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Carbonyl reductase mutants and application thereof

A mutant, reductase technology, applied in the fields of genetic engineering and enzyme engineering, can solve problems such as large steric hindrance, and achieve the effects of improved space-time efficiency, good industrial application prospects, and a wide range of catalysis

Pending Publication Date: 2017-10-17
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

The wild-type carbonyl reductase ChKRED20 can catalyze a variety of acetophenone derivatives to generate chiral alcohols with high optical purity (T.-X.Tang, Y.Liu, Z.-L.Wu.Characterization of a robust anti- Prelog short-chain dehydrogenase / reductase ChKRED20from Chryseobacterium sp.CA49.J MolCatal B-Enzym.2014,105:82-88; patent ZL 201310399109.2, ZL 201410103481.9), but for ortho-substituted acetophenone derivatives such as 1-( 3-Chloro-2,6-difluorophenyl)ethanone, 1-(2-(trifluoromethyl)phenyl)ethanone and 1-(2,4-dimethylphenyl)ethanone Difficult to convert, possibly due to greater steric hindrance in some parts of the catalytic pocket of the enzyme

Method used

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  • Carbonyl reductase mutants and application thereof
  • Carbonyl reductase mutants and application thereof
  • Carbonyl reductase mutants and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The mensuration of embodiment 1 wild-type and mutant crude enzyme liquid enzyme activity

[0031] 1.1 Preparation of crude enzyme solution

[0032] Pick a single clone into LB (containing kanamycin 50 μg / mL) medium, culture overnight at 37 ° C, transfer to TB (containing kanamycin 50 μg / mL) medium with 1% inoculum size, 37 Cultivate at ℃ for 3h, add 0.5mM IPTG for induction, and continue to culture at 30°C for 18h. The bacterial solution was centrifuged at 4°C and 8000rpm to collect the bacteria, the cell homogenizer was crushed, and the supernatant was centrifuged to obtain a crude enzyme solution.

[0033] 1.2 Determination of crude enzyme activity

[0034] Crude enzyme activity assay reaction conditions: the reaction system consists of two phases, the aqueous phase is potassium phosphate buffer (0.1M, pH8.0) containing 3g / L crude enzyme solution (total protein concentration)), 0.2g / L NAD + ; The organic phase is isopropanol and substrate (substrate dissolved in i...

Embodiment 2

[0035] Example 2 Alanine scanning of the catalytic pocket of carbonyl reductase ChKRED20

[0036] 2.1 Construction method of alanine scanning mutants

[0037] Based on the analysis of the crystal structure of the carbonyl reductase ChKRED20, we tried to perform alanine scanning on the 9 amino acid residues (I144, H145, K160, P186, Y188, I189, L194, L197, M201) in the catalytic pocket. Site-directed mutagenesis to alanine, all mutations were carried out using the carbonyl reductase ChKRED20 gene as a template, and the primers used are shown in Table 1.

[0038] Table 1 Primer sequences for alanine scanning

[0039]

[0040] The PCR conditions are: 10×Buffer 5 μL, each primer (10 mM) 6 μL, dNTP (2.5 mM) 4 μL, pfu enzyme (2.5U / mL) 1 μL, plasmid 10 ng, and ultrapure water to make up 50 μL. Conditions: Pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 68°C for 6 min, a total of 16 cycles. The PCR product was treated wi...

Embodiment 3

[0052] Embodiment 3 distance from the 145th histidine site randomization within

[0053] 3.1 Distance from the 145th histidine Saturation mutagenesis library construction method within

[0054] Saturation mutations were performed on the 143rd, 144th, 151st, 153rd, 160th, 186th, 188th, 196th, and 205th positions on the mutant H145A / M201A, respectively. Using mutant H145A / M201A as a template, NNS degenerate primers (N represents A, T, C, G; S represents G, C) are used. The degenerate primers used are shown in Table 4.

[0055] Table 4 The first round of single-site saturation mutation degenerate primers

[0056]

[0057] The PCR conditions are: 10×Buffer 5 μL, each primer (10 mM) 6 μL, dNTP (2.5 mM) 4 μL, pfu enzyme (2.5U / mL) 1 μL, plasmid 10 ng, and ultrapure water to make up 50 μL. Conditions: Pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 68°C for 6 min, a total of 16 cycles. The PCR product was treated wi...

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Abstract

The invention belongs to the technical field of gene engineering and enzyme engineering and particularly relates to carbonyl reductase mutants and an application thereof. Carbonyl reductase ChKRED20 is subjected to molecular modification by the aid of crystal structure based semi-rational design, one or multiple amino acids is / are replaced, a series of mutants wide in substrate spectrum are obtained, and application potential of the mutants in biological catalysis is displayed.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and enzyme engineering, and specifically relates to a carbonyl reductase mutant and its application in asymmetric reduction of carbonyl compounds. Background technique [0002] Chiral alcohols are important intermediates in the synthesis of chiral drugs. Biocatalytic asymmetric reduction of carbonyl compounds to produce chiral alcohols is one of the important methods for preparing chiral alcohols. Biocatalysis usually uses carbonyl reductase (or ketoreductase)-producing microorganisms or recombinant enzymes coupled with coenzyme regeneration systems as biocatalysts. Since recombinant enzymes have almost no side reactions in the reaction, most of the current large-scale industrial production uses recombinant enzymes. However, with the rapid growth of the demand for customization of biocatalysts, the existing basic enzyme libraries are far from meeting the needs in terms of quantity and...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12P41/00C12P7/40
CPCC12N9/0006C12P7/40C12P41/00C12Y101/01
Inventor 吴中柳赵凤佼李同彪李孜一王刚刚刘忠川金赟
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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