一种转葡萄糖苷酶基因的克隆表达及其生物催化合成α-熊果苷

doi:10.13218/j.cnki.csdc.2016.08.009

摘要/Abstract

摘要： 通过一步法克隆试剂盒将来自野油菜黄单胞菌的转葡萄糖苷酶基因与表达载体pET28a同源重组,构建质粒pET28a-agl,并转化到不同感受态细胞中,筛选出高效表达转葡萄糖苷酶的基因工程菌E.coli BL21 Gold (DE3) plysS pET28a-agl,以催化合成α-熊果苷。利用IMAC亲和层析,获得转葡萄糖苷酶用于其酶学性质的研究。催化合成α-熊果苷的较佳反应条件为:转葡萄糖苷酶的质量浓度0.272 5 g/L,对苯二酚质量浓度11 g/L,麦芽糖浓度1.1 mol/L,磷酸盐缓冲液浓度100 mmol/L (pH=6.5),30 ℃下摇床反应3 h。研究发现,反应产物α-熊果苷对转葡萄糖苷酶反应有轻微的抑制作用;1 mmol/L K⁺对转葡萄糖苷酶的相对酶活有提高作用,1 mmol/L Cu²⁺几乎能完全抑制该酶活性。

关键词: 化妆品添加剂, α-熊果苷, 生物催化

Abstract: The transglucosidase gene from Xanthomonas campestris pv.campestris was ligated with expression vector pET28a to construct plasmids pET28a-agl by using One Step Cloning Kit,transformed into different competent cells and screened out the resulting recombinants E.coli BL21 Gold (DE3) plysS pET28a-agl that successfully expresses the transglucosidase and its apparent high enzymatic activity for catalyzing enzymatic synthesis of α-arbutin.Enzymatic properties of the transglucosidase obtained were studied with IMAC affinity chromatograph.The suitable conditions for synthesis of α-arbutin are as follows:mass concentration of the transglucosidase,0.272 5 g/L;mass concentration of hydroquinone,11 g/L;concentration of maltose,1.1 mol/L;concentration of phosphate buffer solution,100 mmol/L (pH=6.5);shaking for 3 h at 30 ℃.It was discovered that the reaction product α-arbutin shows slight inhibition effect on the action of the transglucosidase.K⁺ ion of 1 mmol/L enhances the enzyme activity while Cu²⁺ of 1 mmol/L almostly inhibits the activity of the transglucosidase completely.

Key words: additive of cosmetics, α-arbutin, biocatalysis

中图分类号:

TQ658

王益栋,魏盼盼,邵梦格,张朝晖,陈小龙. 一种转葡萄糖苷酶基因的克隆表达及其生物催化合成α-熊果苷[J]. 日用化学工业, 2016, 46(8): 467-472.

WANG Yi-dong,WEI Pan-pan,SHAO Meng-ge,ZHANG Chao-hui,CHEN Xiao-long. Cloning and expression characterization of transglucosidase gene for biological catalytic synthesis of α-arbutin[J]. China Surfactant Detergent & Cosmetics, 2016, 46(8): 467-472.

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