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ε-ketoester reductase mutants and their application in the synthesis of (r)-α-lipoic acid precursors

A reductase and mutant technology, applied in the field of biochemical engineering technology in Ming Dynasty, can solve the problems of poor stability and insufficient catalytic activity

Active Publication Date: 2020-10-20
EAST CHINA UNIV OF SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Aiming at the problems of insufficient catalytic activity and poor stability of the existing ε-ketoester reductase in the application process, the present invention provides a series of ε-ketoester reductase mutants with improved catalytic activity and improved thermal stability , recombinant expression plasmids and recombinant expression transformants containing the enzyme mutant gene, recombinant cells co-expressing ε-ketoester reductase mutants and BmGDH, and recombinant cells catalyzing the preparation of chiral ε-hydroxycaprylate (R )-Applied method in ECHO

Method used

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  • ε-ketoester reductase mutants and their application in the synthesis of (r)-α-lipoic acid precursors
  • ε-ketoester reductase mutants and their application in the synthesis of (r)-α-lipoic acid precursors
  • ε-ketoester reductase mutants and their application in the synthesis of (r)-α-lipoic acid precursors

Examples

Experimental program
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Effect test

Embodiment 1

[0047] Example 1 Construction and screening of random mutation library of ε-ketoester reductase CpAR2

[0048] The carbonyl reductase mutant CpKR described in the invention patent with the publication number CN 106164260A G86E Named ε-ketoester reductase CpAR2. The carbonyl reductase mutant CpKR G86E It can be encoded by the nucleic acid sequence shown in SEQ ID No.1. The carbonyl reductase mutant CpKR G86E It is obtained by mutation of carbonyl reductase CpKR. Carbonyl reductase CpKR comes from a kind of Candida parapsilosis (Candida parapsilosis). The strain was originally named ECU 6481. Classification experiments and 16S rDNA analysis identified Candida parapsilosis. The strain was patent deposited in the China General Microorganism Culture Collection and Management Center on September 1, 2014, with the preservation number CGMCC9630, and it has been disclosed in the invention patent with the publication number CN 106164260A.

[0049] Using pET28a-CpAR2 as a template,...

Embodiment 2

[0056] Example 2 Site-directed saturation mutation of serine 131

[0057] The MEGAWHOP (Megaprimer PCR of Whole Plasmid) method was used for operation. The primers designed for PCR are listed in Table 1.

[0058] Table 1 PCR primers for whole plasmid amplification of site-directed saturation mutation at position 131

[0059]

[0060]

[0061] Using the recombinant expression plasmid pET28a-CpAR2 as a template, the site-directed mutagenesis of the whole plasmid was carried out by PCR.

[0062] The PCR amplification reaction system (50 μL) for site-directed mutagenesis was: 1 ng of recombinant plasmid template, 0.25 μM each for a pair of mutation primers, 0.2 mM dNTPs and 25 μL PrimeSTAR HS DNA Polymerase (1.25U / 25 μL) (TAKARA, JAPAN), plus Sterilize distilled water to 50 μL. PCR reaction program: (1) Pre-denaturation at 98°C for 3min; (2) Denaturation at 98°C for 10sec, (3) Annealing at 55°C for 5s, (4) Extension at 72°C for 6min, steps (2) to (4) were performed for...

Embodiment 3

[0066] Example 3 Site-directed mutagenesis of CpAR2

[0067] Mutant CpAR2 obtained in Example 1 with improved thermostability S131L / Q252I On the basis of using the primer pair S131Y_For, S131Y_Rev and S131F_For, S131F_Rev described in Example 2 as primers, the recombinant expression plasmid pET28a-CpAR2 S131L / Q252I As a template, PCR site-directed mutagenesis was performed on the whole plasmid, and amino acid residues at position 131 were site-directedly mutated into tyrosine and phenylalanine respectively.

[0068] The PCR amplification reaction system (50 μL) for site-directed mutagenesis was: 1 ng of recombinant plasmid template, 0.25 μM each for a pair of mutation primers, 0.2 mM dNTPs and 25 μL PrimeSTAR HS DNA Polymerase (1.25U / 25 μL) (TAKARA, JAPAN), plus Sterilize distilled water to 50 μL. PCR reaction program: (1) Pre-denaturation at 98°C for 3min; (2) Denaturation at 98°C for 10sec, (3) Annealing at 55°C for 5s, (4) Extension at 72°C for 6min, steps (2) to (4) w...

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Abstract

The invention relates to an epsilon-ketone ester reductase mutant with improved catalysis performance after molecular engineering modification, a recombinant expression vector and a recombinant expression transformed body which contain reductase mutant genes, a preparing method of the recombinant reductase, and a method of using the recombinant reductase or recombinant cells to catalyze and prepare the lipoic acid synthesis precursor (R)-8-chlorine-6-ethyl 3-hydroxyoctanoate. Compared with the prior art, the epsilon-ketone ester reductase CpAR2 mutant has the advantages of being good in thermal stability and good in catalytic activity, shows obvious advantages of being high in substrate concentration, small in use amount of catalyst and the like in catalysis and preparation reaction of the(R)-8-chlorine-6-ethyl 3-hydroxyoctanoate, and has extremely high industrial application potential.

Description

technical field [0001] The invention belongs to the technical field of biochemical engineering, and specifically relates to an ε-ketoester reductase mutant and its gene sequence, a recombinant expression plasmid and a recombinant expression transformant containing the mutant gene sequence, which co-express the ε-ketoester reductase mutation lyophilized cells with glucose dehydrogenase, and the ε-ketoester reductase mutant and co-expressed ε-ketoester reductase mutant and glucose dehydrogenase in catalyzed production of (R)-α-lipoic acid Application of the precursor (R)-8-chloro-6-hydroxyoctanoic acid ethyl ester. Background technique [0002] Alpha-lipoic acid is a B vitamin and a safe and effective antioxidant that can efficiently remove free radicals that are prone to disease and accelerate aging. Its antioxidant effect is far stronger than that of vitamin C and vitamin E, and it can It can realize the reduction and regeneration of vitamin C, vitamin E, glutathione, coenz...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N1/21C12P7/64
CPCC12N9/001C12N15/70C12P7/6436C12Y103/01031
Inventor 郑高伟许尧许建和潘江钱小龙
Owner EAST CHINA UNIV OF SCI & TECH
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