氧化葡萄糖酸杆菌老黄酶Go的酶学性质鉴定  

Characterization of an old yellow enzyme from Gluconobacter oxydans H24

作　　者：陈晶晶[1] 盛茜茜 钱姝文 严明[1] 许琳[1] 

机构地区：[1]南京工业大学生物与制药工程学院,江苏南京211800

出　　处：《生物加工过程》2017年第1期20-27,共8页Chinese Journal of Bioprocess Engineering

基　　金：国家重点基础研究计划(973计划)(2011CBA00804)

摘　　要：从氧化葡萄糖酸杆菌(Gluconobacter oxydans)H24中克隆出老黄酶Go的基因,通过构建重组表达质粒,在大肠杆菌BL21中诱导表达可溶性的蛋白,使用离子交换柱(Hi Trap DEAE FF)对重组酶进行纯化,并对其酶学性质进行表征。结果表明:老黄酶Go的温度和p H耐受性都较好,在40℃条件下,12 h后能保持40%的活性;在p H10的条件下,12 h后能保持40%的活性;Go的底物谱十分广泛,对酰亚胺类、α,β-不饱和醛酮、萜类都有一定的活性。老黄酶Go有良好的有机溶剂耐受性,在体积分数20%的乙酸丁酯中可以保持90%左右的活性,但是金属离子对其影响较大,除Mg^(2+)和K^+外,所选的其他离子对Go都有着不同程度的抑制作用。最后,在催化性质的实验中发现,Go对柠檬醛的转化率有60%,并且其对映体过量值(e.e.)可以达到99%。Go,a novel old yellow enzyme from Gluconobacter oxydans H24, was successfully cloned and overexpressed in BL21（DE3）. Then the recombinant protein was identified and characterized after purified with HiTrap DEAE FF column. Go can reduce various activated alkenes imides, a,, S-unsaturated ketones, and terpene and exhibits high temperature （ retaining 40% catalytic activity, 40 ℃ for 12 h） and pH stability （ retaining 40% activiy,pH 10 for 12 h ） , indicating that it has a great potential for biocatalysis. Also,it shows high tolerance toward organic solvents and maintain 90% activity in 20% ethyl acetate. However,Go has low tolerance towards metal ions. Tested metal ions all existed inhibitions toward Go except Mg2＋ and K＋. Besides, Go） can catalyze citral to （ S ） -citronellal with 60% conversion and but high enantiomeric excess value （e.e.〉99%）.

关 键 词：老黄酶 不对称还原碳碳双键 氧化葡萄糖酸杆菌H24 酶学性质 

分 类 号：Q554[生物学—生物化学]