We found a match
Your institution may have rights to this item. Sign in to continue.
- Title
Engineering of Family-5 Glycoside Hydrolase (Cel5A) from an Uncultured Bacterium for Efficient Hydrolysis of Cellulosic Substrates.
- Authors
Telke, Amar A.; Zhuang, Ningning; Ghatge, Sunil S.; Lee, Sook-Hee; Ali Shah, Asad; Khan, Haji; Um, Youngsoon; Shin, Hyun-Dong; Chung, Young Ryun; Lee, Kon Ho; Kim, Seon-Won
- 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.
- Subjects
GLYCOSIDASES; BIOENGINEERING; HYDROLYSIS; CELLULOSE; CHEMICAL reactions; BIOCHEMISTRY; HYDROLASES
- Publication
PLoS ONE, 2013, Vol 8, Issue 6, p1
- ISSN
1932-6203
- Publication type
Article
- DOI
10.1371/journal.pone.0065727