CN108220261B - Ketoreductase, nucleic acid, recombinant expression vector, recombinant expression strain and application - Google Patents
Ketoreductase, nucleic acid, recombinant expression vector, recombinant expression strain and application Download PDFInfo
- Publication number
- CN108220261B CN108220261B CN201711481250.1A CN201711481250A CN108220261B CN 108220261 B CN108220261 B CN 108220261B CN 201711481250 A CN201711481250 A CN 201711481250A CN 108220261 B CN108220261 B CN 108220261B
- Authority
- CN
- China
- Prior art keywords
- ketoreductase
- recombinant expression
- tert
- hydroxypiperidine
- induction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/10—Nitrogen as only ring hetero atom
- C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
Abstract
The invention provides a ketoreductase, nucleic acid for coding the ketoreductase, a recombinant expression vector containing the nucleic acid, a recombinant expression strain containing the recombinant expression vector, a preparation method of the recombinant expression strain, and application of the ketoreductase in asymmetric reduction preparation of (S) -1-tert-butyloxycarbonyl-3-hydroxypiperidine. Compared with other existing preparation methods, the (S) -1-tert-butoxycarbonyl-3-hydroxypiperidine prepared by using the ketoreductase of the invention has high concentration and optical purity, mild reaction conditions, environmental friendliness, simple and convenient operation, easy industrial amplification and good industrial application prospect.
Description
Technical Field
The invention belongs to the technical field of biochemical engineering, and particularly relates to ketoreductase, nucleic acid, a recombinant expression vector, a recombinant expression strain and application of ketoreductase in asymmetric synthesis of (S) -1-tert-butyloxycarbonyl-3-hydroxypiperidine.
Background
(S) -1-tert-butyloxycarbonyl-3-hydroxypiperidine is an important medical intermediate and is widely applied to medicaments for analgesia, antipsychotic, anti-tumor and the like, such as ibrutinib.
The (S) -1-tert-butyloxycarbonyl-3-hydroxypiperidine can be obtained by chemical or resolution, but the chemical method or resolution method is limited by a relatively low yield, and the resolution method has a theoretical maximum yield of 50%.
Biologically, chiral hydroxy compounds can be synthesized through enzyme catalysis, and the reaction conditions are mild, so that the corresponding selectivity of the product can reach 99%. Currently, more and more drugs or intermediates are synthesized by biocatalytic means. The biocatalytic method is to asymmetrically reduce precursor N-Boc-3-piperidone into (S) -1-tert-butyloxycarbonyl-3-hydroxypiperidine.
Patent CN201310173088.2 discloses an asymmetric reduction of N-Boc-3-piperidone using recombinant Ketoreductase (KRED) enzyme powder, but does not disclose the gene sequence or amino acid sequence of Ketoreductase (KRED). Patent CN201310054684.9 discloses asymmetric synthesis of (S) -1-tert-butoxycarbonyl-3-hydroxypiperidine by using alcohol dehydrogenase PAR, but the coenzyme circulation is performed by using an organic reagent isopropanol, and the organic reagent has great damage to the enzyme activity and obvious inhibition effect. Patent CN201610132936.9 discloses an asymmetric Ketoreductase (KRED) reduction using carbonyl reductase ReCR enzyme liquid, but the enzyme liquid needs to be purified by Ni-NTA and is a two-phase reaction of sec-octanol-water, which is not favorable for scale-up production or has high production cost.
Disclosure of Invention
Aiming at the reported problems of low yield, unfriendly reaction conditions and higher cost in the reaction for preparing (S) -1-tert-butyloxycarbonyl-3-hydroxypiperidine by asymmetric reduction, the invention aims to provide a ketoreductase with high catalytic activity, strong enantioselectivity and higher yield, a nucleic acid for coding the ketoreductase, a recombinant expression vector containing the nucleic acid, a recombinant expression strain containing the recombinant expression vector, a preparation method of the recombinant expression strain and application of the ketoreductase in asymmetric synthesis of (S) -1-tert-butyloxycarbonyl-3-hydroxypiperidine.
The technical scheme adopted by the invention is as follows:
a ketoreductase which is a protein of the following (a), (b) or (c):
(a) 1, polypeptide consisting of an amino acid sequence shown in SEQ ID NO;
(b) a polypeptide having ketoreductase properties which is mutated, deleted or added with one or several amino acids on the basis of the polypeptide of (a); the mutation is 1-10 amino acid mutation on the basis of the polypeptide, namely the polypeptide still has the property of ketoreductase after mutation, and several of the mutation refer to 1-30 amino acids, such as increasing His Tag or adding a signal peptide for secretory expression;
(c) a polypeptide having at least 90% identity to the polypeptide of (a) and ketoreductase activity.
A nucleic acid encoding a ketoreductase enzyme of the invention.
A recombinant expression vector comprising a nucleic acid of the invention.
A recombinant expression strain comprising the recombinant expression vector of the invention.
The preparation method of the recombinant expression strain comprises the following steps:
(a) transforming the recombinant expression vector into a host cell, coating the host cell on an LB plate containing 50 mu g/mL ampicillin, culturing overnight at 37 ℃, and then selecting a positive colony from the host cell to inoculate into 50mL of liquid LB culture medium for culturing; after culturing for 4h, sucking 10ml of seed liquid and transferring the seed liquid into 1L of fresh LB liquid culture medium;
(b) when the concentration of the culture reached OD600When the concentration is 0.6-1.0, adding isopropyl-beta-D-thiogalactopyranoside (IPTG) with the final concentration of 0.05-1.0 mmol/L for induction, carrying out induction culture at the induction temperature of 16-35 ℃ for 8-16h, centrifuging or concentrating after the induction culture to obtain wet thalli with higher concentration, carrying out ultrasonic crushing or high-pressure homogenizer crushing, and centrifuging to collect supernatant, namely the crude enzyme lysate of the recombinant expression strain.
The preparation method of the recombinant expression strain comprises the following steps of (a) preparing a liquid LB culture medium: 10g/L of peptone, 5g/L of yeast extract, 10g/L of NaCl and 7.2 of pH; coli BL21(DE 3); adding isopropyl-beta-D-thiogalactopyranoside (IPTG) with the final concentration of 0.15mmol/L into the step (b) for induction, wherein the induction temperature is 28 ℃, and the induction culture is carried out for 12 h.
The ketoreductase is applied to asymmetric reduction preparation of (S) -1-tert-butyloxycarbonyl-3-hydroxypiperidine, and performs asymmetric reduction reaction on a precursor carbonyl compound in a buffer solution with pH of 6.0-8.0 in the presence of glucose, Glucose Dehydrogenase (GDH) and coenzyme to form (S) -1-tert-butyloxycarbonyl-3-hydroxypiperidine.
The application of the ketoreductase in the asymmetric reduction preparation of (S) -1-tert-butyloxycarbonyl-3-hydroxypiperidine comprises the following steps:
(1) adding 500ml of 50mM phosphate buffer solution into the flask, wherein the pH value of the phosphate buffer solution is 6.0-8.0, and stirring;
(2) then adding the ketoreductase, glucose dehydrogenase and glucose, adding NAD coenzyme and a precursor carbonyl compound, then diluting to constant volume by using a phosphate buffer solution, reacting at the temperature of 25-35 ℃, controlling the pH by using 2mol/L sodium hydroxide, and detecting the reaction completion by using a TLC point plate after reacting for 16 h;
(3) heating the reaction system to 50-70 ℃ for 1-2h, adding diatomite to filter and remove protein, extracting a water phase by using equal volume of ethyl acetate and washing a filter cake;
(4) extracting twice, combining organic phases, drying by using anhydrous sodium sulfate, and decompressing and whirling to obtain an oily crude product, namely the (S) -1-tert-butyloxycarbonyl-3-hydroxypiperidine.
The ketoreductase is applied to asymmetric reduction preparation of (S) -1-tert-butyloxycarbonyl-3-hydroxypiperidine, wherein NAD coenzyme in step (2) is NAD+Or NADP+The amount of the NAD coenzyme is 0.01-0.1 g/L; the precursor carbonyl compound is N-Boc-3-piperidone, and the concentration of the N-Boc-3-piperidone in the reaction liquid is 10-200 g/L; the dosage of the ketoreductase is 100-1000U/L; the dosage of the glucose is 20-200 g/L, the dosage of the glucose dehydrogenase is 100-2000U/L, and the concentration of the glucose is 1.2-1.5 equivalents of the precursor carbonyl compound.
The ketoreductase is applied to the asymmetric reduction preparation of (S) -1-tert-butyloxycarbonyl-3-hydroxypiperidine, wherein the pH of the phosphate buffer solution in the step (1) is 7.0, and the reaction is carried out at the temperature of 30 ℃ in the step (2).
The invention has the beneficial effects that:
the ketoreductase can convert a substrate N-Boc-3-piperidone into (S) -1-tert-butyloxycarbonyl-3-hydroxypiperidine, and the amino acid sequence of the ketoreductase is obviously different from that of other known ketoreductases.
The ketoreductase is applied to the asymmetric reduction preparation of (S) -1-tert-butyloxycarbonyl-3-hydroxypiperidine, a precursor carbonyl compound can reach 200g/L, and the reaction time is less than 24 h; NAD (nicotinamide adenine dinucleotide)+Coenzyme NAD is added in liquid enzyme reaction, and expensive coenzyme NAD is not required to be added in whole cells+(ii) a The yield of the product (S) -1-tert-butyloxycarbonyl-3-hydroxypiperidine is 95.8%, and the e.e. value thereof>99% by using glucose dehydrogenase to realize NAD+The circulation between NADH avoids the harm of organic reagents such as isopropanol and the like to the environment and ketoreductase. Compared with other existing preparation methods, the (S) -1-tert-butoxycarbonyl-3-hydroxypiperidine prepared by using the ketoreductase of the invention has high concentration and optical purity, mild reaction conditions, environmental friendliness, simple and convenient operation, easy industrial amplification and good industrial application prospect.
Drawings
FIG. 1 is a photograph of electrophoresis of PCR amplification in example 2;
FIG. 2 is an SDS-PAGE electrophoretogram in example 3.
The invention will be further illustrated with reference to specific embodiments and the accompanying drawings.
Detailed Description
Example 1
Acquisition of ketoreductase MT-KRED Gene
(1) Collecting a soil sample from China science and technology university in Hefei city, Anhui province, extracting DNA (the gene extraction method refers to Chroma Spin TE-1000, Clontech Laboratories, Inc., USA), cutting the extracted DNA sample with Sau3AI, cutting gel after electrophoresis, recovering a 0.5-4 kb DNA fragment, and connecting the DNA fragment to a pUC19 plasmid through a BamHI site to obtain a plasmid library of a genome;
(2) transforming the plasmid library in the step (1) into Escherichia coli E.coli BL21(DE3) (purchased from Beijing Quanji Biotechnology Co., Ltd.), coating the Escherichia coli E.coli BL21 on an LB plate with corresponding ampicillin, selecting positive clones, inoculating the positive clones to a 96 deep-hole plate with 500 mu LLB, culturing at 37 ℃ for 4h, adding isopropyl-beta-D-thiogalactopyranoside (IPTG) with the final concentration of 0.2mmol/L, and continuously culturing at 28 ℃ for overnight induction; centrifuging to collect deep-well culture, adding 50ul of 10mmol/LpH7.5 sodium phosphate buffer solution, repeatedly freezing and thawing at-80 deg.C for three times, and lysing bacteria;
(3) adding 1mmol/L precursor carbonyl compound N-Boc-3-piperidone as substrate, 20mmol/L glucose, 1U Glucose Dehydrogenase (GDH) and 0.001% phenol red, culturing at 30 ℃ for 4h, selecting deep-hole culture corresponding to the hole with most obvious red color, extracting plasmid and sequencing; the Open Reading Frame (ORF) of the sequence is analyzed by an ORF Finder of the National Center for Biotechnology Information (NCBI) to obtain an ORF nucleotide sequence, then the ketoreductase gene is screened out, and the encoded amino acid sequence SEQ ID NO 1 is further obtained.
Example 2
Cloning of ketoreductase MT-KRED Gene
Synthesizing a primer pair F1 and F2;
the PCR 20ul system was as follows: 2. mu.L of 2mmol/L dNTP, 2. mu.L of 10 XPCR buffer, 0.5. mu.L of PCR Hi-Fi enzyme, 1. mu.L of DNA template obtained in example 1, ddH2O12. mu.L, F1 and F2 each 1. mu.L (5 mmol/L). The PCR amplification step is as follows: firstly, pre-denaturation is carried out for 5min at 95 ℃; ② denaturation at 98 ℃ for 15 s; ③ annealing at 56 ℃ for 30 s; extension for 50s at 72 ℃; repeating the steps II to IV for 32 times; extending for 10min at 72 deg.C, and cooling to 16 deg.C. The PCR product is purified by agarose electrophoresis, the PCR amplification electrophoresis pattern is shown in figure 1, a target band appears at about 1000bp, the target band is recovered by an agarose gel recovery kit to obtain a complete sequence, and the PCR product of the ketoreductase gene is obtained after DNA sequencing and the full length of 1026 bp.
Example 3
Expression of ketoreductase MT-KRED Gene
The PCR product and pET21a vector described in example 2 were digested simultaneously with NdeI/XhoI, purified by agarose gel electrophoresis, and the target fragment was recovered using agarose gel recovery kit; and under the action of T4DNA ligase, the target fragment is connected with a pET21a vector subjected to double enzyme digestion to obtain a recombinant expression vector pET21 a-MT-KRED.
The recombinant expression vector pET21a-MT-KRED is transformed into E.coli BL21(DE3) competent cells, spread on LB plate containing 50. mu.g/mL ampicillin, cultured overnight at 37 ℃, and then positive colonies are selected from the competent cells and inoculated into 50mL of liquid LB medium for culture, wherein the liquid LB medium comprises the following components: 10g/L of peptone, 5g/L of yeast extract, 10g/L of NaCl and 7.2 of pH; after culturing for 4h, 10ml of seed liquid is sucked and transferred into 1L of fresh LB liquid culture medium, and the seed liquid is cultured until OD is reached600And after the temperature reaches 0.6-1.0 ℃, cooling to 28 ℃, adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.15mmol/L for induction culture for 12 hours, centrifugally collecting thalli at 5000rpm (namely, wet thalli of the recombinant expression strain is obtained), washing the thalli once by using 50mmol/L sodium phosphate buffer solution with the pH of 7.0, suspending each gram of thalli in 10mL of the phosphate buffer solution, ultrasonically crushing the thalli in an ice bath, centrifugally collecting supernatant, and obtaining crude enzyme lysate of the recombinant expression strain. And (3) taking partial supernatant to carry out SDS-PAGE protein electrophoresis to check the expression level, wherein the SDS-PAGE electrophoresis pattern is shown in figure 2, and the size of the target protein is consistent with the expected size from figure 2.
Example 4
The application of ketoreductase in preparing (S) -1-tert-butyloxycarbonyl-3-hydroxypiperidine by asymmetric reduction comprises the following steps:
(1) adding 500ml of 50mmol/L phosphate buffer solution into a 2L three-neck flask, wherein the pH value of the phosphate buffer solution is 7.0, and stirring;
(2) then 200ml of the crude enzyme lysate described in example 3, 2000U of Glucose Dehydrogenase (GDH) (purchased from sigmaa) and 100g of glucose were added, 0.05g of NAD coenzyme and 100g of N-Boc-3-piperidone as a precursor carbonyl compound were added, the volume was adjusted to 1L with a phosphate buffer, the reaction was carried out at 30 ℃ with pH controlled with 2mol/L sodium hydroxide, and the completion of the reaction was detected by TLC plate after 16 hours; the dosage of the ketoreductase is 500U/L;
(3) heating the reaction system to 70 ℃ for 2h, adding 10g of diatomite, filtering to remove protein, extracting a water phase by using equal volume of Ethyl Acetate (EA) and washing a filter cake;
(4) extracting twice, combining organic phases, drying by using anhydrous sodium sulfate, and decompressing and whirling to obtain an oily crude product, namely the (S) -1-tert-butyloxycarbonyl-3-hydroxypiperidine.
The crude product of this example was determined to have a purity of 98.0%, a molar yield of 95%, and an e.e. value of > 99%.
The specific analysis conditions for the product e.e. values were: the chromatographic column is a CP-Chirasil-DEX CB chiral capillary column and a FID detector. The temperature of the chromatographic column is 120 ℃, the gasification and detection temperatures are 280 ℃, and the carrier gas is N2。
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention by those skilled in the art should fall within the protection scope defined by the claims of the present invention without departing from the spirit of the present invention.
Sequence listing
<110> Anhui Co-wound biomedical products Ltd
<120> ketoreductase, nucleic acid, recombinant expression vector, recombinant expression strain and application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 341
<212> PRT
<213> incognita
<400> 1
Met Ala Asp Leu Pro Lys Thr Gln Tyr Gly Trp Lys Tyr Asp Lys Ser
1 5 10 15
Ile Asn Asn Leu Lys Leu Val Glu Asp Leu Lys Val Pro Thr Pro Ser
20 25 30
Ala Thr Gln Val Val Val Lys Ile Glu Ala Ala Gly Leu Cys His Ser
35 40 45
Asp Leu His Val Leu Glu Gly Leu Asp Val Gly Asp Asp Tyr Val Met
50 55 60
Gln His Glu Ile Phe Gln His Ile Val Leu Glu Ile Gly Asp Ser Val
65 70 75 80
Asn Pro Glu Val Phe Lys Val Gly Gly Arg Tyr Ala Val His Gly Leu
85 90 95
Asn Ser Cys Gly Ser Cys Glu Met Cys Arg Thr Gly His Asp Asn Asp
100 105 110
Cys Thr Gly Asn Glu Ser Lys Trp Tyr Gly Leu Gly Ile Ser Gly Gly
115 120 125
Tyr Gln Gln Tyr Leu Leu Val Pro Asn Ser His His Leu Leu Pro Ile
130 135 140
Pro Asp Asn Val Ser Tyr Glu Val Ala Ala Ala Thr Ser Asp Ala Val
145 150 155 160
Ser Ala Pro His His Ala Ile Lys Asn Ser Gly Val Thr Pro Ser Ser
165 170 175
Lys Val Leu Met Phe Gly Leu Gly Gly Leu Gly Ser Asn Ala Leu Gln
180 185 190
Ile Leu Lys Ala Phe Gly Ala Tyr Val Val Ala Val Asp Val Lys Pro
195 200 205
Ala Ser Lys Ala Ile Ala Asp Glu Phe Lys Ala Asp Glu Phe Tyr Thr
210 215 220
Asp Ile Ser Gln Ser Ser Trp Lys Pro Ala Ser Phe Asp Tyr Cys Phe
225 230 235 240
Asp Phe Val Ser Leu Gln Val Thr Phe Asp Ile Cys Gln Lys Tyr Ile
245 250 255
Lys Ser His Gly Val Cys His Pro Val Gly Leu Gly Ser Ser Lys Leu
260 265 270
Thr Phe Asp Leu Gly Asn Leu Ala Leu Arg Glu Val Lys Ile Val Gly
275 280 285
Asn Phe Trp Gly Thr Ser Gln Glu Gln Ile Glu Ala Met Glu Leu Val
290 295 300
Ser Ser Gly Arg Val Lys Pro Gln Val His Thr Thr Val Lys Leu Phe
305 310 315 320
Asp Gly Ala Lys Lys Glu Thr His Ser Gly Lys Phe Phe Asn Val Asp
325 330 335
Gly Thr Phe Leu Pro
340
Claims (10)
1. A ketoreductase enzyme, characterized by: the amino acid sequence of the ketoreductase is shown as SEQ ID NO. 1.
2. A nucleic acid encoding the ketoreductase enzyme of claim 1.
3. A recombinant expression vector comprising the nucleic acid of claim 2.
4. A recombinant expression strain comprising the recombinant expression vector of claim 3.
5. The method for producing the recombinant expression strain according to claim 4, wherein: the method comprises the following steps:
(a) transforming the recombinant expression vector of claim 3 into a host cell, plating the host cell on an LB plate containing 50. mu.g/mL of ampicillin, culturing overnight at 37 ℃, and then selecting a positive colony therefrom to inoculate 50mL of liquid LB medium for culture; after culturing for 4h, sucking 10ml of seed liquid and transferring the seed liquid into 1L of fresh LB liquid culture medium;
(b) when the concentration of the culture reached OD600When the concentration is 0.6-1.0, adding isopropyl-beta-D-thiogalactopyranoside with the final concentration of 0.05-1.0 mmol/L for induction, carrying out induction culture at the induction temperature of 16-35 ℃ for 8-16h, centrifuging or concentrating after the induction culture to obtain wet thalli with higher concentration, and centrifuging and collecting supernate after ultrasonic crushing or high-pressure homogenizer crushing to obtain crude enzyme lysate of the recombinant expression strain.
6. The method for producing a recombinant expression strain according to claim 5, wherein: the liquid LB culture medium in the step (a) comprises the following components: 10g/L of peptone, 5g/L of yeast extract, 10g/L of NaCl and 7.2 of pH; coli BL 21; adding isopropyl-beta-D-thiogalactopyranoside with the final concentration of 0.15mmol/L into the step (b) for induction, wherein the induction temperature is 28 ℃, and the induction culture is carried out for 12 h.
7. Use of the ketoreductase of claim 1 in the asymmetric reduction of (S) -1-tert-butoxycarbonyl-3-hydroxypiperidine, characterized in that: in a buffer solution with the pH value of 6.0-8.0, under the existence of glucose, glucose dehydrogenase and coenzyme, the ketoreductase carries out asymmetric reduction reaction on a precursor carbonyl compound to form (S) -1-tert-butyloxycarbonyl-3-hydroxypiperidine.
8. Use of the ketoreductase enzyme of claim 7 in the asymmetric reduction of (S) -1-tert-butoxycarbonyl-3-hydroxypiperidine, characterized in that: the method comprises the following steps:
(1) adding 500ml of 50mM phosphate buffer solution into the flask, wherein the pH value of the phosphate buffer solution is 6.0-8.0, and stirring;
(2) then adding the ketoreductase, glucose dehydrogenase and glucose, adding NAD coenzyme and a precursor carbonyl compound, then diluting to constant volume by using a phosphate buffer solution, reacting at the temperature of 25-35 ℃, controlling the pH by using 2mol/L sodium hydroxide, and detecting the reaction completion by using a TLC point plate after reacting for 16 h;
(3) heating the reaction system to 70 ℃ for 2h, adding diatomite to filter and remove protein, extracting a water phase by using equal volume of ethyl acetate, and washing a filter cake;
(4) extracting twice, combining organic phases, drying by using anhydrous sodium sulfate, decompressing and rotary steaming to obtain an oily crude product, namely the (S) -1-tert-butyloxycarbonyl-3-hydroxypiperidine.
9. Use of the ketoreductase enzyme of claim 8 in the asymmetric reduction of (S) -1-tert-butoxycarbonyl-3-hydroxypiperidine in which the NAD coenzyme in step (2) is NAD+Or NADP+The amount of the NAD coenzyme is 0.01-0.1 g/L; the precursor carbonyl compound is N-Boc-3-piperidone, and the concentration of the N-Boc-3-piperidone in the reaction liquid is 10-200 g/L; the dosage of the ketoreductase is 100-1000U/L; the dosage of the glucose is 20-200 g/L, the dosage of the glucose dehydrogenase is 100-2000U/L, and the concentration of the glucose is 1.2-1.5 equivalents of the precursor carbonyl compound.
10. Use of a ketoreductase enzyme according to any one of claims 8 to 9 in the asymmetric reduction of (S) -1-tert-butoxycarbonyl-3-hydroxypiperidine, characterized in that: the pH of the phosphate buffer solution in the step (1) is 7.0, and the reaction is carried out at the temperature of 30 ℃ in the step (2).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711481250.1A CN108220261B (en) | 2017-12-29 | 2017-12-29 | Ketoreductase, nucleic acid, recombinant expression vector, recombinant expression strain and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711481250.1A CN108220261B (en) | 2017-12-29 | 2017-12-29 | Ketoreductase, nucleic acid, recombinant expression vector, recombinant expression strain and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108220261A CN108220261A (en) | 2018-06-29 |
| CN108220261B true CN108220261B (en) | 2021-05-14 |
Family
ID=62647199
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711481250.1A Active CN108220261B (en) | 2017-12-29 | 2017-12-29 | Ketoreductase, nucleic acid, recombinant expression vector, recombinant expression strain and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108220261B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112251478B (en) * | 2020-10-26 | 2022-08-30 | 宁波酶赛生物工程有限公司 | Ketoreductase and enzyme catalysis preparation method of S-1-BOC-3 hydroxypiperidine |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103276027A (en) * | 2013-05-10 | 2013-09-04 | 苏州汉酶生物技术有限公司 | Method for biologically preparing chiral N-protective pipradrol |
| CN105567652A (en) * | 2014-10-14 | 2016-05-11 | 南京博优康远生物医药科技有限公司 | Ketoreductase and its use in asymmetric synthesis of chiral hydroxyl compound |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE10327454A1 (en) * | 2003-06-18 | 2005-01-20 | Juelich Enzyme Products Gmbh | Oxidoreductase from Pichia capsulata |
| HUE031334T2 (en) * | 2006-09-22 | 2017-07-28 | Pharmacyclics Llc | Inhibitors of bruton's tyrosine kinase |
-
2017
- 2017-12-29 CN CN201711481250.1A patent/CN108220261B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103276027A (en) * | 2013-05-10 | 2013-09-04 | 苏州汉酶生物技术有限公司 | Method for biologically preparing chiral N-protective pipradrol |
| CN105567652A (en) * | 2014-10-14 | 2016-05-11 | 南京博优康远生物医药科技有限公司 | Ketoreductase and its use in asymmetric synthesis of chiral hydroxyl compound |
Non-Patent Citations (3)
| Title |
|---|
| (S)-1-叔丁氧羰基-3-羟基哌啶的化学-酶法合成;竺伟等;《中国医药工业杂志》;20151231;第46卷(第4期);第349-350页 * |
| Characterization of a novel carbonyl reductase from Kuraishia capsulata，ID：I1V8K5_9ASCO;Ning C et al.;《EMBL》;20170215;第1页 * |
| uncharacterized protein KUCA_T00000094001 [Kuraishia capsulata CBS 1993],NCBI Reference Sequence：XP_022456152.1;Morales,L et al.;《GenBank》;20170927;第1-2页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108220261A (en) | 2018-06-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109593750B (en) | Nitrile hydratase mutant, genetic engineering bacterium containing same and application thereof | |
| CN109312375B (en) | Preparation method of hesperetin, preparation method of hesperetin intermediate and biological enzyme for preparing hesperetin | |
| CN108728421B (en) | Carbonyl reductase mutant and application thereof | |
| CN109266595B (en) | Construction and application of recombinant bacterium for producing L-2-aminobutyric acid by converting L-threonine | |
| CN107142251B (en) | Serratia carbonyl reductase and application thereof in preparation of optically active alkyl lactone | |
| CN113621600B (en) | High-activity nitrile hydratase mutant and application thereof | |
| CN104152505A (en) | Method for transforming and preparing 4-hydroxyl-L-isoleucine by using recombinant bacterial strains | |
| CN105349503A (en) | Carbonyl reductase AcCR and encoding gene and application thereof | |
| CN113337450A (en) | Escherichia coli genetic engineering bacterium, construction method and method for producing (R) -citronellal through whole-cell catalysis | |
| CN108220261B (en) | Ketoreductase, nucleic acid, recombinant expression vector, recombinant expression strain and application | |
| CN108570439A (en) | The fusion protein of oxidoreducing enzyme, genetic engineering bacterium and its preparation method and application | |
| CN114507650B (en) | Leucine dehydrogenase mutant and application thereof in synthesis of (S) -o-chlorophenylglycine | |
| CN109706189B (en) | Preparation method of D-chiro-inositol | |
| CN109943583B (en) | Method for preparing ribavirin by using genetically engineered bacteria | |
| CN112481320B (en) | Method for preparing (-) gamma-lactam with high catalytic efficiency | |
| CN112831532B (en) | Method for enzymatic synthesis of D-leucine | |
| CN114891707A (en) | Recombinant strain and method for producing bilirubin through whole-cell catalysis of recombinant strain | |
| CN110857444B (en) | Preparation method of scyllo-inositol | |
| CN109295021A (en) | Ketoreductase and its application in bis- (trifluoromethyl) benzyl carbinols of asymmetric syntheses R-3,5- | |
| CN114806999B (en) | Genetically engineered bacterium and application thereof in preparing dihydrodaidzein | |
| CN106636019B (en) | High-efficiency catalytic synthesis of (S) -phenyl glycol by using (S) -carbonyl reductase oligomer mediated by Sortase A | |
| CN106367426A (en) | Old yellow enzyme gene and application thereof | |
| CN112442474B (en) | Preparation method of (-) gamma-lactam | |
| CN113583992B (en) | Reductive amination enzyme and application thereof in secondary amine synthesis | |
| CN109337891B (en) | Phenylalanine aminomutase mutant with improved thermal stability |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |