Open AccessEngineering of Family-5 Glycoside Hydrolase (Cel5A) from an Uncultured Bacterium for Efficient Hydrolysis of Cellulosic Substrates
Author(s) -
Amar A. Telke,
Ningning Zhuang,
Sunil Ghatge,
Sook-Hee Lee,
Asad Ali Shah,
Haji Muhammad Shoaib Khan,
Youngsoon Um,
HyunDong Shin,
Young Ryun Chung,
Kon Ho Lee,
Seon-Won Kim
Publication year - 2013
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0065727
Subject(s) - cellulase , cellobiose , thermostability , glycoside hydrolase , biochemistry , hydrolysis , chemistry , carbohydrate binding module , cellulose , trichoderma reesei , enzyme
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.