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.
Highlights
Plant cell walls are important resources for the production of ethanol as a next-generation biofuel
The most challenging technological and economical obstacle involves the release of fermentable soluble sugars at prices competitive with those used in breaking down sugarcane and corn kernels [2,3], which can be achieved by increasing the rate of cellulose hydrolysis using wild-type enzymes and protein engineering
Periplasmic Secretion of Cel5A E. coli BL21 harboring pTCel5A showed a clear halo zone on Luria broth (LB) agar plates containing 0.5% carboxymethyl cellulose (CMC), whereas no halo zone was observed for E. coli BL21 harboring pTw/o-ssCel5A (Figure S2)
Summary
Plant cell walls are important resources for the production of ethanol as a next-generation biofuel. A b-1-4-glucose polymer, is a major polysaccharide present in the plant cell wall. The synergistic action of various cellulases, such as cellobiohydrolase, endoglucanase, cellodextrinase, and b-glucosidase, are required for the production of fermentable sugar from cellulose substrates [1]. The most challenging technological and economical obstacle involves the release of fermentable soluble sugars at prices competitive with those used in breaking down sugarcane and corn kernels [2,3], which can be achieved by increasing the rate of cellulose hydrolysis using wild-type enzymes and protein engineering. One approach for obtaining efficient cellulases is isolating novel cellulases from cellulolytic microorganisms or metagenomic libraries of uncultured microorganisms, followed by cellulase engineering to enhance cellulose degradation
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