Carbonyl reductase and application of same in synthesis of chiral hydroxyl compound

A technology of carbonyl reductase and reductase, which is applied in the field of bioengineering, can solve the problems of harsh reaction conditions and low yield, and achieve the effects of mild reaction conditions, high product concentration and easy operation

Active Publication Date: 2015-11-04
ABIOCHEM BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is to provide a high catalytic activity, strong enantioselectivity, substrate tolerance, etc. Good carbonyl reductase and method for using the carbonyl reductase to catalyze the synthesis of chiral aromatic alcohols

Method used

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  • Carbonyl reductase and application of same in synthesis of chiral hydroxyl compound
  • Carbonyl reductase and application of same in synthesis of chiral hydroxyl compound
  • Carbonyl reductase and application of same in synthesis of chiral hydroxyl compound

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Cloning of Example 1 Carbonyl Reductase Gene

[0043] The sequence was synthesized according to the known sequence of 3-oxoacyl-ACP reductase of S. albus (see SEQ ID No: 1 for the amino acid sequence), and the corresponding DNA sequence was obtained as shown in SEQ ID No: 2. The gene was cloned into the NdeI / HindIII site of pET28a to construct the corresponding expression plasmid pABI-ADH-SA, which was verified by PCR and electrophoresis. The primers used were shown in SEQ ID No:3 and 4. Transform into Escherichia coli BL21 (DE3) competent cells to obtain the corresponding recombinant strain sABI-ADH-SA.

Embodiment 2

[0044] Embodiment 2 Expression of recombinant carbonyl reductase

[0045] Inoculate the recombinant strain sABI-ADH-SA into LB shake flasks containing kanamycin and cultivate overnight at 37°C, and inoculate the overnight culture into 1L LB shake flasks at a ratio of 5%, and cultivate to OD at 37°C 600 0.6-0.8, add IPTG to a final concentration of 1 mM, lower the temperature to 30°C and continue culturing for 16 hours. The bacterial cells were collected by centrifugation at 4000rpm, resuspended with 100mL PBS (pH 7.5) buffer solution, ultrasonically disrupted, and centrifuged at 12000rpm for 30min to obtain the supernatant which was the crude enzyme solution.

[0046] The method of measuring enzyme activity (U) is: in 2mL reaction solution, add 2mM acetophenone as substrate, 0.1mM NADH as cofactor, then add 20μL crude enzyme solution, measure OD within 1 minute 340 The decrease is ΔA 340 . Specific enzyme activity per mL of enzyme solution U=ΔA 340 ×1000 / (6220×20), that i...

Embodiment 3

[0050] The asymmetric reduction reaction of embodiment 3 prochiral carbonyl substrates

[0051]

[0052] Suspend 40 g of methyl quinoline-3-carboxylate in 500 mL of sodium phosphate buffer and wash with saturated Na 2 CO 3 The pH of the solution was adjusted to 6.0, and 200 mL of the crude enzyme solution prepared in Example 2, 100 g of glucose and 1000 U of glucose dehydrogenase (purchased from sigma), and 0.2 mmol / L of NAD were added + , add sodium phosphate buffer to 1L, stir and react at 30°C for 24 hours, saturate with Na 2 CO 3 The pH of the reaction was controlled at about 6.0, and the progress of the reaction was detected by TLC. After the reaction, adjust the pH to 2.0, raise the temperature to 70°C and heat for 1 hour to denature the protein, add diatomaceous earth to filter to remove the denatured protein, and then adjust the pH to 13.0; extract twice with an equal volume of n-butanol, and wash with an equal volume of n-butanol The residue was filtered once, ...

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Abstract

The invention provides a novel carbonyl reductase and a method for asymmetric reduction by using recombinant carbonyl reductase. Compared with asymmetric ketone reduction catalyzed other enzymes, the carbonyl reductase provided by the invention has the following advantages in the asymmetric reduction: the carbonyl reductase has wide substrate selectivity, can catalyze reduction of a plurality of substituted aromatic ketones like a benzene ring, a naphthalene ring, pyridine and benzopyridine and enables the optical purity of a product to be as high as more than 99% under the condition of catalysis of a substrate with a concentration as high as 100 g/L. A preparation method for the carbonyl reductase in the invention has the advantages of high concentration and high optical purity of the produced carbonyl reductase, mild reaction conditions, environment friendliness, simple operation and easy industrial scaling-up.

Description

technical field [0001] The invention belongs to the field of bioengineering, and specifically relates to a carbonyl reductase, a recombinant expression vector containing a nucleotide sequence encoding the enzyme, a recombinant expression transformant, an expressed recombinant enzyme and a preparation method of the recombinant enzyme, and the carbonyl reduction Application of enzymes as catalysts in the asymmetric synthesis of chiral hydroxyl compounds. Background technique [0002] Optically active chiral alcohols containing specific functional groups are important chiral building blocks for the synthesis of drugs, pesticides and other fine chemicals. Among them, chiral aromatic alcohols are important intermediates for the synthesis of chiral drugs, alkaloids and other chiral compounds. They are a large class of organic compounds with important industrial potential and play an important role in today's pharmaceutical field. For example, (S)-(+)-1,1-diphenyl-2-propanol and (...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P7/02
CPCC12N9/0006C12P7/02C12Y101/01184
Inventor 罗煜丁时澄瞿旭东田振华
Owner ABIOCHEM BIOTECH CO LTD
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