不同来源寡聚-1,6-葡糖苷酶异源表达及酶学特性  

Heterologous Expression and Enzymatic Properties of Oligo-1,6-Glucosidases from Different Bacteria

作　　者：华烨 刘鹏[1] 王永红[1] HUA Ye;LIU Peng;WANG Yonghong(State Key Laboratory of Bioreactor Engineering,East China University of Science and Technology,Shanghai 200237,China)

机构地区：[1]华东理工大学生物反应器工程国家重点实验室,上海200237

出　　处：《华东理工大学学报（自然科学版）》2021年第4期445-454,共10页Journal of East China University of Science and Technology

基　　金：国家自然科学基金(21878084)。

摘　　要：寡聚-1,6-葡糖苷酶(EC 3.2.1.10)对于乳酸发酵液残糖中含量较高的异麦芽糖有较高的利用活性。基于前期研究工作基础并经BRENDA数据库搜索挑选了5个可能耐高温、耐酸的寡聚-1,6-葡糖苷酶基因(Bacillus coagulans ATCC 7050的malL BF292011(BC1)和malL BF292004(BC2)、Bacillus subtilis 168的malL yvdL BSU34560(BS1)和yugT BSU31290(BS2)以及Bacillus thermoglucosidasius KP 1006的malL(KP)),首次利用pET表达系统在大肠杆菌中进行重组表达、纯化及酶学性质的系统研究。结果显示:BC1、BC2、KP的最适pH为5.0,BS1、BS2的最适pH为6.0。最适温度分别为:KP(60℃),BC1(56℃),BC2(56℃),BS1(43℃),BS2(43℃)。Mg^(2+)、NH_(4)^(+)对于BC1的酶催化活性具有激活作用,Co^(2+)、K+、NH_(4)^(+)对于KP有激活作用,但这些离子对于BC2、BS1和BS2都没有明显的激活作用。Zn^(2+)、Cu^(2+)、Ni^(+)对这5种寡聚-1,6-葡糖苷酶都有明显的抑制作用。以对硝基苯-α-D-葡萄糖苷(pNPG)为底物时,酶的转换数和催化效率从高到低依次为:KP>BC1>BS1>BS2>BC2。以乳酸发酵液中的残糖成分作为参考,在以麦芽糖、异麦芽糖、蔗糖、乳糖、海藻糖以及可溶性直链淀粉作为底物测定酶活时,BC1、BC2、KP、BS1的最适底物都是异麦芽糖,BS2的最适底物为麦芽糖。Oligo-1,6-glucosidase(EC 3.2.1.10)shows a good hydrolytic activity for isomaltose existed in the residual sugar of lactic acid fermentation broth.On the basis of previous work and a validation by BRENDA database search,five possible heat-resistant and acid-resistant oligo-1,6-glucosidase enzyme genes(Bacillus coagulans ATCC7050:malL BF292011(BC1)and malL BF292004(BC2);Bacillus subtilis 168:yvdL BSU34560(BS1)and yugT BSU31290(BS2);Bacillus thermoglucosidasius KP 1006:malL(KP))were selected for recombinant expression in E.coli.The expressed oligo-1,6-glucosidases were purified and their enzymatic properties were tested.The optimal working pH for BC1,BC2 and KP was determined to be 5.0,and that for BS1 and BS2 was 6.0.The working temperature of the oligo-1,6-glucosidases was in an order of KP(60℃),BC1(56℃),BC2(56℃),BS1(43℃),BS2(43℃).In addition,Mg^(2+)and NH_(4)^(+)could enhance the enzymatic activity of BC1,while Co^(2+),K+,NH_(4)^(+)could enhance the enzymatic activity of KP.However,these ions showed minimal effect on the activity of BC2,BS1 and BS2.Zn^(2+),Cu^(2+)and Ni^(+)showed inhibitory effects on the activity of the five oligo-1,6-glucosidases.Using p-nitrobenzene-α-Dglucoside as the substrate,the catalytic efficiency of the enzymes was in an order of KP>BC1>BS1>BS2>BC2.The substrate specificity of the oligo-1,6-glucosidases was tested for maltose,isomaltose,sucrose,lactose,trehalose and the soluble amylose isolated from the residual sugar in lactic acid fermentation broth.The most suitable substrate for BC1,BC2,KP and BS1 was isomaltose,and that for BS2 was maltose.

关 键 词：寡聚-1 6-葡糖苷酶 Bacillus coagulans ATCC 7050 Bacillus subtilis 168 Bacillus thermoglucosidasius KP 1006 

分 类 号：Q71[生物学—分子生物学]