Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (6): 245-251.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0927

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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 Online:2022-06-26 Published:2022-07-11
  • Contact: WANG Ya-wei E-mail:1196840811@qq.com;253182556@qq.com

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