Food and Fermentation Industries >

2022 , Vol. 48 >Issue 2: 59 - 64

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.027246

Stability of CpCR mutants from Candida parapsilosis ATCC 7330 carbonyl reductase

  • GONG Dachun ,
  • WANG Delin ,
  • WAN Li ,
  • LIU Run ,
  • LYU Yucai ,
  • LUO Huajun ,
  • SONG Ting
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  • 1(China Key Laboratory of Light Industry Functional Yeast, China Three Gorges University, Yichang 443002, China)
    2(College of Biological and Pharmaceutical, China Three Gorges University,Yichang 443002, China)

Received date: 2021-03-26

  Revised date: 2021-05-05

  Online published: 2022-02-28

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Abstract

Using the protein rational design and site-directed mutagenesis technology, five mutants with A98N, S307N, G262N, S216N and S258N substitution in carbonyl reductase (wtCpCR) from Candida parapsilosis ATCC 7330 were constructed. And their stability in organic solvents, temperature, shear resistance, oxygen resistance, coenzyme NADPH concentration were studied. The results showed that compared with the enzymatic activity of wtCpCR in potassium dihydrogen phosphate/dibasic potassium phosphate (PB) buffer, the enzymatic activity of the A98N mutant increased by 10%, but the mutant S307N had no enzymatic activity and the activity of other mutants slightly decreased. The interfacial stability of A98N and G262N mutant in the PB/methyl tert-butyl ether biphasic media increased 1.7 and 1.4-fold compared to than that of the wtCpCR, respectively. The thermal resistance properties of four mutants slightly reduced, with T50 values ranging from 31-36 ℃. Under the conditions of 100 and 200 r/min, the shear resistance of the four mutants was enhanced. When the speed was 300 r/min, the stability of the G262N mutant was 1.3 times higher than that of wtCpCR. The G262N mutant enzyme had better stability under aerobic conditions, with a t1/2 value of 11.87 h, which was 1.4 times higher than that of the wild-type enzyme. When the NADPH concentration was 0-0.4 mmol/L, the G262N mutant was much more stable. This study has provided very important scientific evidences for multiple mutations to improve stability modification of the carbonyl reductase from Candida parapsilosis (CpCR), which can improve its economy of biocatalysis process in biphasic reaction media.

Key words： Candida parapsilosis; carbonyl reductase; rational design; site-directed; enzymes stability

Cite this article

GONG Dachun , WANG Delin , WAN Li , LIU Run , LYU Yucai , LUO Huajun , SONG Ting . Stability of CpCR mutants from Candida parapsilosis ATCC 7330 carbonyl reductase[J]. Food and Fermentation Industries, 2022 , 48(2) : 59 -64 . DOI: 10.13995/j.cnki.11-1802/ts.027246

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