Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China.
Appl Environ Microbiol. 2012 Nov;78(21):7519-26. doi: 10.1128/AEM.01307-12. Epub 2012 Aug 3.
This work aims to improve the oxidative stability of alkaline amylase from Alkalimonas amylolytica through structure-based site-directed mutagenesis. Based on an analysis of the tertiary structure, five methionines (Met 145, Met 214, Met 229, Met 247, and Met 317) were selected as the mutation sites and individually replaced with leucine. In the presence of 500 mM H(2)O(2) at 35°C for 5 h, the wild-type enzyme and the M145L, M214L, M229L, M247L, and M317L mutants retained 10%, 28%, 46%, 28%, 72%, and 43% of the original activity, respectively. Concomitantly, the alkaline stability, thermal stability, and catalytic efficiency of the M247L mutant were also improved. The pH stability of the mutants (M145L, M214L, M229L, and M317L) remained unchanged compared to that of the wild-type enzyme, while the stable pH range of the M247L mutant was extended from pH 7.0 to 11.0 for the wild type to pH 6.0 to 12.0 for the mutant. The wild-type enzyme lost its activity after incubation at 50°C for 2 h, and the M145L, M214L, M229L, and M317L mutants retained less than 14% of the activity, whereas the M247L mutant retained 34% of the activity under the same conditions. Compared to the wild-type enzyme, the k(cat) values of the M145L, M214L, M229L, and M317L mutants decreased, while that of the M247L mutant increased slightly from 5.0 × 10(4) to 5.6 × 10(4) min(-1). The mechanism responsible for the increased oxidative stability, alkaline stability, thermal stability, and catalytic efficiency of the M247L mutant was further analyzed with a structure model. The combinational mutants were also constructed, and their biochemical properties were characterized. The resistance of the wild-type enzyme and the mutants to surfactants and detergents was also investigated. Our results indicate that the M247L mutant has great potential in the detergent and textile industries.
本工作旨在通过基于结构的定点突变来提高来自 Alkalimonas amylolytica 的碱性淀粉酶的氧化稳定性。基于对三级结构的分析，选择了五个蛋氨酸（Met145、Met214、Met229、Met247 和 Met317）作为突变位点，并分别将其突变为亮氨酸。在 35°C 下用 500mM H2O2 处理 5 小时后，野生型酶和 M145L、M214L、M229L、M247L 和 M317L 突变体分别保留了原始活性的 10%、28%、46%、28%、72%和 43%。同时，M247L 突变体的碱性稳定性、热稳定性和催化效率也得到了提高。与野生型酶相比，突变体（M145L、M214L、M229L 和 M317L）的 pH 稳定性保持不变，而 M247L 突变体的稳定 pH 范围从野生型的 7.0 到 11.0 扩展到突变体的 6.0 到 12.0。野生型酶在 50°C 孵育 2 小时后失去活性，而 M145L、M214L、M229L 和 M317L 突变体保留的活性不足 14%，而 M247L 突变体在相同条件下保留了 34%的活性。与野生型酶相比，M145L、M214L、M229L 和 M317L 突变体的 kcat 值降低，而 M247L 突变体的 kcat 值略有增加，从 5.0×104 增加到 5.6×104 min-1。进一步利用结构模型分析了 M247L 突变体氧化稳定性、碱性稳定性、热稳定性和催化效率提高的机制。还构建了组合突变体，并对其生化特性进行了表征。还研究了野生型酶和突变体对表面活性剂和洗涤剂的抗性。我们的结果表明，M247L 突变体在洗涤剂和纺织工业中有很大的应用潜力。
