地衣芽胞杆菌α-淀粉酶酸性pH稳定性提升突变体的生化特征

摘要

The pH stability of Bacillus licheniformis α-amylase (BLA) has important value in its manufacturing by a fermentation process,especially at downstream recovery stage,improvement of recovery yield,easy storage conditions and extended shelf life of enzyme product.In the present studies,the cloning,sequence analysis and biochemical characterization of an α-amylase gene from B.licheniformis strain B186 1-1,a wild type isolate with BLA activity and improved pH stability were reported.In comparison with B.licheniformis ATCC 14580,four residues were different,including N4K,R134L,E243D and S320A.This BLA was then expressed in Escherichia coli and the recombinant enzyme was prepared and purified to SDS-PAGE-grade purity.The biochemical properties of the recombinant enzyme were investigated.The recombinant enzyme exhibited the highest activity at 80 ℃ and pH 6.0-7.5 and retained more than 80％ of the activity after incubating at pH 5.0 for 1 h,which is significantly improved compared with BLA from B.licheniformis ATCC14580 retaining only about 10％ of the enzyme activity.The 3D structure prediction and analysis were further performed and the relationship between the mutated amino acid residues and acidic pH stability was discussed.%地衣芽胞杆菌α-淀粉酶(Bacillus licheniformis α-amylase,BLA)的pH稳定性在提升发酵生产过程特别是后提取工段的操作稳定性,提高产品得率,弱化酶成品贮存条件和延长货架期等方面具有重要价值.该研究对1株能够合成更好pH稳定性BLA的地衣芽胞杆菌B186 1-1的BLA进行了分子克隆、序列分析及性质研究,与模式菌株ATCC14580来源的BLA氨基酸序列相比发现,有4个位点的差异,包括:N4K、R134L、E243D和S320A.进一步克隆表达与纯化了该菌株的BLA,重组酶在80℃、pH6.0～7.5下显示最高活性;在pH 5.0下放置1h,该酶保留80％以上酶活力,而来源于菌株ATCC14580的BLA在此相同条件下仅保留约10％酶活力;通过3D结构模拟分析显示BLA中的差异性碱基与其pH稳定性存在相关关系.