食品与发酵工业2024，Vol.50Issue(16)：125-131,7.DOI:10.13995/j.cnki.11-1802/ts.037187
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 and determining its enzymatic properties
摘要
Abstract
Cellobiose phosphorylase(CBP)phosphorolyzes cellobiose into glucose-1-phosphate and plays a crucial role in the conver-sion 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 expres-sion conditions were optimized,the enzyme activity increased from 0.18 IU/mL to 0.47 IU/mL when the induction temperature,isopropy-lthio-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-inacti-vation time of 60 min.Furthermore,Cu2+exhibited the greatest inhibitory effect on the enzyme,followed by Zn2+,Fe3+,Ca2+,and Mn4+in descending order(P＜0.05).In contrast,no significant difference in enzyme activity was observed with K+,Mg2+,and Na+(P＞0.05).Finally,the kinetic parameters of the recombinant CBP were determined,with a Km value of 19.61 μmol/mL,a Vmax value of 1.19 µmol/(mL·min),and a Kcat 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Key words
cellobiose phosphorylase/recombinant expression/Escherichia coli/enzymatic properties/biocatalysis引用本文复制引用
彭念,陈婷婷,王璐,樊俊萍,高玉杉,冉小琴,刘露露,李勇昊..纤维二糖磷酸化酶表达条件的优化及其酶学性质研究[J].食品与发酵工业,2024,50(16):125-131,7.基金项目
国家自然科学基金面上项目(22378033) （22378033）
重庆市自然科学基金面上项目(CSTB2022 NSCQ-MSX0544) （CSTB2022 NSCQ-MSX0544）
重庆市教委科学技术研究项目(KJQN202301546) （KJQN202301546）
重庆科技学院科研启动基金项目(ckrc2021070) （ckrc2021070）
重庆市研究生科研创新项目(CYS23747) （CYS23747）
