Abstract: Microbial amylases play a crucial role in the hydrolysis of rice starch (the starch content of rice is more than 70%) during the brewing of Hongqu glutinous rice wine, which influences or even determines the conversion of the raw material and therefore the product quality of Chinese Hongqu glutinous rice wine. Two Bacillus strains able to hydrolyze starch, numbered BHQ03 and BHQ06, were isolated from a fermentation starter for Hongqu glutinous rice wine. The strains were identified using molecular biological methods, and 4 type I pullulanase encoding genes, namely pulL1 and pulL2, pulL3 and pulL4, were cloned from each strain. pulL1 and pulL3 each encoded 713 amino acids and showed a sequence similarity of 96.31%, while pulL2 and pulL4 each encoded 852 amino acids and had a sequence similarity of 99.77%. The proteins PulL1 and PulL2 both belonged to the G13 subfamily, and had 4 characteristic conservative regions. PulL2 contained a signal peptide of 32 amino acids at the N-terminal, and exhibited a mutation at the catalytic site (D407G). Based on previous studies combined with 3D structure homologous modeling, the catalytic process of the pullulanase was analyzed. The above results provide a basis for scientific understanding of the hydrolysis process of starch during the brewing of Chinese Hongqu glutinous rice wine.

Key words: Hongqu glutinous rice wine, Bacillus, starch hydrolysis, pullulanase, bioinformatic analysis