Abstract: In this study, site-directed mutagenesis of non-conserved amino acid residues in the conserved regions of thermoacidophilic type III pullulan hydrolase TK-PUL was performed, and the effect of key amino acid residues in the conserved regions on its catalytic properties was determined by comparing the enzymatic properties of TK-PUL with those of its mutants. The I500W mutation in the conserved sequence region II of TK-PUL did not affect the optimum pH, pH stability, optimum temperature or thermal stability of TK-PUL, but significantly decreased the α-amylase and pullulanase activity. The kinetic parameters of TK-PUL and the mutant I500W TK-PUL were determined. The kcat/Km value of the mutant I500W for maltotriose was basically unchanged, and the kcat/Km value of the mutant I500W for isopanose was about 64.78% of TK-PUL. The results showed that Ile at residue 500 was important for the preference for α-1,4- and α-1,6-glycosidic linkages of TK-PUL. The I500W mutation did not affect the α-1,4-activity of TK-PUL, but significantly reduced the α-1,6-activity. This study is helpful for an in-depth understanding of the bifunctional catalytic mechanism of TK-PUL, and can also provide a theoretical basis and design ideas for the molecular modification of TK-PUL.

Key words: type III pullulan hydrolase; conserved regions; site-directed mutagenesis; catalytic activity; homology modelling