Xu Huan, Feng Xiangyuan, Yang Qi, Zheng Ke, Yi Le, Duan Shengwen, Cheng Lifeng
Institute of Bast Fiber Crops, Chinese Academy of Agricultural Science, Changsha 410205, China.
Polymers (Basel). 2022 Jul 15;14(14):2878. doi: 10.3390/polym14142878.
In order to obtain a thermostable pectate lyase for ramie degumming, a rational design based on structural analysis was carried out on a novel pectate lyase (Pel419) derived from the DCE-01 for high-efficiency ramie degumming. A total of five potential amino acid sites were chosen to replace residues. Then, the mutant enzymes were subjected to the heterologous expressions in and their enzymatic characteristics were determined. The optimal reaction temperature for the five mutants kept consistent with that for the wild type. The enzyme activity and thermal stability of mutant V52A were significantly improved. Meanwhile, the weight loss rate obtained by V52A with the best enzymatic characteristics in the ramie degumming process at 50 °C is comparable with that obtained by commercial cotton-ramie processing pectinases, indicating that V52A was a potential industrial enzyme that could be applied to large-scale ramie degumming. In this study, the biological functions of conservative residues of Pel419 were preliminarily explored. The mutant V52A with both enzymatic activity and improved heat resistance was acquired, providing a superior material for developing enzyme preparations of ramie degumming, and rendering an effective method for the rational design aiming to improve the thermostability of pectate lyase.
为了获得用于苎麻脱胶的热稳定果胶酸裂解酶，基于结构分析对源自DCE - 01的新型果胶酸裂解酶（Pel419）进行了合理设计，以实现高效苎麻脱胶。总共选择了五个潜在的氨基酸位点来替换残基。然后，对突变酶进行异源表达并测定其酶学特性。五个突变体的最佳反应温度与野生型保持一致。突变体V52A的酶活性和热稳定性显著提高。同时，在50℃苎麻脱胶过程中具有最佳酶学特性的V52A所获得的失重率与商业棉麻加工果胶酶所获得的失重率相当，这表明V52A是一种可应用于大规模苎麻脱胶的潜在工业酶。在本研究中，初步探索了Pel419保守残基的生物学功能。获得了具有酶活性和耐热性提高的突变体V52A，为开发苎麻脱胶酶制剂提供了优良材料，并为旨在提高果胶酸裂解酶热稳定性的合理设计提供了有效方法。
