Carbonyl reductase mutant, preparation method and use thereof, and preparation method of ethyl (R)-6-hydroxy-8-chlorooctanoate
The present invention provides a carbonyl reductase mutant, preparation method and use thereof, and a preparation method of ethyl (R)-6-hydroxy-8-chlorooctanoate. The carbonyl reductase mutant is a carbonyl reductase with amino acid mutation; the carbonyl reductase comprises an amino acid sequence as set forth in SEQ ID NO: 2; the amino acid mutation includes E101V, F214R or E101V/F214R. In the present invention, by introducing mutations on the basis of the original carbonyl reductase sequence, the enzyme activity is improved, the stereoselectivity is improved, and ethyl (R)-6-hydroxy-8-chlorooctanoate can be obtained with high yield and high purity under relatively mild conditions, which reduces the production cost and is suitable for industrial production.
1 . A carbonyl reductase mutant comprising an amino acid mutation;
wherein the carbonyl reductase comprises the amino acid sequence of SEQ ID NO: 2, and the amino acid mutation comprises E101V, F214R or both E101V and F214R.
2 . The carbonyl reductase mutant according to claim 1 , which is derived from a carbonyl reductase derived from Pseudohyphozyma bogoriensis.
3 . The carbonyl reductase mutant of claim 1 , which is expressed by a genetically engineered bacterial strain.
4 . The carbonyl reductase mutant of claim 3 , wherein the genetically engineered bacterial strain comprises any one of Escherichia coli, Pichia pastoris or Bacillus subtilis.
5 . A nucleotide sequence encoding the carbonyl reductase mutant according to claim 1 .
6 . The nucleotide sequence according to claim 5 , which comprises, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12.
7 . An expression vector comprising at least one copy of the nucleotide sequence according to claim 5 .
8 . A bacteria strain transformed with the nucleotide sequence of claim 5 .
9 . A bacteria strain comprising the expression vector of claim 7 .
10 . A method for preparing a carbonyl reductase mutant comprising an amino acid mutation;
wherein the carbonyl reductase comprises the amino acid sequence of SEQ ID NO: 2, and the amino acid mutation comprises E101V, F214R or both E101V and F214R;
the method comprising:
constructing an expression vector according to claim 7 , and transforming the expression vector into a recipient cell to generate a carbonyl reductase mutant transformant;
culturing the carbonyl reductase mutant transformant in a culture medium and collecting the culture medium; and
obtaining the carbonyl reductase mutant from the culture medium.
11 . The method of claim 10 , wherein the expression vector comprises a pET series expression vector.
12 . The method of claim 11 , wherein the pET series expression vector comprises a pET-28a expression vector.
13 . A method for catalyzing a carbonyl reduction reaction, comprising:
contacting a substrate with a carbonyl reductase mutant according to claim 1 .
14 . The method of claim 13 , wherein the substrate comprises ethyl 6-oxo-8-chlorooctanoate.
15 . A method for preparing ethyl (R)-6-hydroxy-8-chlorooctanoate, comprising:
mixing a carbonyl reductase mutant according to claim 1 with a reaction solution containing ethyl 6-oxo-8-chlorooctanoate, and reacting to obtain ethyl (R)-6-hydroxyl-8-chlorooctanoate.
16 . The method according to claim 15 , wherein:
the ethyl 6-oxo-8-chlorooctanoate has a mass percent concentration of 4% to 30%; the carbonyl reductase mutant is used in an amount of 5% to 30% by the weight of ethyl 6-oxo-8-chlorooctanoate;
the reaction solution further comprises glucose dehydrogenase, a coenzyme and glucose; the glucose dehydrogenase in the reaction system is used in an amount of 3% to 10% by the weight of ethyl 6-oxo-8-chlorooctanoate;
the coenzyme is NADP+;
the coenzyme is used in an amount of 1/10,000 to 5/10,000 of the weight of ethyl 6-oxo-8-chlorooctanoate;
the glucose is used in an amount 0.9-2 times the weight of ethyl 6-oxo-8-chlorooctanoate;
the reaction is carried out in a solvent of Tris-HCl buffer, phosphate buffer, triethanolamine hydrochloride buffer, sodium acetate buffer or Tris-phosphate buffer; the reaction is carried out at pH 6.0 to 7.5;
the reaction comprises a cosolvent comprising any one or a combination of at least two of ethanol, propanol, isopropanol, DMF, DMSO, polyethylene glycol or polysorbate 80;
the cosolvent in the reaction is used in a volume percentage of 5% to 10% by the volume of the solvent;
the reaction is carried out at a temperature of 20° C. to 35° C.; and
the reaction is carried for a time of 4 to 24 hours.
17 . A method for manufacturing (R)-α-lipoic acid, comprising the following steps:
(1) preparing ethyl (R)-6-hydroxy-8-chlorooctanoate by using the carbonyl reductase mutant according to claim 12 , and
(2) preparing (R)-α-lipoic acid from the ethyl (R)-6-hydroxy-8-chlorooctanoate.
18 . A method for manufacturing (R)-α-lipoic acid, comprising the following steps:
(1) preparing ethyl (R)-6-hydroxy-8-chlorooctanoate by using the carbonyl reductase mutant transformant according to claim 8 , and
(2) preparing (R)-α-lipoic acid from the ethyl (R)-6-hydroxy-8-chlorooctanoate.
19 . A method for manufacturing (R)-α-lipoic acid, including the following steps:
(1) preparing ethyl (R)-6-hydroxy-8-chlorooctanoate via the method according to claim 14 , and
(2) preparing (R)-α-lipoic acid from the ethyl (R)-6-hydroxy-8-chlorooctanoate.
20 . A method for manufacturing (R)-α-lipoic acid, including the following steps:
(1) preparing ethyl (R)-6-hydroxy-8-chlorooctanoate via the method according to claim 15 , and
(2) preparing (R)-α-lipoic acid from the ethyl (R)-6-hydroxy-8-chlorooctanoate.