Structure-guided systems-level engineering of oxidation-prone methionine residues in catalytic domain of an alkaline α-amylase from Alkalimonas amylolytica for significant improvement of both oxidative stability and catalytic efficiency.
Structure-guided systems-level engineering of oxidation-prone methionine residues in catalytic domain of an alkaline α-amylase from Alkalimonas amylolytica for significant improvement of both oxidative stability and catalytic efficiency.
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DOI：
10.1371/journal.pone.0057403
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Chen J
中科院分区：
文献类型：
--
作者：
Yang H;Liu L;Shin HD;Li J;Du G;Chen J
在洗涤剂工业中，碱性α-淀粉酶必须具有较高的氧化稳定性和催化效率，才能在苛刻的条件下保持酶的活性。在这项工作中，我们试图显着提高氧化稳定性和催化效率的碱性α-淀粉酶从Alkalimonas amylolytica通过工程的催化结构域周围的五个氧化倾向的蛋氨酸残基通过系统的方法。具体地，基于三级结构分析，五种甲硫氨酸（Met 145、Met 214、Met 229、Met 247和Met 317）分别被抗氧化的苏氨酸、异亮氨酸和丙氨酸单独取代。在所产生的15个突变体中，7个突变体M145 A、M145 I、M214 A、M229 A、M229 T、M247 T和M317 I显示出显著增强的氧化稳定性或催化效率。在以前的工作中，我们发现用亮氨酸取代M247可以显著提高氧化稳定性。因此，这8个阳性突变体（M145 A、M145 I、M214 A、M229 A、M229 T、M247 T、M247 L和M317 I）用于进行第二轮组合突变。在构建的85个突变体（25个两点突变体、36个三点突变体、16个四点突变体和8个五点突变体）中，突变体M145 I-214 A-229 T-247 T-317 I显示氧化稳定性增加5.4倍，催化效率增加3.0倍。有趣的是，该突变体的比活性、碱稳定性和热稳定性也有所提高。野生型碱性α-淀粉酶及其突变体M145 I-214 A-229 T-247 T-317 I的三维结构模型表明，催化结构域周围盐桥和氢键的增加有助于催化效率和稳定性的显著提高。突变体M145 I-214 A-229 T-247 T-317 I的氧化稳定性和催化效率显著提高，具有作为洗涤剂添加剂的巨大潜力，这种结构导向的系统工程策略可用于其他微生物酶的蛋白质工程以满足工业需求。
High oxidative stability and catalytic efficiency are required for the alkaline α-amylases to keep the enzymatic performance under the harsh conditions in detergent industries. In this work, we attempted to significantly improve both the oxidative stability and catalytic efficiency of an alkaline α-amylase from Alkalimonas amylolytica by engineering the five oxidation-prone methionine residues around the catalytic domain via a systematic approach. Specifically, based on the tertiary structure analysis, five methionines (Met 145, Met 214, Met 229, Met 247 and Met 317) were individually substituted with oxidation-resistant threonine, isoleucine and alaline, respectively. Among the created 15 mutants, 7 mutants M145A, M145I, M214A, M229A, M229T, M247T and M317I showed significantly enhanced oxidative stability or catalytic efficiency. In previous work, we found that the replacement of M247 with leucine could significantly improve the oxidative stability. Thus, these 8 positive mutants (M145A, M145I, M214A, M229A, M229T, M247T, M247L and M317I) were used to conduct the second round of combinational mutations. Among the constructed 85 mutants (25 two-point mutants, 36 three-point mutants, 16 four-point mutants and 8 five-point mutants), the mutant M145I-214A-229T-247T-317I showed a 5.4-fold increase in oxidative stability and a 3.0-fold increase in catalytic efficiency. Interestingly, the specific activity, alkaline stability and thermal stability of this mutant were also increased. The increase of salt bridge and hydrogen bonds around the catalytic domain contributed to the significantly improved catalytic efficiency and stability, as revealed by the three-dimensional structure model of wild-type alkaline α-amylase and its mutant M145I-214A-229T-247T-317I. With the significantly improved oxidative stability and catalytic efficiency, the mutant M145I-214A-229T-247T-317I has a great potential as a detergent additive, and this structure-guided systems engineering strategy may be useful for the protein engineering of the other microbial enzymes to fulfill industrial requirements.
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