CN108504618B

CN108504618B - Recombinant bacterium for expressing lipase with R configuration selectivity and application thereof

Info

Publication number: CN108504618B
Application number: CN201810526361.8A
Authority: CN
Inventors: 王永华; 蓝东明; 袁红; 黄伟谦; 杨博
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2018-05-29
Filing date: 2018-05-29
Publication date: 2020-08-18
Anticipated expiration: 2038-05-29
Also published as: CN108504618A

Abstract

The invention discloses a recombinant bacterium for expressing lipase with R configuration selectivity and application thereof, wherein the lipase gene with R configuration selectivity is inserted into a multiple cloning site of a pET28a plasmid to obtain a recombinant plasmid; then, the recombinant plasmid is transformed into escherichia coli Shuffle T7 to obtain a recombinant bacterium; the lipase gene selective for the R configuration is the DNA sequence of the following 1) or 2): 1) a DNA sequence shown as SEQ ID NO.2 in the sequence table; 2) a DNA sequence which has more than 90 percent of homology with the DNA sequence limited by 1) and codes the protein with ester hydrolase activity. The recombinant lipase has higher R-configuration stereoselectivity and catalytic activity, the enzyme activity retention is more than 85% under the higher temperature condition, and the recombinant lipase can be used for resolving racemic 2-aryl methyl propionate substances to obtain optically pure S-type 2-aryl methyl propionate substances.

Description

Recombinant bacterium for expressing lipase with R configuration selectivity and application thereof

Technical Field

The invention relates to a lipase AflB with selective R configuration, a coding gene thereof, a vector containing the coding gene, an engineering bacterium and application thereof in chiral resolution of racemic 2-arylpropionic acid substances.

Background

Lipases are widely found in animals, plants and microorganisms, and are enzymes that catalyze hydrolysis or formation of ester bonds, and the substrates for action are usually esters with fatty acid chains greater than ten carbon atoms. The lipase can catalyze various chemical reactions such as hydrolysis, esterification, transesterification and the like, and is an important industrial catalyst. In addition, the broad spectrum/special specificity of the lipase to the substrate endows the lipase with great application value in the industries of ester bond compound synthesis, chiral drug synthesis and the like.

Due to the growing demand for chiral drugs and intermediates, the synthesis or resolution of chiral compounds by biological or enzymatic methods is gaining increasing attention and industrial application. For example, the 2-aryl propionic acid non-steroidal anti-inflammatory drug has the effects of relieving fever, diminishing inflammation and easing pain, is mainly used for treating rheumatic spondylitis, rheumatoid arthritis, postpartum and postoperative pain and the like, and is widely applied clinically. However, the research finds that the 2-aryl propionic acid substance has a pair of optically active enantiomers, and an S-configuration molecule with a real therapeutic effect (for example, the drug effect of (S) -naproxen is 28 times that of (R) -naproxen, and the (R) -naproxen and coenzyme A can form ester, disturb normal membrane function and lipid metabolism of a human body, and bring certain toxic and side effects to the human body). Therefore, pure (S) -2-arylpropionic acid is clinically used for safety of medication. Therefore, it is very important to develop a method for the resolution of 2-arylpropionic acid compounds by an efficient and low-cost enzymatic method.

The key problem in enzymatic resolution of chiral compounds is the search for biocatalysts with highly stereoselective catalytic function. Although lipases are widely distributed in the microbial world, the stereoselective catalytic functions of the lipases are different, and a single enzyme preparation obtained by separating and purifying a microbial fermentation product is often used for catalytic reaction, so that a complex process and high cost are required, and the lipases are not easy to adopt in production.

Disclosure of Invention

The object of the present invention is to solve at least one of the technical problems of the prior art. Therefore, the invention needs to provide an R configuration selective lipase, a vector containing the encoding gene, an engineering bacterium and application thereof.

The technical scheme adopted by the invention is as follows:

a lipase AflB with R-isomer stereoselectivity has an amino acid sequence shown in SEQ ID NO. 1.

Due to the specificity of the amino acid sequence, any fragment of the peptide protein containing the amino acid sequence shown in SEQ ID NO.1 or its variants, such as conservative variants, bioactive fragments or derivatives thereof, as long as the homology of the fragment of the peptide protein or the variant of the peptide protein with the aforementioned amino acid sequence is above 90%, falls into the protection scope of the present invention. Particular such alterations may include deletions, insertions or substitutions of amino acids in the amino acid sequence; where conservative changes to a variant are made, the substituted amino acid has similar structural or chemical properties as the original amino acid, e.g., replacement of isoleucine with leucine, and the variant may also have non-conservative changes, e.g., replacement of glycine with tryptophan.

A plasmid pET28a-AflB containing a lipase AflB coding gene with R configuration selectivity is disclosed, wherein the sequence of the lipase AflB coding gene with R configuration selectivity is shown as SEQ ID NO.2 or a DNA sequence which has homology of more than 90% with SEQ ID NO.2 and codes protein with ester hydrolase activity, and the lipase gene is cloned to pET28a plasmid through whole gene synthesis to obtain the plasmid pET28 a-AflB.

A recombinant bacterium for expressing lipase with R configuration selectivity is obtained by transforming an Escherichia coli host bacterium E.coli Shuffle T7 with a plasmid pET28a-AflB to obtain the recombinant bacterium E.coli Shuffle T7/pET28 a-AflB.

A preparation method of lipase AflB with selective R configuration comprises the following steps: the production strain E.coli Shuffle T7/pET28a-AflB is cultured, and the recombinant lipase AflB with selective R configuration is obtained from the culture.

The preparation method can adopt a culture method which is conventional in the field. For E.coli, it is preferred that: inoculating the recombinant expression transformant to an LB culture medium containing 50 mu g/ml kanamycin, culturing at 37 ℃ and 200rpm until the optical density OD600 of a culture solution reaches 0.8, adding 0.05mM isopropyl-beta-D-thiogalactopyranoside (IPTG) for induction at the induction temperature of 20 ℃, and inducing for 24 hours to obtain the high-efficiency expressed recombinant R configuration selective lipase AflB.

The recombinant R configuration selective lipase AflB is applied to degradation of esters. When the lipase degrades esters, the reaction temperature is 40 +/-10 ℃, and the reaction pH is 7.5 +/-0.5.

The recombinant R configuration selective lipase AflB can hydrolyze any one of the following esters: p-nitrophenol acetate (p-nitrophenylacetate; pNC2), p-nitrophenol butyrate (p-nitrophenylbutyrate; pNC4), p-nitrophenol hexanoate (p-nitrophenylcaproate; pNC6), p-nitrophenol octanoate (p-nitrophenylbutyrate; pNC8), p-nitrophenol decanoate (p-nitrophenylcaprate; pNC10), p-nitrophenol laurate (p-nitrophenyllaurate, pNC12), glyceryl triacetate (Triacetin, TriC2), glyceryl tributyrate (Tributyrin, TriC4), glyceryl tricaaproate (Tricaproin, TriC6), glyceryl tricaprylate (Tricaprylin, TriC8), glyceryl trilaurate (Trilaurin, TriC12), glyceryl tristearate (Tricaprylin, trice 18).

When the recombinant R configuration selective lipase AflB hydrolyzes the esters, the optimal reaction temperature is 40-50 ℃, and the optimal reaction pH is 7.0-8.0. Preference is given to hydrolysis of short-chain fatty glyceride, 2h incubation at 50 ℃ keeps the enzyme activity more than 85%, and the enzyme is resistant to organic solvents such as n-hexane, toluene, diethyl ether and the like.

The application of the recombinant R configuration selective lipase AflB in preparing chiral compounds by stereoselectively catalyzing and resolving racemic substrates.

The racemic substrate is: one or more of naproxen methyl ester ((±) -2- (6-methoxy-2-naphthyl) propionic acid methyl ester), ibuprofen methyl ester ((±) -2- (4-isobutylphenyl) propionic acid methyl ester), flurbiprofen methyl ester ((±) -2- (2-fluoro-4-biphenyl) propionic acid methyl ester), and the like.

Compared with the prior art, the invention has the following advantages and beneficial effects:

(1) advantages with regard to lipases: the lipase has higher R-configuration stereoselectivity and catalytic activity, favors the hydrolysis of short-chain fatty glyceride, has the enzyme activity retention of more than 85% under the condition of higher temperature, and resists organic solvents such as n-hexane, toluene, ether and the like.

(2) The recombinant lipase AflB can be used for resolving racemic 2-aryl methyl propionate substances to obtain optically pure S-type 2-aryl methyl propionate substances (optical purity is more than 98.5%).

(3) The invention provides a method for preparing lipase with high R-configuration stereoselectivity and catalytic activity, which has the advantages that an escherichia coli expression system is applied, and enzyme protein can be obtained efficiently at low cost.

Drawings

FIG. 1 shows the electrophoresis results of the expression and purification process of recombinant R-configuration selective lipase AflB: 1: obtaining a broken whole bacterial liquid after the induction expression of the recombinant bacteria; 2: obtaining a crushing supernatant after the induction expression of the recombinant bacteria; 3: obtaining a crushing precipitation resuspension after the induction expression of the recombinant bacteria; 4: after the recombinant bacteria are induced and expressed, obtaining an eluent which is obtained after the crushed supernatant passes through a Ni column; 5: the eluent obtained after the Ni column eluent passes through Q Sepharose FF; m: low molecular weight protein molecule Marker.

FIG. 2 shows the specific activity of the recombinant R-configuration selective lipase AflB at different pH.

FIG. 3 shows the specific activity of the recombinant R configuration-selective lipase AflB at different temperatures.

FIG. 4 shows the specific activity of the recombinant R-configuration selective lipase AflB on different substrates.

FIG. 5 shows the thermostability of recombinant R configuration-selective lipase AflB at various temperatures.

FIG. 6 is a graph of (S) naproxen methyl ester, (R) naproxen and (S) naproxen separated by chromatography after the catalytic reaction of the lipase AflB with the recombinant R configuration selectivity.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Example 1

Discovery of lipase AflB with selective R configuration

Through the mining of protein databases, an amino acid sequence of Aspergillus fumigatus Af293lipase (GenBank: XP-749106) is found, and the lipase shows lower sequence Identity of 31 percent with the lipase CalB widely used in industry and contains an N-terminal alpha helical domain which is not possessed by CalB. Through homology modeling and substrate molecule docking, the N-terminal alpha helical domain is found to be beneficial to the combination of R-configuration naproxen ((-) -2- (6-methoxy-2-naphthyl) propionic acid). And on the basis of the molecular docking, the corresponding amino acid residues of the substrate pocket are replaced by computer simulation, and Aspergillus fumigatus Af293lipase is further modified to be more prone to be combined with R-configuration naproxen. Finally, the modified Aspergillus fumigatus Af293lipase was named as R configuration selective lipase AflB, which consists of 438 amino acid residues and has a theoretical isoelectric point of 5.4.

The gene coding the lipase AflB with the R configuration selectivity is named as lipase AflB, and the open reading frame of the lipase AflB is shown as SEQ ID NO.2 and consists of 1314 nucleotides.

Example 2

Expression and purification of lipase AflB with R-configuration selectivity

Construction of recombinant plasmid

The sequence of the lipase AflB coding gene is shown as SEQ ID NO.2, the lipase AflB gene is obtained through whole gene synthesis, and then the lipase AflB gene is cloned to pET28a plasmid to obtain plasmid pET28 a-AflB.

II, obtaining of recombinant R configuration selective lipase AflB producing strain

Coli Shuffle T7Competent E.coli (Cat. C3026, NEB) Competent cells were thawed on ice, 50ul was transferred to a pre-cooled 1.5ml centrifuge tube with a pre-cooled pipette tip, then the recombinant plasmid pET28a-AflB 1ul was added, gently shaken and mixed well, and left to stand in ice for 30 minutes. Quickly transferred to a 42 ℃ water bath and accurately placed for 40 seconds. Transfer quickly to ice and cool for 2 minutes. 500ul of LB medium was added to each tube and incubated at 37 ℃ for 45 minutes. 20ul of the suspension was spread on antibiotic-containing plates (50mg/m kanamycin) and cultured overnight. Obtain recombinant E.coli Shuffle T7/pET28 a-AflB.

Expression and purification of recombinant R configuration selective lipase AflB

1. Preparation of lipase AflB protein solution with selective R configuration

(1) Selecting recombinant bacteria E.coli Shuffle T7/pET28a-AflB, adding into 2L LB liquid culture medium (containing 50ug/ml kanamycin), and shake culturing at 37 ℃ until OD600 is about 0.8; adding inducer IPTG (final concentration is 0.05mM), and inducing for 24h at 20 ℃; the cells were centrifuged at 6000rpm for 10 minutes, and then the cells were collected, and 50mM phosphate buffer (pH7.4) (10 mL buffer per gram of cells) was added, and then they were sonicated for 10 minutes (60W, sonication for 2 seconds, stop for 2 seconds), and centrifuged at 10000rpm for 20 minutes, and then the supernatant was collected, i.e., a crude enzyme solution (protein concentration 12mg/mL, total volume about 250 mL).

(2) Loading the crude enzyme solution on 5mL of IDA agarose with Ni, which is produced by GE Healthcare, pre-packed column; the column was then washed with 20ml of solution A (20mM pH7.4PB, 0.5M NaCl, 25mM imidazole) to remove most of the hetero-proteins; then eluted with 20ml of solution B (20mM pH7.4PB, 0.5M NaCl, 125mM imidazole) and the eluate was collected (protein concentration 1.5mg/ml, total volume about 25 ml).

(3) Desalting the eluate with a fast protein liquid chromatography system (FPLC; GE Healthcare): the eluate with the protein peak, i.e., desalted eluate, was collected using a G25 desaling column (GE Healthcare) with equilibration and elution buffer 20mM pH7.9Tris-Cl at a flow rate of 2 mL/min.

(4) And (3) carrying out anion exchange chromatography on the desalted eluent, wherein the specific parameters are as follows: q Sepharose FF (1.0X 5 cm); the column was first washed with 20ml of solution QA (20mM pH7.9Tris-Cl) to remove part of the hetero proteins; then eluted with 20ml of solution B (20mM pH7.9Tris-Cl, 50mM NaCl) and the eluate (protein concentration 1mg/ml, total volume about 20ml) was collected.

(5) And (3) passing the eluent obtained in the step (4) through an ultrafiltration tube AmiconUltra-15(Millipore) with the cutoff molecular weight of 10KDa, concentrating the volume to 1ml, namely obtaining the lipase AflB protein solution with the R configuration selectivity (the protein concentration is 20mg/ml), and subpackaging the solution into 100ul by using an EP tube for preservation at the temperature of-80 ℃ for later use.

2. The solutions obtained in step 1 were subjected to polyacrylamide gel electrophoresis, as shown in FIG. 1.

Fourthly, enzyme activity determination

1. Colorimetric method for measuring esterase hydrolysis activity

Determination of the standard curve: mu.L, 2 mu.L, 4 mu.L, 6 mu.L, 8 mu.L and 10 mu.L of 10mM p-nitrophenol are respectively put on a 96-well enzyme label plate, each well is filled to 100 mu.L by using a corresponding buffer solution, then 100 mu.L of isopropanol is added, and after uniform mixing, the absorbance at 405nm is measured, so that the relation between the absorbance (Y axis) and the concentration (X axis) of the p-nitrophenol is obtained.

And (3) determination of a sample: to a 96-well plate, 80. mu.L of 50mM phosphate buffer (pH7.0), 10. mu.L of 10mM p-nitrophenol octanoate (p-nitrophenyl captylate; pNC8) (Sigma) were sequentially added, and the plate was placed in an incubator at 40 ℃ and preheated for 5min, followed by addition of 10. mu.L of an appropriately diluted enzyme solution, and after 15min of reaction, 100. mu.L of isopropanol was added to terminate the reaction, and the absorbance at 405nm was measured. The blank group was boiled enzyme solution. Three replicates were used and the average was recorded.

Enzyme activity (U): under certain conditions, the enzyme quantity required by the lipase to generate 1 mu mol of p-nitrophenol in a unit time is one lipase activity unit (U).

The specific enzyme activity of the lipase AflB with the R configuration selectivity of the eluent after the p-nitrophenol caprylate is taken as a substrate and the Q Sepharose FF purification is 500 +/-40U/mg.

2. Determination of esterase hydrolysis Activity by alkali titration

Adding 4mL of tributyrin emulsion (wherein the content of tributyrin is 25% (v/v), the content of 4% polyvinyl alcohol is 75% (v/v) and homogenizing at 10000rpm for 10min) and 5mL of 50mM phosphate buffer (pH7.0) into a triangular flask, preheating for 5min in a constant-temperature magnetic stirrer at 40 ℃, then adding 1mL of appropriately diluted enzyme solution, starting timing, and immediately adding 15mL of 95% ethanol after carrying out heat preservation reaction for 15min to terminate the reaction; adding phenolphthalein indicator, titrating free fatty acid released by hydrolysis with a calibrated solution of about 50mM KOH, and recording the volume of KOH consumed; the blank test is that 95% ethanol is added into the preheated reaction system, then 1mL of enzyme solution subjected to heat inactivation treatment is added, the reaction is carried out for 15min after the reaction system is cooled to room temperature, and other operations are the same as the previous operations.

The definition of enzyme activity is: under the above conditions, the amount of enzyme required to produce 1. mu. mol of titratable fatty acid per unit time (per minute) was 1 activity unit (U).

The specific enzyme activity of the lipase AflB with the R configuration selectivity of the eluent after Q Sepharose FF purification by using tributyrin as a substrate is 11000 +/-320U/mg.

Example 3

Determination of enzymatic Properties of recombinant R configuration-Selective Lipase AflB

First, recombination R configuration selective lipase AflB optimum reaction pH value

The relative enzyme activities of different pH's for the recombinant R configuration-selective lipase AflB of example 2 were determined at 40 ℃ using tributyrin as substrate at pH 3.0, 4.0 and 5.0(50mM sodium acetate buffer), pH 6.0 and pH7.0(50mM phosphate buffer), pH 8.0(50 mM Tris-HCl buffer), pH 9.0 and pH10.0(50mM glycine buffer), respectively (FIG. 2).

Secondly, the optimal reaction temperature of the recombinant R configuration selective lipase AflB

The relative enzyme activity of the recombinant R-configuration selective lipase AflB of example 2 was determined by performing catalytic reactions at 20 ℃, 30 ℃, 40 ℃, 45 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃ respectively, using tributyrin as a substrate, at pH7.0(50mM phosphate buffer) (FIG. 3).

Thirdly, the selectivity of the lipase AflB with the selective recombinant R configuration on substrates with different chain lengths

The relative enzyme activity of the recombinant R configuration-selective lipase AflB of example 2 was determined by performing catalytic reactions at 40 ℃ under pH7.0(50mM phosphate buffer) using respective emulsions of glyceryl triacetate (TC2), tributyrin (TC4), glyceryl trioctanoate (TC8), glyceryl trilaurate (TC12) and Olive oil (Olive oil) as substrates (FIG. 4).

Temperature stability of recombined R configuration selective lipase AflB

An amount of the recombinant R configuration-selective lipase AflB of example 2 was incubated at 30 ℃ at 50 ℃ at 60 ℃ at 70 ℃, and samples were taken at the time points of 0min, 10min, 20min, 40min and 70min of incubation, and the relative enzyme activity of each sample was determined using tributyrin as a substrate at 40 ℃ and pH7.0(50mM phosphate buffer) (FIG. 5).

Fifthly, tolerance of the recombinant R configuration selective lipase AflB to different organic solvents

The recombinant R configuration selective lipase AflB of example 2 was incubated at a ratio of 50% with various organic solvents at 4 ℃ for 2h, and then the relative enzyme activity of each sample was determined using tributyrin as a substrate at 40 ℃ and pH7.0(50mM phosphate buffer) (Table 1).

TABLE 1 Effect of organic solvents on the Activity of recombinant R configuration-selective Lipase AflB

From the results, when the lipase AflB with the selective recombinant R configuration hydrolyzes the esters, the optimal reaction temperature is 40-50 ℃, the optimal reaction pH is 7.0-8.0, short-chain fatty glyceride is preferentially hydrolyzed, the enzyme activity is kept more than 85% after 2h of incubation at 50 ℃, and the lipase AflB is resistant to organic solvents such as n-hexane, toluene, ether and the like.

Example 4 application of recombinant R configuration selective lipase AflB in stereoselective catalytic resolution of racemic 2-arylpropionic acid substances

Taking the recombinant R configuration selective lipase AflB in example 2, hydrolyzing racemic naproxen methyl ester ((+ -) -2- (6-methoxy-2-naphthyl) methyl propionate), ibuprofen methyl ester ((+ -) -2- (4-isobutylphenyl) methyl propionate) and flurbiprofen methyl ester ((+ -) -2- (2-fluoro-4-biphenyl) methyl propionate) and other substrates, wherein the reaction solvent is 50mM pH7.0 phosphoric acid-hydrochloric acid buffer solution, the substrate concentration is respectively 5% (volume ratio), the enzyme dosage is 0.05mg/mL, and the reaction temperature is 25 ℃. After 150min of reaction, the reaction product was analyzed for the yield and optical purity of the (S) -type ester substrate by high performance liquid chromatography and chiral column (chiralcl OJ, Japan, Daicel chemical,4.6mmx250mm), as shown in fig. 5, in which the liquid chromatography retention times of (S) -naproxen methyl ester ((S) - (+) -2- (6-methoxy-2-naphthyl) propionic acid), R) -naproxen ((R) - (-) -2- (6-methoxy-2-naphthyl) propionic acid), and (S) -naproxen ((S) - (+) -2- (6-methoxy-2-naphthyl) propionic acid) were 6.9min, 11.4min, and 12.3min, respectively, by the following specific operating methods: mobile phase n-hexane/isopropanol (95/5, v/v, 1.0mL/min), column temperature: the sample concentration is 0.1-1.0mmol/mL at 28 ℃, the pressure range is 0-9.0MPa, and the detection wavelength is UV254 nm.

TABLE 2 results of reactions for the catalytic resolution of different racemic 2-arylmethyl propionates by recombinant ester hydrolases

From the results, different racemic 2-aryl propionic acid substrates are catalyzed and hydrolyzed by lipase AflB with selective recombination R configuration, and corresponding S-configuration methyl ester products with optical purity of more than 98.6% are obtained. The recombinant R configuration selective lipase AflB has good application prospect in the field of preparing optically pure S configuration 2-aryl propionic acid medicines.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the technical scope of the present invention in any way. Any simple modification, equivalent change and modification of the above embodiments according to the technical spirit of the present invention fall within the scope of the present invention.

Sequence listing

<110> university of southern China's science

<120> recombinant bacterium for expressing lipase with R configuration selectivity and application thereof

<160>2

<170>SIPOSequenceListing 1.0

<210>1

<211>438

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>1

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

15 10 15

Leu Thr Val Ile Thr Thr Gly Gly Gln Glu Thr Thr Leu Asp Thr Asp

20 25 30

Ala Lys Ser Ile Ala Ser Leu Ile Ala Ser Glu Ile Val Ser Lys Ile

35 40 45

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

50 55 60

Glu Ala Phe Asp Lys Ile Gln Ser Ile Phe Ala Asp Gly Thr Pro Asp

65 70 75 80

Phe Leu Lys Met Thr Arg Glu Ile Leu Thr Val Gly Leu Ile Pro Ala

85 90 95

Asp Ile Leu Ser Phe Leu Asn Gly Tyr Leu Asn Leu Asp Leu Asn Ser

100 105 110

Ile His Asn Arg Asn Pro Ser Pro Lys Gly Gln Ala Ile Tyr Pro Val

115 120 125

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

130 135 140

Ala Ala Ile His Ile Pro Ala Ser Phe Gly Tyr Gly Lys Asn Gly Lys

145 150 155 160

Lys Pro Val Ile Leu Val Pro Gly Thr Ala Thr Pro Ala Gly Thr Thr

165170 175

Tyr Tyr Phe Asn Phe Gly Lys Leu Gly Ser Ala Ala Asp Ala Asp Val

180 185 190

Val Trp Leu Asn Ile Pro Gln Ala Ser Leu Asn Asp Val Gln Ile Asn

195 200 205

Ser Glu Tyr Val Ala Tyr Ala Ile Asn Tyr Ile Ser Ala Ile Ser Glu

210 215 220

Ser Asn Val Ala Val Leu Ser Trp Ser Gln Gly Gly Leu Asp Thr Gln

225 230 235 240

Trp Ala Leu Lys Tyr Trp Pro Ser Thr Arg Lys Val Val Asp Asp Phe

245 250 255

Ile Ala Ile Ser Pro Asp Phe His Gly Thr Val Leu Ala Gly Pro Leu

260 265 270

Asp Ala Leu Ala Val Ser Ala Pro Ser Leu Trp Gln Gln Gly Trp Asn

275 280 285

Thr Glu Phe Ile Arg Thr Leu Arg Gly Gly Gly Gly Asp Ser Ala Tyr

290 295 300

Val Pro Thr Thr Thr Ile Tyr Ser Thr Phe Asp Glu Ile Val Gln Pro

305 310 315 320

Met Ser Gly Ser Gln Ala Ser Ala Ile Leu Ser Asp Ser Arg Ala Val

325330 335

Gly Val Ser Asn Asn His Leu Gln Thr Ile Cys Gly Gly Lys Pro Ala

340 345 350

Gly Gly Val Tyr Thr His Glu Gly Val Leu Tyr Asn Pro Leu Ala Trp

355 360 365

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

370 375 380

Arg Leu Asp Leu Asp Val Val Cys Gly Arg Val Leu Pro Pro Gln Leu

385 390 395 400

Thr Pro Glu Gln Lys Val Ala Ala Ala Ala Leu Leu Ala Pro Ala Ala

405 410 415

Ala Ala Ile Val Ala Gly Tyr Lys Pro Lys Thr Phe Gly Glu Pro Ala

420 425 430

Ile Ala Ser Tyr Ala His

435

<210>2

<211>1314

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

gcggtgattc cgcgtggagc ggtgccggtt gccagcgacc tgaccatact gaccgtgatt 60

accaccggag ggcaggaaac caccctggac accgatgcga aatctattgc gtctctgatt 120

gcgagcgaga ttgtgagcaa aataggcaag acggaattta gccgtagcac caaagatgca 180

aaatctgtgc aggaagcgtt tgataagatt caaagcattt ttgcggatgg caccccggat 240

tttcttaaaa tgacccgtga aattctgacg gtgggcctta tacccgctga tattcttagc 300

tttctgaatg gctatctgaa tctggatctt aatagcattc ataatcgtaa ccctagcccg 360

aaaggccagg ccatttatcc ggtgaaagcg cctggcgacg cacgttatag cgtagcggag 420

aacgccttac gtgcagctat tcacatcccg gcgagctttg gctatggaaa gaatggcaaa 480

aaaccggtga tcctggttcc ggggaccgcg accccagcgg gcaccaccta ttacttcaat 540

tttggtaaac tgggctcggc tgcagatgcg gacgtggtgt ggttaaacat accgcaggcg 600

tcactgaatg acgtccaaat aaacagcgaa tatgtggcgt atgcgataaa ctatattagc 660

gccatttccg aaagtaacgt ggcagttctg agctggagcc aaggcggcct ggatacccag 720

tgggcgctga aatattggcc gagtacccgt aaagtggtgg atgatttcat agcgattagc 780

ccggacttcc atggtaccgt gctggcaggg ccgctggatg cgctggcggt gagcgcaccc 840

agcctgtggc aacagggctg gaataccgag tttatacgga ccctgcgtgg cggagggggc 900

gacagcgcgt atgtgccgac caccacgatt tattcgacct ttgatgaaat tgtgcaaccg 960

atgtccggct cccaggcgag cgcgattttg tcggattcac gtgcggtggg agttagtaac 1020

aaccatttgc agacaatttg cgggggtaaa cctgctgggg gcgtgtatac ccatgaaggc 1080

gtgctgtata atccgcttgc atgggcactg gcagtggatg ccttaagcca tgatggccca 1140

ggggatccga gccgtctgga tttggatgtg gtttgcggcc gtgttttgcc tccacaactg 1200

actccggaac agaaagtggc ggcagcggcg cttctggctc ctgccgcagc cgcgattgtg 1260

gctggctaca aaccgaaaac cttcggtgaa ccggcgattg ctagttatgc acat 1314

Claims

1. A recombinant bacterium for expressing an R configuration selective lipase is characterized in that the R configuration selective lipase gene is inserted into a recombinant plasmid obtained by a multiple cloning site of a pET28a plasmid; then, the recombinant plasmid is transformed into escherichia coli Shuffle T7 to obtain a recombinant bacterium;

the lipase gene selective for the R configuration is as follows: the DNA sequence shown in SEQ ID NO. 2.

2. The use of the recombinant bacterium according to claim 1 for degrading esters, wherein the esters are any one or more of p-nitrophenol acetate, p-nitrophenol butyrate, p-nitrophenol hexanoate, p-nitrophenol octanoate, p-nitrophenol decanoate, p-nitrophenol laurate, glycerol triacetate, glycerol tributyrate, glycerol trihexanoate, glycerol trioctanoate, glycerol trilaurate, and glycerol tristearate.

3. The use according to claim 2, wherein the lipase is used for the degradation of esters at a reaction temperature of 40 ± 10 ℃ and a reaction pH of 7.5 ± 0.5.

4. The recombinant strain of claim 1 is applied to stereoselective catalytic resolution of racemic 2-aryl propionic acid substances.

5. The use according to claim 4, wherein the racemic 2-arylpropionic acid-based substance is any one or more of naproxen methyl ester ((±) -2- (6-methoxy-2-naphthyl) propionic acid methyl ester), ibuprofen methyl ester ((±) -2- (4-isobutylphenyl) propionic acid methyl ester), and flurbiprofen methyl ester ((±) -2- (2-fluoro-4-biphenyl) propionic acid methyl ester).