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

ABSTRACT: BACKGROUND:Bioethanol production from various starchy materials has received much attention in recent years. ?-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 ?-amylase (FSA) was identified and characterized from genomic analysis of a novel Flavobacteriaceae species. It is closely related with archaeal ?-amylases in the GH13_7 subfamily, but is evolutionary distant with other bacterial ?-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 ?-amylases. The recombinant ?-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.

SUBMITTER: Li C

PROVIDER: S-EPMC3922116 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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

Li Chunfang C Du Miaofen M Cheng Bin B Wang Lushan L Liu Xinqiang X Ma Cuiqing C Yang Chunyu C Xu Ping P

Biotechnology for biofuels 20140131 1

<h4>Background</h4>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.<h4>Results</h4>A novel Flavobacteriaceae Sinomicrobium α-amylase (FSA) was identified and characterized from genomic analysis of a novel Fl ...[more]

PMID: 24485248

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Project description:Background-amylases catalyze the endo-hydrolysis of -1,4-D-glycosidic bonds in starch into smaller moieties. While industrial processes are usually performed at harsh conditions, -amylases from mainly the bacteria, fungi and yeasts are preferred for their stabilities (thermal, pH and oxidative) and specificities (substrate and product). Microbial -amylases can be purified and characterized for industrial applications. While exploring novel enzymes with these properties in the nature is time-costly, the advancements in protein engineering techniques including rational design, directed evolution and others have privileged their modifications to exhibit industrially ideal traits. However, the commentary on the strategies and preferably mutated residues are lacking, hindering the design of new mutants especially for enhanced substrate specificity and oxidative stability. Thus, our review ensures wider accessibility of the previously reported experimental findings to facilitate the future engineering work.Survey methodology and objectivesA traditional review approach was taken to focus on the engineering of microbial -amylases to enhance industrially favoured characteristics. The action mechanisms of - and -amylases were compared to avoid any bias in the research background. This review aimed to discuss the advances in modifying microbial -amylases via protein engineering to achieve longer half-life in high temperature, improved resistance (acidic, alkaline and oxidative) and enhanced specificities (substrate and product). Captivating results were discussed in depth, including the extended half-life at 100C, pH 3.5 and 10, 1.8 M hydrogen peroxide as well as enhanced substrate (65.3%) and product (42.4%) specificities. These shed light to the future microbial -amylase engineering in achieving paramount biochemical traits ameliorations to apt in the industries.ConclusionsMicrobial -amylases can be tailored for specific industrial applications through protein engineering (rational design and directed evolution). While the critical mutation points are dependent on respective enzymes, formation of disulfide bridge between cysteine residues after mutations is crucial for elevated thermostability. Amino acids conversion to basic residues was reported for enhanced acidic resistance while hydrophobic interaction resulted from mutated hydrophobic residues in carbohydrate-binding module or surface-binding sites is pivotal for improved substrate specificity. Substitution of oxidation-prone methionine residues with non-polar residues increases the enzyme oxidative stability. Hence, this review provides conceptual advances for the future microbial -amylases designs to exhibit industrially significant characteristics. However, more attention is needed to enhance substrate specificity and oxidative stability since they are least reported.

Dataset Information

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

Publications

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

Similar Datasets

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