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Title:
KLUYVEROMYCES MARXIANUS ALDO-KETO REDUCTASE KMAKR MUTANT AND APPLICATION THEREOF
Document Type and Number:
WIPO Patent Application WO/2021/057014
Kind Code:
A1
Abstract:
Disclosed are a Kluyveromyces marxianus aldo-keto reductase KmAKR mutant and application thereof, said aldo-keto reductase mutant being obtained by site-directed saturation mutagenesis at position 63 of the amino acid sequence shown in SEQ. ID NO. 2. The specific enzymatic activity of the aldo-keto reductase mutants M5-A, M5-L, and M5-M constructed by the present invention is increased by 1.1 times, 3.2 times, and 4.1 times, respectively, compared with a control group. The mutants KmAKR-Y296W/W297H/K29H/Y28A/T63M have significantly improved catalytic activity on 6-cyano-(5R)-hydroxy-3-carbonylhexanoate and 6-chloro-(5S)-hydroxy-3-carbonylhexanoate. The maximum substrate 6-cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl dosage can reach 450 g/L, the substrate conversion rate is higher than 99%, the product dep value always remains above 99.5%, and the space–time yield of the biocatalytic process is as high as 1224.3 g/L d.

Inventors:
WANG YAJUN (CN)
QIU SHUAI (CN)
LI SHUFANG (CN)
CHENG FENG (CN)
WENG CHUNYUE (CN)
ZHENG YUGUO (CN)
Application Number:
PCT/CN2020/085538
Publication Date:
April 01, 2021
Filing Date:
April 20, 2020
Export Citation:
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Assignee:
UNIV ZHEJIANG TECHNOLOGY (CN)
International Classes:
C12N9/04; C12P7/02
Foreign References:
CN110577940A2019-12-17
CN110004120A2019-07-12
CN110004120A2019-07-12
CN107058248A2017-08-18
CN107058248A2017-08-18
Other References:
YU HAN; QIU SHUAI; CHENG FENG; CHENG YING-NAN; WANG YA-JUN; ZHENG YU-GUO: "Improving the catalytic efficiency of aldo-keto reductase KmAKR towards t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate via semi-rational design", BIOORGANIC CHEMISTRY, ACADEMIC PRESS INC., NEW YORK, NY., US, vol. 90, 8 June 2019 (2019-06-08), US, XP085760173, ISSN: 0045-2068, DOI: 10.1016/j.bioorg.2019.103018
YU HAN; QIU SHUAI; CHENG FENG; CHENG YING-NAN; WANG YA-JUN; ZHENG YU-GUO: "Improving the catalytic efficiency of aldo-keto reductase KmAKR towards t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate via semi-rational design", BIOORGANIC CHEMISTRY, ACADEMIC PRESS INC., NEW YORK, NY., US, vol. 90, 8 June 2019 (2019-06-08), US, XP085760173, ISSN: 0045-2068, DOI: 10.1016/j.bioorg.2019.103018
SHI LIZHEN, BINGBING YING, YAJUN WANG: "Structure function and biocatalytic applications of microbial aldo-keto reductases", BULLETIN OF FERMENTATION SCIENCE AND TECHNOLOGY, vol. 46, no. 4, 1 November 2017 (2017-11-01), pages 198 - 204, XP055796131, DOI: 10.16774/j.cnki.issn.1674-2214.2017.04.002
SHEN WEI; CHEN YI; QIU SHUAI; WANG DAN-NA; WANG YA-JUN; ZHENG YU-GUO: "Semi-rational engineering of a Kluyveromyces lactis aldo-keto reductase KlAKR for improved catalytic efficiency towards t-butyl 6-cyano-(3R, 5R)-dihydroxyhexanoate", ENZYME AND MICROBIAL TECHNOLOGY, STONEHAM, MA, US, vol. 132, 20 August 2019 (2019-08-20), US, XP085905050, ISSN: 0141-0229, DOI: 10.1016/j.enzmictec.2019.109413
Attorney, Agent or Firm:
TIANZHENG PATENT ATTORNEYS (CN)
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