PLoS ONE (Jan 2013)

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,
  • Youngsoon Um,
  • Hyun-Dong Shin,
  • Young Ryun Chung,
  • Kon Ho Lee,
  • Seon-Won Kim

DOI
https://doi.org/10.1371/journal.pone.0065727
Journal volume & issue
Vol. 8, no. 6
p. e65727

Abstract

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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.