热解糖高温厌氧杆菌β-葡萄糖苷酶的重组表达与酶学性质  

Recombinant expression and characterization ofβ-glucosidase from Thermoanaerobacterium thermosaccharolyticum

作　　者：刘洋 张汆[1] 彭惠 董艺凝[1] 赵维萍 LIU Yang;ZHANG Cuan;PENG Hui;DONG Yining;ZHAO Weiping(College of Biological and Food Engineering,Chuzhou University,Chuzhou 239000,China;Collaborative Innovation Center of Modern Bio-Manufacture,Anhui University,Hefei 230601,China)

机构地区：[1]滁州学院生物与食品工程学院,滁州239000 [2]安徽大学现代生物制造协同创新中心,合肥230601

出　　处：《应用与环境生物学报》2021年第4期1062-1068,共7页Chinese Journal of Applied and Environmental Biology

基　　金：安徽省高等学校自然科学研究重点项目(KJ2018A0426,KJ2019A0639,KJ2020A0715);安徽省高校优秀青年人才支持计划重点项目(gxyq ZD2021133);安徽大学现代生物制造协同创新中心实验室开放课题(BM2017003);农业农村部华东作物基因资源与种质创制重点实验室开放基金(ECG2018001);滁州学院科研启动基金(2018qd14)资助。

摘　　要：β-葡萄糖苷酶是纤维素分解酶系中的重要组成部分,为从嗜热微生物中挖掘酶学性质优良的新型β-葡萄糖苷酶,解决β-葡萄糖苷酶在工业应用方面普遍存在的活力偏低、稳定性不足、易受产物抑制等问题,通过密码子优化、基因合成和分子克隆技术,从热解糖高温厌氧杆菌(Thermoanaerobacterium thermosaccharolyticum)基因组中挖掘新型β-葡萄糖苷酶基因bglY,构建重组表达载体pET22b-bglY,并转化至大肠杆菌BL21(DE3)中,经IPTG诱导获得可溶性表达,采用Ni-NTA亲和层析法获得纯酶,并探究其酶学性质.结果显示,BglY属于GH1家族成员,分子量为52×10^(3),最适反应温度65℃,70℃的半衰期达1 h;最适反应pH 6.5,在pH 5-10范围内稳定;p-nitrophenyl-β-D-glucopyranoside(pNPG)为底物时比活为420.2±5.6 U/mg,米氏常数K_(m)值和最大反应速率V_(max)分别为2.1±0.4 mmol/L和909.1±6.2μmol min^(-1) mg^(-1);该酶对β-1,4糖苷键的底物具有水解偏好性,也可以水解纤维二糖和乳糖;5 mmol/L Fe^(3+)、Fe^(2+)、Cr^(2+)、Ca^(2+)、Mn^(2+)和EDTA对酶有激活作用,1%SDS完全抑制其活性;该酶可以抵抗产物葡萄糖的反馈抑制,且0.1-0.6 mol/L浓度的葡萄糖对酶具有激活作用,其中0.4 mol/L葡萄糖可提升活性至1.3倍,当葡萄糖浓度超过0.6 mol/L时,酶的活性才开始出现抑制.本研究表明BglY是一个葡萄糖激活型β-葡萄糖苷酶,其酶学性质优异,反应pH和温度范围较广且稳定,同时能水解天然底物纤维二糖和乳糖,可在提高纤维素生物质向葡萄糖的酶促转化等方面发挥应用潜力.The hydrolytic enzymeβ-glucosidase is an important component of the cellulase system.The aim of this study was to obtain and characterize newβ-glucosidases with excellent enzymatic properties from thermophilic microorganisms in order to solve the problems of low activity,insufficient stability,and susceptibility to product inhibition ofβ-glucosidase in industrial applications.A newβ-glucosidase gene,bglY,was extracted from the genome of Thermoanaerobacterium thermosaccharolyticum and the recombinant expression vector pET22 b-bglY was constructed by codon optimization,gene synthesis,and molecular cloning.The vector was then transformed into Escherichia coli BL21(DE3).Soluble expression was determined by induction with IPTG.High-purity β-glucosidase BglY was obtained by Ni-NTA affinity chromatography.The results showed that BglY belongs to the GH1 family and has a molecular weight of 52 kDa.The optimal reaction temperature was 65℃,and the half-life at 70℃ was 1 h.The optimal reaction pH was 6.5,and the protein was stable in the pH range of 5-10.The specific activity of p-nitrophenyl-β-D-glucopyranoside(pNPG)is 420.2±5.6 U/mg;Km and Vmaxare 2.1±0.4 mmol/L and 909.1±6.2μmol min^(-1) mg^(-1),respectively;the enzyme has a hydrolytic preference for β-1,4 glycosidic substrates,and can also hydrolyze cellobiose and lactose;5 mmol/L Fe^(3+),Fe^(2+)Cr^(2+),Ca^(2+),Mn^(2+),EDTA,and 1%SDS have completely inhibited its activity.The enzyme can resist the inhibition of the of this study was to obtain and characterize new β-glucosidases with excellent enzymatic properties from thermophilic microorganisms in order to solve the problems of low activity,insufficient stability,and susceptibility to product inhibition ofβ-glucosidase in industrial applications.A newβ-glucosidase gene,bglY,was extracted from the genome of Thermoanaerobacterium thermosaccharolyticum and the recombinant expression vector pET22 b-bglY was constructed by codon optimization,gene synthesis,and molecular cloning.The vector was then transform

关 键 词：热解糖高温厌氧杆菌 Β-葡萄糖苷酶 重组表达 酶学性质 

分 类 号：Q55[生物学—生物化学] Q78