N端截短CBM41对枯草芽孢杆菌来源普鲁兰酶酶学性质的影响  

Effects of CBM41 N-terminal Truncation on the Enzymological Properties of the Pullulanase from Bacillus subtilis 168

作　　者：付巧 林啟兰 薛强 熊海容 王亚伟[1,2] FU Qiao;LIN Qi-lan;XUE Qiang;XIONG Hai-rong;WANG Ya-wei(College of Life Science,South-Central University for Nationalities,Wuhan 430074;School of Life Science and Technology,Wuhan Polytechnic University,Wuhan 430048)

机构地区：[1]中南民族大学生命科学学院,武汉430074 [2]武汉轻工大学生命科学与技术学院,武汉430048

出　　处：《生物技术通报》2022年第6期245-251,共7页Biotechnology Bulletin

基　　金：湖北省技术创新重大项目(2018ABA093)。

摘　　要：采用N端截短方式对Bacillus subtilis 168来源普鲁兰酶进行蛋白结构改造,构建不同形式的截短突变体,考察N端结构对酶学特性的影响。利用基因工程的手段分别删去CBM41结构域N端前2、4和6个氨基酸,获得突变体M1(ΔN2)、M2(ΔN4)和M3(ΔN6)。3种突变体最适反应温度(40-45℃)和最适pH(6.0)均与WT一致,WT、M1、M2和M3的T_(m)值分别为48.57℃、50.03℃、48.43℃和49.50℃,M1、M2和M3的比活力均高于野生型,是WT的1.18、1.60和2.44倍,WT、M1、M2和M3的K_(m)值分别为23.89、29.01、17.29和19.08 mg/mL。上述结果表明,通过截短普鲁兰酶N端氨基酸可获得性质得到改良的突变体,为提高该酶的热稳定性、比活力和底物结合能力提供新的方法和思路。Different N-terminal truncated variants were constructed using N-terminal truncation to modify the protein structure of Bacillus subtilis 168 pullulanase,the effects of truncated mutation on enzymatic properties were studied.Three variants,M1(ΔN2),M2(ΔN4)and M3(ΔN6),by deleting the first 2,4 and 6 residues from the N-terminus respectively,were cloned and expressed in Escherichia coli BL21 cells by genetic engineering method.The optimal temperature of three variants was 40-45℃,and the optimal pH was 6.0,which was consistent with the wild-type pullulanase(WT).T_(m) values of WT,M1,M2 and M3 were 48.57,50.03,48.43 and 49.50℃,respectively.The specific activity of M1,M2 and M3 were all higher than those of wild-type,increasing by 1.18,1.60 and 2.44 times,respectively.K_(m) values of WT,M1,M2,and M3 were 23.89,29.01,17.29 and 19.08 mg/mL,respectively.These results indicate that variants with the improved properties can be obtained by N-terminal truncation of pullulanase,which provides new methods and ideas for improving the thermal stability,specific activity and substrate binding capacity of this enzyme.

关 键 词：N端截短 普鲁兰酶 Bacillus subtilis168 酶学特性 

分 类 号：Q78[生物学—分子生物学]