CN116814572A - Carbonyl reductase and mutant thereof and application of carbonyl reductase and mutant in preparation of chiral (R) -8-chloro-6-hydroxy ethyl octanoate - Google Patents

Carbonyl reductase and mutant thereof and application of carbonyl reductase and mutant in preparation of chiral (R) -8-chloro-6-hydroxy ethyl octanoate Download PDF

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CN116814572A
CN116814572A CN202310704732.8A CN202310704732A CN116814572A CN 116814572 A CN116814572 A CN 116814572A CN 202310704732 A CN202310704732 A CN 202310704732A CN 116814572 A CN116814572 A CN 116814572A
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carbonyl reductase
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夏娜娜
魏婷
黄选
韩晓龙
张朋
陈航
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Hangzhou Xinhai Enzyme Source Biotechnology Co ltd
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Abstract

The invention relates to the technical field of genetic engineering of enzymes, and provides carbonyl reductase and a mutant thereof and application thereof in preparing chiral (R) -8-chloro-6-hydroxyoctanoate, wherein the amino acid sequence of the carbonyl reductase is shown as SEQ ID NO.1, the nucleotide sequence of the carbonyl reductase is shown as SEQ ID NO.2, the amino acid sequence of the mutant is obtained by mutating the amino acid sequence shown as SEQ ID NO.1, and the mutation comprises at least one mutation site: 126 bits, 129 bits, 194 bits. The carbonyl reductase and the mutant thereof have good substrate tolerance and catalytic activity, can improve the reaction efficiency, and can be applied to catalyzing asymmetric reduction reaction of the latent chiral carbonyl compound.

Description

Carbonyl reductase and mutant thereof and application of carbonyl reductase and mutant in preparation of chiral (R) -8-chloro-6-hydroxy ethyl octanoate
Technical Field
The invention relates to the technical field of genetic engineering of enzymes, in particular to carbonyl reductase, a mutant thereof and application thereof in preparation of chiral (R) -8-chloro-6-hydroxy ethyl octanoate.
Background
Alpha-lipoic acid (formula C) 8 H 14 O 2 S 2 Molecular weight 206.33, cas number: 1200-22-2) belongs to vitamin medicines, and is a universal antioxidant with both fat solubility and water solubility. Is mainly used for treating liver diseases such as diabetes, acute hepatitis, fatty liver and the like. The physiological activity of lipoic acid is limited to dextrorotatory lipoic acid, i.e., (R) -alpha-lipoic acid, while (S) -alpha-lipoic acid has substantially no physiological activity.
At present, the industrial preparation of (R) -alpha-lipoic acid mainly adopts chemical resolution, takes 8-chloro-6-hydroxy ethyl octanoate and chiral auxiliary (R) -alpha-methylbenzylamine as raw materials, and finally obtains (R) -alpha-lipoic acid through multiple recrystallization, acidification and hydrolysis. Or 8-chloro-6-hydroxy ethyl octanoate and chiral auxiliary (S) -alpha-methylbenzylamine are used as raw materials, and the (R) -8-chloro-6-hydroxy ethyl octanoate is obtained after recrystallization, hydrolysis and esterification, and the product is subjected to the action of thionyl chloride, pyridine and toluene to generate (R) -6, 8-dichloro octanoate, then sodium disulfide is used for substitution, and then organic acid is used for hydrolysis reaction to generate the final product (R) -alpha-lipoic acid. However, the above method has problems of high price and low yield of chiral auxiliary for resolution reaction.
Compared with chemical synthesis methods, the biocatalysis method has the advantages of mild reaction conditions, environmental friendliness, strong stereoselectivity and the like. At present, the biological method is mainly used for preparing the (R) -8-chloro-6-hydroxy ethyl octanoate by alcohol dehydrogenase and carbonyl reductase. Patent US7157253B2 reports that the reduction conversion of ethyl 8-chloro-6-oxooctanoate to ethyl 8-chloro-6-hydroxyoctanoate using alcohol dehydrogenase TbADH from Thermoanaerobium brokii gives an optical purity of 99.5%, but only 85% conversion and the addition of 0.5mM coenzyme and 1mM Dithiothreitol (DTT) to the reaction system is required. Patent W02005049816A2 reports that the substrate can be converted into the product (R) -8-chloro-6-hydroxyoctanoate ethyl ester by using an oxidoreductase from Metschnikowia zobellii, the ee value can reach 97%, but the conversion rate after 24 hours of reaction is only 55% by adding 0.1mM coenzyme to the reaction system. The existing (R) -alpha-lipoic acid synthesis method is difficult to meet the increasing market demand for the optical pure (R) -alpha-lipoic acid, so that the synthesis method of the (R) -alpha-lipoic acid still needs to be further expanded.
Disclosure of Invention
The invention provides carbonyl reductase and a mutant thereof, which have good substrate tolerance and catalytic activity and can improve the reaction efficiency, so as to solve the problems of slow reaction rate and low production efficiency of preparing chiral (R) -8-chloro-6-hydroxy ethyl octanoate by a biocatalysis method in the prior art. The invention also provides a preparation method of chiral (R) -8-chloro-6-hydroxy ethyl octanoate, which has the advantages of mild reaction conditions, environment friendliness, high preparation efficiency and high product purity.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a carbonyl reductase is carbonyl reductase Cat, the amino acid sequence of which is shown as SEQ ID NO.1, and the nucleotide sequence of which is shown as SEQ ID NO. 2.
The carbonyl reductase is derived from Candida tea (Candida they), and can be used for efficiently catalyzing the asymmetric reduction of 6-carbonyl-8-chlorooctanoate to generate optically pure (R) -6-hydroxy-8-chlorooctanoate.
Preferably, the amino acid sequence of the mutant is obtained by mutating the amino acid sequence shown as SEQ ID NO.1, and the mutation comprises at least one mutation site as follows: 126 bits, 129 bits, 194 bits.
The carbonyl reductase mutant has better catalytic activity compared with the wild type carbonyl reductase.
Preferably, the mutation is one or a combination of several of the following mutation sites: serine at position 126 is mutated to alanine, arginine at position 129 is mutated to glutamine, arginine at position 129 is mutated to alanine, arginine at position 129 is mutated to leucine, arginine at position 129 is mutated to cysteine, valine at position 194 is mutated to alanine, valine at position 194 is mutated to cysteine.
A coding gene which is a coding gene of the above carbonyl reductase or mutant.
A recombinant expression vector containing the coding gene.
An engineering bacterium obtained by transforming the recombinant expression vector into host bacteria.
A chiral (R) -8-chloro-6-hydroxy ethyl octanoate is prepared from 8-chloro-6-oxo ethyl octanoate as substrate, and the carbonyl reductase or mutant, dehydrogenase and NADP + Glucose is added into a reaction solvent to obtain a conversion system, and chiral (R) -8-chloro-6-hydroxy ethyl octanoate is obtained through reaction.
The carbonyl reductase and the mutant thereof are used for catalyzing the asymmetric reduction reaction of the 8-chloro-6-oxo-ethyl octanoate, the concentration of the substrate is high, the conversion rate is high, the concentration of the substrate reaches 1mol/L (or 220 g/L), the conversion rate can reach 96%, and the ee value of the product is higher than 99%. Compared with other asymmetric reduction preparation methods for synthesizing precursors of (R) -alpha-lipoic acid, the method has the advantages of short reaction time and high optical purity, is favorable for realizing high-efficiency and low-cost production of (R) -alpha-lipoic acid, and has industrial application prospect.
Preferably, the carbonyl reductase or the mutant is added in the form of wet bacterial form after the induction culture of the engineering bacteria.
Preferably, the wet bacterial cells are obtained by culturing engineering bacteria at 37 ℃ until the OD600 is 2.5-3.5, adding 0.05-1.0mmol/L isopropyl-P-D-thiopyran galactose, inducing for 12-24 hours at 16-25 ℃, and centrifuging.
Preferably, the reaction solvent is a mixed solution of a buffer solution and an organic cosolvent, the buffer solution is Tris-HCl buffer solution, sodium phosphate buffer solution or potassium phosphate buffer solution, the concentration range of the buffer solution is 10-200mM, and the pH value of the buffer solution is 6.0-7.5; the organic cosolvent is one or more of ethanol, isopropanol and methanol, and the volume ratio of the buffer solution to the organic cosolvent is (900-950): (50-100).
Preferably, the initial concentration of the substrate in the conversion system is 200-220g/L, the dosage of the carbonyl reductase or the mutant is 100-120g/L calculated by wet thalli, and the addition ratio of the carbonyl reductase or the mutant to the dehydrogenase is (1-2): 1, NADP + The mass fraction of (2) is 8% -10%, the mol ratio of glucose to substrate is (0.8-1.8): 1.
preferably, the preparation method further comprises the steps of purifying the product after reaction of a conversion system to obtain chiral (R) -8-chloro-6-hydroxy ethyl octanoate with high chemical purity and optical purity, wherein the purification process comprises the steps of extracting by using an equivalent amount of water-insoluble organic solvent, adding anhydrous sodium sulfate into an extract, drying, removing the solvent, dissolving again, and distilling under reduced pressure to obtain the product; the water-insoluble organic solvent is one or more of ethyl acetate, butyl acetate, toluene, methylene dichloride, chloroform, isopropyl ether and methyl tertiary butyl ether.
Therefore, the invention has the following beneficial effects: (1) Provided are carbonyl reductase and mutants thereof that are useful for catalyzing asymmetric reduction reactions of potentially chiral carbonyl compounds; (2) When being applied to the reaction of catalyzing and esterifying (R) -8-chloro-6-hydroxy ethyl octanoate, the catalyst has the advantages of high concentration of catalytic substrate, high reaction rate, high optical purity of the product and the like, is beneficial to realizing the high-efficiency and low-cost production of (R) -alpha-lipoic acid, and has industrial application prospect.
Drawings
FIG. 1 is a schematic diagram of the process of synthesizing (R) -6-hydroxy-8-chloroethyl octanoate by carbonyl reductase Cat catalysis.
FIG. 2 is a liquid chromatogram after benzoylation of racemic ethyl 8-chloro-6-hydroxyoctanoate.
FIG. 3 is a gas chromatogram of ethyl (R) -8-chloro-6-hydroxyoctanoate obtained in example 12.
FIG. 4 is a liquid chromatogram of the (R) -8-chloro-6-hydroxyoctanoate prepared in example 12 after benzoylation.
Detailed Description
The invention is further described below with reference to the drawings and detailed description.
Example 1
Engineering bacteria for constructing carbonyl reductase Cat:
synthesizing a carbonyl reductase Cat coding gene fragment according to a nucleotide sequence SEQ ID NO.2, introducing enzyme cutting sites EcoR I and Xho I at two ends of the coding gene, connecting into a pET28a vector, constructing a recombinant expression plasmid pET28a-Cat, converting the plasmid pET28a-Cat into competent cells (kanamycin resistance) of escherichia coli BL21 (DE 3), coating an LB plate containing the kana to obtain positive clones, picking single colonies, inoculating the single colonies into a 5ml LB test tube (containing 50mg/L kanamycin) for culture, and obtaining engineering bacteria of the carbonyl reductase Cat.
Example 2
Construction of carbonyl reductase mutant Cat R129Q Is the engineered bacterium of (a), carbonyl reductase mutant Cat R129Q Is obtained by mutating arginine at position 129 into glutamine in the amino acid sequence shown in SEQ ID NO. 1:
PCR amplification was performed using the nucleotide sequence shown as SEQ ID NO.2 as a template and the primers R129Q-F and R129Q-R having the nucleotide sequence shown as Table 1 to obtain a carbonyl reductase mutant Cat R129Q After verification of error-free construction of recombinant expression plasmid pET28a-Cat R129Q And carbonyl reductase mutant Cat R129Q The construction method of the engineering bacteria is the same as that of the example 1, and after the engineering bacteria are cultured for a certain time, whether the engineering bacteria are the target single-point mutant R129Q is verified by sequencing.
Example 3
Construction of carbonyl reductase mutant Cat R129A Is the engineered bacterium of (a), carbonyl reductase mutant Cat R129A The amino acid sequence shown in SEQ ID NO.1 is obtained by mutating 129 th arginine into alanine:
PCR amplification was performed using the nucleotide sequence shown as SEQ ID NO.2 as a template and the primers R129A-F and R129A-R having the nucleotide sequence shown as Table 1 to obtain a carbonyl reductase mutant Cat R129A After verification of error-free construction of recombinant expression plasmid pET28a-Cat R129A And carbonyl groupReductase mutant Cat R129A The construction method of the engineering bacteria is the same as that of the example 1, and the engineering bacteria are verified by sequencing after being cultured for a certain time.
Example 4
Construction of carbonyl reductase mutant Cat R129L Is the engineered bacterium of (a), carbonyl reductase mutant Cat R129L The amino acid sequence shown in SEQ ID NO.1 is obtained by mutating 129 th arginine into leucine:
PCR amplification was performed using the nucleotide sequence shown as SEQ ID NO.2 as a template and the primers R129L-F and R129L-R shown as Table 1 to obtain a carbonyl reductase mutant Cat R129L After verification of error-free construction of recombinant expression plasmid pET28a-Cat R129L And carbonyl reductase mutant Cat R129L The construction method of the engineering bacteria is the same as that of the example 1, and the engineering bacteria are verified by sequencing after being cultured for a certain time.
Example 5
Construction of carbonyl reductase mutant Cat R129C Is the engineered bacterium of (a), carbonyl reductase mutant Cat R129C The amino acid sequence shown in SEQ ID NO.1 is obtained by mutating 129 th arginine into cysteine:
PCR amplification was performed using the nucleotide sequence shown as SEQ ID NO.2 as a template and the primers R129C-F and R129C-R having the nucleotide sequence shown as Table 1 to obtain a carbonyl reductase mutant Cat R129C After verification of error-free construction of recombinant expression plasmid pET28a-Cat R129C And carbonyl reductase mutant Cat R129C The construction method of the engineering bacteria is the same as that of the example 1, and the engineering bacteria are verified by sequencing after being cultured for a certain time.
Example 6
Construction of carbonyl reductase double mutant Cat S126A/R129Q Is the engineered bacterium of (a), carbonyl reductase mutant Cat S126A/R129Q The amino acid sequence shown in SEQ ID NO.1 is obtained by mutating serine at position 126 into alanine and mutating arginine at position 129 into glutamine: PCR amplification was performed using the nucleotide sequence shown as SEQ ID NO.2 as a template and primers S126A-F, S A-R, R129Q-F and R129Q-R having the nucleotide sequence shown as Table 1 to obtain a carbonyl reductase mutant Cat S126A/R129Q After verification of error-free construction of recombinant expression plasmid pET28a-Cat S126A/R129Q And carbonyl reductase mutant Cat S126A/R129Q The construction method of the engineering bacteria is the same as that of the example 1, and the engineering bacteria are verified by sequencing after being cultured for a certain time.
Example 7
Construction of carbonyl reductase three-point mutant Cat S126A/R129Q/V194A Is the engineered bacterium of (a), carbonyl reductase mutant Cat S126A/R129Q/V194A The amino acid sequence shown in SEQ ID NO.1 is obtained by mutating serine at position 126 into alanine, mutating arginine at position 129 into glutamine and mutating valine at position 194 into alanine:
PCR amplification was performed using the nucleotide sequence shown in SEQ ID NO.2 as a template and primers S126A-F, S A-R, R129Q-F, R129Q-R, V194A-F and V194A-R having the nucleotide sequence shown in Table 1 to obtain a carbonyl reductase mutant Cat S126A/R129Q/V194A After verification of error-free construction of recombinant expression plasmid pET28a-Cat S126A/R129Q/V194A And carbonyl reductase mutant Cat S126A/R129Q/V194A The construction method of the engineering bacteria is the same as that of the example 1, and the engineering bacteria are verified by sequencing after being cultured for a certain time.
Example 8
Construction of carbonyl reductase mutant Cat S126A/R129Q/V194C Is the engineered bacterium of (a), carbonyl reductase mutant Cat S126A/R129Q/V194C The amino acid sequence shown in SEQ ID NO.1 is obtained by mutating serine at position 126 into alanine, mutating arginine at position 129 into glutamine and mutating valine at position 194 into cysteine:
PCR amplification was performed using the nucleotide sequence shown in SEQ ID NO.2 as a template and primers S126A-F, S A-R, R129Q-F, R129Q-R, V194C-F and V194C-R having the nucleotide sequence shown in Table 1 to obtain a carbonyl reductase mutant Cat S126A/R129Q/V194C After verification of error-free construction of recombinant expression plasmid pET28a-Cat S126A/R129Q/V194C And carbonyl reductase mutant Cat S126A/R129Q/V194C The construction method of the engineering bacteria is the same as that of the example 1, and the engineering bacteria are verified by sequencing after being cultured for a certain time.
TABLE 1 nucleotide sequences of primers
Primer name Nucleotide sequence
S126A-F GTTACCTCTTCTACCGCTGCGGTTCA
S126A-R TGAACCGCAGCGGTAGAAGAGGTAAC
R129Q-F CTTCTACCTCTGCGGTTCAAGATGTTAGCGG
R129Q-R GACGTTTACCGCTAACATCTTGAACCGCAGAG
R129A-F CTCTTCTACCTCTGCGGTTGCTGATGTTAGCG
R129A-R CGCTAACATCAGCAACCGCAGAGGTAGAAGAG
R129L-F CTTCTACCTCTGCGGTTCTTGATGTTAGC
R129L-R GTTTACCGCTAACATCAAGAACCGCAGAG
R129C-F CTACCTCTGCGGTTTGTGATGTTAGC
R129C-R TTACCGCTAACATCACAAACCGCAG
V194A-F CCCGACCTACGCTTTCGGCCCG
V194A-R CGGGCCGAAAGCGTAGGTCGG
V194C-F GTTAACCCGACCTACTGTTTCGGCCCGCAG
V194C-R GCGGGCCGAAACAGTAGGTCGGGTTAACG
Example 9
Inducible expression of carbonyl reductase Cat and carbonyl reductase mutants:
the carbonyl reductase engineering bacteria and carbonyl reductase mutant engineering bacteria constructed in examples 1-8 were inoculated into 50. Mu.g/mL of kanamycin TB medium, cultured overnight at 37℃at 220rpm, then inoculated into 50. Mu.g/mL of kanamycin-containing TB medium at 2% of inoculum size (v/v), cultured at 37℃at 200rpm until the cell concentration OD600 was 3.0, respectively, isopropyl-P-D-thiopyran galactose (IPTG) was added at a final concentration of 0.1mM, and after induction culture at 25℃for 16 hours, the cells were collected by centrifugation at 4000rpm for 10min, respectively.
Example 10
Effect of carbonyl reductase Cat and carbonyl reductase mutant on the preparation of ethyl (R) -6-hydroxy-8-chlorooctoate, ethyl (R) -8-chloro-6-hydroxyoctoate was prepared by the reaction procedure as shown in fig. 1:
(1) Synthesizing a glucose dehydrogenase GDH gene fragment according to a nucleotide sequence shown in SEQ ID NO.3, introducing enzyme cutting sites EcoR I and Xho I at two ends of the gene, connecting the gene into a pET28a vector, constructing a recombinant expression plasmid pET28a-GDH, and converting the plasmid pET28a-GDH into escherichia coli BL21 (DE 3) to construct a glucose dehydrogenase engineering bacterium; inoculating glucose dehydrogenase engineering bacteria into LB culture medium of 50 mug/mL kanamycin at the inoculum size of 2% (v/v), culturing at 37 ℃ at 200rpm until the bacterial concentration OD600 is about 0.6, respectively adding IPTG with the final concentration of 0.1mM, performing induction culture at 25 ℃ for 16 hours, and respectively collecting GDH wet bacterial bodies after centrifugation at 4000rpm for 10 minutes;
(2) Reaction system (1 mL): carbonyl reductase Cat and mutant thereof (100 g) Wet cell L) and GDH (100 g Wet cell After mixing/L) and ethyl 8-chloro-6-oxooctanoate (220 g/L), glucose 0.144g, 1% (v/v) NADP was added + 50. Mu.L of ethanol and 950. Mu.L of Tris-HCl (100 mM, 7.0) were reacted in a shaker at 35℃for 17h.
The yield of the product (R) -8-chloro-6-hydroxyoctanoate after the reaction of example 10 was analyzed using gas chromatography under HP-5MS column with nitrogen as carrier gas at inlet temperature of 230℃and detector temperature of 230℃and column temperature of 200 ℃. The product obtained in example 10 was derivatized with benzoyl chloride and pyridine as derivatizing agents, and then analyzed by liquid chromatography for enantiomeric excess (ee) of the product, wherein the ee was calculated as: ee= (R-S)/(r+s), where R and S are the amounts of (R) -8-chloro-6-hydroxycaprylate and (S) -8-chloro-6-hydroxycaprylate, respectively. The liquid chromatography conditions are Amy-D chromatographic column, mobile phase n-hexane: isopropanol=95; 5, the flow rate is 1.0mL/min, the wavelength lambda=254 nm, the column temperature is 5 ℃, and the benzoyl chromatogram of the racemization 8-chloro-6-hydroxy ethyl octoate obtained under the liquid chromatographic condition is shown in figure 2. The results of the detection calculations are shown in Table 2, and the results of the combined mutation S126A/R129Q/V194A are optimal.
TABLE 2 Effect of carbonyl reductase Cat wild-type and mutant on (R) -8-chloro-6-hydroxyoctanoate conversion ratio and ee value
Example 11
Effect of temperature on (R) -8-chloro-6-hydroxyoctanoate preparation:
reaction system (1 mL): taking carbonyl reductase mutant Cat S126A/R129Q/V194A (100g Wet cell /L)、GDH(100g Wet cell After mixing/L) and ethyl 8-chloro-6-oxooctanoate (220 g/L), glucose 0.144g, 1% (v/v) NADP was added + 50. Mu.L of ethanol and 950. Mu.L of LTris-HCl (100 mM, 7.0) were reacted for 5h in a shaker at 16-40 ℃.
The yields of ethyl (R) -8-chloro-6-hydroxyoctanoate, which was obtained by the reaction at various temperatures in example 11, were examined under the same conditions, and the results are shown in Table 3, and the reaction was optimal at 20 ℃.
TABLE 3 influence of temperature on yield
Temperature (temperature) Yield is good
16 95％
20 96％
25 90％
30 72％
35 46％
40 37％
Example 12
Asymmetric reduction of (R) -8-chloro-6-hydroxyoctanoate by 100 mL-scale reductase Cat mutant:
reaction system (100 mL): taking reductase Cat mutant Cat S126A/R129Q/V194A (100g Wet cell /L)、GDH(100g Wet cell After mixing/L) and ethyl 8-chloro-6-oxooctanoate (220 g/L), glucose 14.4g, 1% (v/v) NADP was added + 50. Mu.L of ethanol and 950. Mu.L of Tris-HCl (100 mM, 7.0) were reacted in a water bath at 20℃for 4 hours.
Example 12 after the completion of the reaction, the yield of the ethyl (R) -6-hydroxy-6-octanoate obtained in example 12 was measured by a gas chromatograph, the ee value was measured by a liquid chromatograph under the same conditions as described above, the gas chromatograph measurement results are shown in FIG. 3, the liquid chromatograph measurement results are shown in FIG. 4, the ratio of ethyl (R) -6-hydroxy-8-chlorooctanoate in the system after the completion of the reaction was large, and the yield of 96% and the ee value of 99% of ethyl (R) -6-hydroxy-8-chlorooctanoate in example 12 were obtained by calculation.
The above examples show that the method provided by the invention has the advantages of high speed and high production efficiency in converting (R) -8-chloro-6-hydroxy ethyl octanoate, and can be applied to large-scale production.

Claims (10)

1. A carbonyl reductase, characterized in that the carbonyl reductase is a carbonyl reductaseCatThe amino acid sequence is shown as SEQ ID NO.1, and the nucleotide sequence is shown as SEQ ID NO. 2.
2. The mutant carbonyl reductase according to claim 1, wherein the amino acid sequence of the mutant is obtained by mutating the amino acid sequence shown in SEQ ID No.1, and the mutation comprises at least one of the following mutation sites: 126 bits, 129 bits, 194 bits.
3. The mutant according to claim 2, wherein the mutation is a combination of one or more of the following mutation sites: serine at position 126 is mutated to alanine, arginine at position 129 is mutated to glutamine, arginine at position 129 is mutated to alanine, arginine at position 129 is mutated to leucine, arginine at position 129 is mutated to cysteine, valine at position 194 is mutated to alanine, valine at position 194 is mutated to cysteine.
4. A coding gene, wherein the coding gene is a gene encoding the carbonyl reductase of claim 1 or the mutant of claim 2 or 3.
5. A recombinant expression vector comprising the coding gene according to claim 4.
6. An engineered bacterium obtained by transforming the recombinant expression vector of claim 5 into a host bacterium.
7. A process for preparing chiral (R) -8-chloro-6-hydroxy ethyl octanoate, which comprises using 8-chloro-6-oxo ethyl octanoate as substrate, reacting the carbonyl reductase according to claim 1 or the mutants according to claim 2 or 3 with dehydrogenase and NADP + Glucose is added into a reaction solvent to obtain a conversion system, and chiral (R) -8-chloro-6-hydroxy ethyl octanoate is obtained through reaction.
8. The method for preparing chiral (R) -8-chloro-6-hydroxyoctanoate according to claim 7, wherein the carbonyl reductase or the mutant is added in the form of wet bacterial form after the induction culture of the engineering bacteria according to claim 6.
9. The preparation method of chiral (R) -8-chloro-6-hydroxy ethyl octanoate according to claim 8, wherein the reaction solvent is a mixed solution of buffer solution and organic cosolvent, the buffer solution is Tris-HCl buffer solution, sodium phosphate buffer solution or potassium phosphate buffer solution, the concentration range of the buffer solution is 10-200mM, and the pH value of the buffer solution is 6.0-7.5; the organic cosolvent is one or more of ethanol, isopropanol and methanol, and the volume ratio of the buffer solution to the organic cosolvent is (900-950): (50-100).
10. The process for preparing chiral (R) -8-chloro-6-hydroxyoctanoate according to claim 8 or 9, wherein the initial concentration of the substrate in the conversion system is 200-220g/L, the amount of carbonyl reductase or mutant is 100-120g/L in terms of wet cells, and the ratio of carbonyl reductase or mutant to dehydrogenase is (1-2): 1, NADP + The mass fraction of (2) is 8% -10%, the mol ratio of glucose to substrate is (0.8-1.8): 1.
CN202310704732.8A 2023-06-14 2023-06-14 Carbonyl reductase and mutant thereof and application of carbonyl reductase and mutant in preparation of chiral (R) -8-chloro-6-hydroxy ethyl octanoate Pending CN116814572A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117230031A (en) * 2023-11-03 2023-12-15 山东理工大学 Carbonyl reductase mutant and application thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117230031A (en) * 2023-11-03 2023-12-15 山东理工大学 Carbonyl reductase mutant and application thereof
CN117230031B (en) * 2023-11-03 2024-01-26 山东理工大学 Carbonyl reductase mutant and application thereof

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