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  

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

作　　者：宋婷[1,2] 王帅静 汪沉 吕育财 罗华军[1,2] 郭金玲 龚大春[1,2] SONG Ting;WANG Shuaijing;WANG Chen;LYU Yucai;LUO Huajun;GUO Jingling;GONG Dachun(China Key Laboratory of Light Industry Functional Yeast,China Three Gorges University,Yichang 443002,China;College of Biological and Pharmaceutical,China Three Gorges University,Yichang 443002,China)

机构地区：[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

出　　处：《食品与发酵工业》2021年第3期18-24,共7页Food and Fermentation Industries

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

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1107 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,T 50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;Cu^2+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。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 1107 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 T 50 value was 37℃.The suitable pH range was 6.2-7.5,and optimal stability was obtained under neutral conditions.Cu^2+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.

关 键 词：近平滑假丝酵母 羰基还原酶CpCR 重组表达 酶学性质 

分 类 号：Q78[生物学—分子生物学] TQ426.97[化学工程]