Close relationship of a novel Flavobacteriaceae α-amylase with archaeal α-amylases and good potentials for industrial applications

Background: Bioethanol production from various starchy materials has received much attention in recent years. alpha-Amylases are key enzymes in the bioconversion process of starchy biomass to biofuels, food or other products. The properties of thermostability, pH stability, and Ca-independency are important in the development of such fermentation process. Results: A novel Flavobacteriaceae Sinomicrobium alpha-amylase (FSA) was identified and characterized from genomic analysis of a novel Flavobacteriaceae species. It is closely related with archaeal alpha-amylases in the GH13_7 subfamily, but is evolutionary distant with other bacterial alpha-amylases. Based on the conserved sequence alignment and homology modeling, with minor variation, the Zn2+- and Ca2+-binding sites of FSA were predicated to be the same as those of the archaeal thermophilic alpha-amylases. The recombinant alpha-amylase was highly expressed and biochemically characterized. It showed optimum activity at pH 6.0, high enzyme stability at pH 6.0 to 11.0, but weak thermostability. A disulfide bond was introduced by site-directed mutagenesis in domain C and resulted in the apparent improvement of the enzyme activity at high temperature and broad pH range. Moreover, about 50% of the enzyme activity was detected under 100 degrees C condition, whereas no activity was observed for the wild type enzyme. Its thermostability was also enhanced to some extent, with the half-life time increasing from 25 to 55 minutes at 50 degrees C. In addition, after the introduction of the disulfide bond, the protein became a Ca-independent enzyme. Conclusions: The improved stability of FSA suggested that the domain C contributes to the overall stability of the enzyme under extreme conditions. In addition, successfully directed modification and special evolutionary status of FSA imply its directional reconstruction potentials for bioethanol production, as well as for other industrial applications.