纤维二糖磷酸化酶表达条件的优化及其酶学性质研究  

Optimizing conditions for expression of cellobiose phosphorylase anddetermining its enzymatic properties

作　　者：彭念 陈婷婷 王璐 樊俊萍 高玉杉 冉小琴 刘露露[1] 李勇昊 PENG Nian;CHEN Tingting;WANG Lu;FAN Junping;GAO Yushan;RAN Xiaoqin;LIU Lulu;LI Yonghao(School of Chemistry and Chemical Engineering,Chongqing University of Science and Technology,Chongqing 401331,China)

机构地区：[1]重庆科技学院化学化工学院,重庆401331

出　　处：《食品与发酵工业》2024年第16期125-131,共7页Food and Fermentation Industries

基　　金：国家自然科学基金面上项目(22378033);重庆市自然科学基金面上项目(CSTB2022 NSCQ-MSX0544);重庆市教委科学技术研究项目(KJQN202301546);重庆科技学院科研启动基金项目(ckrc2021070);重庆市研究生科研创新项目(CYS23747)。

摘　　要：纤维二糖磷酸化酶(cellobiose phosphorylase,CBP)磷酸化裂解纤维二糖为葡萄糖-1-磷酸,它是木质纤维素转化淀粉的关键酶。该研究将来源于Clostridium thermocellum YM4的cbp基因通过表达载体pET-28a(+)构建重组菌Rosetta(DE3)/pET-28a-CBP用于表达CBP。对其诱导表达条件进行优化,结果显示当诱导温度、异丙基硫代-β-D-半乳糖苷(isopropylthio-β-D-galactoside,IPTG)浓度和诱导时间分别为15℃、0.2 mmol/L和12 h时CBP酶活力最高,为0.47 IU/mL。进一步利用Ni柱分离纯化出带有His标签的CBP,结果表明该重组酶分子质量为95 kDa,比酶活1.03 IU/mg;最适反应温度60℃,最适反应pH 6,半失活时间为60 min;金属离子Cu^(2+)显示出对该酶有强烈的抑制作用,然后依次是Zn^(2+)、Fe^(3+)、Ca^(2+)、Mn^(4+)(P<0.05);而K^(+)、Mg^(2+)和Na^(+)对该酶活力无显著性影响(P>0.05);最后对重组CBP的动力学参数进行了表征,K_(m)值为19.61μmol/mL,V_(max)值为1.19μmol/(mL·min)且K_(cat)值为21.38 s^(-1)。Cellobiose phosphorylase(CBP)phosphorolyzes cellobiose into glucose-1-phosphate and plays a crucial role in the conversion of lignocellulose to starch.The cbp gene derived from Clostridium thermocellum YM4 was utilized to express CBP by engineering the recombinant Escherichia coli,Rosetta(DE3)/pET-28a-CBP,with the aid of a pET-28a(+)expression vector.After the induced expression conditions were optimized,the enzyme activity increased from 0.18 IU/mL to 0.47 IU/mL when the induction temperature,isopropylthio-β-D-galactoside(IPTG)concentration,and induction time were set at 15℃,0.2 mmol/L,and 12 h,respectively.Subsequently,the His-tagged CBP was isolated and purified using a Ni column.Results revealed that the recombinant enzyme had a molecular mass of 95 kDa,a specific enzyme activity of 1.03 IU/mL,an optimal reaction temperature of 60℃,an optimal reaction pH of 6,and a half-inactivation time of 60 min.Furthermore,Cu^(2+)exhibited the greatest inhibitory effect on the enzyme,followed by Zn^(2+),Fe^(3+),Ca^(2+),and Mn^(4+)in descending order(P<0.05).In contrast,no significant difference in enzyme activity was observed with K^(+),Mg^(2+),and Na^(+)(P>0.05).Finally,the kinetic parameters of the recombinant CBP were determined,with a K_(m)value of 19.61μmol/mL,a V max value of 1.19μmol/(mL·min),and a K_(cat)value of 21.38 s^(-1).

关 键 词：纤维二糖磷酸化酶 重组表达 大肠杆菌 酶学性质 生物催化 

分 类 号：TS201.25[轻工技术与工程—食品科学] Q78[轻工技术与工程—食品科学与工程]