CN110951711B - Esterase with activity of degrading chiral ester and coding gene and application thereof - Google Patents

Abstract

The invention provides esterase with activity of degrading chiral ester, and a coding gene and application thereof. The gene for coding esterase E19-3664 is obtained by screening and cloning from Antarctic soil, and the research on the expressed protein shows that the protein can selectively degrade L-methyl lactate, shows higher degradation activity on short-chain esters, keeps the enzyme activity above 80 percent within the temperature range of 60-100 ℃, has good thermal stability and extremely strong high temperature resistance. Therefore, the method has good practical application value.

Description

Esterase with activity of degrading chiral ester and encoding gene and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to esterase with activity of degrading chiral ester, and a coding gene and application thereof.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Esterases (esterases) are important ester bond hydrolysis and synthesis enzymes, can catalyze the hydrolysis of triglyceride to generate fatty acid and glycerol, can also catalyze the reverse reaction thereof, and play an important role in the processes of esterification, ester exchange, acidolysis, alcoholysis and the like. The esterase has wide sources and can be separated from microorganisms, animals and plants, wherein the esterase from the microorganisms has good separation effect and is more widely applied. Compared with other catalysts, the esterase has higher catalytic efficiency. Because of wide substrate specificity, high catalytic efficiency and good stability of the esterase, the esterase has wide application in the fields of food, medicine, chemical industry and the like. Some esterases can also specifically catalyze and synthesize chiral molecules, and have great potential in the industrial production of chiral compounds.

Nowadays, it has become a general consensus of the scientific community that different enantiomers in an organism are to be treated with caution as different compounds. In natural and synthetic drugs, a large proportion of compounds belong to the chiral class of compounds. Although chiral compounds have the same molecular formula, due to conformational differences, the activity, toxicity and metabolic pathways in organisms may vary greatly, even in the exact opposite. The single isomer administration has more accurate treatment effect and can reduce the risk brought by different conformations. At present, the single isomer is obtained mainly by a common separation and analysis method, so that the cost is high and the efficiency is extremely low. The esterase has high substrate selectivity, regioselectivity and enantioselectivity, and has a very strong application prospect in the fields of drug synthesis and chiral compound resolution. The utilization of esterase to obtain chiral compounds does not produce other side reactions, does not need coenzyme, has mild reaction conditions, and has become a hot point of research in recent years.

The unique climatic characteristics and environmental conditions of the Antarctic pole endow the Antarctic microorganism with unique physiological characteristics. The south polar microorganisms have evolved physiological structural features and growth metabolic pathways different from those of tropical and temperate microorganisms, can adapt to the severe climatic conditions of the south pole, and become a biological resource treasury. Thus, the esterase isolated from the Antarctic soil-derived microorganism has more unique characteristics than microbial esterases under normal environmental conditions. By exploring the enzymatic properties, a wider application field can be developed.

Disclosure of Invention

In view of the prior art, the invention aims to provide esterase with activity of degrading chiral ester, and a coding gene and application thereof. The gene for coding esterase E19-3664 is obtained by screening and cloning from Antarctic soil, and the research on the expressed protein shows that the protein can selectively degrade L-methyl lactate and has no activity on D-methyl lactate; meanwhile, the enzyme has higher degradation activity to short-chain esters, and the enzyme activity is still kept above 80% within the range of 60-100 ℃, so that the enzyme has good thermal stability and extremely high resistance to high temperature. Therefore, the method has good practical application value.

In the first aspect of the invention, a protein named as E19-3664 is provided, and experiments prove that the protein has the capability of selectively degrading L-methyl lactate and short-chain esters and simultaneously has good thermal stability.

Specifically, the E19-3664 is (a1) or (a2) as follows:

(a1) protein composed of amino acid sequence shown in SEQ ID NO. 1;

(a2) and (b) a protein derived from (a1) by substitution and/or deletion and/or addition of one or more amino acid residues and having the same function.

Wherein SEQ ID NO.1 consists of 317 amino acid residues.

In a second aspect of the present invention, there is provided a gene encoding the E19-3664 protein. The coding gene of the protein named E19-3664 is obtained by screening and cloning from Antarctic soil.

Wherein the gene has the nucleotide sequence of any one of (b1) - (b 3):

(b1) a nucleotide sequence shown as SEQ ID NO. 2;

(b2) a nucleotide sequence complementary to (b 1);

(b3) a nucleotide sequence having a sequence identity of 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (complete) sequence) or more to the nucleotide sequence set forth in (b1) or (b2) and encoding a protein of the same function.

In the third aspect of the present invention, a recombinant expression vector, an expression cassette or a recombinant cell containing the gene is also within the scope of the present invention.

In the fourth aspect of the present invention, the use of the protein E19-3664 as esterase is also within the scope of the present invention.

In a fifth aspect of the present invention, there is provided a primer pair for amplifying the above-mentioned coding gene, the nucleotide sequences of which are shown as SEQ ID NO.3 and SEQ ID NO.4, respectively.

In a sixth aspect of the present invention, there is provided the use of the above-mentioned protein E19-3664, a coding gene, a recombinant expression vector, an expression cassette or a recombinant cell as described in (c1) to (c 3):

(c1) synthesizing chiral ester;

(c2) production of a detergent;

(c3) and degrading short-chain ester.

The invention has the beneficial technical effects that:

the antarctic soil-derived esterase still maintains better enzyme activity at higher temperature, has higher enzyme activity under alkaline condition, and has stronger application prospect in the aspect of production of detergents.

The antarctic soil-derived esterase E19-3664 can efficiently and selectively degrade chiral ester, and has industrial application potential for synthesizing important chiral compounds, so that the antarctic soil-derived esterase has good practical application value.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are included to illustrate an exemplary embodiment of the invention and not to limit the invention.

FIG. 1 shows the electrophoresis pattern of pure esterase E19-3664 after nickel column affinity chromatography purification in example 2 of the present invention, M, protein molecular weight marker (marker);

FIG. 2, histogram of substrate degradation activity analysis of esterase E19-3664 in example 3 of the present invention;

FIG. 3 is a graph showing the temperature profile of the enzyme activity of the southern soil-derived esterase in example 3 of the present invention;

wherein: (A) the effect of temperature on enzyme activity; (B) the effect of temperature on enzyme stability;

FIG. 4 is a pH chart showing the enzymatic activity of the southern Pole soil-derived esterase in example 3 of the present invention.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise. It is to be understood that the scope of the invention is not to be limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. The experimental procedures, if specific conditions are not indicated in the following detailed description, are generally in accordance with conventional procedures and conditions of molecular biology within the skill of the art, which are fully explained in the literature. See, e.g., Sambrook et al, "molecular cloning: the techniques and conditions described in the laboratory Manual, or according to the manufacturer's recommendations.

In one embodiment of the invention, a protein named E19-3664 is provided, which has the capability of selectively degrading L-methyl lactate and short-chain esters and good thermal stability through experimental verification.

Specifically, the E19-3664 is (a1) or (a2) as follows:

(a1) protein composed of amino acid sequence shown in SEQ ID NO. 1;

(a2) and (b) a protein derived from (a1) by substitution and/or deletion and/or addition of one or more amino acid residues and having the same function.

Wherein SEQ ID NO.1 consists of 317 amino acid residues.

In another embodiment of the present invention, there is provided a gene encoding the E19-3664 protein. The coding gene of the protein named E19-3664 is obtained by screening and cloning from Antarctic soil.

In still another embodiment of the present invention, the gene has the nucleotide sequence of any one of (b1) to (b 3):

(b1) a nucleotide sequence shown as SEQ ID NO. 2;

(b2) a nucleotide sequence complementary to (b 1);

(b3) a nucleotide sequence which has > 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (complete) sequence) identity to the nucleotide sequence shown in (b1) or (b2) and encodes the same functional protein.

In yet another embodiment of the present invention, SEQ ID NO.2 consists of 954 nucleotides, wherein nucleotides 1-951 are coding sequences, and nucleotides 952-954 are transcribed as a stop codon to terminate peptide chain synthesis.

In another embodiment of the present invention, a recombinant expression vector, an expression cassette or a recombinant cell containing the gene is also within the scope of the present invention.

In another specific embodiment of the invention, the recombinant expression vector is a recombinant prokaryotic expression vector, and the recombinant prokaryotic expression vector is obtained by inserting the coding gene into an expression vector pET22 b.

In another embodiment of the present invention, the recombinant cell is a prokaryotic cell, preferably a bacterium, further selected from escherichia coli, bacillus, and the like; furthermore, the recombinant cell is Escherichia coli BL21(DE3) containing the above gene and/or recombinant expression vector.

In yet another embodiment of the present invention, the transformant comprises a prokaryote.

In another embodiment of the present invention, the use of the protein E19-3664 as esterase is also within the scope of the present invention.

In another embodiment of the present invention, a primer pair for amplifying the above-mentioned coding gene is provided, wherein the nucleotide sequences of the primer pair are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

In still another embodiment of the present invention, there is provided the protein E19-3664, a coding gene, a recombinant expression vector, an expression cassette or a recombinant cell described above, having the following applications (c1) to (c 3):

(c1) synthesizing chiral ester;

(c2) production of a detergent;

(c3) and degrading short-chain ester.

Specifically, in the application (c1), the protein E19-3664 can selectively degrade L-methyl lactate and has no activity on D-methyl lactate;

in the application of (c3), the short-chain ester is a short-chain ester compound with a carbon chain length of 2-10 carbon atoms, and is more preferably a short-chain ester compound with a carbon chain length of 2-4 carbon atoms.

The above application may be performed in a high temperature environment. The high temperature environment is not lower than 60 ℃ (such as 60 ℃ -70 ℃); further, it is not lower than 70 ℃ (e.g., 70 ℃ -75 ℃, 75 ℃ -80 ℃, 80 ℃ -85 ℃, 85 ℃ -90 ℃ or higher temperature, or 70 ℃, 71 ℃, 72 ℃, 73 ℃, 74 ℃, 75 ℃, 76 ℃, 77 ℃, 78 ℃, 79 ℃, 80 ℃, 81 ℃, 82 ℃, 83 ℃, 84 ℃, 85 ℃, 86 ℃, 87 ℃, 88 ℃, 89 ℃, 90 ℃).

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The test methods in the following examples, which are not specified under specific conditions, are generally carried out under conventional conditions.

Culture medium:

LB liquid medium: 1 wt% of peptone, 0.5 wt% of yeast powder, 1 wt% of NaCl and distilled water.

LB solid medium: 1 wt% of peptone, 0.5 wt% of yeast powder, 1 wt% of NaCl, 1.5 wt% of agar and distilled water.

Example 1: obtaining of encoding gene sequence of antarctic soil source esterase and sequence analysis thereof

The strain source is as follows: the gene E19-3664 of the antarctic microorganism esterase is from soil at a site E19 in the area near the Antarctic great wall station.

The method comprises the following specific steps:

1.1 construction of subclone libraries

The large fragment plasmid fosmid in E.coli EPI300 clone E19-3664 was extracted using the BAC/PAC DNA extraction kit from OMEGA according to the instructions. The extracted fosmid was then partially digested with the restriction enzyme Sau3AI (from Fermentas) to obtain a2,000-5,000 bp DNA fragment, which was ligated to a BamHI-digested and dephosphorylated pUC19 plasmid (from NEB). E.coli Top10 competent cells were electroporated with the ligation reaction solution, plated on LB solid plates containing 100. mu.g/ml ampicillin and 1% (v/v) tributyrin (purchased from Sigma), and inverted cultured at 37 ℃ for 12-16 hours to construct a subclone library of fosmid DNA of clone E19-3664.

1.2 determination of the sequence of the ester hydrolase Gene

Selecting subclones which generate transparent degradation circles on the solid plate, extracting plasmids and sequencing. The NCBIORFFinder software was used to predict possible open reading frames on DNA sequences. Similarity search is carried out on the predicted open reading frames in NCBInr library by using BLASTX to determine the esterase gene sequence E19-3664 carried by the clone E19-3664, the gene E19-3664 is 954bp in total, wherein the gene contains an open reading frame of 954bp, the open reading frame encodes the esterase E19-3664 from Antarctic soil, the start codon is 1bp, the stop codon is 952bp, and the protein encodes 317 amino acids in total. The amino acid sequence of the precursor protein of the antarctic soil-derived esterase E19-3664 is shown in SEQ ID NO. 1. The nucleotide sequence of the obtained encoding gene E19-3664 of the Antarctic soil esterase E19-3664 is shown as SEQ ID NO. 2.

1.3 sequence analysis of Antarctic soil-derived esterase

The sequence most similar to Antarctic soil source E19-3664 in GenBank is alpha/beta hydrolase from Pseudomonas sp.IB20, and the sequence similarity is 99%. Meanwhile, the similarity between the sequence given by BLAST and the Antarctic soil-derived esterase E19-3664 is between 81% and 99%, the sequences are genes predicted based on gene sequences, and the biochemical properties of the genes are not researched yet.

Example 2: cloning, heterologous expression and separation and purification of esterase

2.1 amplification of Gene sequences by PCR

(1) Two specific primers are designed according to the sequence of the gene E19-3664:

3664F：AAGAAGGAGA TATACATATG GCGCACACCC CCTGGCCTGCCAG(SEQ ID NO.3)；

3664R：TCGAGTGCGG CCGCAAGCTT CGAAGACTTT CCACCTGTGTAGC(SEQ ID NO.4)；

the primers were synthesized by jinan platane biotechnology limited.

(2) Amplifying a target gene fragment by using Fastpfu DNA polymerase (purchased from Transgen company) by using 3664F and 3664R as primers and fosmid where the gene E19-3664 is located as a template;

the PCR reaction conditions were: pre-denaturation at 95 ℃ for 2 min; then denaturation at 95 ℃ for 20sec, annealing at 50 ℃ for 20sec, extension at 72 ℃ for 1min, and 30 cycles; extension at 72 ℃ for 10 min.

(3) The PCR amplification product was subjected to 1 wt% agarose gel electrophoresis, and the result showed that a DNA fragment of about 1,000bp was obtained. Then, the amplified DNA fragment was recovered using a DNA recovery kit of Omega according to the instructions thereof.

(4) The recovered E19-3664 gene fragment was ligated to the pET22b vector using a seamless cloning kit (purchased from nearshore protein technologies, Inc.).

(5) Coli DH 5. alpha. competence was prepared according to the procedure for E.coli competence described in molecular cloning protocols.

(6) The ligated recombinant pET22b vector was transformed into E.coli DH 5. alpha. competent cells by heat shock transformation as described in molecular cloning protocols.

(7) The transformed E.coli DH 5. alpha. was plated on LB solid medium containing 100. mu.g/ml ampicillin and cultured overnight at 37 ℃. Selecting positive clones, transferring the positive clones to an LB liquid culture medium for culture, and extracting plasmids.

The LB solid medium comprises the following components:

1 wt% of peptone, 0.5 wt% of yeast powder, 1 wt% of NaCl, 1.5 wt% of agar and distilled water.

The LB liquid medium comprises the following components:

1 wt% of peptone, 0.5 wt% of yeast powder, 1 wt% of NaCl and distilled water.

2.2 transformation of the recombinant expression vector pET22b-E19-3664 into E.coli BL21(DE 3).

(1) Preparing escherichia coli BL21 competence according to a method for preparing escherichia coli competence on molecular cloning experimental guidelines;

(2) transferring a recombinant vector pET22b-E19-3664 with correct sequencing into escherichia coli BL21 competence according to a heat shock transformation method on a molecular cloning experimental manual;

(3) the transformed E.coli BL21 was streaked on LB medium containing 100. mu.g/ml ampicillin and cultured overnight at 37 ℃.

2.3 inducible expression and purification of the Gene in E.coli

(1) Scraping lawn on a plate, inoculating the lawn into 100ml LB liquid culture medium containing 100 mug/ml ampicillin, and culturing for 2-3 h at 37 ℃;

(2) transferring into 50ml LB liquid culture medium containing 100 μ g/ml ampicillin at 1% (v/v), culturing at 37 deg.C and 180rpm until OD value is 0.6-0.8 at 600nm, adding IPTG to final concentration of 0.05mM, and culturing at 16 deg.C and 180rpm for 24 hr;

(3) collecting LB culture solution induced by IPTG, centrifuging at 11000rpm and 5 deg.C for 5min, and collecting thallus;

(4) adding 50mM Tris-HCl buffer solution (pH 8.0) containing 100mM NaCl to resuspend the thalli;

(5) carrying out ultrasonic crushing on the resuspended bacterial liquid;

(6) centrifuging the crushed bacterial liquid at 11000rpm and 4 ℃ for 30min, and collecting supernatant; centrifuging the supernatant at 11000rpm and 4 deg.C for 20min, and collecting the supernatant;

(7) filtering the supernatant with 22 μm filter membrane, and performing nickel column affinity chromatography according to the specification;

(8) the purity of the samples collected after chromatography was checked by SDS-PAGE, which confirmed that an electrophoretically pure enzyme of Antarctic esterase E19-3664 had been obtained (see FIG. 1). The residual imidazole was removed by dialysis and finally stored at-20 ℃ until use.

Example 3: determination of the Properties of Antarctic esterase E19-3664

3.1 substrate specificity assay

pNP ester substrates C2, C4, C8, C10, C12, C14 (from Sigma) were formulated with isopropanol at different carbon chain lengths.

The standard reaction is:

mu.l of 10mM substrate was pre-heated with 960. mu.l of 50mM Tris-HCl (pH 8.0) at 40 ℃ for 3min, 20. mu.l of enzyme solution was added and reacted at 40 ℃, the reaction was terminated after 5min by adding 100. mu.l of 20 wt% SDS (sodium dodecyl sulfate), and the OD at 405nm was measured with the reaction without the addition of enzyme solution as a blank. The standard curve was plotted with different concentrations of pNP (from Sigma).

Enzyme activity is defined as the amount of enzyme required to catalyze the hydrolysis of a pNP ester substrate to produce 1. mu.M pNP per minute at a certain temperature as one unit of enzyme activity (U). The results show that esterase E19-3664 can efficiently degrade short-chain pNP ester substrates (C2 and C4), wherein the degradation capability to the C2 substrate is strongest, and the activity reaches 641U/mg.

3.2 analysis of optimum temperature and temperature stability

Measurement of optimum reaction temperature: the enzyme activities of the antarctic esterase at 0 ℃, 10 ℃, 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃ and 100 ℃ are respectively detected in 50mM Tris-HCl (pH 8.0) buffer solution by taking C2 as a substrate. The highest enzyme activity was defined as 100%.

The result shows that the optimal enzyme activity temperature of the enzyme is 70 ℃, and the enzyme retains over 70 percent of high activity at 50-100 ℃ (as shown in figure 3A).

Temperature stability analysis: the enzyme solution was incubated at 70 ℃ and the same enzyme amount was removed every 15min over 2 hours to determine the residual activity of the Antarctic esterase E19-3664 in 50mM Tris-HCl (pH 8.0) buffer at 40 ℃. The enzyme activity at 0 ℃ is defined as 100%, and the result shows that more than 80% of the enzyme activity is still maintained after the temperature of 70 ℃ is incubated for 2h (as shown in figure 3B).

3.3 optimum pH

Determination of optimum reaction pH: preparing Britton-Robinson buffer solution with the pH value within the range of 4.0-11.0 and at intervals of 1 or 0.5 pH units. The enzyme activity of the Antarctic esterase E19-3664 under different pH conditions is determined, and the highest enzyme activity is defined as 100%.

The results show that esterase E19-3664 is an alkaline esterase, and the optimum pH of the esterase is 8.0 (shown in figure 4).

3.4 Activity assay for chiral ester substrates

Various chiral ester substrates (purchased from Sigma) were formulated with acetonitrile. The standard reaction is:

mu.l of 143mM chiral ester was added to 200. mu.l of 5mM EPPS buffer (pH 8.0) containing 0.455mM Phenol red, 10. mu.l of 0.5mg/ml pure enzyme was further added, and reacted at 40 ℃ for 3 hours to detect OD 550. The reaction without the addition of the enzyme solution was used as a control. The difference value of the OD550 after the control and the reaction is used for reflecting the activity of the enzyme, and the larger the difference value is, the higher the activity of the enzyme is.

Results show that esterase E19-3664 can selectively degrade L-methyl lactate and has no activity on D-methyl lactate, so esterase E19-3664 has industrial application potential for synthesizing important chiral compounds.

4. Results

Through the construction of a subclone library and later sequencing, the nucleic acid sequence of the esterase gene E19-3664 carried on fosmid in the Escherichia coli clone E19-3664 was determined. Specific primers are designed according to the E19-3664 gene sequence, a gene segment for coding the antarctic soil-derived esterase E19-3664 is cloned from fosmid DNA of a clone E19-3664 by utilizing a PCR technology, and an expression vector containing the antarctic soil-derived esterase E19-3664 and an escherichia coli recombinant cell containing the expression vector are constructed. The gene E19-3664 contains an 954bp open reading frame, encodes a novel esterase E19-3664, has an initiation codon at 1bp and a termination codon at 952bp, and encodes 317 amino acid precursor proteins. The gene E19-3664 is heterologously expressed and purified in Escherichia coli to obtain mature active esterase E19-3664.

The purified esterase E19-3664 was characterized. The result shows that the enzyme has stronger degradation activity on short-chain esters with carbon chain length of 2-4 carbon atoms (figure 2). The enzyme can keep high enzyme activity within the range of 50-100 ℃, and can stably exist at the temperature higher than 70 ℃ (figure 3). The optimum pH value is 8.0 and the pH value is in the range of 7.0 to 8.5 (FIG. 4). Activity analysis of various chiral ester substrates showed that esterase E19-3664 is an important precursor for industrially important chiral compounds: methyl lactate shows excellent chiral selectivity, which indicates that esterase E19-3664 has industrial application potential for synthesizing important chiral compounds.

It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can modify the technical solution of the present invention as needed or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention.

SEQUENCE LISTING

<110> university of Qilu Industrial science

<120> esterase with activity of degrading chiral ester and coding gene and application thereof

<130>

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 317

<212> PRT

<213> Artificial sequence

<400> 1

Met Ala His Thr Pro Trp Pro Ala Ser Glu Pro Glu Leu Ala Glu Met

1 5 10 15

Arg Gly Phe Asn Lys Lys Leu Ala Trp Leu Pro Arg Phe Lys Ile Arg

20 25 30

Asn Arg Ile Thr Pro Arg Val Ile Gln Ala Leu Leu Arg Val Ser Gln

35 40 45

Thr Leu Lys Lys Ala Pro Ala Ala Gln Thr His Leu Val Gly Ser Val

50 55 60

Pro Val Arg Ile Leu Arg Pro Ala Gly Lys Pro Lys Gly Val Val Leu

65 70 75 80

Asp Ile His Gly Gly Gly Trp Val Ile Gly Asn Ala Gln Met Asp Asp

85 90 95

Asp Leu Asn Leu Gly Met Val Gln Ala Cys Gly Val Ala Val Val Ser

100 105 110

Val Asp Tyr Arg Leu Ala Val Asp Thr Pro Val Glu Gly Leu Met Glu

115 120 125

Asp Cys Leu Ala Ala Ala Arg Trp Leu Leu Gly Asp Cys Pro Glu Phe

130 135 140

Ala Asp Leu Pro Val Ile Ile Val Gly Glu Ser Ala Gly Gly His Leu

145 150 155 160

Ala Ala Thr Thr Leu Leu Ala Leu Lys Gln Trp Pro Gln Leu Leu Ala

165 170 175

Arg Val Ser Gly Ala Val Leu Tyr Tyr Gly Val Tyr Asp Leu Thr Gly

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Gly Leu Pro Pro Ala Leu Met Phe Val Gly Glu Leu Asp Pro Leu Lys

245 250 255

Asp Asp Thr Leu Leu Leu Ala Glu Arg Trp Gly Ala Val Ala Gln Val

260 265 270

Glu Ala His Leu Leu Pro Glu Ala Ala His Gly Phe Ile His Phe Pro

275 280 285

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

290 295 300

Gln Gln Ile Asn Gly Arg Tyr Thr Gly Gly Lys Ser Ser

305 310 315

<210> 2

<211> 954

<212> DNA

<213> Artificial sequence

<400> 2

atggcgcaca ccccctggcc tgccagcgag ccggagctgg ccgagatgcg cggctttaat 60

aaaaagctcg cctggttgcc gcgctttaaa atccgcaacc gcatcacgcc gcgtgtgatc 120

caggcgctgc tgcgcgtcag tcaaacgctc aagaaggccc cggcggcgca aacccatctg 180

gtgggctcgg tacccgtgcg catcctgcgg ccggcgggta aacccaaggg cgtggtgctg 240

gatatccacg gcggtggctg ggtgatcggc aatgcgcaga tggacgatga cctgaacctg 300

ggcatggtgc aggcgtgcgg cgtggcggtg gtgtcggtgg attatcggct ggcggtcgat 360

acgccggtcg aggggttgat ggaagactgc ttggccgccg ctcgctggct gttgggcgac 420

tgtcctgagt ttgccgacct gccggtgatc attgttggtg aatcagcggg cggccatctg 480

gcggcgacga cgttgctggc gctcaagcaa tggccgcagt tgctcgcgcg ggtgagcggg 540

gcggtgttgt attacggggt ttatgatttg accggcacgc cgagcgtacg cgcagccggg 600

ccggacacgt tattactgga tggcccgggg atggtcgagg ccttgcgtat gctcacgccg 660

gggttgagtg atgagcaaag gcggcagccg ccgttgtcac cgttgtatgg cgactttacg 720

gggttgccgc cggcgttgat gtttgtcggc gagctggacc cgctgaagga tgacacgctg 780

ttgctggcag agcggtgggg ggcggtggcg caggttgagg cgcacttgct gccggaagct 840

gcccatgggt ttattcattt tccggtggcg ctggcaaata gtgtgctggt ctacagcagg 900

gcgtggatca ctcaacagat caatggccgc tacacaggtg gaaagtcttc gtag 954

<210> 3

<211> 43

<212> DNA

<213> Artificial sequence

<400> 3

aagaaggaga tatacatatg gcgcacaccc cctggcctgc cag 43

<210> 4

<211> 43

<212> DNA

<213> Artificial sequence

<400> 4

tcgagtgcgg ccgcaagctt cgaagacttt ccacctgtgt agc 43

Claims (10)

1. The application of the protein E19-3664 in degradation of L-methyl lactate and C2-C4 short-chain ester is characterized in that the amino acid sequence of the protein E19-3664 is shown as SEQ ID No. 1.

2. The use according to claim 1, wherein the use is carried out in a high temperature environment, the high temperature environment being 60-100 ℃.

3. The use according to claim 1, wherein the nucleotide sequence of the gene encoding said protein E19-3664 is shown in SEQ ID No. 2.

4. The application of a recombinant expression vector, an expression cassette or a recombinant cell containing the gene coding the protein E19-3664 in degrading L-methyl lactate and C2-C4 short-chain ester, wherein the nucleotide sequence coding the E19-3664 gene is shown as SEQ ID No. 2.

5. The use of claim 4, wherein the recombinant expression vector is a recombinant prokaryotic expression vector.

6. The use of claim 5, wherein said recombinant prokaryotic expression vector comprises the coding gene inserted into expression vector pET32a, wherein the nucleotide sequence of said coding gene is shown in SEQ ID NO. 2.

7. The use of claim 4, wherein the recombinant cell is a prokaryotic cell.

8. The use of claim 7, wherein the prokaryotic cell is a bacterium.

9. The use of claim 8, wherein the bacteria are selected from the group consisting of escherichia coli and bacillus sp.

10. The use according to claim 4, wherein said recombinant cell is BL21(DE3) comprising a gene encoding said protein E19-3664 and/or a recombinant expression vector comprising a gene encoding said protein E19-3664.

Images