Carbohydrate Enzyme Biotechnology Laboratory, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India; DBT PAN-IIT Center for Bioenergy, Indian Institute of Technology Guwahati, India.
Carbohydrate Enzyme Biotechnology Laboratory, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India.
Bioresour Technol. 2019 Jun;282:494-501. doi: 10.1016/j.biortech.2019.03.051. Epub 2019 Mar 12.
Site-directed mutagenesis of β-1,4-endoglucanase from family 5 glycoside hydrolase (CtGH5) from Clostridium thermocellum was performed to develop a mutant CtGH5-F194A that gave 40 U/mg specific activity against carboxymethyl cellulose, resulting 2-fold higher activity than wild-type CtGH5. CtGH5-F194A was fused with a β-1,4-glucosidase, CtGH1 from Clostridium thermocellum to develop a chimeric enzyme. The chimera (CtGH1-L1-CtGH5-F194A) expressed as a soluble protein using E. coli BL-21cells displaying 3- to 5-fold higher catalytic efficiency for endoglucanase and β-glucosidase activities. TLC analysis of hydrolysed product of CMC by chimera 1 revealed glucose as final product confirming both β-1,4-endoglucanase and β-1,4-glucosidase activities, while the products of CtGH5-F194A were cellobiose and cello-oligosaccharides. Protein melting studies of CtGH5-F194A showed melting temperature (T), 68 °C and of CtGH1, 79 °C, whereas, chimera showed 78 °C. The improved structural integrity, thermostability and enhanced bi-functional enzyme activities of chimera makes it potentially useful for industrial application in converting biomass to glucose and thus bioethanol.
对来自梭菌属（Clostridium thermocellum）的糖苷水解酶家族 5 中的β-1,4-内切葡聚糖酶（CtGH5）进行了定点突变，得到了突变体 CtGH5-F194A，其对羧甲基纤维素的比活性为 40 U/mg，比野生型 CtGH5 高 2 倍。CtGH5-F194A 与来自梭菌属（Clostridium thermocellum）的β-1,4-葡萄糖苷酶 CtGH1 融合，开发出一种嵌合酶。该嵌合体（CtGH1-L1-CtGH5-F194A）在大肠杆菌 BL-21 细胞中表达为可溶性蛋白，其内切葡聚糖酶和β-葡萄糖苷酶活性的催化效率提高了 3-5 倍。TLC 分析表明，该嵌合体水解 CMC 的产物为葡萄糖，证实了其同时具有β-1,4-内切葡聚糖酶和β-1,4-葡萄糖苷酶活性，而 CtGH5-F194A 的产物为纤维二糖和纤维寡糖。CtGH5-F194A 的蛋白质熔融研究表明，其熔融温度（T）为 68°C，CtGH1 为 79°C，而嵌合体则为 78°C。该嵌合体具有更好的结构完整性、热稳定性和增强的双功能酶活性，使其在将生物质转化为葡萄糖从而生产生物乙醇方面具有潜在的工业应用价值。
