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

Biotechnology for Biofuels

Chunfang Li²  Miaofen Du²  Bin Cheng²  Lushan Wang²  Xinqiang Liu²  Cuiqing Ma²  Chunyu Yang²  Ping Xu¹ 

[1] State Key Laboratory of Microbial Metabolism & School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China

[2] State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People’s Republic of China

关键词: Domain C;    Thermostability;    Site-directed mutagenesis;    Evolutionary position;    α-Amylases;   

Others  :  793778
DOI  :  10.1186/1754-6834-7-18

 received in 2013-09-02, accepted in 2014-01-21,  发布年份 2014

【 摘 要 】

Background

Bioethanol 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.

Results

A 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.

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.

【 授权许可】

   
2014 Li et al.; licensee BioMed Central Ltd.

【 图 表 】

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