Prabmark Kanoknart, Boonyapakron Katewadee, Bunterngsook Benjarat, Arunrattanamook Nattapol, Uengwetwanit Tanaporn, Chitnumsub Penchit, Champreda Verawat
Enzyme Technology Research Team, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, 113 Thailand Science Park, Phahonyothin Road, Khlong Luang, Pathum Thani, 12120 Thailand.
Microarray Research Team, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, 113 Thailand Science Park, Phahonyothin Road, Khlong Luang, Pathum Thani, 12120 Thailand.
3 Biotech. 2022 Oct;12(10):269. doi: 10.1007/s13205-022-03339-4. Epub 2022 Sep 9.
Alkaline cellobiohydrolases have the potential for application in various industries, including pulp processing and laundry where operation under high pH conditions is preferred. In this study, variants of Cel6A cellobiohydrolase from were generated by structural-based protein engineering with the rationale of increasing catalytic activity and alkaline stability. The variants included removal of the carbohydrate-binding module (CBM) and substitution of residues 173 and 200. The CBM-deleted enzyme with Y200F mutation predicted to mediate conformational change at the N-terminal loop demonstrated increased alkaline stability at 60 °C, pH 8.0 for 24 h up to 2.25-fold compared with the wild-type enzyme. Another CBM-deleted enzyme with L173E mutation predicted to induce a new hydrogen bond in the substrate-binding cleft showed enhanced hydrolysis yield of pretreated sugarcane trash up to 4.65-fold greater than that of the wild-type enzyme at the pH 8.0. The variant enzymes could thus be developed for applications on cellulose hydrolysis and plant fiber modification operated under alkaline conditions.
The online version contains supplementary material available at 10.1007/s13205-022-03339-4.
碱性纤维二糖水解酶有潜力应用于各种行业，包括纸浆加工和洗衣业，在这些行业中，在高pH条件下操作更为可取。在本研究中，通过基于结构的蛋白质工程技术，以提高催化活性和碱性稳定性为目的，对来自[具体来源未提及]的Cel6A纤维二糖水解酶进行了变体改造。这些变体包括去除碳水化合物结合模块（CBM）以及替换173位和200位残基。预测Y200F突变的CBM缺失酶可介导N端环构象变化，在60°C、pH 8.0条件下24小时的碱性稳定性比野生型酶提高了2.25倍。另一种预测L173E突变的CBM缺失酶可在底物结合裂隙中诱导形成新的氢键，在pH 8.0时，预处理甘蔗渣的水解产率比野生型酶提高了4.65倍。因此，这些变体酶可用于碱性条件下的纤维素水解和植物纤维改性应用。
在线版本包含可在10.1007/s13205-022-03339-4获取的补充材料。
