Lipase mutant and its application

Abstract

This invention discloses mutants of Candida antarcticis lipase (PAL) and S. nigra lipase (SRL) from corn and their applications in the asymmetric hydrolysis of diester substrates to prepare a series of optically pure products. Utilizing a semi-rational design approach, this invention investigates the influence of a series of amino acid sites on enantioselectivity through three-dimensional structural analysis of the lipase. Furthermore, it constructs a series of combined mutants by combining positive mutation sites and applies them to an asymmetric hydrolysis process. Under room temperature (10-40℃) and pure aqueous phase conditions, a series of S-monoester products are prepared with ee > 95%, overcoming the limitation that wild-type lipases can only achieve high selectivity at lower temperatures and with the addition of solubilizers. This lays the foundation for the industrialization of pharmaceutical intermediates prepared by asymmetric hydrolysis of lipases.

Description

Technical Field

[0001] The invention relates to lipase mutants derived from Antarctic Candida and corn head smut and application thereof in preparing optically pure pharmaceutical intermediates by asymmetric hydrolysis, belonging to the technical field of bioengineering. Background Art

[0002] Lipase is a multifunctional biocatalyst that can catalyze reactions such as hydrolysis, esterification, and transesterification. Due to its advantages of high catalytic activity, good stability, good stereoselectivity, and broad-spectrum substrate specificity in aqueous and organic phases, it is widely used in enzyme-catalyzed synthesis processes to prepare various optically pure advanced pharmaceutical intermediates. Lipase is used as a biocatalyst in the preparation of key intermediates such as paroxetine, escitalopram, duloxetine, rivastigmine, and ibuprofen. Studies have shown (US20100087525) that lipase has a wide substrate spectrum, and adjusting the reaction temperature and cosolvent can significantly affect its selectivity and catalytic activity, indicating that its active pocket has strong plasticity. Therefore, lipase can be modified using technologies such as directed evolution and semi-rational design to improve its stereoselectivity and catalytic activity, further expanding the scope of application of lipase.

[0003] Directed evolution simulates the natural evolution process in experiments, artificially generates genetic diversity through rapid mutation and recombination, and then applies screening pressure according to specific needs and purposes to screen out enzyme proteins that meet application expectations. This technology has been widely used in enzyme engineering and other biological and other cross-disciplinary fields, but the research on stereoselective modification of enzyme catalysis has developed slowly compared with other enzymatic property modifications. The reason is that it is difficult to construct a screening model for stereoselective modification of enzymes, and the efficiency of analysis and detection using HPLC or GC is difficult to match the screening of large-volume mutation libraries. Therefore, the research on directed evolution of enzymes with stereoselective modification is still a challenge. With the advancement of the understanding of protein structure and function, the modification of enzymes has gradually shifted to small, functionally rich mutation libraries constructed based on semi-rational design technology. Semi-rational design refers to the use of relevant information such as protein sequence, structure, function, and computer assistance to pre-screen possible beneficial targets and amino acid residues for enzyme modification, thereby constructing a high-quality mutation library, reducing the workload of screening, and improving the efficiency of enzyme modification.

[0004] There are two main schemes for preparing chiral intermediates using lipase: chiral resolution and asymmetric hydrolysis. Since the resolution process requires optical resolution of the racemate, the theoretical yield of the target configuration product is less than 50%; the asymmetric hydrolysis process does not need to discard one of the optical isomers, so it fundamentally solves the problem of low production capacity and racemization reaction yield caused by enantiomer racemization recovery in the resolution process, and the theoretical yield can reach 100%. In recent years, lipase asymmetric hydrolysis has attracted more and more researchers' attention, but the actual application effect is not satisfactory. Zaida et al. used immobilized lipase to asymmetric hydrolyze 3-phenylglutaric acid diethyl ester to prepare (R)-5-ethoxy-5-oxo-3-phenylpentanoic acid (Tetrahedron: Asymmetry 22 (2011) 2080-2084), using dioxane as a cosolvent and reacting at low temperature, and the product ee value was 92%. Wei et al. used lipase to asymmetric hydrolyze 3-(4-fluorophenyl) glutaric acid dimethyl ester to prepare (R)-3-(4-fluorophenyl)-5-methoxy-5-oxopentanoic acid (Journal of Molecular Catalysis B: Enzymatic 97 (2013) 270-277). With dioxane as a cosolvent, the reaction was carried out at 30°C, and the ee value of the product was only 71.6%. Liu et al. selected 3-(4-fluorophenyl)-glutaric acid dimethyl ester as a model substrate and screened commercial lipase for asymmetric hydrolysis process research. The results are shown in Table 1. When 10% acetonitrile was added as a cosolvent and the reaction was carried out at room temperature, the stereoselectivity of the product could not meet the process requirements (Process Biochemistry 47 (2012) 1037-1041).

[0005] Table 1 Lipase screening reported in the literature

[0006] Lipase ee(%) Configuration PPL 56.3 S CRL 11.1 S RNL 3.6 R HLE 26.4 R Lipase AK 0 - Lipase PS 3.7 S Lipase AYS 0 - Lipozyme TL 63.0 S Novozym 435 91.8 R

[0007] Teva (US20100087525) reported a process route for asymmetric hydrolysis of substrate 3-isobutyl glutaric acid diester using commercial lipase. By adding substrate 3-isobutyl glutaric acid dimethyl ester and co-solvent tert-amyl alcohol, the reaction was carried out at a low temperature of -2°C. The final substrate concentration was about 135g / L, the conversion rate was 99%, the product was S configuration, and the ee value was 96%. When 3-isobutyl glutaric acid dimethyl ester was added at a low temperature of -2°C for reaction, the product ee value dropped to 89%. If the co-solvent tert-amyl alcohol was removed from the reaction system, the product ee value under the same reaction conditions dropped to 70%. Based on the above cases, the lipase asymmetric hydrolysis process still has the following problems: 1) The source of asymmetric hydrolysis lipase with strict stereoselectivity is insufficient to meet the process development requirements; 2) The stereoselectivity is unstable and is greatly affected by the reaction temperature, cosolvent and substrate addition mode; 3) Due to the low temperature environment conversion, the catalytic activity of lipase is far lower than the optimal reaction conditions, the cost of enzyme use increases, and the production capacity is greatly reduced; 4) The addition of cosolvents also increases the process cost and the difficulty of product separation and extraction, and cannot be achieved for industrial scale-up.

[0008] In this study, semi-rational design technology was used, with diethyl 3-isobutylglutarate as the model substrate and Pseudozyma antarctia lipase (PAL) as the research object to analyze the mutation sites, modify the stereoselectivity, and obtain positive single mutants; a series of mutant strains were constructed by combining the positive mutation sites, which can perform stereoselective and strict asymmetric hydrolysis of various diesters in an all-aqueous system at room temperature; gene mining technology was used to clone and express Sporisorium reilianum lipase (SRL), and after integrating the PAL forward mutation site, it showed strict stereoselectivity in the asymmetric hydrolysis of diester substrates, providing an efficient and stable biocatalyst for the lipase asymmetric hydrolysis process, which has great potential for industrial application. Summary of the invention

[0009] The present invention provides a series of Antarctic Candida PAL and Zea mays Smut SRL lipase mutants through semi-rational design technology, which can avoid low-temperature reaction conditions and eliminate the dependence on co-solvents, significantly improve the reaction stereoselectivity and process stability, and provide efficient and stable biocatalysts for the asymmetric hydrolysis process of a series of diester substrates.

[0010] The technical solution adopted by the present invention is:

[0011] The selection of the key mutation sites of the Antarctic Candida lipase PAL of the present invention is determined by constructing and screening a mutant library.

[0012] The present invention provides a series of Candida antarctica lipase PAL mutants. Relative to the wild-type Candida antarctica lipase PAL, the corresponding mutation sites and mutation forms of the mutants are composed of single point mutation forms and all possible combinations shown in Table 2.

[0013] Table 2 Beneficial mutation sites and their mutation forms

[0014] Amino acid position Parent amino acid Beneficial mutations 140 L V,I,M,A,F,W,G,S,C,T,D,N,E,P,H,K 154 V A,I,L,M,F,W,S,C,T,Q,Y 281 A I,L,M,F,V,S,C,T 282 A V,I,L,M,F,G,S,C,N,K,Y,R

[0015] The amino acid sequence of the wild-type Antarctic Candida lipase PAL of the present invention is shown in SEQ ID NO: 1, and the corresponding encoding gene is shown in SEQ ID NO: 2. The mutants L140X1, V154X2, A281X3, A282X4, L140X1 / V154X2, L140X1 / A281X3, L140X1 / A282X4, V154X2 / A281X3, V154X2 / A282X4, A281X3 / A282X4, L140X1 / V154X2 / A281X3, L140X1 / V154X2 / A282X4, L140X1 / A281X3 / A282X4, V154X2 / A281X3 / A282X4, L140X1 / V154X2 / A281X3 / A282X4, wherein X1 represents the beneficial mutation form of the L140 site, X2 represents the beneficial mutation form of the V154 site, X3 represents the beneficial mutation form of the A281 site, and X4 represents the beneficial mutation form of the A282 site.

[0016] The amino acid sequence of the lipase SRL of the wild-type corn head smut fungus of the present invention is shown in SEQ ID NO: 3, and the corresponding encoding gene is shown in SEQ ID NO: 4. According to the comparison results of the primary and tertiary structures of SRL and PAL lipases, positions 145, 159, 287 and 288 of the SRL lipase correspond to amino acid positions 140, 154, 281 and 282 of the PAL lipase, respectively. The SRL lipase mutants L145X1, V159X2, A287X3, G288X4, L145X1 / V159X2, L145X1 / A287X3, L145X1 / G288X4, V159X2 / A287X3, V159X2 / G288X4, A287X3 / G288X4, L145X1 / V159X2 / A287X3, V159X1 / V159X2 9X2 / G288X4, L145X1 / A287X3 / G288X4, V159X2 / A287X3 / G288X4, L145X1 / V159X2 / A287X3 / G288X4, wherein X1 represents the beneficial mutation form of the L145 site, X2 represents the beneficial mutation form of the V159 site, X3 represents the beneficial mutation form of the A287 site, and X4 represents the beneficial mutation form of the G288 site.

[0017] The present invention also relates to a recombinant vector containing the coding gene, and a recombinant genetic engineering bacterium transformed by the recombinant vector. The recombinant vector is constructed by connecting the mutant nucleotide sequence of the present invention to various vectors by conventional methods. The vector can be various conventional vectors in the art, such as various plasmids, bacteriophages or viral vectors.

[0018] The present invention also provides a genetically engineered bacterium containing the coding gene or the recombinant vector. The genetically engineered bacterium can be obtained by transforming the recombinant expression vector of the present invention into a host microorganism. The host microorganism can be various conventional host microorganisms in the art, as long as the recombinant expression vector can stably self-replicate and the mutant gene of the present invention carried by it can be effectively expressed, such as various yeasts, Escherichia coli, Bacillus, actinomycetes or molds.

[0019] The present invention also relates to the application of the lipase and its mutant in asymmetric hydrolysis of X-substituted glutaric acid diester to prepare S-monoester product. The application is to put substrate, mutant enzyme and buffer into a reactor for catalytic reaction; the substrate concentration is 1-300g / L; the buffer is citric acid-sodium citrate, phosphate and Tris-HCl buffer with pH 4.0-9.0; the reaction temperature of the application is 10-40°C. The reaction formula is as follows.

[0020]

[0021] The beneficial effects of the present invention are mainly reflected in: obtaining a series of highly selective lipases and mutants thereof that can asymmetrically hydrolyze prochiral substrates under industrially acceptable conditions such as room temperature (10-40° C.) and pure water phase to prepare intermediates such as (S)-3-isobutyl-5-methoxy-5-oxopentanoic acid, (S)-3-isobutyl-5-ethoxy-5-oxopentanoic acid, (S)-3-(4-fluorophenyl)-5-methoxy-5-oxopentanoic acid and (S)-3-(4-chlorophenyl)-5-methoxy-5-oxopentanoic acid, with reaction ee>95% and conversion rate>99%, which is the first report of a lipase that specifically asymmetrically hydrolyzes a series of diester substrates to generate S-monoester products under room temperature water phase conditions. After the mutants are applied to the asymmetric hydrolysis process of various pharmaceutical intermediates, the "split-racemization recovery" process can be avoided in the preparation process, the production capacity and substrate utilization rate are improved, the stereoselectivity is strict, the production efficiency can be significantly improved, and the lipase has great industrial application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 For active pocket structure analysis and mutation site selection DETAILED DESCRIPTION

[0023] Gene source: The Pseudozyma antarctia lipase PAL gene and the Sporisorium reilianum lipase SRL gene involved in this patent are obtained by whole gene synthesis, and the mutants involved are all obtained by site-directed mutagenesis.

[0024] Analysis of the three-dimensional structure of enzymes: The three-dimensional structure of enzymes was analyzed using Pymol visualization software.

[0025] Determination of enantiomeric selectivity: The reaction products (S)-3-isobutyl-5-methoxy-5-oxopentanoic acid, (S)-3-isobutyl-5-ethoxy-5-oxopentanoic acid, (S)-3-(4-fluorophenyl)-5-methoxy-5-oxopentanoic acid and (S)-3-(4-chlorophenyl)-5-methoxy-5-oxopentanoic acid and their corresponding enantiomers were determined by high performance liquid chromatography. The detection conditions refer to US20100087525.

[0026] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0027] Example 1: Selection of mutation sites

[0028] The active pocket of lipase PAL consists of an acyl pocket and an alcohol pocket, with a catalytic triad D187-H224-S105 and an oxygen anion hole at the junction of the two pockets. The amino acid residues T138, L140, V154 and I189 that constitute the acyl pocket and the amino acid residues L278, A281, A282 that constitute the alcohol binding pocket may affect the binding conformation of the enzyme and the substrate, such as Figure 1 As shown, the above 7 sites were selected as candidate mutation sites.

[0029] Example 2: Construction of recombinant expression vector and engineered bacteria

[0030] The PAL (SEQ ID NO: 1) and SRL (SEQ ID NO: 3) lipase sequences were selected, EcoR I and Hind III were designed according to the characteristics of the expression vector pET22b, and the lipase genes pal (SEQ ID NO: 2) and srl (SEQ ID NO: 4) were synthesized by the conventional operation of genetic engineering and the method of total gene synthesis. The pal and srl gene fragments synthesized in Example 1 were double-digested and recovered using EcoR I and Hind III restriction endonucleases, and the fragments were connected to the commercial vector pET22b treated with the same restriction endonuclease using T4 DNA ligase at 16°C overnight, thereby constructing the recombinant expression vectors pET22b-pal and pET22b-srl. The constructed recombinant expression vector was transformed into E. coli BL21 (DE3) competent cells, spread on LB plates containing a final concentration of 50 μg / mL ampicillin, and cultured overnight at 37°C. Colonies grown on the plates were randomly picked for colony PCR identification and positive clones were sequenced for verification. The results showed that the recombinant bacteria E. coli BL21 (DE3) / pET22b-pal and E. coli BL21 (DE3) / pET22b-srl were successfully constructed.

[0031] Example 3: Preparation of bacterial cells containing recombinant lipase or its mutants

[0032] The genetically engineered bacteria E. coli BL21 (DE3) / pET22b-pal and E. coli BL21 (DE3) / pET22b-srl or their mutants were inoculated into 2 × YT medium containing 50 μg / mL ampicillin and cultured at 37 ° C until the bacterial concentration OD 600 The value was 0.4-0.6, and IPTG with a final concentration of 0.1 mmol / L was added to the 2×YT liquid culture medium. After induction culture at 18°C ​​for 18-24 hours, the culture solution was centrifuged at 4°C and 8000 rpm for 5 minutes, the supernatant was discarded, and the wet cells containing recombinant PAL and SRL lipases or their mutants were collected.

[0033] Example 4: Preliminary screening of the effects of candidate amino acids on enantiomeric selectivity

[0034] For the 7 candidate sites determined in Example 1, a preliminary screening mutation library was constructed by site-directed mutagenesis, amplified using the high-fidelity enzyme PrimerStar, and the PCR product was directly digested with Dpn I, transformed into E.coli BL21 (DE3), coated with Amp plates for screening, and obtained mutants after sequencing verification. The corresponding mutant enzymes were obtained as described in Example 3, and 3-isobutyl glutaric acid diethyl ester was used as a model substrate to explore the effect of amino acid changes at each candidate site on enantiomeric selectivity. Using wild-type PAL as a control, the enantiomeric selectivity of PAL mutants was detected, and the results showed that the enantiomeric selectivity of 140, 154, 281 and 282 sites was significantly affected by the modification, so further site-directed saturation mutations were performed on the above 4 sites. For SRL lipase, the candidate sites were also determined by the results of PAL enantiomeric selectivity studies, and the sites with significant effects were 145, 159, 287 and 288.

[0035] Example 5: Construction of a lipase site-directed saturation mutation library and selectivity assay

[0036] Site-directed mutagenesis was used to construct a series of point mutants at positions 140, 154, 281, 282 of PAL lipase and positions 145, 159, 287, 288 of SRL lipase. The pET22b-pal and pET22b-srl plasmids were used as templates, respectively, and the high-fidelity enzyme PrimerStar was used for amplification. The PCR products were directly digested with Dpn I, transformed into E. coli BL21 (DE3), coated on Amp plates for screening, and obtained after sequencing verification. The corresponding mutant enzymes were obtained as described in Example 3. Using diethyl 3-isobutylglutarate as a model substrate, the enantiomeric selectivity of the obtained series of mutants was compared, and the results are shown in Table 3. The enantiomeric selectivity changes of the single-point mutants at positions 145, 159, 287, and 288 of SRL lipase were the same as those of the single-point mutations at positions 140, 154, 281, and 282 of PAL lipase, respectively. Under the same conditions for the amino acids at the corresponding positions, the enantiomeric selectivity of the SRL mutants was slightly worse than that of the PAL mutants.

[0037] Table 3 Lipase enantioselectivity determination

[0038]

[0039] Note: If the product configuration is R type, the ee value is marked as "-"; if the product configuration is S type, the ee value of 0-30% is marked as "+"; the ee value of 30-60% is marked as "++"; the ee value of 60-90% is marked as "+++"; the ee value >90% is marked as "++++"

[0040] Example 6: Preparation of (S)-3-isobutyl-5-methoxy-5-oxovaleric acid by transformation of lipase combination mutants

[0041] According to the single point mutation of Example 5, a combined mutant strain E. coli BL21 (DE3) / pET22b-pal (L140V / A281V) was constructed, and the bacterial cells were prepared by the method of Example 3 for catalytic reaction. 9.9 mL of 100 mM buffer (pH 8.0) was added to a 50 mL transformation bottle, 100 μL of substrate 3-isobutyl glutaric acid dimethyl ester, 0.39 g of wet bacterial cells were added, the reaction temperature was 30° C., the shaking speed was 150 rpm, and after the reaction was completed, it was extracted with n-hexane, and the conversion rate and the enantiomeric ratio of (S)-3-isobutyl-5-methoxy-5-oxovaleric acid in the reaction extract were analyzed by HPLC. The results showed that the conversion rate was >99%, and the product (S)-3-isobutyl-5-methoxy-5-oxovaleric acid ee was >95%.

[0042] Example 7: Preparation of (S)-3-isobutyl-5-ethoxy-5-oxovaleric acid by transformation of lipase combination mutants

[0043] According to the single point mutation of Example 5, the combined mutant strain E. coli BL21 (DE3) / pET22b-srl (L145M / V159L / A287V) was constructed, and the bacterial cells were prepared by the method of Example 3 for catalytic reaction. 9.9 mL of 100 mM buffer (pH 8.0) was added to a 50 mL transformation bottle, 100 μL of substrate 3-isobutyl glutaric acid diethyl ester, 0.39 g of wet bacterial cells were added, and the reaction conditions and sample treatment methods were the same as those of Example 6. The test results showed that the conversion rate was >99%, and the product (S)-3-isobutyl-5-ethoxy-5-oxopentanoic acid ee was >95%.

[0044] Example 8: Preparation of (S)-3-(4-fluorophenyl)-5-methoxy-5-oxopentanoic acid by transformation of lipase combination mutants

[0045] According to the single point mutation of Example 5, the combined mutant strain E. coli BL21 (DE3) / pET22b-pal (L140I / A281V) was constructed, and the bacterial cells were prepared by the method of Example 3 for catalytic reaction. 9.9 mL of 100 mM buffer (pH 8.0) was added to a 50 mL transformation bottle, 0.1 g of substrate 3-(4-fluorophenyl)-dimethyl glutarate, and 0.39 g of wet bacterial cells were added, and the reaction conditions and sample treatment methods were the same as those of Example 6. The test results showed that the conversion rate was >99%, and the product (S)-3-(4-fluorophenyl)-5-methoxy-5-oxopentanoic acid ee was >95%.

[0046] Example 9: Preparation of (S)-3-(4-chlorophenyl)-5-methoxy-5-oxopentanoic acid by transformation of lipase combination mutants

[0047] According to the single point mutation of Example 5, the combined mutant strain E. coli BL21 (DE3) / pET22b-srl (V159A / A287I / G288W) was constructed, and the bacterial cells were prepared by the method of Example 3 for catalytic reaction. 9.9 mL of 100 mM buffer (pH 8.0) was added to a 50 mL transformation bottle, 0.1 g of substrate 3- (4-chlorophenyl) -dimethyl glutarate, 0.39 g of wet bacterial cells were added, and the reaction conditions and sample treatment method were the same as in Example 6. The test results showed that the conversion rate was > 99%, and the product (S) -3- (4-chlorophenyl) -5-methoxy-5-oxopentanoic acid ee was > 95%. Sequence Listing <110> Shanghai Aobo Biopharmaceutical Co., Ltd. <120> Lipase mutant and its application <130> 2020.2.26 <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 317 <212> PRT <213> Pseudozyma antarctica <400> 1 Leu Pro Ser Gly Ser Asp Pro Ala Phe Ser Gln Pro Lys Ser Val Leu 1 5 10 15 Asp Ala Gly Leu Thr Cys Gln Gly Ala Ser Pro Ser Ser Val Ser Lys 20 25 30 Pro Ile Leu Leu Val Pro Gly Thr Gly Thr Thr Gly Pro Gln Ser Phe 35 40 45 Asp Ser Asn Trp Ile Pro Leu Ser Thr Gln Leu Gly Tyr Thr Pro Cys 50 55 60 Trp Ile Ser Pro Pro Pro Phe Met Leu Asn Asp Thr Gln Val Asn Thr 65 70 75 80 Glu Tyr Met Val Asn Ala Ile Thr Ala Leu Tyr Ala Gly Ser Gly Asn 85 90 95 Asn Lys Leu Pro Val Leu Thr Trp Ser Gln Gly Gly Leu Val Ala Gln 100 105 110 Trp Gly Leu Thr Phe Phe Pro Ser Ile Arg Ser Lys Val Asp Arg Leu 115 120 125 Met Ala Phe Ala Pro Asp Tyr Lys Gly Thr Val Leu Ala Gly Pro Leu 130 135 140 Asp Ala Leu Ala Val Ser Ala Pro Ser Val Trp Gln Gln Thr Thr Gly 145 150 155 160 Ser Ala Leu Thr Thr Ala Leu Arg Asn Ala Gly Gly Leu Thr Gln Ile 165 170 175 Val Pro Thr Thr Asn Leu Tyr Ser Ala Thr Asp Glu Ile Val Gln Pro 180 185 190 Gln Val Ser Asn Ser Pro Leu Asp Ser Ser Tyr Leu Phe Asn Gly Lys 195 200 205 Asn Val Gln Ala Gln Ala Val Cys Gly Pro Leu Phe Val Ile Asp His 210 215 220 Ala Gly Ser Leu Thr Ser Gln Phe Ser Tyr Val Val Gly Arg Ser Ala 225 230 235 240 Leu Arg Ser Thr Gly Gln Ala Arg Ser Ala Asp Tyr Gly Ile Thr 245 250 255 Asp Cys Asn Pro Leu Pro Ala Asn Asp Leu Thr Pro Glu Gln Lys Val 260 265 270 Only Only Only Only Leu Only Only Only Only Only Only Only Only Only Only Only Only Val Onst Gly 275 280 285 Pro Lys Gln Asn Cys Glu Pro Asp Leu Met Pro Tyr Ala Arg Pro Phe 290,295,300 Ala Val Gly Lys Arg Thr Cys Ser Gly Ile Val Thr Pro 305 310 315 <210> 2 <211> 954 <212> DNA <213> Pseudozyme Antarctica <400> 2 ttgccttccg gatctgaccc agctttttcc cagccaaagt ccgtcttgga tgctggtttg 60 acttgtcaag gtgcctcccc atctccgtc tccaagccaa tttgttggt cccggtacc 120 ggtaccactg gtcctcaatc ctttgactcc aactggatcc ctttgtccac ccagttggtt 180 tacactcctt gttggatctc cccaccacca ttcatgttga acgacaccca agttaacacc 240 gagtacatgg tcaacgccat taccgccttg tacgctggtt ctggaaacaa caagttgcca 300 gtcttgactt ggtctcaagg tggtttggtt gcccaatggg gtttgacctt cttcccatct 360 attagatcta aggttgaccg tttgatggct ttcgctccag actacaaggg taccgttttg 420 gctggtcctt tggatgcttt ggctgtctct gccccatccg tttggcaaca gactactggt 480 tctgccttga ccactgcttt gagaaacgcc ggtggtttga cccagatcgt tccaaccacc 540 aacttgtact ccgctaccga cgagatcgtt caaccacaag tttccaactc cccattggac 600 tcctcctact tgttcaacgg taagaacgtt caagctcaag ctgtctgcgg tccattgttc 660 gtcattgacc acgctggttc tttgacctcc cagttttcct acgtcgtcgg tagatccgct 720 ttgagatcta ccaccggtca agctagatct gctgactacg gaatcaccga ctgcaatcca 780 ttgccagcca acgacttgac cccagaacaa aaggttgccg ccgctgcttt gttggctccc 840 gctgctgctg ccattgttgc tggtcctaag cagaactgcg agcccgattt gatgccttac 900 gctagaccat tcgctgtcgg taagagaact tgttccggta ttgtcactcc ataa 954 <210> 3 <211> 322 <212> PRT <213> Sporisorium reilianum <400> 3 Thr Pro Leu Val Lys Arg Leu Pro Ser Gly Ser Asp Pro Ala Tyr Thr 1 5 10 15 Leu Ser Lys Ala Gln Leu Asp Ser Val Leu Ala Cys Gln Asn Gly Ser 20 25 30 Pro Ser Ser Gln Lys Asn Pro Ile Leu Leu Val Pro Gly Thr Gly Thr 35 40 45 Thr Gly Pro Gln Ser Phe Asp Ser Asn Trp Ile Pro Leu Ser Thr Gln 50 55 60 Leu Gly Tyr Ser Pro Cys Trp Val Ser Pro Pro Pro Phe Met Leu Asn 65 70 75 80 Asp Thr Gln Val Asn Ala Glu Tyr Ile Val Asn Ala Val Lys Val Leu 85 90 95 Ser Ser Ala Ser Gly Ala Lys Val Pro Val Leu Thr Trp Ser Gln Gly 100 105 110 Gly Leu Ala Ala Gln Trp Ala Leu Thr Phe Phe Pro Ser Ile Arg Thr 115 120 125 Gln Val Asp Arg Leu Met Ala Phe Ala Pro Asp Tyr Lys Gly Thr Val 130 135 140 Leu Ala Ala Phe Leu Thr Thr Pro Gly Leu Ala Ser Glu Ser Val Trp 145 150 155 160 Gln Gln Gln Ala Gly Ser Ala Leu Thr Thr Ala Leu Ala Asn Ala Gly 165 170 175 Gly Leu Thr Lys Ile Val Pro Thr Thr Asn Leu Tyr Ser Ala Thr Asp 180 185 190 Asp Ile Val Gln Pro Gln Thr Phe Asn Gly Pro Leu Asp Ser Gly Tyr 195 200 205 Leu Asn Gly Gly Ala Lys Asn Ile Gln Ala Gln Ser Val Cys Gly Pro 210 215 220 Leu Phe Val Val Asp His Ala Gly Thr Leu Thr Ser Gln Phe Ser Phe 225 230 235 240 Val Val Gly Arg Ser Ala Leu Arg Ser Thr Thr Gly Gln Ala Gln Ser 245 250 255 Lys Asp Tyr Gly Val Thr Asp Cys Asn Pro Leu Pro Ala Asp Ser Leu 260 265 270 Thr Pro Asp Gln Lys Leu Arg Ala Glu Gly Leu Leu Leu Val Ala Gly 275 280 285 Ala Asn Val Ala Ala Gly Pro Lys Gln Asn Cys Glu Pro Asp Leu Met 290 295 300 Pro Tyr Ala Arg Gln Tyr Ala Val Gly Lys Arg Thr Cys Ser Gly Val 305 310 315 320 Leu Island <210> 4 <211> 969 <212> DNA <213> Sporisorium reilianum <400> 4 actccattgg ttaagagatt gccatccggt tctgacccag cttacacttt gtctaaggct 60 caattggact ctgttttggc ttgtcagaac ggatctccat cctcccaaaa gaacccaatt 120 ttgttggttc caggaactgg tactactggt ccacaatcttt ttgattctaa ctggattcct 180 ttgtctactc agttgggtta ttccccatgt tgggtttctc caccaccatt tatgttgaac 240 gatactcaag ttaacgctga atacattgtt aacgctgtta aggttttgtc ttctgcttct 300 ggtgctaaag ttccagtttt gacttggtct caaggtggtt tggctgctca gtgggctttg 360 actttttttc catctattag aactcaggtt gatagattga tggcttttgc tccagattat 420 aagggtactg ttctagctgc ttttttgact actccaggtc tagcttctga atctgtgtgg 480 cagcagcaag ctggatccgc tttgactact gctttggcta atgctggtgg tttgactaag 540 attgttccaa ctactaactt gtactctgct acagatgata ttgttcaacc acaaactttc 600 aacggacctt tggattctgg ttacttgaac ggtggtgcta agaatattca ggctcaatcc 660 gtttgtggac cattgtttgt tgttgatcat gctggtactt tgacctctca attttctttt 720 gttgttggta gatctgcttt gagatctact actggtcagg ctcagtctaa agactacggt 780 gttactgatt gtaatccatt gccagctgat tctttgactc cagatcaaaa gttgagagct 840 gagggtttgt tgttggttgc tggtgctaac gttgctgctg gtccaaaaca aaactgtgag 900 ccagatttga tgccatacgc tagacaatat gctgttggta aaagaacatg ttccggagtt 960 attttgtaa 969

Claims

1. Candida antarctica lipase mutants for asymmetric hydrolysis of diester compounds S -type monoester products, wherein the mutant is relative to a wild-type antarctic Candida lipase, the amino acid sequence of the wild-type antarctic Candida lipase is as shown in SEQ ID NO: 1, the leucine residue at position 140 is mutated to valine, and the alanine residue at position 281 is mutated to valine; The diester compound is selected from 3-isobutyl glutarate dimethyl ester, 3-isobutyl glutarate diethyl ester, 3-(4-fluorophenyl)-glutarate dimethyl ester, or 3-(4-chlorophenyl)-glutarate dimethyl ester.

2. A recombinant vector constructed by the coding gene of the Antarctic Candida lipase mutant as defined in claim 1 for use in the asymmetric hydrolysis of diester compounds to prepare S -type monoester product, wherein the diester compound is as defined in claim 1.

3. The recombinant genetically engineered bacteria transformed by the recombinant vector defined in claim 2 are used to prepare the asymmetric hydrolysis of diester compounds. S -type monoester product, wherein the genetically engineered bacteria include Escherichia coli, Pichia pastoris or Bacillus subtilis, and the diester compound is as defined in claim 1.

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