CN117821429A - Recombinant protease mutant, coding gene, vector, engineering bacterium and application of recombinant protease mutant in resolution of (R, S) -2-tetrahydrofurfuryl acid ethyl ester - Google Patents

Abstract

The invention discloses a recombinant protease mutant, a coding gene, a vector, engineering bacteria and resolutionR,S) -the use of ethyl 2-tetrahydrofurfuryl acid, said use being: by bacillus subtilisThe supernatant crude enzyme solution obtained by fermenting and culturing SCK6 or the purified pure enzyme solution obtained by purifying the crude enzyme solution is used as a catalyst, and racemization is carried outR,S) The tetrahydrofurfuryl acid ethyl ester is taken as a substrate, phosphate buffer with pH of 8.0 is taken as a reaction medium, and resolution preparation is carried out under the conditions of 10-60 ℃ and 500-1500rpmS) -2-tetrahydrofurfuryl acid ethyl ester and%R) -2-tetrahydrofurfuryl acid. The enzyme-producing microorganism strain bacillus subtilis B.subtilisSCK6/pWB980-BLAP (Y310E) provided by the invention has strong stereoselectivityS) -2-tetrahydrofurfuryl acid ethyl ester with ee value (optical purity) not less than 99.9%, and process for preparing the sameR) -2-tetrahydrofurfuryl acid with ee value (optical purity) > 68.63% conversion 59.3% and E value 105.5. The chiral auxiliary is avoided in chemical production, the energy consumption is low, the reaction time is short, the environmental pollution is small, and the method has potential of industrial production.

Description

Recombinant protease mutant, coding gene, vector, engineering bacterium and application of recombinant protease mutant in resolution of (R, S) -2-tetrahydrofurfuryl acid ethyl ester

Technical Field

The invention relates to the field of molecular biology and biotechnology, in particular to a method for preparing a protease mutant with stereoselectivity and a coding gene thereof by utilizing a molecular transformation technology, and a carrier, engineering bacteria and application of the coding gene.

Background

The antibiotic faropenem has broad-spectrum activity on gram-positive bacteria, gram-positive bacteria and the like except pseudomonas aeruginosa, and is used for urinary system infection, respiratory system infection, adnexitis, intrauterine infection and bartholinitis; superficial skin infections, deep skin infections, and the like. The (S) -2-tetrahydrofurfuryl acid ethyl ester and the (R) -2-tetrahydrofurfuryl acid can be used for synthesizing cephalosporin antibiotics and azolidines, and in addition, the (R) -2-tetrahydrofurfuryl acid can also be used as an intermediate for synthesizing carbapenem antibiotics faropenem, so that the method has wide market prospect in the fields of medicine and chemical industry. At present, the synthesis of optically pure (S) -tetrahydrofurfuryl acid ethyl ester is mainly based on a chemical method. However, the chiral auxiliary used by the chemical method is expensive, the recycling rate of the resolving agent is low, and the reaction steps are complicated, so that the commercialized application of the chiral auxiliary is limited. Enzymatic resolution is increasingly favored by virtue of its mild reaction conditions, high stereoselectivity and environmental friendliness.

At present, the synthesis of the (S) -2-tetrahydrofurfuryl acid ethyl ester and the (R) -2-tetrahydrofurfuryl acid mainly comprises a chiral auxiliary recrystallization method and a kinetic enzyme method resolution method. Chiral auxiliary recrystallization method: the principle of recrystallization is to use an optically active compound as an auxiliary agent to selectively precipitate a specific optical isomer as a salt and separate the other optical isomer to obtain a pure product. Alajorin uses strychnine and ephedrine to catalytically decompose tetrahydrofuran into diastereoisomeric salts, and finally recrystallizes according to the solubility difference of the two enantiomer salts to obtain (R) -tetrahydrofurfuryl acid with the eep value of 95% and (S) -tetrahydrofurfuryl acid with the ees value of 98%. Hμ et al select O, O' -dibenzoyltartaric acid as resolving agent, and after recrystallization, the (S) enantiomer eS reaches 99.3%, yield 55.2%, and because calcium oxide is consumed very much in the reaction process, serious pollution problems exist, and the subsequent treatment cost is high, the chiral auxiliary recrystallization method cannot be used for large-scale production of (S) -2-tetrahydrofurfuryl acid ethyl ester, (R) -2-tetrahydrofurfuryl acid

Kinetic enzymatic resolution method: enzymatic resolution in kinetic resolution relies primarily on the difference in the reaction rates of the two enantiomers relative to the enzyme to effect resolution, and when one configuration has a much greater reaction rate than the other, the enantiomer monomer is rapidly formed in the reaction. The Aspergillus protease is obtained by screening YoshitoF mu jima and the like, the reaction time is more than 20 hours, the keeper value can reach 94.4 percent, then the keeper value can reach more than 99 percent through precipitation crystallization of methanol/methyl ethyl ketone (1:5), and the total yield in the reaction liquid is 23 percent. The method has the advantages of low cost, small pollution and mild reaction conditions, is suitable for large-scale industrial production, and can gradually replace chemical reagents in dynamic resolution due to excellent substrate specificity of enzyme as an important tool in chiral resolution.

Along with the development and updating of biotechnology, enzymatic catalysis becomes a research hotspot in biochemical industry, and at present, the acquisition of a target product by enzymatic resolution is becoming popular, and the resolution effect of a substrate can be remarkably improved by using genetically engineered enzyme. In addition, the enzymatic resolution has the advantages of simple reaction steps, low energy consumption, few byproducts, no pollution and the like. Therefore, the engineering enzyme after transformation is used for resolving the (R, S) -2-tetrahydrofurfuryl acid ethyl ester, which is hopeful to replace a chemical method to realize industrial application.

Disclosure of Invention

The invention aims to provide a high-stereoselectivity recombinant protease mutant, a coding gene, a recombinant vector containing the gene, recombinant genetic engineering bacteria obtained by transforming the recombinant vector, and application of the recombinant genetic engineering bacteria in resolution of (R, S) -2-tetrahydrofurfuryl acid ethyl ester by a biological method, and solves the technical problem that ee. value is low in the process of preparing (S) -2-tetrahydrofurfuryl acid ethyl ester and (R) -2-tetrahydrofurfuryl acid by using wild recombinant protease. When the conversion rate of the recombinant protease mutant reaches 59.3%, the ees of the (S) -2-tetrahydrofurfuryl acid ethyl ester reaches 99.9%, and the yield is 5.86g/L; the keep of (R) -2-tetrahydrofurfuryl acid above 68.63% and the yield is 5.79g/L.

The technical scheme adopted by the invention is as follows:

the invention provides a high stereoselectivity recombinant protease mutant, wherein the amino acid sequence of the recombinant protease mutant is shown as BLAPIDNO. 1:

MMRKKSFWLGMLTAFMLVFTMAFSDSASAAQPAKNVEKDYIVGFKSGVKTASVKK

DIIKE

SGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIK

ADK

VQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTV

AALD

NTTGVLGVAPSVSLYAVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTA

MK

QAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVG

AEL

EVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSST

ATY

LGSSFYYGKGLINVEAAAQLEHHHHHH

because of the specificity of the amino acid sequence, all polypeptide fragments and variant types thereof, such as conservative variants, biologically active fragments or derivatives, comprising an amino acid sequence as set forth in BLAPIDNO.1 are within the scope of the invention if the polypeptide fragment or polypeptide variant has more than 90% homology to the amino acid sequence and has the same enzymatic activity. In particular, such alterations as insertions, deletions, substitutions of amino acids; or the amino acid replaced has a similar structure or chemical nature to the original amino acid, for example, replacement of leucine with isoleucine; or a non-conservative change, such as the replacement of methionine with glycine.

The protein may be in the form of a natural protein, a recombinant protein, a synthetic protein, or synthesized from a prokaryotic or eukaryotic host using recombinant techniques. Depending on the host used for recombinant production, the protein of the invention may be glycosylated in its form. The proteins of the invention may or may not contain an initial methionine residue.

The invention also relates to a coding gene of the recombinant protease mutant, and the nucleotide sequence of the coding gene is shown as BLAPIDNO. 2:

ATGATGCGGAAGAAGAGCTTTTGGCTGGGCATGTTAACAGCATTTATGTTGGTGTTTACAATGGCGTTTTCAGATAGCGCGTCAGCAGCACAACCGGCGAAAAACGTGGAAAAGGATTATATCGTTGGATTTAAAAGCGGCGTTAAAACGGCATCAGTTAAGAAGGATATTATTAAAGAATCAGGCGGCAAAGTTGATAAACAATTTAGAATCATCAACGCAGCAAAAGCAAAACTGGATAAAGAAGCACTGAAAGAAGTGAAAAATGATCCGGATGTGGCATATGTGGAAGAAGATCATGTAGCACATGCACTTGCACAAACAGTTCCGTATGGCATTCCGCTGATTAAAGCAGATAAAGTGCAAGCCCAAGGATTTAAAGGCGCAAATGTGAAAGTTGCGGTGCTGGATACAGGCATTCAAGCCTCACATCCGGATTTAAATGTTGTTGGAGGCGCAAGCTTTGTGGCAGGCGAAGCTTATAATACAGATGGCAATGGACATGGCACACATGTGGCAGGAACAGTGGCAGCACTGGATAATACAACAGGCGTGCTGGGCGTGGCACCGTCAGTTTCACTTTATGCAGTAAAAGTTCTGAACTCATCAGGATCAGGATCATATTCAGGAATTGTTAGCGGAATTGAATGGGCAACAACAAATGGCATGGATGTTATTAATATGTCACTGGGCGGCGCTAGCGGATCAACAGCAATGAAACAAGCGGTGGATAATGCGTATGCAAGAGGCGTGGTTGTGGTTGCGGCGGCAGGAAATAGCGGAAGCTCAGGAAATACAAATACGATTGGATATCCGGCAAAATATGATTCAGTTATTGCAGTTGGCGCAGTGGATAGCAATTCAAATAGAGCATCATTTTCATCAGTGGGCGCAGAACTGGAAGTTATGGCACCGGGCGCAGGCGTTTATTCAACATATCCGACAAATACATATGCAACACTTAATGGCACATCAATGGCGAGCCCGCATGTTGCGGGCGCAGCAGCATTAATTCTTTCAAAACATCCGAATTTGAGCGCATCACAAGTCAGAAATAGACTGTCATCAACAGCAACATATCTGGGATCAAGCTTTTATTATGGCAAAGGACTGATTAACGTTGAAGCAGCGGCTCAACTGGAACATCATCATCATCATCATTAA

the recombinant esterase mutant coding gene is obtained by the following method: a pair of mutation primers Y310E-F (5'-CGGGCGCAGGCGTTGAATCAACATATCCGACAAA-3'), Y310E-R (5'-TGTCGGATATGTTGATTCAACGCCTGCGCCCGGT-3') were designed using pWB980-BLAP as a template plasmid, and whole plasmid amplification was performed on a PCR apparatus. The amplified product obtained was transformed into Bacillus subtilis SCK6, which was then spread on a LB liquid medium plate containing 0.5-2. Mu.g/mL of erythromycin resistance and 40-60. Mu.g/mL of kanamycin, and placed in a constant temperature incubator at 37℃for 12-16 hours. And (3) picking single colonies from the cultured flat plates for colony PCR verification, selecting positive single colonies, and sending to sequencing to obtain the pWB980-BLAP (Y310E) recombinant vector with correct sequencing, wherein the stereoselective recombinant protease mutant gene is positioned in the recombinant vector.

Because of the specificity of the nucleotide sequence, all variants of the polynucleotide shown in BLAPIDNO.2 that have more than 70% homology and identical function to the polynucleotide are within the scope of the invention. Variants of the polynucleotides refer to polynucleotide sequences that alter one or more nucleotides therein.

The invention further relates to a recombinant plasmid pWB980-BLAP (Y310E) containing the recombinant protease mutant coding gene and a recombinant genetic engineering bacterium B.subtilisSCK6/pWB980-BLAP (Y310E) obtained by transforming a host bacterium with the recombinant plasmid; the recombinant plasmid takes pWB980 as a basic plasmid, and the host bacteria are preferably bacillus subtilis SCK6.

The invention further relates to application of the recombinant protease mutant coding gene in synthesis of recombinant protease mutants, and the application is as follows: construction of recombinant plasmid containing the recombinant protease mutant GenepWB980BLAP (Y310E), the recombinant plasmid was transformed into B.subtilisSCK6, and the resulting recombinant B.subtilisSCK6/pWB980-BLAP (Y310E). After the recombinant bacillus subtilis is fermented and induced, the fermentation liquor is separated by centrifugation to obtain supernatant containing the protease mutant. Passing the supernatant through Ni-NTA affinity chromatography column, and the target protein is purifiedAnd (3) carrying out column adsorption, passing through a hetero-protein flow, and finally eluting by using an eluent to obtain purified recombinant protease mutant BLAP (Y310E) pure enzyme.

The invention further relates to application of the recombinant protease mutant BLAP (Y310E) in resolution of (R, S) -2-tetrahydrofurfuryl acid ethyl ester, wherein the application is to use a supernatant crude enzyme solution obtained after fermentation induction of engineering bacteria containing recombinant protease mutant coding genes, pure enzyme obtained by purifying the crude enzyme solution is used as a catalyst, the (R, S) -2-tetrahydrofurfuryl acid ethyl ester is added as a substrate, the reaction is carried out in PB buffer with pH value of 8.0 and 100mM at the reaction condition of 10-60 ℃ and 500-1500rpm (preferably 30 ℃ and 1000rpm for 3 hours), and finally 0.4M trichloroacetic acid 200 mu L is added to inactivate the enzyme in a reaction system, and the reaction process is stopped. 100. Mu.L of the reaction mixture was sampled every 1 hour, and 500. Mu.L of ethyl acetate was added for extraction. And after the extraction is finished, 400 mu L of an upper organic phase is added with a proper amount of anhydrous magnesium sulfate for water removal, 200 mu L of supernatant fluid is taken through an organic film and placed in an inner cannula of a gas-phase bottle, and the content of each component in a sample is detected by utilizing gas chromatography. After the reaction is finished, the reaction liquid is separated and purified to obtain (S) -tetrahydrofurfuryl acid ethyl ester and (R) -2-tetrahydrofurfuryl acid. The final concentration of the substrate added is 14.4mg/mL calculated by the buffer volume; the catalyst was added in an amount of 15mg/50mL buffer based on the crude enzyme concentration and 1.5mg/50mL buffer based on the pure enzyme concentration.

The gas phase detection condition of the final product comprises: chromatographic column: CP-Chirasil-DexCB (25 m,0.25mm,0.25 μm, agilent), hydrogen flow rate: 36mL/min, air flow rate: 350mL/min, nitrogen flow rate 2mL/min, injector temperature: 250 ℃, detector temperature: 250 ℃, column temperature: 100 ℃, split ratio 50:1 (substrate)/20:1 (product), column equilibration time: 7min. Substrate enantiomeric excess (ees), product enantiomeric excess (eep), conversion (C), enantiomer ratio (E).

The crude enzyme solution and the pure enzyme solution are obtained according to the following steps: inoculating engineering bacteria bacillus subtilis SCK6/pWB980-BLAP (Y310E) containing recombinant protease mutant coding genes into an LB liquid culture medium containing erythromycin resistance of 1 mug/mL and kanamycin of 50 mug/mL, and culturing for 12-16h at 37 ℃ and 200rpm to obtain seed liquid; transferring the seed solution into TB liquid culture medium containing erythromycin of 1 mug/mL and kanamycin of 50 mug/mL according to the inoculation amount of 2% of volume concentration, carrying out shaking culture at 37 ℃ and 200rpm for 48 hours, centrifuging the fermentation liquid (at 4 ℃ and 12000rpm for 10 minutes), and taking the supernatant as crude enzyme liquid. The crude enzyme solution was concentrated in a 10kD ultrafiltration tube (4 ℃ C., 4000Xg for 40 min), and purified by an AKTA protein purifier to obtain a purified enzyme solution.

Compared with the prior art, the invention has the beneficial effects that: the invention carries out site-directed mutagenesis on wild protease BLAP to obtain mutants with improved stereoselectivity. The mutant recombinant plasmid is transformed into bacillus subtilis SCK6 to construct bacillus subtilis SCK6/pWB980-BLAP (Y310E) engineering bacteria; the engineering bacteria are subjected to low-temperature centrifugation, ultrafiltration and purification to obtain recombinant protease mutant BLAP (Y310E); the bacillus subtilis SCK6/pWB980-BLAP (Y310E) engineering bacteria or recombinant protease mutant BLAP (Y310E) is utilized to split (R, S) -2-tetrahydrofurfuryl acid ethyl ester by a biocatalyst to obtain products (S) -2-tetrahydrofurfuryl acid ethyl ester and (R) -2-tetrahydrofurfuryl acid, the ees of the products can reach more than 99.9 percent, the eep is 68.63 percent, the E value is 105.5, the conversion rate is 59.3 percent,

drawings

FIG. 1 shows linearized pWB980-BLAP (Y310E) agarose gel electrophoresis.

FIG. 2 is a map of the constructed recombinant plasmid pWB980-BLAP (Y310E).

FIG. 3 is a diagram of protein electrophoresis after purification of recombinant protease mutant BLAP (Y310E).

FIG. 4 is a high pressure gas chromatography of crude enzyme solution of Bacillus subtilis SCK6/pWB980-BLAP for resolution of racemic (R, S) -2-tetrahydrofurfuryl acid ethyl ester to give (S) -2-tetrahydrofurfuryl acid ethyl ester.

FIG. 5 is a high pressure gas phase chromatogram of crude enzyme solution resolution of Bacillus subtilis SCK6/pWB980-BLAP (Y310E) for the production of ethyl (S) -2-tetrahydrofurfuryl acid from racemic (R, S) -2-tetrahydrofurfuryl acid ethyl ester.

FIG. 6 is a high pressure gas phase chromatogram of pure enzyme solution resolution of Bacillus subtilis SCK6/pWB980-BLAP (Y310E) for the production of ethyl (S) -2-tetrahydrofurfuryl acid from racemic (R, S) -2-tetrahydrofurfuryl acid ethyl ester.

FIG. 7 is a graph showing the progress of the catalysis of racemic ethyl tetrahydrofurfuryl by Bacillus subtilis SCK6/pWB980-BLAP (Y310E).

Detailed Description

The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:

example 1: construction of the pWB980-BLAP (Y310E) plasmid

1. Construction of the recombinant vector pWB980-BLAP.

(1) The shuttle plasmid puc57 containing the target gene BLAP was extracted with a plasmid extraction kit, and the BLAP gene was amplified by PCR using puc57-BLAP plasmid as a DNA template and a primer BLAP-F (5'-GGTGGTGGTTGGGTTCAGGGTAGTCTGGATAC-3') with homology arms, BLAP-R (5'-AACCCAACCACCACCATGATAATAAACCAGTGCC-3').

The PCR system (50. Mu.L) was as follows:

1. Mu.L each of the upstream primer BLAP-F and the downstream primer BLAP-R, 1. Mu.L of plasmid puc57-BLAP, 25. Mu.L of 2 XKeyPo Master Mix high-fidelity enzyme, and ddH ₂ O was added to 50. Mu.L.

PCR reaction conditions: pre-denaturation at 98℃for 5min; denaturation at 98 ℃,10s; annealing at 55 ℃ for 15s; extending at 72 ℃ for 10s; a total of 30 cycles; finally, the temperature is reduced to 4 ℃ for preservation after the extension is carried out for 2min at 72 ℃. And mixing 2 mu L of PCR reaction liquid with 1 mu L of loading buffer liquid uniformly, and performing agarose gel electrophoresis detection by spotting, wherein the electrophoresis result shows that the band is single, and the amplified fragment size accords with the expectation and is about 1164bp.

(2) The linearized plasmid gene was amplified by PCR using the empty vector pWB980 as template and the primer pWB980-F (5'-GGTGGTGGTTGGGTTCAGGGTAGTCTGGATAC-3'), pWB980-R (5'-AACCCAACCACCACCATGATAATAAACCAGTGCC-3') with homology arms.

The PCR system (50. Mu.L) was as follows:

1. Mu.L each of the upstream primer pWB980-F and the downstream primer pWB980-R, 1. Mu.L each of the empty vector pWB980, 25. Mu.L each of the 2 XKeyPo Master Mix high-fidelity enzyme, and ddH ₂ O was added to 50. Mu.L.

The PCR conditions were as follows: pre-denaturation at 98℃for 5min; denaturation at 98 ℃,10s; annealing at 55 ℃ for 15s; extending at 72 ℃ for 60s; a total of 30 cycles; finally, the temperature is reduced to 4 ℃ for preservation after the extension is carried out for 2min at 72 ℃. After mixing 2 mu LPCR reaction solution and 1 mu L of loading buffer solution, spotting and carrying out agarose gel electrophoresis detection, the electrophoresis result shows that the band is single, and the amplified fragment size accords with the expectation and is about 4153bp.

(3) The PCR amplified fragments (the two ends of the fragment already contain 15bp homologous fragments) prepared in the step (1) and the step (2) are subjected to homologous recombination and self-ligation by using a one-step cloning kit (ClonExpress II OneTepclon kit, nanjinopran biotechnology Co., ltd.) according to the specification, so as to obtain a recombinant vector pWB980-BLAP.

2. The whole plasmid fragment was amplified by overlap extension PCR (genesplicingbyov erlapextension PCR), i.e.OverlapPCR, using the recombinant vector pWB980-BLAP as template and the primers Y310E-F (5'-CGGGCGCAGGCGTTGAATCAACATATCCGACAAA-3'), Y310E-R (5'-TGTCGGATATGTTGATTCAACGCCTGCGCCCGGT-3') with homology arms.

The PCR system (50. Mu.L) was as follows:

1. Mu.L each of the upstream primer Y310E-F and the downstream primer Y310E-R, 1. Mu.L of the recombinant vector pWB980-BLAP, 25. Mu.L of the 2 XKeyPo Master Mix high-fidelity enzyme, and ddH were added ₂ O was added to 50. Mu.L.

PCR reaction conditions: pre-denaturation at 98℃for 5min; denaturation at 98 ℃,10s; annealing at 65 ℃ for 5s; extending at 72 ℃ for 55s; a total of 30 cycles; finally, the temperature is reduced to 4 ℃ for preservation after the extension is carried out for 2min at 72 ℃. After mixing 2 mu LPCR reaction solution and 1 mu L of loading buffer solution, spotting and carrying out agarose gel electrophoresis detection, the result is shown in figure 1, the electrophoresis result shows that the band is single, and the amplified fragment size accords with the expectation, namely, about 5217bp.

3. And (3) treating the PCR amplified fragment (the two ends of the fragment already contain 15bp homologous fragments) of the recombinant vector pWB980-BLAP in the step (2) with a PCR product purification kit to remove impurities and improve the purity of the product. The size of the product fragment was then measured using an ultra-micro ultraviolet spectrophotometer and the absorbance ratio A260/A280 of the PCR purified product at 260nm and 280nm was 1.81 at a concentration of 257 ng/. Mu.l. The competent cells of Bacillus subtilis SCK6 were removed from the-80℃refrigerator and placed on ice. (2) In the super clean bench, after the competent cells were thawed, 5. Mu.L of PCR purified product was pipetted into the competent cells. (3) The ep tube of competent cell mixture was put in a metal bath at 37℃and 500rpm for 1.5h of shaking recovery. (4) After the completion of the shaking, the competent cell mixture was completely aspirated in an ultra clean bench, uniformly spread on a 1. Mu.g/mL erythromycin-resistant and 50. Mu.g/mL kanamycin-resistant LB liquid medium plate, and cultured overnight in a constant temperature incubator at 37 ℃. And collecting the cultured bacterial liquid, sequencing, and extracting plasmids from positive clones with correct sequencing results. The plasmid is recombinant plasmid pWB980-BLAP (Y310E) containing protease BLAP mutant, the amino acid sequence of the protease BLAP mutant is shown as BLAPIDNO.1, the coding gene sequence is shown as BLAPIDNO.2, and figure 2 is a map of the recombinant vector. The bacillus subtilis SCK6 transferred into the recombinant vector is recombinant bacillus subtilis SCK6/pWB980-BLAP (Y310E).

Meanwhile, the recombinant bacillus subtilis SCK6/pWB980-BLAP is taken as a wild type, and when the recombinant bacillus subtilis SCK6/pWB980-BLAP is prepared, whether the recombinant vector pWB980-BLAP is different from the engineering bacterium is the recombinant vector pWB980-BLAP or not, the PCR amplification process is not adopted by the upstream primer Y310E-F and the downstream primer Y310E-R, and other conditions are unchanged.

Composition of LB medium per liter: 5g of yeast powder, 10g of peptone and 10g of sodium chloride are dissolved in 1L of purified water, and the pH is natural.

The electrophoretogram of the purified recombinant protease mutant BLAP (Y310E) of the present invention is shown in FIG. 3.

Example 2: preparation of crude enzyme solution of recombinant protease BLAP mutant

The recombinant Bacillus subtilis SCK6/pWB980-BLAP (Y310E) constructed in example 1 was inoculated into LB liquid medium containing 1. Mu.g/mL of erythromycin resistance and 50. Mu.g/mL of kanamycin, and cultured at 37℃for 16 hours, which was a seed solution. The seed solution was inoculated in an inoculum size of 2% (v/v) into TB medium containing 1. Mu.g/mL of erythromycin resistance and 50. Mu.g/mL of kanamycin, cultured at 200rpm at 37℃for 48 hours, centrifuged at 12000rpm at 4℃for 10 minutes, and the supernatant was collected to obtain a crude enzyme solution containing the recombinant protease BLAP (Y310E) mutant.

A crude enzyme solution containing wild-type recombinant protease BLAP was prepared under the same conditions, except that "recombinant Bacillus subtilis SCK6/pWB980-BLAP (Y310E) was replaced with recombinant Bacillus subtilis SCK6/pWB980-BLAP", and the other conditions were unchanged.

Composition per liter of TB medium: peptone 12g, yeast powder 24g, KH ₂ PO ₄ 2.31g，K ₂ HPO ₄ 16.43g,5g glycerol. Adding deionized water to a constant volume of 1L, naturally adjusting pH, and sterilizing.

Example 3: preparation of pure enzyme solution of recombinant protease BLAP mutant

The recombinant bacillus subtilis SCK6/pWB980-BLAP (Y310E) crude enzyme solution prepared by the method of the example 2 is concentrated by an ultrafiltration centrifuge tube with the specification of 10kDa, and the method comprises the following steps: the residual ethanol in the ultrafiltration tube was removed by centrifugation at 4000Xg for 35 minutes, and then washed by centrifugation at 4000Xg with ultra-pure water for 5 minutes. Adding 4000Xg crude enzyme solution into an ultrafiltration tube, centrifuging for 40 minutes, and obtaining ultrafiltration concentrated solution without passing through a membrane. The ultrafiltration concentrate was collected and prepared for purification by immersing it in 0.1mol NaOH aqueous solution for 2 hours, then adding 20% ethanol and centrifuging at 4000Xg for 20 minutes and preserving it at 4 ℃.

In the process of constructing an expression system, a primer design is utilized after a stop codon of the blap gene, a6 XHis-tag purification tag is added, and an AKTA protein purifier can be used for purifying recombinant protease.

Preparing a reagent: imidazole buffer solution (eluent) with concentration of 500mM, imidazole buffer solution (balance solution) with concentration of 0mM, deionized water, 20% ethanol and Tris-HCl buffer solution with pH of 8.0; nickel column packing (column volume 5 mL); the detection wavelength was 280nm.

Before using the machine, the machine is washed by deionized water for a first time, excessive impurity components are washed off, the balance is carried out for 10 minutes, then the same operation is carried out by Tris buffer solution with pH of 8.0, and a nickel column is connected after the process is finished. The nickel column was then equilibrated with imidazole buffer at a concentration of 0mM at a flow rate of 2 mL/min. The ultrafiltration concentrate to be purified was subjected to loading treatment using a flow rate of 0.5 mL/min. And (3) after the ultrafiltration concentrated solution is loaded, regulating the concentration gradient by using imidazole buffer solution with the concentration of 500mM, eluting target protein by using the concentration gradient of 50mM, and placing the collected enzyme solution, namely the pure enzyme solution containing the recombinant protease BLAP (Y310E) mutant, in a refrigerator at the temperature of minus 20 ℃ for preservation.

Pure enzyme solutions containing the wild-type recombinant protease BLAP were prepared under the same conditions.

Example 4: determination of recombinant protease BLAP mutant Activity

The method comprises the following steps: the buffer system is boric acid buffer solution with pH of 10.5, the concentration of casein is 10g/L, 100 mu L of enzyme solution is absorbed, the buffer solution is added to dilute the buffer solution to the range of 10U/mL-15U/mL, after the operation according to the specification is finished, the buffer solution is placed in a spectrophotometer at 680nm to measure absorbance, the absorbance value is brought into a tyrosine standard concentration curve to calculate enzyme activity, and finally the enzyme activity of the original enzyme solution is obtained by multiplying the dilution multiple.

Definition of enzyme activity: in the environment of 40 ℃ and pH10.5 solution, 1mL of the solution to be tested hydrolyzes casein to generate 1 mu g of tyrosine within 60s, namely 1 enzyme activity unit expressed as U/mL. The concentration of the l-tyrosine is set as an abscissa, the value of absorbance of A680 is set as an ordinate, the drawing of an l-tyrosine concentration standard curve is carried out, and a regression equation of 0.0414+0.0101x is obtained by linear regression fitting, wherein R is adjusted ² 0.9997. After enzyme activity detection, the enzyme activity of the purified wild enzyme BLAP (namely a control) is 1512.6U/mL, the enzyme activity of the BLAP (Y310E) (namely a target) is 1876.3U/mL, and the enzyme activity of a mutant is improved by 24%.

Determination of specific enzyme activity of recombinant protease BLAP mutant:

100. Mu.L of the enzyme solution was added to 890. Mu.L of phosphate buffer having pH8.0, 14.4mg of racemic ethyl 2-tetrahydrofurfuryl acid (100 mM) was weighed and added to the solution for uniform mixing, reacted at 30℃and 1000rpm for 0.5 hours, and after adding 2mL of ethyl acetate for sufficient shaking, centrifuged at 12000rpm and 4℃for 2 minutes, 800. Mu.L of the upper organic phase was taken and subjected to gas phase detection.

Specific enzyme activity definition: in the above case, the amount of enzyme required for synthesizing 1. Mu. Mol of (S) -2-tetrahydrofurfuryl acid ethyl ester per minute is defined as one unit of enzyme activity (U).

The specific enzyme activity of wild recombinant protease BLAP is 2557.3U/mg, the specific enzyme activity of recombinant protein mutant enzyme BLAP (Y310E) is 133.3U/mg, and the catalytic performance of the mutant is obviously improved by calculating the yield of (S) -2-tetrahydrofurfuryl acid ethyl ester.

Example 5: stereoselectivity of recombinant protease BLAP mutant to racemic tetrahydrofurfuryl acid ethyl ester

1mL of reaction system is constructed, and the stereoselectivity of the target protein is detected by a fermentation liquid catalysis method. The reaction system: 800. Mu.L of the upper layer broth (i.e., the supernatant obtained in example 2, containing about 0.239ug of pure enzyme) and 14.4mg of racemic ethyl 2-tetrahydrofurfuryl acid (0.1M) were mixed with 200. Mu.L of buffer (pH 8.0, 100 mM) added thereto. Reaction conditions: the reaction was carried out at 30℃in a metal bath at 1000rpm for 3 hours, and 200. Mu.L of 0.4M trichloroacetic acid was finally added to deactivate the enzyme in the reaction system, thereby stopping the progress of the reaction. Under the same conditions, a blank control was obtained without catalyst. 200. Mu.L of the reaction mixture was sampled every 1 hour, and 1000. Mu.L of ethyl acetate was added for extraction. And after the extraction is finished, 800 mu L of the upper organic phase is added with a proper amount of anhydrous magnesium sulfate for water removal, 200 mu L of supernatant fluid is taken through an organic film and placed in an inner cannula of a gas-phase bottle, and the content of each component in a sample is detected by utilizing gas chromatography.

Gas phase detection conditions: chromatographic column: CP-Chirasil-DexCB (25 m,0.25mm,0.25 μm, agilent), hydrogen flow rate: 36mL/min, air flow rate: 350mL/min, nitrogen flow rate 2mL/min, injector temperature: 250 ℃, detector temperature: 250 ℃, column temperature: 100 ℃, split ratio 50:1 (substrate)/20:1 (product), column equilibration time: 7min. Substrate enantiomeric excess (ees), product enantiomeric excess (eep), conversion (C), enantiomer ratio (E). Calculated according to the following formula:

in the formula, [ S ] _S1 ]And [ S ] _S2 ]Respectively represent peak areas of (S) -2-tetrahydrofurfuryl acid ethyl ester and (R) -2-tetrahydrofurfuryl acid ethyl ester, [ P ] _P1 ]And [ P ] _P2 ]The peak areas of the (S) -2-tetrahydrofurfuryl acid and (R) -2-tetrahydrofurfuryl acid constituent products are shown, respectively.

The gas phase results are shown in FIG. 4 and FIG. 5, and the enantiomeric excess value e.e of the recombinant protease BLAP (Y310E) for ethyl (S) -2-tetrahydrofurfuryl acid. _s 99.9%. E.e of wild-type recombinant protease BLAP under identical conditions. _s Only 68.5% showed that the recombinant protease BLAP mutant was enantioselective for (S) -2-tetrahydrofurfuryl acid ethyl ester over the wild-type recombinant protease BLAP.

Gas chromatography: instrument model Waters1525; chiral gas chromatography column CP-Chirasil-DexCB (25 m,0.25mm,0.25 μm, agilent); hydrogen flow rate: 36mL/min, air flow rate: 350mL/min, nitrogen flow rate 2mL/min, injector temperature: 250 ℃, detector temperature: 250 ℃, column temperature: 100 ℃, split ratio 50:1 (substrate)/20:1 (product), column equilibration time: 7min.

Example 6: optimization of resolution process of racemic tetrahydrofurfuryl acid ethyl ester by BLAP (Y310E) pure enzyme solution

The resolution reaction system was 50mL: 44.5mL of deionized water, 0.72g of racemic (R, S) -2-tetrahydrofurfuryl acid ethyl ester as a substrate, and 5mL of pure enzyme solution (namely, the pure enzyme solution containing the recombinant protease BLAP (Y310E) mutant obtained in example 3).

Reaction conditions: the pH of the reaction system was always adjusted to about 8.0 with a 3M NaOH solution at 35℃and pH 8.0.

Detection of (S) -2-tetrahydrofurfuryl acid ethyl ester: 200 mu L of the solution is taken every 30min, 1mL of ethyl acetate solution is added into a 1.5mL ep tube, the mixture is fully and evenly vibrated, and then the mixture is centrifuged at 12000rpm and 4 ℃ for 2min, and 800 mu L of the upper organic phase is taken, thus obtaining the sample for detecting (S) -2-tetrahydrofurfuryl acid ethyl ester. Adding a proper amount of anhydrous magnesium sulfate for dewatering, then taking 200 mu L of supernatant through an organic film into an inner cannula of a gas phase bottle, and detecting the content of each component in a sample by utilizing gas chromatography. The results of the pure enzyme solution catalysis of the recombinant protease BLAP (Y310E) mutant of the present invention are shown in FIG. 6, and it can be seen that the enantioselectivity for (S) -2-tetrahydrofurfuryl acid ethyl ester is very high.

Detection of (R) -2-tetrahydrofurfuryl acid: extracting the water phase with 800 μl of ethyl acetate again to remove impurities, sucking the upper organic phase with a pipetting gun, adjusting pH to about 2 with 3M HCl, acidifying, extracting with 1mL ethyl acetate again, centrifuging under the same conditions, collecting 600 μl supernatant in new 2mL ep tube, adding 800 μl of absolute ethanol, adding 100 μl of acetyl chloride in a fume hood, shaking in a metal bath at 30deg.C and 900rpm for 30min, centrifuging in a centrifugal concentrator at 60deg.C under 100mbar for 40min to remove ethanol, adding the rest solution into saturated NaHCO ₃ After the solution has reached a pH of about 7-8 (slow addition of saturated NaHCO) ₃ The solution, during which a large amount of bubbles emerge), is then shaken in a metal bath at 30℃and 500rpm for 10min, ensuring NaHCO ₃ After the reaction of the solution is completed, 1mL of ethyl acetate solution is added, and after sufficient shaking and centrifugation, 800 mu L of supernatant is sucked, namely the sample for detecting (R) -2-tetrahydrofurfuryl acid. Adding a proper amount of anhydrous magnesium sulfate for dewatering, then taking 200 mu L of supernatant through an organic film into an inner cannula of a gas phase bottle, and detecting the content of each component in a sample by utilizing gas chromatography.

The (S) -2-tetrahydrofurfuryl acid ethyl ester and (R) -2-tetrahydrofurfuryl acid were detected as in example 5, the substrate enantiomer excess value ees, the product enantiomer excess value eep, the conversion C and the enantiomer ratio E were calculated, and the reaction progress was plotted on the abscissa with time (h). As shown in FIG. 7, the eEs gradually increased as the reaction time progressed, and when the reaction time reached 2h, ee _s Can reach more than 99.9 percent and then always keep the peak value. As the reaction proceeds, ee _p Also gradually rise, reaching a peak 68.63% at 3.5h, and then rapidly fall. The E value also peaks 105.05 at 3.5h with increasing reaction time, and then drops rapidly as the other configuration is also hydrolyzed. The optimal resolution time is 3.5h, the E value is 105.5, the conversion rate is 59.3 percent, the ees of the (S) -2-tetrahydrofurfuryl acid ethyl ester can reach more than 99.9 percent, and the yield is 5.86g/L; (R) -2-tetrahydrofurfuryl acid eep can also reach 68.63 percent, and the yield is 5.79g/L.

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

Claims (10)

1. A recombinant protease mutant is characterized in that the recombinant protease mutant is alkaline protease, and the amino acid sequence of the recombinant protease mutant is shown as SEQ ID NO. 1.

2. A gene for coding a recombinant protease mutant according to claim 1, wherein the nucleotide sequence of the coding gene is shown in SEQ ID No. 2.

3. A recombinant vector constructed from the coding gene of claim 2.

4. A recombinant genetically engineered bacterium transformed with the recombinant vector of claim 3.

5. An application of the coding gene of the recombinant protease mutant in preparing the recombinant protease mutant, which is characterized in that a recombinant plasmid pWB980-BLAP (Y310E) containing the recombinant protease mutant gene is constructed, the recombinant plasmid is transformed into bacillus subtilis B.subtilis SCK6, the obtained recombinant bacillus subtilis B.subtilis SCK6/pWB980-BLAP (Y310E) is marked as engineering bacteria, and after fermentation and induction of the engineering bacteria, the fermentation liquor is centrifugally separated to obtain supernatant containing the protease mutant, namely supernatant crude enzyme liquid; purifying the supernatant crude enzyme solution to obtain the pure enzyme solution.

6. Use of a recombinant protease mutant according to claim 1 for resolution of (R, S) -2-tetrahydrofurfuryl acid ethyl ester.

7. The application of the method according to claim 6, wherein the engineering bacteria containing the coding genes of the recombinant protease mutants are subjected to resolution reaction at 10-60 ℃ and 500-1500rpm by taking a crude enzyme solution of a supernatant obtained by fermenting and culturing the engineering bacteria or a pure enzyme solution obtained by purifying the crude enzyme solution as a catalyst and taking racemized (R, S) -2-tetrahydrofurfuryl acid ethyl ester as a substrate and a PB buffer solution with pH of 8.0 and 100mM as a reaction medium, and after the reaction is completed, separating and purifying the reaction solution to obtain (S) -2-tetrahydrofurfuryl acid ethyl ester and (R) -2-tetrahydrofurfuryl acid;

the addition amount of the substrate is 10-20mg/mL based on the volume of the buffer solution; the catalyst addition amount is calculated as the enzyme activity of the supernatant crude enzyme liquid obtained by fermenting and culturing engineering bacteria or the pure enzyme liquid purified by the crude enzyme liquid to be 20-40U/mL of reaction system.

8. The application of claim 7, wherein the host of the engineering bacteria is bacillus subtilis SCK6, and the preparation method of the engineering bacteria comprises the following steps:

1) Using pWB980-BLAP as a template plasmid, and using an upstream primer Y310E-F and a downstream primer Y310E-R with homology arms, performing full plasmid fragment amplification by overlap extension PCR to construct a recombinant plasmid pWB980-BLAP (Y310E) containing the coding gene of the recombinant protease mutant;

upstream primer Y310E-F:5'-CGGGCGCAGGCGTTGAATCAACATATCCGACAAA-3';

downstream primer Y310E-R:5'-TGTCGGATATGTTGATTCAACGCCTGCGCCCGGT-3';

2) Converting the recombinant plasmid pWB980-BLAP (Y310E) into bacillus subtilis B.subtilisSCK6 to obtain recombinant bacillus subtilis B.subtilisSCK6/pWB980-BLAP (Y310E), and obtaining the engineering bacterium.

9. The use according to claim 8, wherein the method for preparing the pWB980-BLAP plasmid comprises the steps of:

(1) Extracting shuttle plasmid puc57 containing target gene BLAP by using a plasmid extraction kit to obtain puc57-BLAP plasmid which is used as a DNA template, designing an upstream primer BLAP-F and a downstream primer BLAP-R with homology arms, and carrying out PCR amplification on the BLAP gene of the DNA template to obtain a PCR amplified fragment 1;

the upstream primer BLAP-F:5'-GGTGGTGGTTGGGTTCAGGGTAGTCTGGATAC-3';

the downstream primer BLAP-R:5'-AACCCAACCACCACCATGATAATAAACCAGTGCC-3';

(2) Designing an upstream primer pWB980-F and a downstream primer pWB980-R with homologous arms by using an empty vector pWB980 as a template, and carrying out PCR reaction on the primers to amplify linearized plasmid genes to obtain a PCR amplified fragment 2;

the upstream primer pWB980-F:5'-GGTGGTGGTTGGGTTCAGGGTAGTCTGGATAC-3';

the downstream primer pWB980-R:5'-AACCCAACCACCACCATGATAATAAACCAGTGCC-3';

(3) And carrying out homologous recombination self-ligation on the PCR amplified fragment 1 and the PCR amplified fragment 2 by using a one-step cloning kit to obtain a recombinant vector pWB980-BLAP.

10. The use according to claim 7, wherein the catalyst is prepared by the following method:

(1) Seed culture: inoculating engineering bacteria containing recombinant protease mutant coding genes into an LB liquid culture medium containing erythromycin resistance of 0.5-2 mug/mL and kanamycin of 40-60 mug/mL, and culturing for 12-16h at 37 ℃ to obtain seed liquid;

(2) Fermentation culture: inoculating the seed solution into a TB culture medium containing 0.5-2 mug/mL erythromycin resistance and 40-60 mug/mL kanamycin at an inoculum size of 2% (v/v), culturing at 37 ℃ for 40-55 hours at 100-300rpm, centrifuging, and collecting supernatant, namely crude enzyme solution containing the recombinant protease mutant;

(3) Preparation of pure enzyme solution: concentrating the crude enzyme solution by using an ultrafiltration centrifuge tube with the specification of 10kDa, and purifying recombinant protease by using an AKTA protein purifier to obtain pure enzyme solution.