Priyadharshini Ramachandran, Gunasekaran Paramasamy
Department of Genetics, Centre for Excellence in Genomic Sciences, School of Biological Sciences, Madurai Kamaraj University, Madurai, 625 021, India.
Biotechnol Lett. 2007 Oct;29(10):1493-9. doi: 10.1007/s10529-007-9428-0. Epub 2007 Jun 28.
Amylases that are active under acidic conditions (pH <6), at higher temperatures (>70 degrees C) and have less reliance on Ca(2+) are required for starch hydrolysis. The alpha-amylase gene of Bacillus licheniformis MTCC 6598 was cloned and expressed in Escherichia coli BL21. The calcium-binding site spanning amino acid residues from 104 to 200 in the loop regions of domain B and D430 in domain C of amylase were changed by site-directed mutagenesis and the resultant mutant amylases were analyzed. Calcium-binding residues, N104, D161, D183, D200 and D430, were replaced with D104 and N161, N183, N200 and N430, respectively. Mutant amylase with N104D had a slightly decreased activity at 30 degrees C but a significantly improved specific activity at pH 5 and 70 degrees C, which is desirable character for a food enzyme. The amylase mutants with D183N or D200N lost all activity while the mutant amylase with D161N retained its activity at 30 degrees C but had significantly less activity at 70 degrees C. On the other hand, the activity of the mutant amylase with D430N was not changed at 30 degrees C but had an improved activity at 70 degrees C.
淀粉水解需要在酸性条件（pH <6）、较高温度（>70摄氏度）下具有活性且对Ca(2+)依赖性较小的淀粉酶。地衣芽孢杆菌MTCC 6598的α-淀粉酶基因被克隆并在大肠杆菌BL21中表达。通过定点诱变改变淀粉酶结构域B的环区中跨越104至200位氨基酸残基的钙结合位点以及结构域C中的D430，并对所得的突变淀粉酶进行分析。钙结合残基N104、D161、D183、D200和D430分别被替换为D104和N161、N183、N200和N430。具有N104D的突变淀粉酶在30摄氏度时活性略有下降，但在pH 5和70摄氏度时比活性显著提高，这是食品酶所需的特性。具有D183N或D200N的淀粉酶突变体失去了所有活性，而具有D161N的突变淀粉酶在30摄氏度时保留了其活性，但在70摄氏度时活性显著降低。另一方面，具有D430N的突变淀粉酶在30摄氏度时活性未改变，但在70摄氏度时活性有所提高。
