Isolation And Characterization Of Cellulases And Xylanases From A Goat Rumen-Derived Metagenome Library

A goat rumen-derived fosmid metagenome library was constructed to screen novel cellulases and xylanases. With about9,500clones and average DNA insert size of30kb, the library including at least285Mb metagenomic DNA.6cellulase-positive clones and4xylanase-positive clones with different EcoRI restriction maps were screened after the Congo-Red stain analysis.Analysis of the terminal sequences of positive clones showed that GRFosCEL-10exhibited the lowest identity with known sequences while all the xylanase-positive clones had low homology with known sequences (26-51%). Functional characterization of the crude enzymes exhibited that4cellulase-positive clones had highest activity at pH4.0, which suggested that they contained putative acid-tolerant cellulases encoding genes in the DNA inserts. However, all the xylanase-positive clones showed similar characterization, with highest activity at pH7.0and50℃. Our priliminary analysis of positive clones sets a sound bases on the deep characterization of the cellulases and xylanases encoding genes in the DNA insert.Whole sequencing of GRFosCEL-1showed that the DNA insert was33,848bp long, had39%G+C content and included31predicted genes sizing at le ast200bp.4carbohydrate-active enzymes encoding genes, orf11, orf12, orfl6and orf18, were found in the DNA insert. BlastP analysis showed that ORF11, ORF12, ORF16and ORF18exhibited the highest homology to an endoglucan ase from Roseburia intestinalis L1-82(55%identity), a family43glycosyl hyd rolase from Prevotella ruminicola23(36%identity), a β-glucosidase-related gly cosidases from Ruminococcaceae bacterium (39%identity) and a β-D-xylosidas e/a-L-arabinofuranosidase from Bryantella formatexigens DSM14469(64%iden ty) respectively.Gene orf11, which was designated as celA, had a coding sequence of1,449bp in length, encoding a protein of482amino acid residues with a calculate d molecular mass of56kDa. Signal peptide analysis of CelA revealed that it had a typical17amino acid long lipoprotein signal peptide at the N-terminus, indicating that CelA was a lipoprotein. Modular architecture analysis revealed t hat CelA was consisted of a N-terminal sequence of～90ami no acid residues with unknown function and C-terminal catalytic domain. Sequence alignment of the catalytic domain of CelA and five GHF5endoglucanases indicated two hi ghly conserved residues, Glu280and Glu412, which were considered to be cru cial for the catalytic activity of GHF5enzymes. When celA was expressed in E.coli BL21at37℃for4h, a truncated protein with a molecular mass of45kDa was highly expressed, and N-terminal amino acid sequence analysis show ed that it began at amino acid112of the full length protein. Truncated CelA was purified to homogeneity and further characterized. The optimal pH and te mperature of the recombinant CelA were7.0and50℃. It was stable over a w ide pH range from4.5to10.0and its activity was highly increased by many commonly used metal ions and chemicals. CelA was a halotolerant enzyme, re maining elevated activity in the presence of0-3.5M NaCl and KC1. It was th e first report about halotolerant endo-β-1,4-endoglucanase derived from rumen. I n addition, CelA degraded a variety of substrates with β-1,4-linkages and displ ayed high activity against mixed β-1,3/4-linkaged lichenan and barley-glucan. T hese properties indicate that CelA has potential values for industrial application.Whole sequencing of clone GRFosXYL-10exhibited a xylanase gene xyn10A, which showed the highest homology (47%identity) to functionally known family10glycosyl hydrolase XynB from Fibrobacter succinogenes subsp. succinogenes S85. Xyn10A is2709bp in length, encoding902amino acid residues with a theoretical molecular mass of98.6kDa and pI of5.36. Modular architecture analysis revealed that Xyn10A composed a N-terminal GH10catalytic domain and two CBM6, which were designated as CBM6-1and CBM6-2. To unravel the function of Xyn10A, two truncated protein, namely Xyn10A-TX1and Xyn10A-TX2, with the truncation of signal peptide and further two CBM6s respectively, were further characterized. The optimal pH and temperature of Xyn10A-TX1were pH7.0and45℃. Stability analysis indicated that Xyn10A-TX1was stable in alkaline environment, but reduced its activity quickly in high temperature. Most metal ions could inhibited the enzyme activity, while1mM Pb2+、Co2+、Cr3+and chemical reagents EDTA、Tween20and DTT stimulated the activity. In addition, Xyn10A-TX1showed binding ability to avicel and insoluble oat spelt xylan. Xyn10A-TX2exhibited the highest activity at pH6.5and55℃.Binding ability analysis showed that the truncation of CBM6-1and CBM6-2significantly reduced its binding ability to avicel and insoluble oat spelt xylan.