Abstract: Thermoacidophilic type III pullulan hydrolase TK-PUL can completely hydrolyze starch to starch sugar under liquefaction condition. It has great application potential in the ''one step liquefaction-saccharification'' starch syrup production process. In this study, error-prone PCR technology was used to improve the catalytic activity of TK-PUL. After two sequential error-prone PCR and high-throughput screening, the mutant L538D with improved catalytic activity was obtained. Compared with TK-PUL, the specific activity of L538D with soluble starch as substrate increased by 50%, the specific activity of L538D toward pullulan increased by 21%. And the k_cat/K_m value of L538D for soluble starch, pullulan increased by 44% and 27%, respectively. In other words, the hydrolytic activity of L538D toward α-1,4-glycosidic bond and α-1,6-glycosidic bond were significantly improved, and the hydrolytic activity of L538D toward α-1,4-glycosidic bond increased to a greater degree. Homology modeling showed that replacing Leu538 with Asp in TK-PUL may improve the flexibility of the loop structure including the catalytic site Glu538 by shortening the length of the side chain of amino acid residues and reducing the hydrophobicity of amino acid residues, thus improving the catalytic activity of the enzyme. The results demonstrated that Leu538 in TK-PUL is vital for its catalytic activity.