Engineering of Family-5 Glycoside Hydrolase (Cel5A) from an Uncultured Bacterium for Efficient Hydrolysis of Cellulosic Substrates
- Engineering of Family-5 Glycoside Hydrolase (Cel5A) from an Uncultured Bacterium for Efficient Hydrolysis of Cellulosic Substrates
- Amar A. Telke; Ningning Zhuang; Sunil S. Ghatge; Sook-Hee Lee; Asad Ali Shah; Haji Khan; 엄영순; Hyun-Dong Shin; Young Ryun Chung; Kon Ho Lee; Seon-Won Kim
- Issue Date
- PLoS ONE
- VOL 8, NO 6, e65727-1-e65727-11
- 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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