Unlock instant, AI-driven research and patent intelligence for your innovation.

A kind of ketoreductase mutant and its application

A technology of reductase and variant ketone, which is applied in the field of ketoreductase and preparation of chiral hydroxyester, and can solve the problems of unsatisfactory reactivity, stability and selectivity

Active Publication Date: 2016-03-16
HANGZHOU NORMAL UNIVERSITY
View PDF8 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although wild-type ketoreductases generally have good reactivity and selectivity for their natural substrates, their reactivity, stability and selectivity for unnatural substrates are often unsatisfactory

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A kind of ketoreductase mutant and its application
  • A kind of ketoreductase mutant and its application
  • A kind of ketoreductase mutant and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1 mutant ketoreductase

[0037] (1) Obtaining wild-type ketoreductase (SEQ ID NO: 7)

[0038] Clone the gene tadh (nucleotide sequence shown in SEQ ID NO: 8) encoding ketoreductase from Thermotogamaritima, connect to the vector pET28a (+) to form a recombinant, and transform the recombinant into Escherichia coli Internal fermentation expression of JM109 (DE3): First culture the recombinant bacteria in liquid LB medium at 37°C for 3-5 hours to obtain seed liquid; then inoculate the seed liquid into the autoinduction medium at a volume ratio of 1:100, After culturing at 20-25°C for 48 hours, cells containing wild-type ketoreductase can be obtained. The amino acid sequence of the wild-type ketoreductase is shown in SEQ ID NO: 7, and the nucleotide sequence of the wild-type ketoreductase has been obtained by gene sequencing verified.

[0039] (2) Mutation and screening

[0040] On the basis of homology modeling and molecular docking, possible key amino acid re...

Embodiment 2

[0073] Embodiment 2 enzyme source

[0074] The 6 double mutant recombinants encoding mutant ketoreductase W21Q / W86E, W21Q / W86V, W21S / W86E, W21S / W86E, W21S / W86H, W21S / W86I, W21S / W86N prepared in Example 1 were respectively transformed into Competent cells (Escherichia coli JM109 (DE3), purchased from Zhejiang Toyobo Biological Co., Ltd.), obtained Escherichia coli containing mutant ketoreductase coding gene, placed in LB solid medium (1wt% NaCl, 1wt% peptone, 0.5wt% yeast powder, 1.5wt% agar, the solvent is water, the pH value is 7.2-7.4) and cultivated in a 37°C incubator for 12h. A single colony was picked and inoculated into the self-inducing medium, and shaken on a shaker at 200 rpm for 48 hours at 25°C. Collect the bacterial solution, centrifuge at 8000rpm for 10min, and wash with PBS buffer solution (NaCl137mmol / l, KCl2.7mmol / l, NaCl2.7mmol / l, 2 HPO 4 10mmol / l, KH 2 PO 4 1.76mmol / l, pH7.2~7.4) to resuspend the bacteria, 200W ultrasonic for 5min for ultrasonic cell di...

Embodiment 3

[0077] Example 3 Mutant ketoreductase catalyzes the reduction of ethyl 2-oxo-4-phenylbutyrate (EOPB)

[0078] The mutant ketoreductase W21S / W86E pure enzyme solution obtained by the method in Example 2 (200 μL, 20 g / L reaction system based on dry weight of bacteria), glucose (final concentration 10 mM), glucose dehydrogenase GDH (final Concentration 200μL, namely 5U / L), reducing coenzyme NADP + (final concentration 0.05mM) mixed with the substrate EOPB (final concentration 7mM), add 15μL DMSO to form 1mL transformation system in water, the pH value of the transformation system is 7.2-7.4, the reaction formula is as follows figure 2 As indicated, shake at 37°C and 200rpm for 12h on a shaker. After the reaction, the reaction solution was extracted with an equal volume of ethyl acetate, and the ethyl acetate layer was taken for high-performance liquid chromatography (chiral chromatography IC column, mobile phase: n-hexane (A) / isopropanol (B)=95 / 5, v / v, 1mL / min, column tempera...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a ketoreductase mutant and an application thereof. Mutant ketoreductase is obtained by mutating a tryptophan residue on 21st-position of an amino acid sequence as shown in SEQ ID NO: 7 into glutamine or serine, and meanwhile mutating a tryptophan residue on 86th-position into glutamic acid, histidine, isoleucine, asparaginate or valine. The mutant ketoreductase disclosed by the invention changes three-dimensional orientation of wild type ketoreductase for catalysis of ketone and ester compounds (such as ethyl 2-oxo-4-phenylbutyrate or ethyl 2,4-dioxo-4-phenylbutanoate); in the generated main product (such as ethyl 2-hydroxy-4-phenylbutyrate or ethyl 2-hydroxy-4-oxo-4-phenylbutanoate), an (S-) conformation is changed into an (R-) conformation and an enantiomer excess (ee value) changes from 76.7% (S-) to 99.4% (R-).

Description

(1) Technical field [0001] The invention relates to a ketoreductase and its application in the preparation of chiral hydroxyester, in particular to a mutant ketoreductase, a coding gene, a carrier, a recombinant engineering bacterium and its application. (2) Background technology [0002] Ketoreductase (Ketoreductase) is an oxidoreductase ubiquitous in nature, which can asymmetrically reduce carbonyl-containing substances (such as ketoesters, ketoacids, etc.) to corresponding chiral alcohols. The reduction of ketones catalyzed by ketoreductase requires a cofactor to transfer hydrogen to the carbonyl group. The commonly used cofactor is reduced nicotinamide adenine dinucleotide phosphate (NADPH) or reduced nicotinamide adenine dinucleotide (NADH). [0003] Compared with the problems of expensive catalysts, environmental pollution, low product purity, and harsh catalytic conditions in the chemical synthesis process, ketoreductase is widely used because of its many advantages s...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P7/62C12R1/01
CPCC12N9/0006C12P7/62C12Y101/01184
Inventor 陈振明张飒王志国周硕赖敦岳张双玲
Owner HANGZHOU NORMAL UNIVERSITY