Laboratory of Applied Biocatalysis, School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, Guangdong, China.
South China Institute of Collabrative Innovation, Xincheng Road, Songshan Lake, Dongguan 523808, China.
Int J Biol Macromol. 2020 May 1;150:9-15. doi: 10.1016/j.ijbiomac.2020.02.033. Epub 2020 Feb 6.
Chitinase is a promising biocatalyst for chitin biotransformation in the field of recalcitrant biomass degradation. Excellent catalytic performance is conducive to its commercial utilization. In this work, sequence- and structure-based semi-rational design was performed to evolve the thermostability and activity of a previously identified chitinase PpChi1 from Paenibacillus pasadenensis CS0611. After combinational mutagenesis, the mutant S244C-I319C/T259P with disulfide bond introduction and proline substitution exhibited higher specific activity at higher temperature, 26.3-fold in half-life value at 50 °C, and a 7.9 °C rise in half-inactivation temperature T compared to the wild-type enzyme. The optimal reaction temperature of the mutant was shifted from 45 °C to 52.5 °C. Molecular dynamic simulation and structure analysis confirmed that these improvements of the mutant were attributed to its stabilized folding form, possibly caused by the decreased entropy of unfolding. This work gives an initial insight into the effect of conserved proline residues in thermostable chitinases and proposes a feasible approach for improving chitinase thermostability to facilitate its application in chitin hydrolysis to valuable oligosaccharides.
几丁质酶是一种很有前途的生物催化剂，可用于难降解生物质领域的几丁质生物转化。优异的催化性能有利于其商业化利用。在这项工作中，我们进行了基于序列和结构的半理性设计，以提高先前鉴定的来自地衣芽孢杆菌 CS0611 的几丁质酶 PpChi1 的热稳定性和活性。经过组合诱变，引入二硫键和脯氨酸取代的突变体 S244C-I319C/T259P 在高温下表现出更高的比活性，在 50°C 时半衰期值提高了 26.3 倍，半衰期 T 提高了 7.9°C。与野生型酶相比。突变体的最佳反应温度从 45°C 转移到 52.5°C。分子动力学模拟和结构分析证实，这些改进归因于突变体折叠形式的稳定化，可能是由于展开熵的降低所致。这项工作初步探讨了耐热几丁质酶中保守脯氨酸残基的作用，并提出了一种提高几丁质酶耐热性的可行方法，以促进其在几丁质水解为有价值的寡糖中的应用。
