Biochemical and synergistic properties of a novel alpha‐amylase from Chinese nong‐flavor Daqu

Abstract

Abstract Background Daqu is the most important fermentation starter for Chinese liquor, with large number of microbes and enzymes being openly enriched in the Daqu system over thousands of years. However, only a few enzymes have been analyzed with crude protein for total liquefying power and saccharifying power of Daqu. Therefore, the complex enzymatic system present in Daqu has not been completely characterized. Moreover, their pivotal and complicated functions in Daqu are completely unknown. Results In this study, a novel α-amylase NFAmy13B, from GH13_5 subfamily (according to the Carbohydrate-Active enZYmes Database, CAZy) was successfully heterologous expressed by Escherichia coli from Chinese Nong-flavor (NF) Daqu. It exhibited high stability ranging from pH 5.5 to 12.5, and higher specific activity, compared to other GH13_5 fungal α-amylases. Moreover, NFAmy13B did not show activity loss and retained 96% residual activity after pre-incubation at pH 11 for 21 h and pH 12 for 10 h, respectively. Additionally, 1.25 mM Ca2+ significantly improved its thermostability. NFAmy13B showed a synergistic effect on degrading wheat starch with NFAmy13A (GH13_1), another α-amylase from Daqu. Both enzymes could cleave maltotetraose and maltopentaose in same degradation pattern, and only NFAmy13A could efficiently degrade maltotriose. Moreover, NFAmy13B showed higher catalytic efficiency on long-chain starch, while NFAmy13A had higher catalytic efficiency on short-chain maltooligosaccharides. Their different catalytic efficiencies on starch and maltooligosaccharides may be caused by their discrepant substrate-binding region. Conclusions This study mined a novel GH13_5 fungal α-amylase (NFAmy13B) with outstanding alkali resistance from Nong-flavor (NF) Daqu. Furthermore, its synergistic effect with NFAmy13A (GH13_1) on hydrolyzing wheat starch was confirmed, and their possible contribution in NF Daqu was also speculated. Thus, we not only provide a candidate α-amylase for industry, but also a useful strategy for further studying the interactions in the complex enzyme system of Daqu.