CN107236718B

CN107236718B - Low-temperature esterase from metagenome, coding gene and application thereof

Info

Publication number: CN107236718B
Application number: CN201710457737.XA
Authority: CN
Inventors: 吴菁; 吴伟; 李卓; 梅江鹏; 杨江科
Original assignee: Wuhan Polytechnic University
Current assignee: Wuhan Polytechnic University
Priority date: 2017-06-16
Filing date: 2017-06-16
Publication date: 2021-04-13
Anticipated expiration: 2037-06-16
Also published as: CN107236718A

Abstract

The invention relates to the fields of biotechnology and genetic engineering, in particular to a low-temperature esterase derived from metagenome, a coding gene and application thereof. The low-temperature esterase is Est18, and the nucleotide sequence of the esterase is shown as SEQ ID NO. 1; the coded amino acid sequence is shown in SEQ ID NO. 2. Est18 is a low temperature alkaline esterase that is relatively stable at temperatures below 40 ℃, with an optimum temperature of 20 ℃, an optimum pH of 8.0, and a maximum activity of >80% at pH 11.0.

Description

Low-temperature esterase from metagenome, coding gene and application thereof

Technical Field

The invention relates to the field of biotechnology and bioengineering, in particular to low-temperature esterase Est18 derived from a soil metagenome, and a coding gene and application thereof.

Background

Esterases are widely found in animals, plants and microorganisms, and are enzymes that catalyze hydrolysis or the formation of ester bonds, and the substrates for the action are usually esters with less than ten carbon atoms in the aliphatic chain. Esterases belong to the alpha/beta sheet hydrolase superfamily, the catalytic center generally consists of serine, aspartate/glutamate and histidine, and the conserved sequence is a pentapeptide (GXSXG) sequence near serine. Esterase can catalyze various chemical reactions such as hydrolysis, esterification, transesterification and the like, is an important industrial biocatalyst, and is widely applied to the fields of fine chemical engineering, washing, medicines, food, papermaking, leather processing, textile, wastewater treatment, feed industry and the like. From the catalytic property, the esterase has high chemoselectivity and stereoselectivity, and the reaction does not need coenzyme, has mild reaction condition and less byproducts. Another significant feature of esterases in production applications is their ability to function in heterogeneous systems (i.e., oil-water interfaces) or in the organic phase. In the aqueous phase, esterases generally catalyze hydrolysis reactions, while in the organic phase, esterases catalyze esterification and transesterification reactions.

Metagenome, also called metagenome, refers to the sum of all the genetic materials of micro-organisms in a habitat. Because the metagenome technology is to clone the directly extracted environmental microorganism genome DNA into different vectors and transfer the vectors into proper hosts, and to screen and obtain new genes or bioactive substances through different screening technologies, the technology actually bypasses the microorganism culture link, can better develop and utilize the uncultured microorganism resources, and provides a wider gene bank for industry, agriculture or all aspects. At present, more and more patent enzymes come from metagenomic libraries, and can be suitable for the fields of food, chemical industry, medicine and the like.

Disclosure of Invention

The invention aims to provide a novel low-temperature alkaline esterase Est18 derived from Mount Taishan soil metagenome, a coding gene and a preparation method thereof, wherein the esterase can be used for hydrolysis of ester bonds, especially hydrolysis of short-chain esters.

The invention screens a strain TS-Est18 which shows esterase activity from a Mount Tai soil metagenome library by a specific substrate tributyrin functional Screening method (Function-drive Screening), and analyzes and obtains an esterase gene by a bioinformatics analysis methodest18. The whole length of the gene is 957bp (from the start codon to the stop codon), and 318 amino acids are coded. Cloning to obtain esterase gene by a PCR methodest18And the recombinant plasmid pEst18 is obtained after the recombinant plasmid is connected with an expression vector pET-30b (+), and the recombinant esterase Est18 is obtained after the induction expression of Escherichia coli BL21(DE3) pLys. Est18 is low temperature alkaline esterase, and is relatively stable at temperature below 40 deg.C, with optimum temperature of 20 deg.C, stable in pH range of 6-11, optimum pH of 8, and maximum activity at pH11>80 percent. The recombinant strain containing the gene is preserved in China center for type culture Collection, CCTCC M2017317, and the strain is classified as Escherichia coli.

Hair brushThe first object of the invention is to provide an esterase gene encoding said esterase Est18est18The esterase geneest18The nucleotide sequence of (A) is shown in SEQ ID NO. 1.

The second purpose of the invention is to provide esterase Est18, the amino acid sequence of which is shown in SEQ ID NO. 2.

The invention also provides a gene containing the esteraseest18The recombinant expression vector of (1). The expression vector is preferably a pET-30b (+) vector.

The invention also provides a gene containing the esteraseest18 in the above-mentioned manner. The genetic engineering bacteria are preferably escherichia coliEscherichia coli BL21 (DE3) pLys。

The third purpose of the invention is to provide a method for preparing low-temperature alkaline esterase Est18, which comprises the following steps:

1) transformation of host cells with the above recombinant vectorsEscherichia coliBL21(DE3) pLys. Obtaining a recombinant strain;

2) culturing the recombinant strain, and inducing the recombinant esterase to express;

3) the expressed esterase Est18 was recovered and purified.

The low-temperature alkaline esterase Est18 is from Mount Tai soil metagenome (host bacteria are unknown), and the amino acid sequence of the low-temperature alkaline esterase Est18 is shown as SEQ ID number 2:

MSTLDPKLFSDKAIAPETRAVNDAIIAAMTGMPEWWDVGAAKTRAARARGEGIFPAAAKSERARWIEIDGPAGKVPLRVIAPENPRGVYLHIHGGGHVLGAADQQDRLLERISNETRLTAISVEYRLAPENPYPAGPDDCEAAALWVNEHAADFGGHKIAIGGESAGAHLSALTILRLRDKHGLTPFNAANLVFGVFDLGMTPSARAFGDERLVLRTRDIEKFGEAFLPGTDDEQKRAPEFSPLYANLAGLCPALFTIGTRDALLDDSLFMHARWVAAGNVGELDIYPGGCHGFIAFPYPQAFASIARQATFLNAALG

the invention also provides application of the low-temperature alkaline esterase Est18 in catalyzing ester hydrolysis, esterification or transesterification. Preferred short chain fatty acid esters are p-nitrophenol esters having a short carbon chain of C2-C8, such as p-nitrophenyl acetate, p-nitrophenyl butyrate, p-nitrophenyl octanoate, etc., wherein the catalytic activity is highest when the substrate is p-nitrophenyl butyrate (C4).

The esterase Est18 is a low-temperature alkaline esterase, contains 318 amino acids in total, has an enzyme activity of more than 80% at a temperature of between 20 and 45 ℃, and has an enzyme activity which is sharply reduced when the temperature is higher than 50 ℃. The optimum pH value is 8.0, more than 80% of enzyme activity is still preserved in the buffer solution with the pH value of 11 after heat preservation for 20 min, and the enzyme activity is higher between the pH value of 7 and 11, preferably 7.0 to 9.0. 1 mM Cu²⁺、Fe²⁺And Zn²⁺Has strong inhibition effect on the enzyme activity of Est18, while Ca²⁺，Mg²⁺，Ni²⁺And Co²⁺And the metal chelating agent EDTA has the function of enhancing the enzyme activity.

The invention obtains a new esterase gene by screening from the Mount Tai soil metagenome library, finds that the protein coded by the gene is stable and efficient at 20 ℃, is quickly inactivated at 50 ℃ (facilitating the control of enzymatic reaction), and shows higher stability and activity in an alkaline environment. Therefore, the low-temperature alkaline esterase of the invention can be applied to the industries of food, chemical industry, washing, pharmacy and the like to reduce the production temperature and reduce the energy consumption.

Drawings

FIG. 1: in thatE. coliSDS-PAGE analysis of the expressed recombinant esterase Est 18. Lane M is the protein Marker, Lane A is the soluble fraction before induction by esterase Est18, Lane B is the soluble fraction after induction by esterase Est18, and Lane C is the purified protein by esterase Est 18.

FIG. 2: the recombinant esterase Est18 is optimal for the reaction pH.

FIG. 3: the recombinant esterase Est18 has the optimum reaction temperature.

FIG. 4: thermostability of the recombinant esterase Est 18.

FIG. 5: divalent cations influence esterase Est18 activity.

FIG. 6: organic solvents, denaturants and chelators had an effect on Est18 activity.

Detailed Description

Example 1: esterase geneest18Obtaining of

Screening and purifying a Mount Tai soil metagenome library by using tributyrin as a substrate to obtain a bacterial strain TS-Est18 showing esterase activity. Extracting the quality of TS-Est18And carrying out subcloning sequencing on the DNA to obtain the sequence information of the insert. Annotating gene information by using a bioinformatics method, analyzing and determining an Open Reading Frame (ORF) of an esterase gene in the gene information, and naming the ORF as an ORFest18The gene sequence is shown as SEQ ID NO.1, the total length is 957bp (ATG is started and TAG is stopped), the amino acid sequence of the coded esterase Est18 is shown as SEQ ID NO.2, and the total number of amino acids is 318.

Example 2: esterase geneest18Cloning of (3), recombinant vector and construction of recombinant Strain

The esterase gene obtained by the inventionest18Cloning to an expression vector to construct a recombinant expression vector. Based on the esterase gene sequence obtained by analysis, primer5 software is utilized to design upstream primer Forwards (5' -CC) for amplifying esterase complete geneAAGCTTTCCGGTTTTTGACGAAGCGC，BamHI) and Reverse primer (5' -CG)GGATCCGAGCGCGATCCGCAAACTAG，HindIII). Amplification of esterase genes Using the following PCR amplification procedureest18: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 deg.C for 1min, renaturation at 56 deg.C for 1min, extension at 72 deg.C for 1min, and performing 25 cycles; after the circulation is finished, the extension is carried out for 10 min at 72 ℃ and 10 min at 4 ℃, and the length of the amplified target fragment is about 1 kb. Purified PCR product is subjected toBamHI andHindIII double enzyme digestion, rubber tapping recovery andBamHI andHinand connecting the plasmid pET-30b (+) subjected to the double enzyme digestion by dIII, transforming escherichia coli DH5 alpha, and screening by kanamycin resistance and tributyrin activity to obtain a positive clone. Extracting positive cloned plasmid to obtain recombinant vector pEst18, and identifying the inserted fragment and SEQ ID NO.1 through double enzyme digestion and sequencing. Escherichia coli DH5 alpha strain containing recombinant vector pEst18 is preserved in China center for type culture Collection, CCTCC M2017317 and the preservation date is 2017, 6 and 8.

Example 3: construction of genetically engineered bacterium for expressing esterase Est18

3.1 preparation of E.coli BL21(DE3) pLys competent cells

1. Inoculating a small amount of Escherichia coli BL21(DE3) pLys strain into 3 ml LB tube solution, and culturing at 200 rpm and 37 ℃ overnight;

2. inoculating the bacterial liquid in the test tube into a 200ml LB shake flask according to the inoculation amount of 1% volume ratio, and carrying out overnight culture at 200 rpm and 20 ℃;

3. rapidly cooling the cultured shake flask to 4 ℃ in ice water, subpackaging the shake flask into a centrifugal tube (50 ml) precooled by ice, and carrying out ice bath for several minutes;

centrifuging at 4.4 deg.C and 4000 rpm for 10 min to recover cells, and discarding supernatant;

5. 10 ml of 0.1M CaCl precooled with ice₂Resuspending the cells, centrifuging at 4000 rpm at 4 ℃ for 10-15 min, and recovering the cells;

6. repeat 5 with 10 ml 0.1M CaCl₂Resuspending the cells, and ice-cooling for more than 30 min;

centrifuging at 7.4 deg.C and 4000 rpm for 10 min to recover cells;

8. every 50 ml of original culture is 1 ml of 0.1M CaCl₂After resuspension, 1 ml of 50% glycerol is added and mixed uniformly, and then the mixture is subpackaged into 1.5 ml centrifuge tubes with 50-100 mu l per tube. Storage at-80 ℃. Coli BL21(DE3) pLys competent cells were thus obtained.

3.2 transformation

Mu.l of the plasmid pEst18 obtained in example 2 was mixed with 50. mu.l of pLys competent cells of Escherichia coli BL21(DE3) in an amount of 0.5 to 1. mu.l, and the mixture was subjected to ice-bath for 30min, heat shock in a water bath at 42 ℃ for 90 s, ice-bath for 2 min, and then added with 500. mu.l of LB liquid medium, and cultured at 37 ℃ for 45 min at 200 rpm. The culture was centrifuged and plated on an LB plate containing 50. mu.g/ml kanamycin, whereby E.coli BL21(DE3) pLys containing pEst18 was obtained.

Example 4: expression and purification of esterase Est18

4.1 protein inducible expression

Escherichia coli BL21(DE3) pLys containing pEst18 was cultured in 200ml of LB medium at 37 ℃ until OD600 became 0.4-0.6, IPTG was added to a final concentration of 0.2 mM, and the mixture was cultured overnight at 20 ℃. Centrifuging at 4000 rpm for 10 min at 4 deg.C per 50 ml of bacterial solution, collecting thallus, and adding 2 ml (20 mM, pH 7.4) of PBS buffer (KH)₂PO₄-K₂HPO₄) Resuspending the cells, sonicating for 15 min, centrifuging, and collecting the supernatant.

4.2 purification of esterase Est18 and SDS-PAGE electrophoresis

The supernatant collected in 4.1 was purified by His affinity column, and the specific embodiment was as follows:

ni of His affinity chromatography column²⁺Binding and Binding Buffer (20 mM PBS Buffer, 5 mM imidazole and 500 mM NaCl and 0.22 μm filter balance). Adding 0.1M NiSO (0.5 column volume)₄Solution of Ni²⁺After filling, dH is added₂O (which has been filtered through a 0.22 μm filter) washes unbound Ni²⁺Then, the column was equilibrated with a Binding Buffer of 5 column volumes.

2. Purification of esterase Est18 protein. After equilibration, 10 ml of the supernatant collected in 4.1 (the salt concentration and imidazole concentration in the supernatant were the same as in the Binding Buffer) was loaded and unbound (hetero-protein) protein was washed off with 5 column volumes of Binding Buffer. Then eluted, the Elution volume being 20 ml (within this 20 ml, the Elution Buffer (20 mM PBS Buffer, 500 mM imidazole and 500 mM NaCl and filtered with a 0.22 μm filter) and Binding Buffer were mixed at this step, the Elution Buffer ratio was gradually increased to increase the imidazole concentration from 5 mM at a constant rate to 500 mM); finally, the eluate was completely eluted with 5-fold volume of Elution Buffer, and the protein peak (1 ml/tube) was collected.

3. The collected protein eluate was examined for esterase activity. Marking small squares on the bottom of an LB culture dish containing tributyrin, then marking each square, taking 10 mu l of collected eluent respectively, adding the eluent into the corresponding small squares according to the serial number, and placing the small squares in an incubator at 37 ℃ for about 3 h for positive culture to obtain the result (the eluent containing the esterase Est18 protein has hydrolysis loops).

4. The collected eluate is desalted and concentrated. Desalting and concentration were carried out using ultrafiltration tubes (Merck Millipore), and the specific procedures were carried out according to the manual of Merck Millipore.

5. SDS-PAGE of esterase Est 18. The purified esterase was subjected to SDS-PAGE gel electrophoresis (FIG. 1) to give purified esterase Est18, a purified protein of about 37kDa in size, according to theoretical expectations.

4.3 esterase Est18 Activity assay

The esterase Est18 activity determination adopts p-nitrophenol ester, and the specific method is as follows:

1 preparing 200 mM p-nitrophenol ester by using dimethyl sulfoxide (DMSO);

2 Add 980. mu.l 50mM Tris-HCl Buffer (100 mM NaCl, 50mM Tris-HCl, 0.3% (V/V) Triton X-100, pH 8.0), 10. mu.l p-nitrophenyl ester, 10. mu.l pure enzyme solution (0.012276. mu.g/. mu.l after 100-fold dilution) to 1 ml reaction;

3 detecting the content of free p-nitrophenol at the absorbance of 400 nm by using a spectrophotometer under the condition that the extinction coefficient of the content of the p-nitrophenol is determined to be 16,642M^-1cm^-1(i.e., ∈ =16,642M^-1cm^-1). The enzyme activity units are defined as the amount of enzyme required to produce 1. mu. mol p-nitrophenol per minute under standard conditions (25 ℃, 101.325 kPa).

Example 5: substrate specificity analysis of esterase Est18

The activities of esterase Est18 for hydrolyzing p-nitrophenol esters of different acyl groups were compared according to the assay conditions of 4.3. The substrate is p-nitrophenyl acetate (C2), p-nitrophenyl butyrate (C4) and p-nitrophenyl caprylate (C8). The result shows that Est18 has catalytic activity on p-nitrophenol ester with short acyl carbon chain, wherein the catalytic activity is the highest when the substrate is p-nitrophenol butyrate (C4).

Example 6: analysis of the optimal reaction conditions for esterase Est18

The optimum reaction pH of esterase Est18 is measured in the range of 4.0-11.0. Preparing different buffer solutions, wherein the pH of the HAc-NaAc buffer solution is 4.0-6.0, and K is₂HPO₃- KH₂PO₃The pH of the buffer solution is 6.0-8.0, H₃BO₃- Na₂B₄O₇The pH of the buffer solution is 7.4-9.0, Na₂CO₃- NaHCO₃The pH of the buffer solution is 9.0-11.0. The concentrations of these buffer solutions at different pH were 50 mM. The buffer solution (Tris-HCl buffer) according to the determination conditions in 4.3 was replaced with the prepared buffer solutionAnd measuring the enzyme activity of the recombinant esterase Est18, wherein the substrate is p-nitrophenol butyrate. The results of the effect of pH on the activity of the recombinant esterase Est18 are shown in FIG. 3. H at 50mM₃BO₃- Na₂B₄O₇The esterase Est18 has the highest enzyme activity when the pH value of the buffer solution is 8.0, and the esterase has higher enzyme activity when the pH value is between 7.0 and 11.0. When the pH is lower than 7.0, the activity is rapidly reduced (FIG. 2)

The optimum reaction temperature of esterase Est18 is measured within the range of 20-70 ℃. The enzyme activity was measured at different temperatures according to the reaction system in 4.3 using 50mM buffer (Tris-HCl buffer) pH8.0 as a buffer and p-nitrophenol butyrate as a substrate as described in the measurement conditions in 4.3. The result shows that Est18 is low-temperature enzyme, the reaction speed is higher when the temperature is lower, the enzyme activity reaches more than 80% between 20 ℃ and 45 ℃, and the enzyme activity is sharply reduced when the temperature is higher than 50 ℃ (figure 3).

Example 7: esterase Est18 enzymatic stability analysis

The thermal stability of esterase Est18 was determined in the range of 20-50 ℃. The purified enzyme solution was incubated at 20, 30, 40, 45 and 50 ℃ respectively, and taken out at regular intervals to measure the enzyme activity according to the detection method shown in 4.3, and the results are shown in FIG. 4. The esterase Est18 has gradually decreased esterase residual enzyme activity along with the increase of the treatment temperature, the enzyme activity is sharply decreased when the temperature is higher than 50 ℃, and the residual enzyme activity is basically lost after the treatment at 50 ℃ for 5min (figure 4).

Determination of the effect of the divalent cation esterase Est18 activity. 1 mM of Fe was added to the reaction system separately²⁺、Zn²⁺、Cu²⁺、Mn²⁺、Ca²⁺、Mg²⁺、Ni²⁺And Co²⁺Incubating at room temperature for 20 min, and determining relative enzyme activity according to a 4.3 determination method (using p-nitrophenol butyrate as a substrate), wherein the enzyme activity in a reaction system without any ions is 100%. The results show that low concentrations of Fe compared to the control²⁺、Zn²⁺And Cu²⁺Has different degrees of inhibition effects on the activity of catalyzing p-nitrophenol butyrate by esterase. And Mn²⁺Has little influence on the enzyme activity of esterase Est18, and the low concentration of Ca²⁺、Mg²⁺、Ni²⁺And Co²⁺Has an enhancing effect on the enzyme activity of esterase Est18 (figure 5).

Determination of the effect of organic solvents and denaturants etc. on the activity of Est 18. 15% (v/v) and 30% (v/v) organic solvents (methanol, ethanol, isopropanol, acetone and dimethyl sulfoxide DMSO), 1% denaturants (sodium dodecyl sulfate SDS, Tween 20) and chelating agents (10% EDTA) are respectively added into a reaction system to be incubated for 20 min, and then the relative enzyme activity is determined according to a determination method of 4.3 (taking p-nitrophenol butyrate as a substrate), and the enzyme activity in the original reaction system is 100%. The results show that the tested organic solvent has different degrees of inhibition on the activity of Est18, and the degree of inhibition is enhanced with the increase of the concentration of the organic solvent. The denaturant inhibited enzyme activity very strongly, leaving only 0.14% activity after 20 min incubation with 1% SDS. However, Est18 showed a greater increase in enzyme activity in the presence of the chelator EDTA (122%) (fig. 6).

SEQUENCE LISTING

<110> Wuhan university of light industry

<120> low-temperature esterase from metagenome, encoding gene and application thereof

<160> 2

<210> SEQ ID NO.1

<211> 957

<212> DNA

<213> environmental DNA

<400> SEQ ID NO.1

atgagcacgc tcgatccgaa actgttcagc gataaagcga ttgccccgga aacgcgcgcg 60

gtgaacgatg ccattatcgc cgcgatgacc ggcatgcctg aatggtggga tgtcggcgcg 120

gccaagacgc gcgcggcgcg cgccaggggc gaaggcattt tcccagcggc ggcgaaatcc 180

gaacgcgcgc gctggatcga gatagatggg cccgccggca aggtgccgtt gcgcgtcatc 240

gcgccggaaa atccgcgcgg cgtatatctg cacattcacg gcggcggcca cgtgttgggc 300

gccgcggatc aacaggatcg gttgctcgag cgcatctcaa acgaaaccag gctgacggcg 360

atcagcgttg agtatcgctt ggcgccggaa aatccctatc ccgctgggcc cgacgattgt 420

gaagcggcgg cgctttgggt gaacgagcat gcggcggatt ttggcggcca caagattgcc 480

atcggcggcg aaagcgcggg cgcgcacctg tcagcgctga ctattctgcg cttgcgcgac 540

aagcatgggc tgacgccctt caatgcggcg aaccttgtgt tcggcgtttt cgatttggga 600

atgacgccaa gtgcgcgcgc gtttggcgat gagcggctgg tcttgcgcac gcgcgacatc 660

gagaagttcg gcgaggcgtt cttgcccggt acggacgacg agcagaagcg cgcgccggaa 720

ttctcgccgc tctacgcgaa cctcgccggc ttgtgccccg cgctcttcac catcggcacg 780

cgcgatgcgc tcctggacga cagtctcttc atgcacgcgc gctgggtggc ggcgggaaat 840

gtcggcgaac tcgacatcta tccgggcggc tgtcatggct tcatcgcctt cccgtacccg 900

caggcgttcg cgtcgatcgc gcgccaagcc acgtttttga acgcggccct gggttag 957

<210> SEQ ID NO.2

<211> 318

<212> PRT

<213> environmental DNA

<400> SEQ ID NO.2

Met Ser Thr Leu Asp Pro Lys Leu Phe Ser Asp Lys Ala Ile Ala Pro

1 5 10 15

Glu Thr Arg Ala Val Asn Asp Ala Ile Ile Ala Ala Met Thr Gly Met

20 25 30

Pro Glu Trp Trp Asp Val Gly Ala Ala Lys Thr Arg Ala Ala Arg Ala

35 40 45

Arg Gly Glu Gly Ile Phe Pro Ala Ala Ala Lys Ser Glu Arg Ala Arg

50 55 60

Trp Ile Glu Ile Asp Gly Pro Ala Gly Lys Val Pro Leu Arg Val Ile

65 70 75 80

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

85 90 95

His Val Leu Gly Ala Ala Asp Gln Gln Asp Arg Leu Leu Glu Arg Ile

100 105 110

Ser Asn Glu Thr Arg Leu Thr Ala Ile Ser Val Glu Tyr Arg Leu Ala

115 120 125

Pro Glu Asn Pro Tyr Pro Ala Gly Pro Asp Asp Cys Glu Ala Ala Ala

130 135 140

Leu Trp Val Asn Glu His Ala Ala Asp Phe Gly Gly His Lys Ile Ala

145 150 155 160

Ile Gly Gly Glu Ser Ala Gly Ala His Leu Ser Ala Leu Thr Ile Leu

165 170 175

Arg Leu Arg Asp Lys His Gly Leu Thr Pro Phe Asn Ala Ala Asn Leu

180 185 190

Val Phe Gly Val Phe Asp Leu Gly Met Thr Pro Ser Ala Arg Ala Phe

195 200 205

Gly Asp Glu Arg Leu Val Leu Arg Thr Arg Asp Ile Glu Lys Phe Gly

210 215 220

Glu Ala Phe Leu Pro Gly Thr Asp Asp Glu Gln Lys Arg Ala Pro Glu

225 230 235 240

Phe Ser Pro Leu Tyr Ala Asn Leu Ala Gly Leu Cys Pro Ala Leu Phe

245 250 255

Thr Ile Gly Thr Arg Asp Ala Leu Leu Asp Asp Ser Leu Phe Met His

260 265 270

Ala Arg Trp Val Ala Ala Gly Asn Val Gly Glu Leu Asp Ile Tyr Pro

275 280 285

Gly Gly Cys His Gly Phe Ile Ala Phe Pro Tyr Pro Gln Ala Phe Ala

290 295 300

Ser Ile Ala Arg Gln Ala Thr Phe Leu Asn Ala Ala Leu Gly

305 310 315

Claims

1. A low temperature alkaline esterase Est18 is characterized in that the amino acid sequence is shown as SEQ ID NO. 2; the low temperature alkaline esterase Est18 is stable in the pH range of 6-11, and the maximum activity at the pH of 11 is more than 80%.

2. A low temperature alkaline esterase gene Est18, encoding the low temperature alkaline esterase Est18 as claimed in claim 1, characterized in that the corresponding nucleotide sequence is shown as SEQ ID No. 1.

3. The recombinant vector carrying the low-temperature alkaline esterase gene est18 of claim 2, wherein the expression vector of the recombinant vector is pET-30b (+).

4. A recombinant strain comprising the low temperature alkaline esterase gene est18 of claim 2.

5. The recombinant strain of claim 4, wherein the strain is a bacterium, a yeast, or an animal cell.

6. Escherichia coli (Escherichia coli) containing a gene encoding Est18 low-temperature alkaline esterase has a preservation number of CCTCC M2017317.

7. A method for preparing low-temperature alkaline esterase Est18, which is characterized by comprising the following steps:

transforming a host cell with the recombinant vector of claim 3 to obtain a recombinant strain;

culturing the recombinant strain, and inducing the recombinant esterase to express;

the expressed esterase Est18 was recovered and purified.

8. The use of a low temperature alkaline esterase Est18 according to claim 1 for catalyzing ester hydrolysis, esterification or transesterification reactions.

9. Use according to claim 8, characterized in that the esters are short-chain esters.