Search

CN-122012428-A - Carbonyl reductase mutant and application thereof in synthesis of R-lipoic acid chiral intermediate

CN122012428ACN 122012428 ACN122012428 ACN 122012428ACN-122012428-A

Abstract

The invention belongs to the technical field of biocatalysis, and particularly relates to a carbonyl reductase mutant and application thereof in synthesizing an R-lipoic acid chiral intermediate (R) -6-hydroxy-8-chlorooctanoic acid ethyl ester. According to the invention, the (R) -6-hydroxy-8-chloroethyl octanoate is asymmetrically synthesized under mild conditions (20-40 ℃ and pH 6-9) by using the non-separated and purified 6-oxo-8-chloroethyl octanoate stock solution as a substrate and using a carbonyl reductase mutant coupled glucose dehydrogenase BtGDH as a catalyst. The enzyme can completely convert 500 g/L of 6-oxygen-8-chlorooctanoic acid ethyl ester stock solution into optically pure (R) -6-hydroxy-8-chlorooctanoic acid ethyl ester in 8 h. The method does not need to pretreat the substrate (remove the solvent), has the synthesis yield reaching 87 percent, and has the advantages of high space-time yield, simple process, environmental protection and the like.

Inventors

  • LIN JUAN
  • DAI YANBIN
  • SU BINGMEI
  • ZHONG FENG
  • LIU LINGLING
  • LIU LIXIA
  • Luo Yihuo
  • CUI CAN
  • LUO HUADONG
  • GUO ZHENMING

Assignees

  • 福建科宏生物工程股份有限公司

Dates

Publication Date
20260512
Application Date
20260313

Claims (10)

  1. 1. A carbonyl reductase mutant, characterized in that the carbonyl reductase mutant is an amino acid residue mutation at any two, three, four, five or six positions on the amino acid sequence shown in SEQ ID No. 3: G94L、H145C、S153I、Y188C、M198L、L206C。
  2. 2. The carbonyl reductase mutant according to claim 1, wherein the carbonyl reductase mutant is a six-point mutant AxSDR mu6 obtained by performing an amino acid residue mutation of G94L/H145C/S153I/Y188C/M198L/L206C on the amino acid sequence shown in SEQ ID No. 3.
  3. 3. A recombinant expression vector comprising a gene encoding the carbonyl reductase mutant according to claim 1 or 2 and a gene encoding glucose dehydrogenase BtGDH; the DNA sequence of the coding gene of the glucose dehydrogenase BtGDH is shown as SEQ ID NO. 2.
  4. 4. The recombinant expression vector of claim 3, wherein the genes encoding glucose dehydrogenase BtGDH and the carbonyl reductase mutant of claim 1 are recombined on a pET-30a plasmid.
  5. 5. A co-expression engineering bacterium, characterized in that it is obtained by transforming the recombinant expression vector according to claim 3 or 4 into a host bacterium.
  6. 6. The co-expression engineering bacterium according to claim 5, wherein the host bacterium is escherichia coli BL21 (DE 3).
  7. 7. A method for synthesizing (R) -ECHO, which is characterized in that 6-oxo-8-chlorooctanoic acid ethyl ester stock solution is used as a substrate, glucose is used as an auxiliary substrate, and the (R) -ECHO is synthesized under the catalysis of the carbonyl reductase mutant coupled glucose dehydrogenase BtGDH as defined in claim 1 or 2; the amino acid sequence of the glucose dehydrogenase BtGDH is shown as SEQ ID NO. 1.
  8. 8. The method for synthesizing (R) -ECHO according to claim 7, wherein the coenzyme NAD + is further added during the enzyme reaction.
  9. 9. The method for synthesizing (R) -ECHO according to claim 7, further comprising purifying (R) -ECHO by extracting the reaction solution with an equal volume of ethyl acetate for 3 times, mixing the organic phases, and concentrating under reduced pressure to obtain an oily liquid (R) -ECHO.
  10. 10. The method for synthesizing (R) -ECHO according to claim 7, wherein the carbonyl reductase mutant, glucose dehydrogenase BtGDH is derived from whole cells or cell disruption solution of recombinant cells coexpressed by genetic engineering means with carbonyl reductase mutant, glucose dehydrogenase BtGDH, or is derived from whole cells or cell disruption solution of recombinant cells each expressing carbonyl reductase mutant, glucose dehydrogenase BtGDH.

Description

Carbonyl reductase mutant and application thereof in synthesis of R-lipoic acid chiral intermediate Technical Field The invention belongs to the technical field of biocatalysis, and particularly relates to a carbonyl reductase mutant and application thereof in enzyme-catalyzed asymmetric synthesis of an R-lipoic acid chiral intermediate (R) -6-hydroxy-8-chlorooctanoic acid ethyl ester. Background αLipoic acid (alpha-LA) is an important medical intermediate and antioxidant, has the effects of resisting oxidation, protecting nerves, promoting energy metabolism, maintaining blood sugar balance, protecting cardiovascular and the like, and is widely applied to the fields of big health, biological medicine, cosmetics and the like. There are two configurations of alpha-LA, namely (R)Alpha-LA and (S) -alpha-LA, of which only (R)Alpha-LA is physiologically active. Since none of the chiral precursors involved in the prior art chemical synthesis of α -LA is selective, the (R)The preparation of alpha-LA is usually obtained by subjecting chemically prepared racemic alpha-LA to 3-step chemical resolution, which greatly improves (R)The cost of synthesis of alpha-LA is 5 times that of racemate, so most of the products on the market are still racemates. However, in order to relieve the burden of human metabolism (S) -alpha-LA, optically pure (R) is prepared with high efficiencyThe alpha-LA has important application value and wide market prospect. The specific configuration of alpha-LA is determined by the configuration of the chiral precursor ethyl 6-hydroxy-8-chlorooctanoate (ECHO), so that the asymmetric synthesis of (R) -ECHO becomes (R)Research hot spot for reducing cost and enhancing efficiency of alpha-LA synthesis. As carbonyl reductase research continues to advance, research into asymmetric reductive synthesis (R) -ECHO using ethyl 6-oxo-8-chlorooctanoate (ECCO) is becoming more mature. CN11004119a was able to catalyze 110 g/L (0.5M) ECCO with a mutant of carbonyl reductase CpAR2 in the presence of 0.1 mM NADP + and 5% (v/v) dimethyl sulfoxide, after reaction 4 h in 85% yield and >99% ee (R) -ECHO. CN 115948356B discloses a carbonyl reductase mutant SsCRL-211H/V127A/L135I, which can asymmetrically reduce 2M ECCO in 3H to obtain (R) -ECHO with 90% yield and 99% ee value with GDH co-expressed cells. Although the reported substrate loadings have met the requirements of large-scale production, none of the carbonyl reductases used are resistant to organic solvents, requiring removal of the organic solvent stabilizing its structure prior to participation in catalysis, and poor compatibility with upstream chemical reactions. However, the substrate after desolvation is unstable and needs to be immediately subjected to a bioreduction process, which results in a biocatalytic reaction requiring a strict pretreatment process. Disclosure of Invention In order to solve the problems, the invention utilizes a protein engineering technology to obtain a carbonyl reductase mutant which can asymmetrically reduce the ethyl 6-oxo-8-chlorooctoate (ECCO) stock solution which is not separated and purified with high efficiency (ECCO stock solution is ECCO prepared by a chemical method and is dissolved in dichloromethane) to synthesize (R) -6-hydroxy-8-chlorooctoate [ (R) -ECHO ], the mutant is applied to the biosynthesis of (R) -ECHO, the ECCO stock solution loading can be up to 500 g/L, the conversion rate of 8 h can be up to 99% after the reaction is carried out, and the ee value is more than 99%. In order to solve the technical problems, the invention adopts the following technical scheme: In a first aspect, the present invention provides a carbonyl reductase mutant obtained by mutating an amino acid residue on the basis of AxSDR (the amino acid sequence is shown as SEQ ID NO. 3, and the DNA sequence is shown as SEQ ID NO. 4). Specifically, the carbonyl reductase mutant is obtained by mutating any two, three, four, five or six amino acid residues on the amino acid sequence shown in SEQ ID NO. 3: G94L、H145C、S153I、Y188C、M198L、L206C。 In some embodiments, the carbonyl reductase mutant is a six-point mutant AxSDR mu6 obtained by making an amino acid residue mutation of G94L/H145C/S153I/Y188C/M198L/L206C over the amino acid sequence shown in SEQ ID NO. 3. In a second aspect, the present invention provides a recombinant expression vector comprising a gene encoding the carbonyl reductase mutant of the first aspect and a gene encoding glucose dehydrogenase BtGDH; the DNA sequence of the coding gene of the glucose dehydrogenase BtGDH is shown as SEQ ID NO. 2. In some embodiments, the gene encoding glucose dehydrogenase BtGDH and the carbonyl reductase mutant as described in the first aspect are recombined on an expression vector to obtain an expression plasmid, preferably the expression plasmid is a pET series plasmid, more preferably a pET-30a plasmid. In a third aspect, the present invention provides a co-expression engineering bacterium obtained