile Asp Leu Met Glu Lys Ser Leu Ala

145 150 155 160

Phe Phe Thr Glu Lys Gly Glu Glu Lys Val Tyr Thr Tyr Phe Met Ile

165 170 175

Ser Glu Gly Ile Asn Asp Tyr Thr His Phe Thr Lys Lys Gly Ala Asn

180 185 190

Glu Met Ala Asn Leu Val Ala Lys Gly Ile Lys Glu Leu Gly Leu Pro

195 200 205

Leu Thr Glu Ser Ile Ile Lys Glu Arg

210 215

<210> 2

<211> 654

<212> DNA

<213> esterase mutant Gene (Est8-XL)

<400> 2

atggcaaatc atatttatct tgccggcgat tcgactgttc aaacgtatgg agacagcaca 60

aatcaagggg gctgggggca gtttctcggc tcgcatctgc cggagcatat tcaagtgatc 120

aacagagcga tcgcgggaag aagctcgaaa acatttgtgg aagagggcag gcttcaggca 180

atcctcgatg tgattgagcc ggatgattgg ctgttcgtgc agatgggcca taatgacgcg 240

tcaaaaaata agccggagcg ctacaccgag ccctatacta cttataaaca atatttaaag 300

cagtatatcg caggcgcgcg ggaaaaaggc gcccatccgc ttctcattac ccccgtagcc 360

cgctttcatt acgaaaacgg cgtgtttttg aacgattttc ctgattactg cattgccatg 420

aagcagacgg ctgaagagga gaatgtccag ctcattgatc tgatggagaa aagtctcgct 480

ttctttactg agaagggcga ggaaaaagtg tacacctatt ttatgatttc agaagggatt 540

aatgattaca cgcattttac aaaaaaaggc gcaaatgaaa tggcgaatct tgtggcaaaa 600

ggcataaagg agctcggcct gccattgaca gaatcgatca tcaaagaaag gtga 654

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Gly Asp Ser Thr Asn Gln Gly Gly Trp Gly Gln Phe Leu Gly Ser His

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Leu Pro Glu His Ile Gln Val Ile Asn Arg Ala Ile Gly Gly Arg Ser

35 40 45

Ser Lys Thr Phe Val Glu Glu Gly Arg Leu Gln Ala Ile Leu Asp Val

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Ile Glu Pro Asp Asp Trp Leu Phe Val Gln Met Gly His Asn Asp Ala

65 70 75 80

Ser Lys Asn Lys Pro Glu Arg Tyr Thr Glu Pro Tyr Thr Thr Tyr Lys

85 90 95

Gln Tyr Leu Lys Gln Tyr Ile Ala Gly Ala Arg Glu Lys Gly Ala His

100 105 110

Pro Leu Leu Ile Thr Pro Val Ala Arg Phe His Tyr Glu Asn Gly Val

115 120 125

Phe Leu Asn Asp Phe Pro Asp Tyr Cys Ile Ala Met Lys Gln Thr Ala

130 135 140

Glu Glu Glu Asn Val Gln Leu Ile Asp Leu Met Glu Lys Ser Leu Ala

145 150 155 160

Phe Phe Thr Glu Lys Gly Glu Glu Lys Val Tyr Thr Tyr Phe Met Ile

165 170 175

Ser Glu Gly Ile Asn Asp Tyr Thr His Phe Thr Lys Lys Gly Ala Asn

180 185 190

Glu Met Ala Lys Leu Val Ala Lys Gly Ile Lys Glu Leu Gly Leu Pro

195 200 205

Leu Thr Glu Ser Ile Ile Lys Glu Arg

210 215

<210> 4

<211> 654

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<213> wild esterase gene (Est8)

<400> 4

atggcaaatc atatttatct tgccggcgat tcgactgttc aaacgtatgg agacagcaca 60

aatcaagggg gctgggggca gtttctcggc tcgcatctgc cggagcatat tcaagtgatc 120

aacagagcga tcgggggaag aagctcgaaa acatttgtgg aagagggcag gcttcaggca 180

atcctcgatg tgattgagcc ggatgattgg ctgttcgtgc agatgggcca taatgacgcg 240

tcaaaaaata agccggagcg ctacaccgag ccctatacta cttataaaca atatttaaag 300

cagtatatcg caggcgcgcg ggaaaaaggc gcccatccgc ttctcattac ccccgtagcc 360

cgctttcatt acgaaaacgg cgtgtttttg aacgattttc ctgattactg cattgccatg 420

aagcagacgg ctgaagagga gaatgtccag ctcattgatc tgatggagaa aagtctcgct 480

ttctttactg agaagggcga ggaaaaagtg tacacctatt ttatgatttc agaagggatt 540

aatgattaca cgcattttac aaaaaaaggc gcaaatgaaa tggcgaaact tgtggcaaaa 600

ggcataaagg agctcggcct gccattgaca gaatcgatca tcaaagaaag gtga 654

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gcaaatcata tttatcttgc 20

<210> 6

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cctttctttg atgatcgatt c 21

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

taatacgact cactataggg 20

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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tgctagttat tgctcagcgg 20

Claims

1. An esterase mutant Est8-XL with improved activity, which is characterized in that the amino acid sequence of the esterase mutant Est8-XL is shown as SEQ ID NO. 1.

2. The gene encoding esterase mutant Est8-XL of claim 1, characterized in that the nucleotide sequence of the encoding gene is shown in SEQ ID No. 2.

3. The method for constructing esterase mutant Est8-XL according to claim 1, comprising the following steps:

3) and recovering and purifying the expressed esterase mutant Est 8-XL.

4. A recombinant plasmid, which is characterized by comprising a gene shown as SEQ ID NO. 2.

5. A recombinant bacterium, which is characterized by comprising a gene shown as SEQ ID NO. 2.

6. Use of the esterase mutant Est8-XL according to claim 1, the coding gene according to claim 2, the recombinant plasmid according to claim 4 or the recombinant bacterium according to claim 5 for preparing cephalosporin antibiotics.

7. Use according to claim 6, for the synthesis of cephalosporin antibiotic intermediates for deacetylation of 7-aminocephalosporanic acid.