DuPont thermophilic bacteria lipase mutant, encoding gene, engineering bacteria and application thereof

Abstract

The present invention belongs to the field of biotechnology, and discloses a DuPont thermophilic bacteria lipase mutant, encoding gene, engineered bacteria and application thereof. The DuPont thermophilic bacteria lipase mutant is obtained by point mutation of a peptide segment as shown in SEQ ID NO.1, and the point mutation is: the 89th W is mutated to L and / or the 90th I is mutated to A. The DuPont thermophilic bacteria lipase mutant provided by the present invention has greatly improved activity compared with the parent, can selectively hydrolyze high-concentration 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester at a higher rate, and can be used for industrial production of (3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid, thereby further synthesizing pregabalin.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a DuPont thermophilic bacteria lipase mutant, a coding gene, an engineering bacterium and applications thereof. Background Art

[0002] Lipase (EC 3.1.1.3), whose systematic name is triacylglycerol acylhydrolase, is a class of enzymes that catalyze the hydrolysis of the ester bond of triacylglycerol. It is widely used in the fields of food, washing, feed, leather making, bioenergy, oil processing, organic synthesis and enantiomeric separation. In addition to the characteristics of high selectivity, few side reactions and high optical purity of products, the reaction catalyzed by lipase also has the advantages of mild catalytic reaction conditions and green environmental protection, which makes lipase have unique advantages in the preparation of optically pure compounds, especially chiral drugs. Lipase biocatalysis has become an important technical means for the synthesis of important chiral chemicals, especially the preparation of chiral drugs.

[0003] Thermostable enzymes have great potential for biotechnology and industrial applications due to their good thermal stability and solvent stability, and have attracted much attention from academia and industry. Thermomyces dupontii is a thermophilic fungus that has been studied more in recent years and is an important source of thermostable enzymes. The lipase TDL2 from Thermomyces dupontii has good thermal stability and shows good stereoselectivity for ethyl 2-carboxyethyl-3-cyano-5-methylhexanoate (I). The (3S)-2-carboxyethyl-3-cyano-5-methylhexanoate (II) generated after hydrolysis is a key chiral intermediate for the preparation of pregabalin. Pregabalin (III) can be synthesized by decarboxylation, alkaline hydrolysis and hydrogenation reduction. However, there is not much research on Thermomyces dupontii lipase, and how to further improve its application effect is an industry problem that needs to be solved urgently.

[0004] Summary of the invention

[0005] The purpose of the present invention is to transform DuPont Thermomyces lipase by site-directed saturation mutagenesis technology, provide a lipase mutant protein, improve its activity on 2-carboxyethyl-3-cyano-5-methylhexanoate, thereby facilitating the application of the lipase in the preparation of pregabalin intermediates.

[0006] The DuPont thermophilic bacteria lipase mutant of the present invention is obtained by point mutation of a peptide segment with a sequence as shown in SEQ ID NO.1, wherein the point mutation is: W at position 89 is mutated to L and / or I at position 90 is mutated to A.

[0007] SEQ ID NO.1: EVSQDLLDLF NLFAQYSAAA YCAENNDAPV GSNVTCSENV CPKV EEADATFLYSFEDSGL GDVTGLLALD NTNKLIVLAF RGSRSIENWI GNLIVGLRDI DDI CSGCEGH TGFVASWKSVANTLRGQVEN AVKEHPDYRV VFTGHSLGGA LATIAGAAL R GNGYNIDVFS YGAPRVGNRA FAEFLTAQTGGTLYRITHTN DIVPRLPPRD WGYSHS SPEY WITSGNDAPV TANDIVKVEG IDSTDGNNQP NIPDIPSHLWYFGAIAECD.

[0008] The present invention uses a site-directed saturation mutagenesis method to mutate a lipase TDL2 coding gene (sequence as shown in SEQ ID NO.2) and then transforms the host Escherichia coli, and after inducing expression, a mutant with improved activity is screened out by a high-throughput screening method. The obtained mutant can greatly improve the activity of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester compared with the parent, and can catalyze high-concentration (500g / L) 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester to prepare pregabalin key chiral intermediate (3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid.

[0009] The specific method is as follows: the lipase TDL2 (GenBank no. MN782511.1) from Thermomyces dupontii has been successfully constructed with the expression vector pET28-TDL2 and expressed in Escherichia coli BL21 (DE3). The site-directed saturation mutagenesis technique was used, and the plasmid pET28-TDL2 was used as a template to mutate sites 89 and 90 respectively, and then transferred into the expression host E. coli BL21 (DE3) to construct a site-directed saturation mutation library. A single clone in the mutation library was selected to induce the expression of enzyme protein in a 96-well culture plate, and after centrifugation and resuspension, the bacterial solution was added with substrate (2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester) and bromothymol blue indicator. After the reaction time is appropriate, strains with significantly higher activity than the control were initially screened out, and then the activity of these positive mutant strains was determined, and the high-activity mutant strains screened were sequenced to obtain the mutant DNA sequence information. By screening the mutant library constructed as above, three mutants with improved activity were obtained, namely W89L, I90A and W89L / I90A.

[0010] The sequence of the DuPont thermophilic bacteria lipase mutant W89L (the tryptophan at position 89 of the enzyme is mutated to leucine) is as follows (SEQ ID NO.3): EVSQDLLDLF NLFAQYSAAA YCAENNDAPV GSNVTCSENV CPK VEEADATFLYSFEDSGL GDVTGLLALD NTNKLIVLAF RGSRSIENLI GNLIVGLRDI DDICSGCEGH TGFVASWKSVANTLRGQVEN AVKEHPDYRV VFTGHSLGGA LATIAGAALR GNGYNIDVFS YGAPRVGNRA FAEFLTAQTGGTLYRITHTN DIVPRLPPRD WGYSHSSPEY WITSGNDAPV TANDIVKVEG IDSTDGNNQP NIPDIPSHLWYFGAIAECD.

[0011] The sequence of the DuPont thermophilic bacteria lipase mutant I90A (the isoleucine at position 90 of the enzyme is mutated to alanine) is as follows (SEQ ID NO.5): EVSQDLLDLF NLFAQYSAAA YCAENNDAPV GSNVTCSENV CP KVEEADATFLYSFEDSGL GDVTGLLALD NTNKLIVLAF RGSRSIENWA GNLIVGLRDIDDICSGCEGH TGFVASWKSVANTLRGQVEN AVKEHPDYRV VFTGHSLGGA LATIAGAALR GNGYNIDVFS YGAPRVGNRA FAEFLTAQTGGTLYRITHTN DIVPRLPPRD WGYSHSSPEY WITSGNDAPV TANDIVKVEG IDSTDGNNQP NIPDIPSHLWYFGAIAECD.

[0012] The sequence of the DuPont thermophilic bacteria lipase mutant W89L / I90A (the tryptophan at position 89 of the enzyme is mutated to leucine, and the isoleucine at position 90 is mutated to alanine) is as follows (SEQ ID NO.7): EVSQDLLDLF NLFAQYSAA AYCAENNDAPV GSNVTCSENV CPKVEEADAT FLYSFEDSGL GDVTGLLALD NTNKLIVLAF RGSRSIENLAGNLIVGLRDI DDICSGCEGH TGFVASWKSV ANTLRGQVEN AVKEHPDYRV VFTGHSLGGA LATIAGAALRGNGYNIDVFS YGAPRVGNRA FAEFLTAQTG GTLYRITHTN DIVPRLPPRD WGYSHSSPEY WITSGNDAPVTANDIVKVEGIDSTDGNNQP NIPDIPSHLW YFGAIAECD.

[0013] The present invention also provides a coding gene related to the above-mentioned DuPont thermophilic bacteria lipase mutant. The sequence of the coding gene is shown in SEQ ID NO.4 (encoding the mutant shown in SEQ ID NO.3), SEQ ID NO.6 (encoding the mutant shown in SEQ ID NO.5) or SEQ ID NO.8 (encoding the mutant shown in SEQ ID NO.7). On this basis, an engineering bacterium containing the above-mentioned coding gene and a method for preparing a whole-cell catalyst of the engineering bacterium are also provided, comprising the following steps: inoculating the positive clone Pichia yeast containing the coding gene into BMGY medium, culturing for 24 hours at 30°C and 220rpm, collecting the cells by centrifugation, and resuspending them in BMMY medium for 5 days, during which 0.5% of the system volume of methanol is added every 12 hours; finally, the culture supernatant is collected, centrifuged and concentrated by ultrafiltration membrane to obtain the whole-cell catalyst.

[0014] Preferably, the BMGY medium comprises the following components at final concentrations: 1.0% glycerol, 1.0% yeast extract, 2.0% peptone, 1.34% yeast nitrogen base, 4*10 -5 % biotin, 100 mM potassium phosphate. BMMY medium includes the following components at final concentrations: 1.0% methanol, 1.0% yeast extract, 2.0% peptone, 1.34% yeast nitrogen base, 4*10 -5 % biotin, 100 mM potassium phosphate.

[0015] BMGY medium is a buffered complete medium containing glycerol, which can secrete and express Pichia recombinant strains to obtain high-density bacteria, which can reduce protease activity. Glycerol is used as a direct carbon source and energy source, and the process does not produce acid. The BMMY medium used for subsequent culture was used to replace glycerol with 1% methanol for continuous methanol-induced promoter-driven protein expression. The use of the above-mentioned medium and the corresponding final concentration components can better induce engineered bacteria to express proteins.

[0016] The DuPont thermophilic bacteria lipase mutant provided by the present invention can be well used in catalyzing 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester to prepare (3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid. The lipase mutant can be used in the form of an unpurified crude enzyme, or in the form of a partially purified or fully purified enzyme, or in the form of an engineered bacteria whole cell. If necessary, the lipase mutant of the present invention can also be made into an immobilized enzyme or an immobilized cell-formed immobilized enzyme using an immobilization technique known in the art.

[0017] Preferably, the reaction conditions in the above catalytic process include: pH=7.0, reaction temperature of 30°C, and addition of 100mM-200mM calcium acetate. Adding 100mM-200mM calcium acetate to the reaction system can be used to relieve product inhibition.

[0018] The invention also provides a kit, which contains the DuPont thermophilic bacteria lipase mutant.

[0019] The beneficial effects of the present invention are as follows: the DuPont Thermomyces lipase mutant provided by the present invention has greatly improved activity compared with the parent, and even if the crude extract of the enzyme is used as a catalyst, high-concentration 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester can be selectively hydrolyzed at a high rate, and can be used for industrial production of (3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid, thereby further synthesizing pregabalin. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The figure shows the reaction process of preparing (3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid by catalyzing 200 g / L of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester by the lipase mutant;

[0021] Figure 2 The figure shows the reaction process of preparing (3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid by catalyzing 500 g / L of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester by the lipase mutant. DETAILED DESCRIPTION

[0022] The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments and drawings to fully understand the purpose, scheme and effects of the present invention.

[0023] Example 1: W89 site-directed saturation mutagenesis of lipase

[0024] The site-directed saturation mutagenesis technique is described in (Current Protocols in Protein Science 2011, 26.6.1-26.6.10; Anal. Biochem. 2008, 375: 376-378). The specific method and process are as follows:

[0025] In order to saturate the tryptophan (W) at position 89 in the amino acid sequence of lipase TDL2, primers W89-F and W89-R were designed (see Table 1), and the plasmid pET28-TDL2 was used as a template for full plasmid amplification. The PCR system was: 5×PSBuffer 10μL, dNTP (2.5mM each) 4μL, mutation primers W89-F and W89-R 0.5μL each, plasmid pET28-TDL2 0.5μL, PrimeSTAR DNA polymerase 0.5μL, and water was added to 50μL. The PCR conditions were 98℃ pre-denaturation for 2min, 25 cycles: 98℃ 10s, 65℃ 10s, 72℃ 6min, and finally 72℃ 10min. After PCR was positive as analyzed by 1.0% agarose gel electrophoresis, 20 μL of PCR solution was taken, 1 μL of DpnⅠ was added, and the template plasmid DNA (pET28-TDL2) was removed by enzyme digestion at 37°C for 2 h, inactivated at 65°C for 10 min, transformed into competent cells E. coli BL21 (DE3), coated with LB plates containing kanamycin (50 μg / mL), and cultured at 37°C overnight to obtain a mutant library of about 400 clones.

[0026] Table 1: Primers

[0027]

[0028]

[0029] Example 2: Screening of the W89 site-directed saturation mutation library of lipase

[0030] The high-throughput screening technology for screening mutant libraries is described in (Bioorgan. Med. Chem., 1999, 7: 2183-2188). The specific process is as follows:

[0031] Single clones from the mutant library in Example 1 were picked and placed in a 96-well plate. 200 μL of LB medium (containing 50 μg / mL kanamycin) was added to each well and cultured at 37°C for about 3 h until OD 600 The cell culture temperature was about 0.6, 0.1mM IPTG (isopropyl-β-D-thiogalactopyranoside) was added, and the cells were induced at 25℃ for 12h. 12000g, 4℃, centrifuged for 2min, the supernatant was discarded, the cells were resuspended with PBS buffer (pH7.2, 10mM), and the reaction solution containing the substrate 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester and the indicator bromothymol blue was added. The wild-type engineered bacterial cells were used as a reference, and the positive clones with improved activity were preliminarily screened on a 96-well plate according to the rate of color change (blue→yellow) of the reaction system. The positive clones screened out were confirmed by repeated screening, and the mutant 1 with significantly improved activity was screened, and the single clone was selected and sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing. The mutant was W89L, and its amino acid sequence is shown in SEQ ID NO.3 in the sequence list, and the nucleic acid sequence is shown in SEQ ID NO.4 in the sequence list.

[0032] Example 3: Construction and screening of the I90 site-directed saturation mutation library of lipase

[0033] In order to saturate the isoleucine (I) at position 90 in the amino acid sequence of lipase TDL2, primers I90-F and I90-R were designed (see Table 1), and plasmid pET28-TDL2 was used as a template to perform full plasmid amplification, construct an I90 site-directed saturation mutation library and screen it. The construction of the site-directed saturation mutation library was the same as in Example 1, except that the mutation primers were I90-F and I90-R. The screening of the I90 site-directed saturation mutation library was the same as in Example 2. The positive clones screened were then confirmed by activity assay, and plasmids were extracted from the positive clones. The introduced point mutations were determined by DNA sequencing. DNA sequencing of the positive clones with the highest activity showed that Ile at position 90 was mutated into Ala (I90A). The amino acid sequence of the mutant I90A is shown in SEQ ID NO.5 in the sequence table, and one of the coding gene sequences is shown in SEQ ID NO.6 in the sequence table.

[0034] Example 4: Site-directed mutagenesis of W89L / I90A

[0035] The site-directed mutagenesis technique is described in (Current Protocols in Protein Science 2011, 26.6.1-26.6.10; Anal. Biochem. 2008, 375: 376-378), and the specific process is as follows:

[0036] In order to mutate lipase TDL2 into double mutant W89L / I90A, mutant primers I90A-F and I90A-R (Table 1) were designed respectively, and the single mutant plasmid pET28-TDL2-W89L obtained in Example 1 was used as a template to perform full plasmid amplification for site-directed mutagenesis. The PCR system was: 5×PS Buffer 10 μL, dNTP (2.5 mM each) 4 μL, mutant primers I90A-F and I90A-R 0.5 μL each, plasmid pET28-TDL2-W89L 0.5 μL, PrimeSTAR DNA polymerase 0.5 μL, and water was added to 50 μL. The PCR conditions were 98°C pre-denaturation for 2 min, 25 cycles: 98°C 10 s, 65°C 10 s, 72°C 6 min, and finally 72°C 10 min. After PCR was positive by 1.0% agarose gel electrophoresis analysis, 20 μL of each PCR solution was taken, 1 μL of Dpn I was added, and the template plasmid (pET28-TDL2-W89L) was removed by enzyme digestion at 37°C for 2h, inactivated at 65°C for 10min, transformed into competent cells E.coli BL21 (DE3), and coated with LB plates containing kanamycin (50 μg / mL). Single clones were picked for colony PCR verification, and positive clones were preserved and sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing, confirming that the double mutant W89L / I90A was obtained. The double mutant W89L / I90A was induced to express (the expression method was the same as in Example 1), and the wild type, single mutant W89L and single mutant I90A were used as controls for screening and comparison (the screening method was the same as in Example 2), and it was found that the activity of W89L / I90A was higher than that of single mutant W89L and single mutant I90A. The amino acid sequence of W89L / I90A is shown in SEQ ID NO.7 in the sequence listing, and the nucleic acid sequence is shown in SEQ ID NO.8 in the sequence listing.

[0037] Example 5: Induced expression of parent lipase and lipase mutant engineered bacteria

[0038] The starting strain E. coli BL21 (DE3) / pET28-TDL2, the mutant strain E. coli BL21 (DE3) / pET28-TDL 2-W89L (see Example 1), E. coli BL21 (DE3) / pET28-TDL2-I90A (Example 3) and E. coli BL21 (DE3) / pET28-TDL2-W89L / I90A (Example 4) were inoculated into LB liquid medium containing 50 mg / L kanamycin at 37 ° C for 12 h, and then inoculated into fresh LB liquid medium containing 50 mg / L kanamycin at a 1% inoculum (v / v) and cultured until the bacterial concentration OD 600About 0.6, then add IPTG with a final concentration of 0.1mM to the LB liquid culture medium, induce and culture at 25℃ for 12h, centrifuge at 4℃ and 8000g for 10min, collect the bacterial cells containing the recombinant lipase, which can be used for enzyme activity determination and biocatalytic preparation of (3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid.

[0039] Example 6: Activity determination of parent lipase and lipase mutants

[0040] The activity of the mutant strain and the starting strain cells obtained in Example 5 was measured respectively. The reaction system composition: Tris-HCl (pH 7.5, 100 mM), 100 mM 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester, 50 mM Ca(OAc) 2 and a certain amount of cells, react at 30℃, 150r / min for 1h, take 200μL of sample, add 50μL of 1M HCl to terminate the reaction, extract with ethyl acetate, treat with anhydrous sodium sulfate, and determine the conversion rate and enantiomeric excess (ee) of the product by gas phase. The external standard quantitative method was used to determine the content of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester and (3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid in the conversion solution. The amount of each component was determined by gas chromatography Shimadzu GC-14C, column type: G-TA capillary column; chromatographic conditions: column temperature 135℃, injection chamber temperature 220℃, FID detector 220℃, helium flow rate 1mL / min, split ratio 30:1. The enzyme activity unit (U) is defined as: the amount of enzyme required to catalyze the production of 1 μmol (3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid from 2-carboxyethyl-3-cyano-5-methylhexanoate within 1 min at 30°C and pH 7.5.

[0041] Table 2: Comparison of lipase activity between lipase mutants and parent lipase

[0042]

[0043] Example 7: Construction and expression of lipase mutant Pichia pastoris engineered bacteria

[0044] TDL2 was heterologously expressed in Pichia pastoris X33 strain using the secretory expression vector pPICZαA of Pichia pastoris. Primers PF and PR were designed and used, and the double mutant plasmid pET28-TDL2-W89L / I90 obtained in Example 4 was used as a template to PCR amplify the coding gene fragment of the lipase double mutant W89L / I90. The PCR system was 50 μL, including 0.5 μL of plasmid template pET28-TDL2-W89L / I90, 1 μL of forward and reverse primers (5 mM), and 1 μL of KOD-Plus-Neo DNA polymerase. PCR program: 94°C 3min, 98°C 10s, 68°C 1min, 30cycles, 68°C 10min, 16°C 5min. Immediately after the amplification was completed, 1 μL of DpnI was added and treated at 37°C for 1h to remove the plasmid template. 1% agarose gel electrophoresis, gel cutting and recovery of the target gene amplification product. The vector pPICZαA was digested with EcoRI and XbaI, and then electrophoresed on 1% agarose gel. The plasmid DNA fragments were recovered by cutting the gel. II On e Step Cloning Kit for splicing, refer to the instructions of the kit. The splicing solution was heat-shocked to transform E. coli DH5α competent cells, coated with LB plates containing 100μg / mL Zeocin, and cultured at 37℃ overnight. The positive clones on the Zeocin plate were picked and sequenced, and the lipase double mutant W89L / I90 encoding gene was cloned between the EcoRI and XbaI sites of pPICZαA to construct the Pichia pastoris secretion expression plasmid pPICZαA-TDL2-W89L / I90. The plasmid pPICZαA-TDL2-W89L / I90 was linearized with SacI and transformed into Pichia pastoris X33 by electroporation using a Bio-Rad gene puncher according to the operating manual. The linearized empty pPICZαA was used as a negative control. Transformants were screened on YPDS medium containing 200mg / L ZeocinTM and confirmed by PCR.

[0045] The positive cloned yeast colony was inoculated into BMGY medium (1.0% glycerol, 1.0% yeast extract, 2.0% peptone, 1.34% yeast nitrogen base, ammonium sulfate without amino acids, 4×10 -5% biotin, 100 mM potassium phosphate, pH 6.0) and grown at 30°C and 220 rpm. After 24 hours, the cells were collected by centrifugation and resuspended in BMMY medium (the ingredients are the same as BMGY, but 1% methanol is used instead of glycerol). After 5 days of cultivation, 0.5% (final volume / volume) methanol was added to the culture every 12 hours for continuous methanol induction. The culture supernatant of the recombinant Pichia was collected, centrifuged at 12000g for 15 minutes, and concentrated using an Amicon Ultra-15 ultrafiltration membrane with a molecular weight cutoff of 10 kDa, and freeze-dried to prepare enzyme powder, which was stored at -20°C for later use.

[0046] Example 8: Application of lipase mutants in the preparation of (3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid (200 g / L)

[0047] The composition and operation of the conversion system are as follows: 100 mL of the reaction system contains 1 g of crude enzyme powder of the lipase mutant W89L / I90 (see Example 7), 150 mM of calcium acetate, and 20 g of ethyl 2-carboxyethyl-3-cyano-5-methylhexanoate (substrate concentration 200 g / L); the reaction temperature is 30° C. During the reaction, the pH value of the reaction solution is controlled to 7.0 by adding 4 M NaOH dropwise, and the reaction progress is monitored by gas chromatography during sampling (see Figure 1 ), the gas phase detection conditions are the same as those in Example 6. Figure 1 It can be seen that 0.5% (W / v) of lipase mutant W89L / I90 enzyme powder can catalyze the selective hydrolysis of 200g / L CNDE, react for 4h, the conversion rate reaches 46.8%, and the product (3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid with an ee value of more than 99% is obtained. After the reaction is completed, centrifugal separation is performed, the aqueous phase is collected, and the volume is reduced to 1 / 3. The remaining substrate is removed by two toluene extractions, and the residual toluene is removed by reduced pressure distillation to obtain the target product (3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid (ee>99%).

[0048] Example 9: Application of lipase mutants in the preparation of (3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid (500 g / L)

[0049] The composition and operation of the conversion system are as follows: 100 mL of the reaction system contains 1 g of lipase mutant W89L / I90 enzyme powder (see Example 7), 150 mM calcium acetate, and 50 g of ethyl 2-carboxyethyl-3-cyano-5-methylhexanoate (substrate concentration 500 g / L); the conversion operation and post-conversion treatment are the same as in Example 8, and the reaction progress is shown in Figure 2 .from Figure 2 It can be seen that under high substrate concentration conditions (500 g / L), the conversion rate can reach 46.1% after 10 hours of conversion, and the product ee>99%.

[0050] The above is only a preferred embodiment of the present invention. The present invention is not limited to the above implementation. As long as the technical effect of the present invention is achieved by the same means, it should belong to the protection scope of the present invention. Within the protection scope of the present invention, its technical scheme and / or implementation method can have various modifications and changes.

Claims

1. A mutant of the DuPont thermophilic bacteria lipase, characterized in that: The peptide sequence is obtained by point mutation of the peptide segment shown in SEQ ID NO.1, wherein the point mutation is: W at position 89 is mutated to L and I at position 90 is mutated to A.

2. A gene encoding the DuPont Thermomyces lipase mutant according to claim 1.

3. The coding gene according to claim 2, characterized in that The sequence of the coding gene is shown in SEQ ID NO.

8.

4. An engineered bacterium containing the coding gene according to claim 2 or 3.

5. A method for preparing the whole-cell catalyst of the engineered bacteria according to claim 4, characterized in that: The following steps are involved: The positive cloned Pichia yeast containing the coding gene was inoculated into BMGY medium and cultured at 30°C and 220 rpm for 24 h. The cells were collected by centrifugation and resuspended in BMMY medium for 5 d, during which 0.5% methanol by volume of the system was added every 12 h. Finally, the culture supernatant was collected, centrifuged and concentrated by ultrafiltration membrane to obtain the whole-cell catalyst.

6. The preparation method according to claim 5, characterized in that: BMGY medium contains the following components at final concentrations: 1.0% glycerol, 1.0% yeast extract, 2.0% peptone, 1.34% yeast nitrogen base, 4*10 -5 % biotin, 100 mM potassium phosphate.

7. The preparation method according to claim 5, characterized in that: BMMY medium contains the following components at final concentrations: 1.0% methanol, 1.0% yeast extract, 2.0% peptone, 1.34% yeast nitrogen base, 4*10 -5 % biotin, 100 mM potassium phosphate.

8. Use of the DuPont Thermomyces lipase mutant according to claim 1 in catalyzing the preparation of (3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid from ethyl 2-carboxyethyl-3-cyano-5-methylhexanoate.

9. The use according to claim 8, characterized in that: The reaction conditions in the catalytic process include: pH=7.0, reaction temperature of 30°C, and addition of 100 mM-200 mM calcium acetate.

10. A kit, characterized in that: It contains the DuPont thermophilic bacteria lipase mutant described in claim 1.

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