食品与发酵工业 >

2021 , Vol. 47 >Issue 3: 18 - 24

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.025002

研究报告

近平滑假丝酵母ATCC 7330羰基还原酶CpCR的表达及酶学性质研究

  • 宋婷 ,
  • 王帅静 ,
  • 汪沉 ,
  • 吕育财 ,
  • 罗华军 ,
  • 郭金玲 ,
  • 龚大春
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  • 1(中国轻工业功能酵母重点实验室(三峡大学),湖北 宜昌,443002)
    2(三峡大学 生物与制药学院,湖北 宜昌,443002)
硕士研究生(龚大春教授为通讯作者,E-mail:185195061@qq.com)

收稿日期: 2020-07-09

  修回日期: 2020-07-31

  网络出版日期: 2021-03-08

基金资助

国家自然基金项目(21776162);湖北省技术创新专项项目(2019ABA114)

收起

Expression and characterization of carbonyl reductase CpCR from Candida parapsilosis ATCC 7330

  • SONG Ting ,
  • WANG Shuaijing ,
  • WANG Chen ,
  • LYU Yucai ,
  • LUO Huajun ,
  • GUO Jingling ,
  • GONG Dachun
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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: 2020-07-09

  Revised date: 2020-07-31

  Online published: 2021-03-08

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摘要

通过构建重组菌Escherichia coli BL21(DE3)/ pACYC Duet-1-cpcr,表达带有His标签的近平滑假丝酵母 Candida parapsilosis ATCC 7330羰基还原酶CpCR基因,并采用Ni-Agarose亲和层析对重组酶CpCR进行分离纯化和酶学性质研究。重组酶CpCR的基因序列全长 1 107 bp,含有368 个氨基酸,分子质量在 41 kDa 左右,比酶活力为 20 U/mg;该酶在4~33 ℃的温度范围稳定性较好,相对酶活力在80%以上,T50值为37 ℃;该酶的pH适宜范围在6.2~7.5,在中性条件下稳定性最好;Cu2+对该酶有强烈的抑制作用,在1 mmol/L条件下,相对酶活力下降30%,在5 mmol/L条件下,相对酶活下降50%;该酶对底物苯甲醛、正丁醛亲和能力强于4-氯-3-酮基-丁酸乙酯(4-chlor-3-keto-butyrate-ethyl ester,COBE),对苯甲醛或正丁醛的催化效率是对COBE的20倍左右;该酶是一种NADPH依赖型的羰基还原酶。本研究为羰基还原酶CpCR分子改造和催化应用提供了重要的基础数据。

关键词： 近平滑假丝酵母; 羰基还原酶CpCR; 重组表达; 酶学性质

本文引用格式

宋婷 , 王帅静 , 汪沉 , 吕育财 , 罗华军 , 郭金玲 , 龚大春 . 近平滑假丝酵母ATCC 7330羰基还原酶CpCR的表达及酶学性质研究[J]. 食品与发酵工业, 2021 , 47(3) : 18 -24 . DOI: 10.13995/j.cnki.11-1802/ts.025002

Abstract

Carbonyl reductases from Candida parapsilosis are valuable to the synthesis of hydroxyl compounds. By constructing recombinant E. coli BL21 (DE3)/pACYCDuet-1-cpcr, the carbonyl reductase CpCR gene (cpcr) from C. parapsilosis. ATCC 7330 was expressed. The recombinant CpCR was separated and purified by Ni-Agarose affinity chromatography from His-tag on the pACYCDuet-1 and its enzymatic properties were investigated. The results showed that the full length of the carbonyl reductase gene cpcr was 1 107 bp,containing 368 amino acids with molecular weight around 41 kDa and specific enzyme activity of 20 U/mg. The recombinant CpCR was stable in the temperature range of 4-33 ℃ with the relative enzyme activity above 80%, and the T50 value was 37 ℃. The suitable pH range was 6.2-7.5, and optimal stability was obtained under neutral conditions. Cu2+ had a strong inhibition on the recombinant CpCR, and lowered the relative enzyme activity by 30% at 1 mmol/L, and by 50% at a 5 mmol/L. The affinity of the enzyme to benzaldehyde and n-butyraldehyde was stronger than that to 4-chlor-3-keto- butyrate-ethyl ester (COBE). The catalytic efficiency for benzaldehyde or n-butyraldehyde was about 20 times of that for COBE. The recombinant CpCR was an NADPH-dependent carbonyl reductase. This study provided basic data for the molecular modification and catalytic application of carbonyl reductase CpCR.

Key words： Candida parapsilosis; carbonyl reductase CpCR; recombinant expression; enzymatic properties

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