红曲黄酒来源芽孢杆菌普鲁兰酶基因克隆和生物信息学分析

红曲黄酒酿造原料为大米，淀粉质量分数在70%以上，微生物来源淀粉酶系对淀粉的水解至关重要，影响甚至决定红曲黄酒酿造的原料转化过程和产品品质。从酒曲中分离具有淀粉水解能力的芽孢杆菌2?株，编号BHQ03和BHQ06，进行分子生物学鉴定并分别从2?株菌中克隆获得pulL1和pulL2，pulL3和pulL4共4?个I型普鲁兰酶基因，其中基因pulL1和pulL3均编码713?个氨基酸，序列相似度96.31%，基因pulL2和pulL4均编码852?个氨基酸，序列相似度99.77%。对基因pulL1和pulL2编码蛋白PulL1和PulL2进一步分析发现，二者均属于G13家族，具有4?段特征性保守区域。PulL2的N-端含有32 个氨基酸残基的信号肽序列，且催化活性位点存在突变（D407G）。结合文献研究和三维结构模拟，分析普鲁兰酶的具体催化过程。上述研究为科学解析红曲黄酒酿造的淀粉水解过程提供参考。

Cloning and Bioinformatic Analysis of the Pullulanase Genes of Bacillus sp. Originated from Chinese Hongqu Glutinous Rice Wine

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.