Gene clone of Gayal rumen bacteria cellulase

ObjectiveRumen bacteria cellulase gene resources of Gayal were selected and enzymatic characteristics were studied from molecular biology level, so as to provide reference for development and utilization of new cellulases, and lay foundation for revealing the machenism of cellulose degradation. MethodLarge fragment genome DNA was extracted from Gayal rumen microorganisms to establish rumen microorganism gene library and select cellulase activity. The selected genes with high activity were sequenced, and studied in bioinformatics and enzymatic characteristics. Result20352 positive clones were obtained, the white spot rate reached 92%, the library capacity was 899.6 Mb, no load rate was 1.82%. Two posi-tive clones with cellulase activity were obtained(B1 and B2). B1 gene sequence length was 1230 bp, encoding 409 amino acids, the coverage rate of its encoding product and encoding product from Ruminococcus albus cellulase gene (β-1,4-endoglucanase, GenBank accession number P23661.1) reached 99%, and their homology was up to 97%. B2 gene sequence length was 1002 bp, encoding 333 amino acids, coverage rate of its encoding product and encoding product from Uncultured microorganism cellulase gene(cellodextrinase, GenBank accession number ADB80112.1) reached 99%and ho-mology was 83%. B1 and B2 genes could be induced in Rosetta prokaryotic expression host bacteria. The optimum conditions for B1 cellulase were pH 6.0 and temperature 40℃, and for B2 cellulase were pH 6.0 and temperature 40-50℃. Conclu-sionTwo strains with high-activity cellulase(B1 and B2) are selected from rumen microorganism gene library established. B1 isβ-1,4-endoglucanase and B2 is cellodextrinase. They can serve as new materials for degradation of crude fiber in vitro.