生物技术通报 ›› 2022, Vol. 38 ›› Issue (6): 245-251.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0927

• 研究报告 • 上一篇    下一篇

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

付巧1(), 林啟兰1, 薛强1, 熊海容1, 王亚伟1,2()   

  1. 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
  • 收稿日期:2021-07-17 出版日期:2022-06-26 发布日期:2022-07-11
  • 作者简介:付巧,女,硕士研究生,研究方向:微生物酶工程;E-mail: 1196840811@qq.com
  • 基金资助:
    湖北省技术创新重大项目(2018ABA093)

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

FU Qiao1(), LIN Qi-lan1, XUE Qiang1, XIONG Hai-rong1, WANG Ya-wei1,2()   

  1. 1. College of Life Science,South-Central University for Nationalities,Wuhan 430074
    2. School of Life Science and Technology,Wuhan Polytechnic University,Wuhan 430048
  • Received:2021-07-17 Published:2022-06-26 Online:2022-07-11

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Tm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Km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。

关键词: N端截短, 普鲁兰酶, Bacillus subtilis 168, 酶学特性

Abstract:

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). Tm 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. Km 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.

Key words: N-terminal truncation, pullulanase, Bacillus subtilis 168, enzymatic property