Telke, Amar A. Zhuang, Ningning Ghatge, Sunil S. Lee, Sook-Hee Shah, Asad Ali Khan, Haji Um, Youngsoon Shin, Hyun-Dong Chung, Young Ryun Lee, Kon Ho Kim, Seon-Won 2024-01-20T12:05:01Z 2024-01-20T12:05:01Z 2021-09-04 2013-06 1932-6203 https://pubs.kist.re.kr/handle/201004/128022 Cel5A, an endoglucanase, was derived from the metagenomic library of vermicompost. The deduced amino acid sequence of Cel5A shows high sequence homology with family-5 glycoside hydrolases, which contain a single catalytic domain but no distinct cellulose-binding domain. Random mutagenesis and cellulose-binding module (CBM) fusion approaches were successfully applied to obtain properties required for cellulose hydrolysis. After two rounds of error-prone PCR and screening of 3,000 mutants, amino acid substitutions were identified at various positions in thermotolerant mutants. The most heat-tolerant mutant, Cel5A_2R2, showed a 7-fold increase in thermostability. To enhance the affinity and hydrolytic activity of Cel5A on cellulose substrates, the family-6 CBM from Saccharophagus degradans was fused to the C-terminus of the Cel5A_2R2 mutant using overlap PCR. The Cel5A_2R2-CBM6 fusion protein showed 7-fold higher activity than the native Cel5A on Avicel and filter paper. Cellobiose was a major product obtained from the hydrolysis of cellulosic substrates by the fusion enzyme, which was identified by using thin layer chromatography analysis. English Public Library of Science Engineering of Family-5 Glycoside Hydrolase (Cel5A) from an Uncultured Bacterium for Efficient Hydrolysis of Cellulosic Substrates Article 10.1371/journal.pone.0065727 1 PLoS ONE, v.8, no.6 PLoS ONE 8 6 Y scie scopus 000321038800077 2-s2.0-84878957507 Multidisciplinary Sciences Science & Technology - Other Topics Article ENDOGLUCANASE CEL5A CRYSTAL-STRUCTURE CELLULASE BINDING CRYSTALLOGRAPHY MECHANISM SYSTEM SUITE