Analysis of Rumen Microbial Protein Abundance of Gayals based on Metaproteomics

Background: Presently, our understanding of the rumen of Gayals is still very shallow, which is recognized as the most effective and developed fiber degradation system in nature, with abundant microorganisms. Molecular biology technology is an effective means to study the microbial resources in the rumen. Methods: Rumen contents of 3 Gayals (Gayals, Bos frontalis; G) and 3 Yellow Cattle (Yunnan Yellow Cattle, Bos taurus; Y) were collected in this study. Rumen microbial proteins were extracted by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), then to analyze the bioinformatics of protein abundance was performed through the bovine rumen transcriptome database (gene.uniGeneset.fasta). Result: The results were as follows: the differences in protein abundance of Gayals rumen bacteria in Firmicutes, Actinobacteria, Ruminococcus and Olsenella were significantly higher than Yellow cattle (P less than 0.05) and the difference in protein abundance of Chytridiomycota and Batrachochytrium in rumen fungus of Gayals was significantly less than that of Yellow cattle. Enrichment analysis by KEGG metabolism pathway of differentially expressed proteins in rumen microorganisms was performed, Gayals have higher abundance of β-glucosidase and 6-phosphate-β-glucosidase than Yellow Cattle.

Characteristic of a GH10 Xylanase From The Anaerobic Rumen Fungus Anaeromyces Robustus and Application in Bread Making

The rumen of ruminants contains a variety of fungi capable of producing xylanases to break down plant cell walls. In this study, a new GH10 xylanase gene ArXyn10c20 from anaerobic rumen microorganism Anaeromyces robustus was successfully expressed in Pichia Pastoris GS115, with a protein molecular weight of approximately 42 kDa and showed the similarity by 64.08% with the β-Xylanase form Neocallimastix Californiae. The optimal pH and temperature for ArXyn10c20 was 5.5 at 40℃. ArXyn10c20 was stable in the pH range 5.0 – 9.0 for 1h which the residual enzyme activity was all above 75%. The activity of recombinant xylanase was significantly enhanced by 1 mM Cu 2+ . The products of ArXyn10c20 hydrolysis of beechwood xylan were xylobiose, xylotriose and xylotetraose by TLC analysis. In food applications, ArXyn10c20 can significantly improve the quality of dough and bread. With the addition of 7.5 mg ArXyn10c20, the hardness, gumminess and chewiness of the bread decreased by 42.24%, 45.33% and 55.36% respectively and the reducing sugar increased by 18.67%. The new discovered xylanase ArXyn10c20 has great potential in food industry.

Catalytic Efficiency Diversification of Duplicate β-1,3-1,4-Glucanases from Neocallimastix patriciarum J11

ABSTRACTFour types of β-1,3-1,4 glucanase (β-glucanase, EC 3.2.1.73) genes, designatedbglA13,bglA16,bglA51, andbglM2, were found in the cDNA library ofNeocallimastix patriciarumJ11. All were highly homologous with each other and demonstrated a close phylogenetic relationship with and a similar codon bias toStreptococcus equinus. The presence of expansion and several predicted secondary structures in the 3′ untranslated regions (3′UTRs) ofbglA16andbglM2suggest that these two genes were duplicated recently, whereasbglA13andbglA16, which contain very short 3′UTRs, were replicated earlier. These findings indicate that the β-glucanase genes fromN. patriciarumJ11 may have arisen by horizontal transfer from the bacterium and subsequent duplication in the rumen fungus. β-Glucanase genes ofStreptococcus equinus,Ruminococcus albus7, andN. patriciarumJ11 were cloned and expressed byEscherichia coli. The recombinant β-glucanases cloned fromS. equinus,R. albus7, andN. patriciarumJ11 were endo-acting and had similar substrate specificity, but they demonstrated different properties in other tests. The specific activities and catalytic efficiency of the bacterial β-glucanases were also significantly lower than those of the fungal β-glucanases. Our results also revealed that the activities and some characteristics of enzymes were changed during the horizontal gene transfer event. The specific activities of the fungal β-glucanases ranged from 26,529 to 41,209 U/mg of protein when barley-derived β-glucan was used as the substrate. They also demonstrated similar pH and temperature optima, substrate specificity, substrate affinity, and hydrolysis patterns. Nevertheless, BglA16 and BglM2, two recently duplicated β-glucanases, showed much higherkcatvalues than others. These results support the notion that duplicated β-glucanase genes, namely,bglA16andbglM2, increase the reaction efficiency of β-glucanases and suggest that the catalytic efficiency of β-glucanase is likely to be a criterion determining the evolutionary fate of duplicate forms inN. patriciarumJ11.