Engineering of Family-5 Glycoside Hydrolase (Cel5A) from an Uncultured Bacterium for Efficient Hydrolysis of Cellulosic Substrates |
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| Authors: | Amar A Telke Ningning Zhuang Sunil S Ghatge Sook-Hee Lee Asad Ali Shah Haji Khan Youngsoon Um Hyun-Dong Shin Young Ryun Chung Kon Ho Lee Seon-Won Kim |
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| Institution: | 1. Division of Applied Life Sciences (BK21), PMBBRC, Gyeongsang National University, Jinju, Republic of Korea.; 2. Center for Environmental Technology Research, KIST, Seoul, Republic of Korea.; 3. School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, Georgia, United States of America.; 4. Department of Microbiology, School of Medicine, Gyeongsang National University, Jinju, Republic of Korea.; Oak Ridge National Laboratory, United States of America, |
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| Abstract: | 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. |
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