Chinese Yak Rumen Source Not Cultivate Microbial Degradation Of Cellulose Enzyme Screening, Cloning And Identification

Cellulose is the most abundant renewable biomass on earth. Saccharification of cellulose to fermentable sugar is an important process in conversion biomass to biofuels and chemicals and is the bottleneck of the application. To hunt for novel, high activity cellulases, the cellulases resources in yak (Bos grunniens) rumen were explored in this study.The community analysis for rumen bacteria showed that66.8%bacteria belonged to Firmicutes and27.9%bacteria belonged to Bacteroidetes.10%bactera there were known species, including typical rumen cellulose-degrading bacteria Ruminococcus albus, Butyrivibrio fibrisolvens and Fibrobacter succinogenes. The other90%were uncultured, a part of which fell into Ruminococcaceae, Lachnospiraceae and Prevotellaceae, and clustered with several known rumen cellulose-degrading bacteria, suggesting they may be novel uncultured cellulose-degrading bacteria. The community analysis for rumen fungi showed that the abundance of fungi is only10%of that of bacteria, and there exist cellulose-degrading fungus Orpinomyces sp. So this rumen sample not only contains known cellulose-degrading microorganism, but also has great possibility to contain many novel uncultured cellulose-degrading microorganism.Considering the majority of the rumen microorganisms were unculturable, we make use of metagenomic technology to construct cosmid library, and functionally screened for cellulases. The screening result5endoglucanase positive clones,24exoglucanase positive clones,50glucosidase positive clones,27xylosidase positive clones and6xylanase positive clones. Three of them, RuBGX2、RuBGX3and Rucel5B were cloned and characterized.Both Rubgx2and Rubgx3have not only β-glucosidase activity, but also β-xylosidase activity, and belong to glycoside hydrolase family (GH)3. They shared48.9%amino acid sequence identity and belonged to glycoside hydrolase family3. Their optimal pH were about5, optimal temperature were about40℃. They had broad-spectrum activity towards β-configurational glycoside substrates, and can hydrolyze both cellooligosaccharide substrates and artificial aryl substrates. Their β-glucosidase activities enable the promotion of cellulose degradation by reducing inhibition of intermediate product, while their β-xylosidase activities enable the promotion of hemicellulose degradation through synergy effect with xylanase. Homology modeling and substrate docking results showed their β-glucosidase activity and β-xylosidase activity have the same active site, indicating these two kinds of activities are due to broad-spectrum specificity of substrates.Ruce15B has both endoglucanase activity and exoglucanase activity, and belongs to GH5. Ruce15B’s endoglucanase activity was219.7U/mg, and its exoglucanase activity was52.9U/mg. It exhibited optimal temperatures at about60℃and was thermostable at55℃, suggesting it’s a thermostable enzyme. Ruce15B can hydrolyze β-1,4-glucosidic linkage, not β-1,3, β-1,6glucosidic linkage, or β-1,4xylosidic linkage. It was further confirmed that Ruce15B can cleave cellobiose from4-methylumbelliferyl-β-D-cellobiopyranoside, cellotetraose and insoluble cellulose (avicel, filter paper and PASC) through exo-mode of action. Time course degradation of soluble amorphous cellulose (CMC, barley glucan, lichenan) by Ruce15B showed that degree of polymerization of products was decreased rapidly at the beginning of the reaction, suggesting Ruce15B’s endo-mode of action.So this research analyzed the diversity of yak rumen microorganism community, constructed the metagenomic library platform to functional screen cellulase, clones and characterized two β-glucosidase/xylosidase RuBGX2and RuBGX3and endo/exoglucanase Ruce15B. RuBGX2and RuBGX3not only exhibited both β-glucosidase and β-xylosidase activity, but also exhibited high conversion on cellooligosaccharides and the synergy effect with xylanase. Rucel5B exhibited endo-and exo-mode in cellulose degradation, high activity and thermostability. These results indicated rumen is a good source for novel cellulose-degrading enzyme and these cellulases had huge potential in saccharification of cellulosic biomass.