Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan, 030006, China.
Institute of Molecular Science, Shanxi University, Taiyuan, 030006, China.
Appl Microbiol Biotechnol. 2018 Nov;102(22):9647-9656. doi: 10.1007/s00253-018-9327-4. Epub 2018 Sep 3.
Escherichia coli phytase appA, which hydrolyzes phytate, has been widely applied as an important feed supplement, but its resistance to trypsin needs to be improved. Six putative solvent-accessible amino acid residues (K74, K75, K180, R181, K183, and K363), which could be easily attacked by trypsin, were selected to improve trypsin tolerance of Escherichia coli phytase appA. Inspection of the three-dimensional structure and computational design via hydrogen bond analysis, six optimal mutation sites of K74D/K75Q/K180N/R181N/K183S/K363N, which strengthened the hydrogen bonding, were performed to generate three mutants. Results showed that the most beneficial mutant appA-M6 had a specific activity of 3262 U/mg with molecular weight of approximately 52-55 kDa. Similar to appA-WT, the optimal pH (4.5) and temperature (60 °C) of appA-M6 were unchanged. Compared with appA-WT, appA-M6 showed a significant enhancement (p < 0.05) in resistance to trypsin and a 3.8 °C increase in melting temperature (T). We concluded that introduction of hydrogen bonds and N-glycosylation modification resulted in decreased enzyme flexibility and increased the enzyme stability against proteolysis and thermal denaturation. The mutant appA-M6 generated in this study could be applied for the large-scale commercial production of phytase and thus could benefit the food and feed industry.
大肠杆菌植酸酶 appA 可水解植酸，被广泛用作重要的饲料添加剂，但它对胰蛋白酶的抗性需要提高。选择了六个可能被胰蛋白酶轻易攻击的假定溶剂可及氨基酸残基（K74、K75、K180、R181、K183 和 K363），以提高大肠杆菌植酸酶 appA 的胰蛋白酶耐受性。通过氢键分析的三维结构检查和计算设计，选择了六个最佳突变位点 K74D/K75Q/K180N/R181N/K183S/K363N，以增强氢键，从而生成三个突变体。结果表明，最有利的突变体 appA-M6 的比活为 3262 U/mg，分子量约为 52-55 kDa。与 appA-WT 相似，appA-M6 的最适 pH（4.5）和温度（60°C）保持不变。与 appA-WT 相比，appA-M6 对胰蛋白酶的抗性显著提高（p<0.05），并且熔点（Tm）升高了 3.8°C。我们得出结论，引入氢键和 N-糖基化修饰导致酶的灵活性降低，增加了酶对蛋白水解和热变性的稳定性。本研究中产生的突变体 appA-M6 可用于植酸酶的大规模商业生产，从而有利于食品和饲料工业。
