CN109182298B - Recombinant lipase mutant, engineering bacterium and application - Google Patents

Abstract

The invention discloses a recombinant lipase mutant, an engineering bacterium and its preparation and application of (2S, 3R)-N-acetyl-piperidine-2,3-dimethyl dicarboxylate. The mutant is SEQ ID NO. .2 The 140th, 141st, 144th, 189th and 190th positions of the amino acid sequence shown in 2 are obtained by single mutation or combined mutation. Compared with the wild-type enzyme, the mutant has greatly improved catalytic activity and substrate tolerance when transforming the above-mentioned reaction, and the time-consuming of the reaction process is significantly shortened. Compared with the chemical preparation of (S,S)-2,8-diazabicyclo[4,3,0]nonane, the technology provided by the invention has high stereoselectivity, milder reaction conditions, and requires equipment low reaction cost, and is environmentally friendly.

Description

Recombinant lipase mutant, engineering bacterium and application

(I) technical field

The invention belongs to the fields of biological pharmacy and biotransformation, and particularly relates to a Candida Antarctica Lipase B (CALB) mutant, a mutant gene, a recombinant vector containing the mutant gene, a recombinant genetic engineering bacterium containing the mutant gene, and application of the Candida antarctica lipase B mutant in preparation of (2S,3R) -N-acetyl-piperidine-2, 3-dimethyl diformate.

(II) background of the invention

Lipase (EC 3.1.1.3), the systematic name of which is triacylglycerol acylhydrolase (triacylglycerol acylhydrolase), mainly catalyzes the hydrolysis of triglyceride into glycerol and free fatty acid in vivo, and is widely used in the industry for transesterification reactions such as esterification and ester exchange, alcoholysis, acid hydrolysis, and aminolysis. The lipase is widely existed in organisms such as microorganisms, plants, animals and the like, wherein the microbial lipase has the characteristics of multiple types, high activity, good stability, excellent selectivity and substrate specificity, wide reaction pH and temperature range and the like, and has important application in industrial production. Currently, the research on microbial lipases has mainly focused on strains having industrial application values, such as Rhizopus (Rhizopus), Aspergillus (Aspergillus), Penicillium (Penicillium), Mucor (Mucor), Geotrichum (Geotrichum), Candida (Candida), Pseudomonas (Pseudomonas), Burkholderia (Burkholderia), and the like.

(S, S) -2, 8-diazabicyclo [4,3,0] nonane is an important chiral intermediate for synthesizing moxifloxacin which is a fourth-generation quinolone antibacterial drug, is developed by German Bayer company, and is a broad-spectrum antibiotic mainly used for treating respiratory diseases such as acute sinusitis, chronic bronchitis and the like. For the preparation of (S, S) -2, 8-diazabicyclo [4,3,0] nonane, the traditional synthetic methods mainly comprise a chemical resolution method, an asymmetric synthetic method and a chiral source method. The chemical resolution method is a main application method at present, and specifically takes 2, 3-pyridine dicarboxylate as a starting raw material, and a target product is obtained through the steps of dehydration, ammonolysis, cyclization, reduction, chemical resolution and the like. However, the method has the problems of low resolution yield, high energy consumption, serious pollution and the like, and does not meet the development trend of modern green chemistry (EP 0550903A1, US 20080221329A 1). In recent years, enzyme methods have been used instead of chemical methods to improve reaction conditions, reduce reaction costs, and increase product selectivity have been the focus of attention. The biological resolution method has high chemical, regional and stereoselectivity, mild reaction conditions and environmental friendliness, and effectively makes up for the defects of the chemical method. By using lipase as catalyst, racemic N-acetyl-piperidine-2, 3-dimethyl dicarboxylate can be efficiently resolved to obtain optically pure (2S,3R) -N-acetyl-piperidine-2, 3-dimethyl dicarboxylate, which can be further used for synthesizing (S, S) -2, 8-diazabicyclo [4,3,0] nonane. Compared with a chemical resolution method, the lipase resolution method has the advantages that the resolution step is advanced, the atom economy is high, the stereo selection is high, and the use of a chemical resolution agent is avoided. At present, few reports are provided about the resolution production of (2S,3R) -N-acetyl-piperidine-2, 3-dimethyl dicarboxylate by lipase method, and patent US 8680276B2 reports that 80g/L of racemic N-acetyl-piperidine-2, 3-dimethyl dicarboxylate is completely resolved within 140h by 40g/L of immobilized Candida antarctica lipase B. Nitin W, Fadnavis et al replaced immobilized Candida antarctica lipase B with 40g/L of liquid Candida antarctica lipase B (Addzyme CALB (5000TBU)), and resolved 80g/L of racemic N-acetyl-piperidine-2, 3-dicarboxylic acid dimethyl ester completely within 16h (ORGANIC PROCESS RESEARCH & DEVOPMENT Vol. 19: p. 1: 296-301).

However, the currently reported technology for producing (2S,3R) -N-acetyl-piperidine-2, 3-dimethyl dicarboxylate by lipase resolution mainly has the problems of low substrate concentration, low lipase catalytic activity, large catalyst usage amount, long-time reaction and the like in the resolution process, and cannot meet the requirements of large-scale industrial production.

Disclosure of the invention

The invention aims to provide a Candida antarctica lipase B mutant, a coding gene, a recombinant vector containing the mutant gene, a recombinant genetic engineering bacterium containing the mutant gene, and application of the Candida antarctica lipase B mutant in preparation of (2S,3R) -N-acetyl-piperidine-2, 3-dimethyl diformate. The mutant has higher substrate tolerance and higher catalytic activity to racemic N-acetyl-piperidine-2, 3-dimethyl dicarboxylate, can effectively solve the problems of low lipase catalytic activity, low substrate concentration of resolution reaction, long reaction time and the like at present, greatly improves the catalytic efficiency, reduces the industrial production period and reduces the production cost.

The technical scheme adopted by the invention is as follows:

the invention improves the catalytic activity of the wild Candida antarctica lipase B to racemic N-acetyl-piperidine-2, 3-dimethyl diformate by mutating single amino acid or multiple amino acids.

In one embodiment of the invention, a lipase coding gene (SEQ ID NO.1) is connected with an expression vector pET22b to construct a recombinant expression plasmid. And (3) transforming the recombinant expression plasmid into E.coli Rosetta (DE3) host bacteria to obtain the gene engineering bacteria containing the recombinant plasmid.

In one embodiment of the invention, the lipase is genetically modified by using a recombinant expression plasmid containing a lipase gene as a template through a single-point saturation mutation technology or a multi-point combined saturation mutation technology. The transformed recombinant expression plasmid is transformed into E.coli Rosetta (DE3) host bacteria to obtain the gene engineering bacteria containing lipase mutant genes. And performing induced expression culture on the obtained genetically engineered bacteria to obtain somatic cells of the lipase mutant, and performing large-scale screening by using bromothymol blue as a color developing agent through a high-throughput screening method to obtain the mutant with excellent performance.

The recombinant lipase mutant is obtained by performing single mutation or combined mutation on the 140 th, 141 th, 144 th, 189 th and 190 th amino acid sequences shown in SEQ ID NO.2, and preferably the mutant is obtained by mutating the amino acid sequences shown in SEQ ID NO.2 into one of the following amino acid sequences: (1) leucine 140 is mutated to glycine; (2) alanine at position 141 is mutated to leucine; (3) leucine at position 144 is mutated to serine; (4) isoleucine at position 189 is mutated to lysine, arginine, alanine, asparagine, histidine or tyrosine; (5) valine at position 190 is mutated to leucine; (6) leucine at position 140 is mutated to glycine, and alanine at position 141 is mutated to leucine; (7) isoleucine at position 189 is mutated to asparagine, and valine at position 190 is mutated to leucine; (8) isoleucine at position 189 is mutated into histidine, and valine at position 190 is mutated into leucine; (9) leucine at position 144 is mutated to serine, and isoleucine at position 189 is mutated to lysine; (10) leucine 140 is mutated to glycine, alanine 141 to leucine and isoleucine 189 to lysine.

In one embodiment of the invention, the lipase mutant gene is integrated into a Pichia pastoris X-33 genome through homologous recombination to obtain the recombinant Pichia pastoris genetically engineered bacteria containing the lipase mutant gene. And (3) carrying out methanol induction culture on the obtained genetically engineered bacteria, and separating the culture solution from the bacteria to obtain lipase mutant primary enzyme solution. Separating the obtained crude enzyme liquid by nickel column affinity chromatography to obtain lipase mutant pure enzyme, and comparing the catalytic activity of the mutant lipase with that of the original lipase to determine the improvement of the catalytic performance. The enzyme activity unit (U) is defined as: the amount of enzyme required to consume 1. mu. mol of (2R,3S) -N-acetyl-piperidine-2, 3-dicarboxylic acid dimethyl ester in 1min at 35 ℃ and pH 6.0 is defined as 1U.

The invention relates to an application of the lipase mutant in preparation of (2S,3R) -N-acetyl-piperidine-2, 3-dimethyl dicarboxylate, and the application specifically comprises the following steps: pure enzyme extracted from crude enzyme liquid obtained by fermentation culture of engineering bacteria containing lipase mutant genes is used as a catalyst, racemic N-acetyl-piperidine-2, 3-dimethyl dicarboxylate is used as a substrate, a buffer solution (preferably sodium phosphate buffer solution) with the pH value of 6 is used as a reaction medium to form a reaction system, the reaction is completed at 35 ℃ and 600rpm, and the reaction liquid is separated and purified to obtain (2S,3R) -N-acetyl-piperidine-2, 3-dimethyl dicarboxylate. In the reaction system, the amount of the catalyst is 0.1-0.8g/L (3000-30000U/L) based on the weight of the pure enzyme, the initial concentration of the substrate is 1-2mol/L (200-500 g/L), and the required conversion time is 5-8 h.

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

the invention obtains a series of lipase mutants with high stereoselectivity and catalytic activity for racemic N-acetyl-piperidine-2, 3-dimethyl dicarboxylate, and the obtained lipase mutants are used for producing (2S,3R) -N-acetyl-piperidine-2, 3-dimethyl dicarboxylate by catalytic resolution and have the advantages of mild reaction conditions, high substrate concentration, less catalyst consumption, short reaction time and high optical purity. 1mol/L (243.26g/L) racemic N-acetyl-piperidine-2, 3-dicarboxylic acid dimethyl ester has a conversion rate of 49.9 percent within 5h under the catalysis of 0.1g/L lipase mutant, e.e._s>99% is obviously superior to the lipase reported in the prior art.

(IV) description of the drawings

FIG. 1: schematic representation of lipase-catalyzed resolution of racemic N-acetyl-piperidine-2, 3-dicarboxylic acid dimethyl ester.

FIG. 2: SDS-PAGE of coli Rosetta (DE3)/pET22b-CALB expression lipase.

Lane 1 is Marker; lane 2 is E.coli Rosetta (DE3)/pET22b-CALB non-induced disruption supernatant; lane 3 is E.coli Rosetta (DE3)/pET22b-CALB non-induced disruption pellet-like; lane 4 is E.coli Rosetta (DE3)/pET22b-CALB induced disruption supernatant; lane 5 is E.coli Rosetta (DE3)/pET22b-CALB induced disruption pellet serum.

FIG. 3: SDS-PAGE (sodium dodecyl sulfate-PAGE) graph of Pichia pastoris X-33/CALB expression and purified lipase.

Lane 1 is Marker; lane 2 is the Pichia pastoris X-33/CALB fermentation supernatant; lane 3 is a Pichia pastoris X-33/CALB purified sample.

FIG. 4: CALB splits 1mol/L substrate process diagram.

FIG. 5: mu-Ile 189Lys is used for resolving a 1mol/L substrate process map.

FIG. 6: mu-Ile 189Lys split 500g/L substrate process map.

FIG. 7: and (3) splitting a 1mol/L substrate process map by mut-Leu144Ser/Ile189 Lys.

FIG. 8: and (3) splitting a 1mol/L substrate process diagram by mut-Leu140Gly/Ala141Leu/Ile189 Lys.

(V) detailed description of the preferred embodiments

The present invention will be described in further detail with reference to specific examples, but the present invention is not limited to the following examples. The implementation conditions adopted in the examples can be further adjusted according to different requirements of specific use, and the implementation conditions not indicated are those in routine experiments.

Example 1: construction of recombinant Lipase Gene engineering bacterium E.coli Rosetta (DE3)/pET22b-CALB

Candida Antarctica Lipase B (CALB) gene sequence is optimized by yeast codon and pGEM-T-CALB plasmid is obtained by whole gene synthesis. Design of expression primer 1 (GG)CCATGGCCTTACCTAGTGGTTCCGACCCTG), primer 2 (GG)GCGG CCGCTCAATGATGATGATGGTGGTGAGGAGTAACAATTCCTGAAC) (restriction sites Nco I and Not I underlined), was amplified with high fidelity Pfu DNA polymerase to obtain 951bp of lipase gene sequence (nucleotide sequence shown IN SEQ IN NO.1)Shown as an amino acid sequence SEQ IN NO. 2). The amplified fragment was digested with Nco I and Not I restriction enzymes, and the fragment was ligated with pET22b treated with the same restriction enzymes using T4DNA ligase to construct an expression vector pET22 b-CALB. The constructed expression vector is transformed into E.coli Rosetta (DE3) competent cells to obtain recombinant lipase gene engineering bacteria E.coli Rosetta (DE3)/pET22 b-CALB.

Example 2: expression and screening of recombinant lipase mutants

A recombinant strain (E.coli Rosetta (DE3)/pET22b-CALB) containing an expression vector pET22b-CALB is used as an original strain, and the catalytic activity of lipase on substrate racemic N-acetyl-piperidine-2, 3-dimethyl diformate is further improved by a site-specific saturation mutation technology. Primers were designed as follows:

Leu/Ala (L/A)140/141 combination:

an upstream primer 5: 5 '-ACTACAAAGGTACCGTGNDTNDTGGTCCACTTGACGCCTT-3'

A downstream primer 6: 5'-GAGAACTGTGATGTCAAAGATCCTGCATGATCGATAAC-3'

Leu(L)144：

An upstream primer 7: 5 '-AGGTACCGTGTTGGCTGGTCCANNKGACGCCTTGGCAGT-3'

A downstream primer 8: 5 '-GGACACTGCCAAGGCGTCMNNTGGACCAGCCAACACGGT-3'

Ile(I)189：

An upstream primer 9: 5' -CTCAGCTACAGACGAANNKGTTCAGCCTCAAGTTAGT-3’

A downstream primer 10: 5' -CTAACTTGAGGCTGAACMNNTTCGTCTGTAGCTGAG-3’

Val(V)190：

An upstream primer 11: 5' -TCAGCTACAGACGAAATTNNKCAGCCTCAAGTTAGTA-3’

A downstream primer 12: 5' -TTACTAACTTGAGGCTGMNNAATTTCGTCTGTAGCTG-3’

Ile/Val (I/V)189/190 combination:

an upstream primer 13: 5' -TACTCAGCTACAGACGAANDTNDTCAGCCTCAAGTTAGT-3’

A downstream primer 14: 5'-GAGAACTGTGATGTCAAAGATCCTGCATGATCGATAAC-3'

Mutations were introduced by PCR using pET22b-CALB plasmid as template, and the PCR reaction procedure was as follows: 3min at 98 ℃; repeating 29 cycles at 98 deg.C for 10s, 58 deg.C for 10s, and 72 deg.C for 6 min; extension was continued for 10min at 72 ℃. The PCR product was treated with DpnI at 37 ℃ for 3 hours, inactivated, transformed into E.coli Rosetta (DE3) competent cells, plated on LB solid plates containing a final concentration of 100mg/L ampicillin resistance and 20mg/L chloramphenicol resistance, cultured at 37 ℃ for 12 hours, and single colonies were randomly picked for selection.

Screening for positive clones: individual colonies on the plates were randomly picked into 96-well plates, and 1mL of liquid LB medium containing 100. mu.g/mLAmp (ampicillin) + 20. mu.g/mLCm (chloramphenicol) was added and cultured overnight at 37 ℃ and 150 rpm. mu.L of the seed solution was transferred to another new 96-well plate to which 1mL of liquid LB medium containing 100. mu.g/mLAmp (ampicillin) + 20. mu.g/mLCm (chloramphenicol) was added, and after culturing at 37 ℃ and 150rpm for 4 hours, IPTG (final concentration of 0.1mM) was added to the plate and then induced-expression culture was carried out at 22 ℃ for 12 hours, as shown in FIG. 2. Centrifuging the obtained bacterial liquid for 30min by a 96-well plate centrifuge to obtain wet thalli, washing the wet thalli once by phosphate buffer solution with the pH of 7.0, storing the wet thalli in a refrigerator at the temperature of minus 80 ℃, repeatedly freezing and thawing for 3 times, adding 200 mu L of lysozyme enzyme liquid with the concentration of 2g/L, treating for 2h at the temperature of 22 ℃, and centrifuging to remove cell fragments to obtain crude enzyme liquid. The mutant enzyme activity is judged by the color change of a pH indicator bromothymol blue, and specifically, a 96-pore plate 220 mu L enzyme activity determination system comprises: 20 μ L of pH indicator, 100 μ L of substrate (40g/L of racemic N-acetyl-piperidine-2, 3-dicarboxylic acid dimethyl ester), 100 μ L of enzyme solution obtained by lysozyme disruption treatment, and color change of the reaction solution was observed. Correspondingly, the higher the enzyme activity of the mutant is, the faster the color of the mutant is changed from blue to yellow, so that the mutant with relatively high activity is screened out.

A series of different lipase mutants were obtained by analysis of the saturation mutations at the Leu140/Ala141, Leu144, Ile189, Val190 and Ile189/Val190 positions. The optimal mutants were determined by comparing the high and low conversion of different lipase mutants in the catalytic substrate (racemic N-acetyl-piperidine-2, 3-dicarboxylic acid dimethyl ester). The transformation reaction was carried out in a 10mL transformation flask, the substrate concentration was 40g/L, the mutant wet cells were 10g/L, the reaction was carried out at 30 ℃ and 150rpm for 30min, and the optimum mutant was determined by determining the conversion rate of the reaction by HPLC analysis of the reaction solution.

The results show that the superior mutants obtained from different mutation sites are mut-Leu140Gly/Ala141Leu (Leu 140 IN the amino acid sequence shown IN SEQ IN NO.2 is mutated to Gly and Ala141 is mutated to Leu), mut-Leu144Ser (Leu 144 IN the amino acid sequence shown IN SEQ IN NO.2 is mutated to Ser), mut-Ile189Lys (Ile 189 IN the amino acid sequence shown IN SEQ IN NO.2 is mutated to Lys), mut-Ile189Arg (Ile 189 IN the amino acid sequence shown IN SEQ IN NO.2 is mutated to Arg), mut-Ile189Ala (Ile 189 IN the amino acid sequence shown IN SEQ IN NO.2 is mutated to Ala), mut-Ile189Asn/Val190Leu (Ile 18 IN the amino acid sequence shown IN SEQ IN NO.2 is mutated to Asn and Val190 is mutated to Leu), mut-Ile189 Val190 Leu/Leu (Ile 189 IN the amino acid sequence shown IN SEQ IN NO.2 is mutated to His and Tyr 190 is mutated to Val), and Tyr/Leu 190 IN the amino acid sequence 189 to Tyr), and Val190 mutated to Leu).

Performing superposition mutation on the better mutation sites to obtain a series of better mutants, wherein the optimal mutants are mut-Leu144Ser/Ile189Lys (Leu at position 144 of the amino acid sequence shown IN SEQ IN NO.2 is mutated into Ser, and Ile at position 189 is mutated into Lys) and mut-Leu140Gly/Ala141Leu/Ile189Lys (Leu at position 140 of the amino acid sequence shown IN SEQ IN NO.2 is mutated into Gly + Ala at position 141 is mutated into Leu + Ile at position 189 is mutated into Lys)

Example 3: construction of wild type and mutant recombinant lipase gene engineering bacteria Pichia pastoris X-33/CALB and Pichia pastoris X-33/CALB-muts

pGEM-T-CALB plasmid is used as PCR template to design expression primer 3 (GG)CTCGAGAAAAGAGAGGCTGAAGCTTTACCTAGTGGTTCCGACC), primer 4 (GGT)CTAGATCAATGATGATGATGGTGGTGAGGAGTAACAATTCCTGAA) (Xho I and Xba I restriction sites are underlined), and the amplification was carried out using high fidelity Pfu DNA polymerase to obtain a 951bp lipase gene sequence (nucleotide sequence shown in SEQ ID NO. 1). The amplified fragment was digested with Xho I and Xba I restriction enzymes, and ligated with pPicz α -A treated with the same restriction enzymes using T4DNA ligase to constructThe vector pPicz alpha-A-CALB is established. The constructed vector is linearized by Sca I, and the linearized pPicz alpha-A-CALB is introduced into Pichia pastoris X-33 by an electric shock transformation method and integrated into a genome to obtain the recombinant lipase gene engineering strain Pichia pastoris X-33/CALB.

The site-directed mutagenesis primer is designed as follows:

Leu140Gly/Ala141Leu：

an upstream primer 15: 5'-TCCTGACTACAAAGGTACCGTGGGGCTTGGTCCACTTGA-3'

A downstream primer 16: 5'-TGCCAAGGCGTCAAGTGGACCAAGCCCCACGGTACCTTT-3'

Leu144Ser：

An upstream primer 17: 5'-GGTACCGTGTTGGCTGGTCCATCTGACGCCTTGGCAGTG-3'

A downstream primer 18: 5'-GGAGCGGACACTGCCAAGGCGTCAGATGGACCAGCCAA-3'

Ile189Lys：

An upstream primer 19: 5'-CTCAGCTACAGACGAAAAGGTTCAGCCTCAAGTTAG-3'

A downstream primer 20: 5'-CTAACTTGAGGCTGAACCTTTTCGTCTGTAGCTGAG-3'

Ile189Arg：

An upstream primer 21: 5'-CTCAGCTACAGACGAACGTGTTCAGCCTCAAGTT-3'

A downstream primer 22: 5'-AACTTGAGGCTGAACACGTTCGTCTGTAGCTGAG-3'

Ile189Ala：

An upstream primer 23: 5'-ACTCAGCTACAGACGAAGCAGTTCAGCCTCAAGTTAG-3'

A downstream primer 24: 5'-CTAACTTGAGGCTGAACTACTTCGTCTGTAGCTGAGT-3'

Ile189Asn/Val190Leu：

An upstream primer 25: 5'-CTCAGCTACAGACGAAAATCTGCAGCCTCAAGTTAGTAA-3'

The downstream primer 26: 5'-GTTACTAACTTGAGGCTGCAGATTTTCGTCTGTAGCTGAG-3'

Ile189His/Val190Leu：

An upstream primer 27: 5'-CTCAGCTACAGACGAACATCTTCAGCCTCAAGTTAGTAA-3'

The downstream primer 28: 5'-GTTACTAACTTGAGGCTGAAGATGTTCGTCTGTAGCTGA-3'

Ile189Tyr/Val190Leu：

An upstream primer 29: 5'-TACTCAGCTACAGACGAATATCTGCAGCCTCAAGTTAG-3'

A downstream primer 30: 5'-CTAACTTGAGGCTGCAGATATTCGTCTGTAGCTGAGTA-3'

The pPicz alpha-A-CALB plasmid is used as a template, and mutation is introduced by PCR, wherein the PCR reaction program is as follows: 3min at 98 ℃; repeating 29 cycles at 98 deg.C for 10s, 58 deg.C for 10s, and 72 deg.C for 4min for 30 s; extension was continued for 10min at 72 ℃. Treating the PCR product with DpnI at 37 ℃ for 3h, inactivating, transforming to E.coliDH5 alpha competent cells, coating on an LB solid plate containing 100mg/L of bleomycin resistance at the final concentration, culturing at 37 ℃ for 12h, and randomly picking out a single colony for sequencing. The constructed vector is linearized by Sca I, the linearized pPicz alpha-A-CALB-muts is led into Pichia pastoris X-33 by an electric shock transformation method, and the recombinant lipase gene engineering bacteria Pichia pastoris X-33/CALB-muts are obtained after the integration into the genome.

Example 4: separation and purification of wild type and mutant recombinant lipase

The wild type and mutant recombinant lipase gene engineering bacteria constructed in the example 3 are inoculated to BMGY culture medium, cultured for 12h at 30 ℃, centrifuged and transferred to BMMY culture medium, and induced with methanol for 72h at 30 ℃. Centrifuging to obtain fermentation supernatant containing the target protein. And ultrafiltering and concentrating the supernatant by using an ultrafiltration membrane to obtain concentrated enzyme liquid. Incubating the concentrated enzyme solution with Ni affinity chromatography resin balanced by binding buffer, washing with washing buffer (50mM, pH 8.0 sodium phosphate buffer containing 300mM NaCl, 15mM imidazole) until the protein is basically free of impurity, eluting with elution buffer (50mM, pH 8.0 sodium phosphate buffer containing 300mM NaCl, 500mM imidazole) and collecting target protein, combining the target protein after electrophoretic identification of purity, dialyzing with dialysis buffer (20mM, pH 7.0 sodium phosphate buffer) for 24h, taking the retentate, and determining the protein content with BCA kit, and frozen in a refrigerator at-80 ℃ (FIG. 3) to obtain pure enzymes of wild type and mutant lipase CALB-WT, mut-Ile189Lys, mut-Ile189Arg, mut-Ile189Ala, mut-Ile189Asn/Val190Leu, mut-Ile189His/Val190Leu, Ile189Tyr/Val190Leu, mut-Leu140Gly/Ala141Leu/Ile189Lys, and mut-Leu144Ser/Ile189 Lys.

Example 5: lipase activity assay

The purified wild-type and mutant lipases CALB-WT, mut-Ile189Lys, mut-Ile189Arg, mut-Ile189Ala, mut-Ile189Asn/Val190Leu, mut-Ile189His/Val190Leu, Ile189Tyr/Val190Leu, mut-Leu144Ser/Ile189Lys, mut-Leu140Gly/Ala141Leu/Ile189Lys in example 4 were used as catalytic substrates (racemic N-acetyl-piperidine-2, 3-dimethyl dicarboxylate)

The enzyme catalysis system comprises the following components under the catalysis conditions: 1mL of phosphate buffer (100mM, pH 6.0) containing 50mM racemic N-acetyl-piperidine-2, 3-dicarboxylic acid dimethyl ester was reacted at 35 ℃ and 800rpm with the addition of wild-type and mutant pure enzymes diluted with the same buffer solution. After reacting for a certain time, adding 30 mu L of 6M HCl to terminate the reaction, uniformly mixing, sampling and extracting, and processing a sample to detect the enzyme activity.

TABLE 1 CALB wild type and mutant Lipase enzyme Activity

The enzyme activity unit (U) is defined as: the amount of enzyme required to consume 1. mu. mol of (2R,3S) -N-acetyl-piperidine-2, 3-dicarboxylic acid dimethyl ester in 1min at 35 ℃ and pH 6.0 is defined as 1U.

Example 6: application of recombinant wild-type lipase CALB in preparation of (2S,3R) -N-acetyl-piperidine-2, 3-dimethyl dicarboxylate

The wild-type CALB pure enzyme obtained in example 4 was used as a biocatalyst, and racemic N-acetyl-piperidine-2, 3-dicarboxylic acid dimethyl ester was used as a substrate to prepare (2S,3R) -N-acetyl-piperidine-2, 3-dicarboxylic acid dimethyl ester by an enzymatic resolution reaction.

The catalytic reaction system and catalytic conditions are as follows: 30mL of sodium phosphate buffer solution (pH 6.0) was added CALB pure enzyme to a final concentration of 0.1g/L and an initial substrate concentration (racemic N-acetyl-piperidine-2, 3-dicarboxylate as substrate) of 1mol/L (243.26g/L), water bath at 35 ℃ was stirred magnetically at 600rpm, pH was controlled to 6.0 by automatic flow-addition of 4M NaOH solution, and samples were taken at regular time and analyzed by HPLC. The catalytic course (fig. 4) results show a catalytic 5h conversion of 1.13%.

Example 7: application of recombinant lipase mutant mut-Ile189Lys in preparation of (2S,3R) -N-acetyl-piperidine-2, 3-dimethyl dicarboxylate

The lipase mutant mut-Ile189Lys pure enzyme obtained in example 4 was used as a biocatalyst, racemic N-acetyl-piperidine-2, 3-dicarboxylic acid dimethyl ester was used as a substrate, and an enzymatic resolution reaction was performed to prepare (2S,3R) -N-acetyl-piperidine-2, 3-dicarboxylic acid dimethyl ester.

The catalytic reaction system and catalytic conditions are as follows: to 30mL of sodium phosphate buffer solution (pH 6.0) was added mut-Ile189Lys pure enzyme to a final concentration of 0.1g/L and an initial substrate concentration (racemic N-acetyl-piperidine-2, 3-dicarboxylate as substrate) of 1mol/L (243.26g/L), water bath at 35 ℃ was added, 600rpm was magnetically stirred, pH was controlled to 6.0 by auto-flowing 4M NaOH solution, and samples were taken at regular time and analyzed by HPLC. The results of the catalytic run (fig. 5) show a catalytic 5h conversion of 49.9%, e.e._s>99％。

Example 8: application of recombinant lipase mutant mut-Ile189Lys in preparation of (2S,3R) -N-acetyl-piperidine-2, 3-dimethyl dicarboxylate

The lipase mutant mut-Ile189Lys pure enzyme obtained in example 4 was used as a biocatalyst, racemic N-acetyl-piperidine-2, 3-dicarboxylic acid dimethyl ester was used as a substrate, and an enzymatic resolution reaction was performed to prepare (2S,3R) -N-acetyl-piperidine-2, 3-dicarboxylic acid dimethyl ester.

The catalytic reaction system and catalytic conditions are as follows: to 30mL of sodium phosphate buffer solution (pH 6.0) was added mut-Ile189Lys pure enzyme to a final concentration of 0.8g/L and an initial substrate concentration (racemic N-acetyl-piperidine-2, 3-dicarboxylic acid dimethyl ester as substrate) of 500g/L, a water bath at 35 ℃ was applied, the mixture was magnetically stirred at 600rpm, the pH was controlled to 6.0 by means of an automatic flow-in 4M NaOH solution, and samples were taken at regular time and analyzed by HPLC. The results of the catalytic run (fig. 6) show a catalytic 8h conversion of 49.9%, e.e._s>99％。

Example 9: application of recombinant lipase mutant mut-Leu144Ser/Ile189Lys in preparation of (2S,3R) -N-acetyl-piperidine-2, 3-dimethyl dicarboxylate

The lipase mutant mut-Leu144Ser/Ile189Lys pure enzyme obtained in example 4 was used as a biocatalyst, and racemic N-acetyl-piperidine-2, 3-dicarboxylic acid dimethyl ester was used as a substrate to prepare (2S,3R) -N-acetyl-piperidine-2, 3-dicarboxylic acid dimethyl ester by an enzymatic resolution reaction.

The catalytic reaction system and catalytic conditions are as follows: to 30mL of sodium phosphate buffer solution (pH 6.0) was added mut-Leu144Ser/Ile189Lys pure enzyme to a final concentration of 0.1g/L and an initial substrate concentration (racemic N-acetyl-piperidine-2, 3-dicarboxylate as a substrate) of 1mol/L (243.26g/L), water bath at 35 ℃ was magnetically stirred at 600rpm, pH was controlled to 6.0 by auto-flowing 4M NaOH solution, reaction was timed and sampled and analyzed by HPLC. The results of the catalytic run (fig. 7) show a catalytic 6h conversion of 49.9%, e.e._s>99％。

Example 10: application of recombinant lipase mutant mut-Leu140Gly/Ala141Leu/Ile189Lys in preparation of (2S,3R) -N-acetyl-piperidine-2, 3-dimethyl dicarboxylate

The lipase mutant mut-Leu140Gly/Ala141Leu/Ile189Lys pure enzyme obtained in example 4 was used as a biocatalyst, and racemic N-acetyl-piperidine-2, 3-dicarboxylic acid dimethyl ester was used as a substrate to prepare (2S,3R) -N-acetyl-piperidine-2, 3-dicarboxylic acid dimethyl ester by an enzymatic resolution reaction.

The catalytic reaction system and catalytic conditions are as follows: to 30mL of sodium phosphate buffer solution (pH 6.0) was added mut-Leu140Gly/Ala141Leu/Ile189Lys pure enzyme to a final concentration of 0.1g/L, an initial substrate concentration (racemic N-acetyl-piperidine-2, 3-dicarboxylic acid dimethyl ester as substrate) of 1mol/L (243.26g/L), a water bath at 35 ℃ was added, stirring was performed magnetically at 600rpm, pH was controlled to 6.0 by auto-flowing 4M NaOH solution, sampling was performed periodically, and analysis was performed by HPLC. The results of the catalytic run (fig. 8) show a catalytic 6h conversion of 49.9%, e.e._s>99％。

Sequence listing

<110> Zhejiang industrial university

<120> recombinant lipase mutant, engineering bacterium and application

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 951

<212> DNA

<213> Unknown (Unknown)

<400> 1

ttacctagtg gttccgaccc tgctttctct cagcctaaga gtgtgctgga tgcaggtctt 60

acatgtcaag gtgcatcccc atcttccgtt agtaaaccta ttttactggt tccaggaact 120

ggtactactg gaccacaaag tttcgattct aattggatcc ctctgtccac ccaactagga 180

tatacgccat gttggatttc tcctccacca ttcatgttaa acgatactca agttaacact 240

gagtacatgg ttaacgctat taccgcactt tacgctggtt caggaaacaa taaattgcct 300

gtcttgacct ggtctcaggg tggcttagtc gcccaatggg gactgacatt cttcccttca 360

atcagatcaa aggtcgacag acttatggcc tttgctcctg actacaaagg taccgtgttg 420

gctggtccac ttgacgcctt ggcagtgtcc gctccttccg tctggcaaca aaccacgggt 480

tcagctttga cgactgccct gcgaaatgct ggaggattga ctcaaatagt gcccactact 540

aacctatact cagctacaga cgaaattgtt cagcctcaag ttagtaacag tccactagat 600

tcatcctatc tatttaacgg caagaatgtt caagcacagg cagtctgtgg tcctcttttc 660

gttatcgatc atgcaggatc tttgacatca cagttctcat acgtagtggg tcgatccgcc 720

ttgaggtcaa caacgggtca agccagatct gccgactacg gtatcaccga ttgtaaccct 780

ctgcctgcaa acgatctgac ccctgaacaa aaggtcgctg ccgcagccct gctggctcca 840

gcagctgccg ctatcgttgc tggtccaaaa caaaattgcg aacctgattt aatgccttac 900

gcaagacctt tcgctgtcgg aaagagaacc tgttcaggaa ttgttactcc t 951

<210> 2

<211> 317

<212> PRT

<213> Unknown (Unknown)

<400> 2

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 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

Ala Ala Ala Ala Leu Leu Ala Pro Ala Ala Ala Ala Ile Val Ala 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

Claims (7)

1. A recombinant lipase mutant, characterized in that the mutant is to mutate the amino acid sequence shown in SEQ ID NO. 2 to one of the following: (1) leucine at position 144 is mutated to serine, and Mutation of isoleucine at position 189 to lysine; (2) mutation of leucine at position 140 to glycine, mutation of alanine at position 141 to leucine and mutation of isoleucine at position 189 to lysine . 2. A gene encoding the recombinant lipase mutant of claim 1. 3. A recombinant vector constructed from the coding gene of claim 2. 4. A recombinant genetically engineered bacteria transformed by the recombinant vector described in claim 3. 5. The application of the recombinant lipase mutant of claim 1 in the preparation of moxifloxacin pharmaceutical intermediate (2S,3R)-N-acetyl-piperidine-2,3-dicarboxylate dimethyl ester. 6. application as claimed in claim 5, it is characterized in that described application is: the fermented liquid centrifugation after fermentation culture of genetically engineered bacteria that is integrated with recombinant lipase mutant coding gene, with fermentation supernatant or separation and purification The latter enzyme is used as a catalyst, racemic N-acetyl-piperidine-2,3-dicarboxylate dimethyl ester is used as a substrate, and a 100mM phosphate buffer with a pH value of 3.0-10.0 is used as a reaction medium to form a reaction system. , the conversion reaction is carried out at 25-50 °C, and after the reaction is completed, the reaction solution of (2S,3R)-N-acetyl-piperidine-2,3-dicarboxylic acid dimethyl ester is obtained. 7. The application according to claim 6, wherein in the reaction system, the initial concentration of the substrate is 1-2 mol/L, and the consumption of the enzyme is 0.1-0.8 g/L.

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