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

Abstract

BackgroundBioethanol production from various starchy materials has received much attention in recent years. α-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.ResultsA novel Flavobacteriaceae Sinomicrobium α-amylase (FSA) was identified and characterized from genomic analysis of a novel Flavobacteriaceae species. It is closely related with archaeal α-amylases in the GH13_7 subfamily, but is evolutionary distant with other bacterial α-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 α-amylases. The recombinant α-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°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°C. In addition, after the introduction of the disulfide bond, the protein became a Ca-independent enzyme.ConclusionsThe 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.

Protein engineering of selected residues from conserved sequence regions of a novel Anoxybacillus α-amylase

“…While the former position is well-conserved as an aromatic one throughout the entire α-amylase family GH13, it is mostly occupied by phenylalanine or tryptophan2324 and the presence of the tyrosine (Figure 2) here in ASKA seems to be unique for the GH13 subfamily11. Because tryptophan is highly characteristic of the GH13 subfamilies 6 and 7, which include plants, archaeons and flavobacteria232425, phenylalanine, which is found in the GH13 subfamilies 15 and 24 (animals) as well as 27, 28, 32 and 36 (various groups of bacteria), was chosen as the substitute for Tyr-187 in the mutant. The latter mutant position in CSR-II, Leu-189, is a conserved position, but if it is replaced then isoleucine (Figure 2) is often found2324.…”

Exploration of extremophiles for high temperature biotechnological processes

“…Engineered FSADSK retained 50% of activity at 100 8C and a 30 min extended half-life at elevated temperatures [12]. Finally, the detailed characterization of another calcium-independent aamylase (Gt-amyII) from G. thermoleovorans revealed that this thermozyme is even capable of hydrolyzing raw starches at industrial sub-gelatinisation conditions [13 ].…”

Sinomicrobium pectinilyticum sp. nov., a pectinase-producing bacterium isolated from alkaline and saline soil, and emended description of the genus Sinomicrobium

“…In contrast to S. oceani SCSIO 03483 T , strain 5DNS001 T was able to hydrolyse pectin and starch in MA and produce novel pectinase and a-amylase (Li et al, 2014).…”