普鲁兰酶N467G突变体的酶学性质分析  

Enzymatic characterization of the pullulanase mutant N467G

作　　者：张亚楠 申培立 牛丹丹 田康明 KUGEN PERMAUL SUREN SINGH 王正祥 ZHANG Yanan;SHEN Peili;NIU Dandan;TIAN Kangming;KUGEN PERMAUL;SUREN SINGH;WANG Zhengxiang(College of Chemical Engineering and Materials Science,Tianjin University of Science and Technology,Tianjin 300457,China;College of Biotechnology,Tianjin University of Science and Technology,Tianjin 300457,China;Department of Biotechnology and Food Technology,Faculty of Applied Sciences,Durban University of Technology,Durban,4001,South Africa)

机构地区：[1]天津科技大学化工与材料学院,天津300457 [2]天津科技大学生物工程学院,天津300457 [3]Department of Biotechnology and Food Technology,Faculty of Applied Sciences,Durban University of Technology,Durban,4001,South Africa

出　　处：《食品与发酵工业》2021年第5期7-11,共5页Food and Fermentation Industries

基　　金：国家重点研发计划政府间国际科技创新合作重点专项项目(2018YFE0100400);天津市高等学校创新团队建设规划项目(TD13-5009)。

摘　　要：在淀粉制糖工业中,普鲁兰酶通常与糖化酶配合使用,其在酸性pH和较高温度下的催化活力是影响淀粉脱支效率的主要因素。该研究通过对长野芽胞杆菌(Bacillus naganoensis)普鲁兰酶蛋白质解折叠自由能的差值(ΔΔG)的计算预测和突变位点稳定性分析,选择突变位点并进行定点突变,获得突变体N467G。与野生型普鲁兰酶分子相比,突变体N467G的最适作用温度提高至60℃,最适作用pH降低至4.5;其在60℃条件下孵育2.5 h或在pH 3.5~5.5孵育1 h残留酶活力均保持在80%以上,稳定性明显提高。此突变体的获得为其后续高效表达与工业化应用奠定了重要基础。Pullulanase is extensively used in preparation of glucose syrup from starch,combined with glucoamylase.However,its catalytic activity at low pH and high temperature is the main factor affecting its efficiency during starch debranching.In this study,the difference of the protein unfolding free energy(ΔΔG)of Bacillus naganoensis pullulanase(PulA)mutants was calculated and the stability of each mutation site was analyzed.A mutant,N467G,was obtained through site-directed mutagenesis of the Asn467 residue of PulA and its biochemical properties were examined.Compared to the wild-type PulA,the optimal temperature of mutant N467G was increased by 5℃to 60℃and the optimal pH was reduced by 0.5 pH value to 4.5.Its relative activity was over 80%after incubating the enzyme at 60℃for 2.5 h or at pH 4.0-5.0 for 1 h,which represented a significantly higher stability.The result lays a foundation for the subsequent high-efficiency protein production and industrial application of this mutant.

关 键 词：普鲁兰酶 定向进化 最适作用pH 最适作用温度 

分 类 号：TS241[轻工技术与工程—制糖工程]