摘要
从海洋细菌假交替单胞菌(Pseudoalteromonas sp.)DL-6中克隆β-N-乙酰己糖胺酶基因(PsHex),扩增至pET-28a(+),并成功在大肠杆菌BL21(DE3)中异源表达,经镍-次氮基三乙酸亲和层析纯化得到纯蛋白。结果表明:PsHex编码876个氨基酸,分子质量约99.46 kDa,理论等电点5.65,序列对比分析表明,Ps Hex属于糖苷水解酶GH20家族;Ps Hex蛋白最适温度30℃,最适pH 8.5,比活力11.30 U/mg,且在20℃、pH 7.5时稳定性最高;重组蛋白可降解琼脂、葡聚糖、可溶性淀粉、卡拉胶等多种底物;Na^(+)、Mg^(2+)、Fe^(3+)、K^(+)、Ca^(2+)、二硫苏糖醇、吐温-80、乙二胺四乙酸、β-巯基乙醇对酶有激活作用;Ps Hex以对硝基苯基-β-D-乙酰氨基葡萄糖苷为底物的米氏常数(K_(m))为2.523 mol/L、转化效率(K_(cat))为79.035 min^(-1)、最大反应速率(v_(max))为2.081 U/mg、K_(cat)/K_(m)=31.326。该研究为酶法降解几丁质产生N-乙酰葡萄糖胺的生产应用提供了理论参考。
The gene(PsHex)encodingβ-N-acetylglucosaminidase from the marine bacterium Pseudoalteromonas sp.DL-6 was cloned into the pET-28a(+)vector and then successfully heterologously expressed in Escherichia coli BL21(DE3).The expressed protein was purified through nickel-nitrilotriacetic acid affinity chromatography.The results showed that PsHex encoded 876 amino acids,and the predicted molecular mass and pI were 99.46 kD and 5.65,respectively.Phylogenetic analysis and multiple sequence alignments showed that PsHex belonged to the glycosyl hydrolase 20(GH20)family.The optimal temperature and pH for the recombinant enzyme were 30℃and 8.5,respectively,its specific activity was 11.30 U/mg and it displayed the highest stability at 20℃and pH 7.5.This enzyme could degrade various substrates,including agarose,chitosan,soluble starch and carrageenan and could be activated by metal ions(Na+,Mg^(2+),Fe^(3+),K^(+),and Ca^(2+)),DL-dithiothreitol,tween-80,ethylenediaminetetraacetic acid andβ-mercaptoethanol.The Michaelis constant(K_(m)),catalytic rate constant(K_(cat)),maximum reaction rate(vmax)and K_(cat)/K_(m) values toward the substrate 4-nitrophenyl 2-acetamido-2-deoxy-β-d-glucopyranoside were 2.523 mol/L,79.035 min-1,2.081 U/mg and 31.326,respectively.This study provides a theoretical reference for the enzymatic degradation of chitin to produce N-acetylglucosamine.
作者
刘向歌
赵子琪
吴家葳
于爽
迟乃玉
王晓辉
LIU Xiangge;ZHAO Ziqi;WU Jiawei;YU Shuang;CHI Naiyu;WANG Xiaohui(College of Life and Health,Dalian University,Dalian 116622,China;Liaoning Marine Microbiology Engineering Technology Research Center,Dalian 116622,China;College of Medicine,Dalian University,Dalian 116000,China;Beijing Biocytogen Co.Ltd.,Beijing 102206,China)
出处
《食品科学》
EI
CAS
CSCD
北大核心
2024年第16期68-76,共9页
Food Science
基金
中央引导地方科技发展资金项目(110013)
国家自然科学基金青年科学基金项目(31500039)
大连市青年科技之星项目(2017RQ155)。
