Site-Directed Mutagenesis of Klebsiella variicola Pullulanase for the Improvement of Its Acid Resistance

Abstract:

By analyzing the active site of Klebsiella variicola SHN-1 pullulanase and alignment of the amino acid sequences of the pullulanases from K. pneumoniae，Bacillus acidopullulyticus and B.naganoensis，eight site-directed mutagenesis including H626D，R756E，H852D，R908E，I619N，F723Y，S850T，and K851S were subjected to improve the acid resistance of K. variicola pullulanase. After analysis of enzymatic properties，the optimum pH of H852D was shifted from 5.0 to 4.7，and its stability was obviously improved when stored at pH 4.5. By the analysis of homology modeling and structure of the pullulanase，the formation of two hydrogen bonds by single-site substitution was supposed to be responsible for the improvement of stability at low pH，and the change of pKa value in the active center of pullulanase caused the optimum pH migration.