Abstract

Thermoacidophilic pullulan hydrolase type III from Thermococcus kodakarensis (TK-PUL) completely hydrolyzes starch under liquefaction conditions. It has great application potential in the process of “one step liquefaction-saccharification” starch syrup production. We employed error-prone PCR to enable the directed evolution of TK-PUL in vitro. After two rounds of error-prone PCR and high-throughput screening, we obtained the L538D mutant TK-PUL with improved catalytic activity. Compared with TK-PUL, the specific activity of the L538D enzyme for soluble starch and pullulan increased by 50 and 21%, respectively. The k_cat/K_M value of the enzyme for soluble starch, pullulan, maltotriose, and isopanose increased by 44, 27, 84 and 37%, respectively. Overall, the hydrolytic activity of L538D TK-PUL for α-1,4-glycosidic and α-1,6-glycosidic bonds was significantly improved, to a greater extend for α-1,4-glycosidic bonds. Homology modeling showed that replacing Leu538 with Asp in TK-PUL might improve the flexibility of the loop structure including the Glu534 catalytic site by shortening the length of the side chain of amino acid residues and reducing their hydrophobicity, thus, improving the catalytic activity of the enzyme.