ABSTRACT
Objective: This study aimed to identify and characterize a novel endo-ß-glucanase, IDSGLUC9-4, from the rumen metatranscriptome of Hu sheep. Methods: A novel endo-ß-glucanase, IDSGLUC9-4, was heterologously expressed in Escherichia coli and biochemically characterized. The optimal temperature and pH of recombinant IDSGLUC9-4 were determined. Subsequently, substrate specificity of the enzyme was assessed using mixed-linked glucans including barley ß-glucan and Icelandic moss lichenan. Thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF) analyses were conducted to determine the products released from polysaccharides and cello-oligosaccharides substrates. Results: The recombinant IDSGLUC9-4 exhibited temperature and pH optima of 40 °C and pH 6.0, respectively. It exclusively hydrolyzed mixed-linked glucans, with significant activity observed for barley ß-glucan (109.59 ± 3.61 µmol·mg-1·min-1) and Icelandic moss lichenan (35.35 ± 1.55 µmol·mg-1·min-1). TLC and HPLC analyses revealed that IDSGLUC9-4 primarily released cellobiose, cellotriose, and cellotetraose from polysaccharide substrates. Furthermore, after 48 h of reaction, IDSGLUC9-4 removed most of the glucose, indicating transglycosylation activity alongside its endo-glucanase activity. Conclusion: The recombinant IDSGLUC9-4 was a relatively acid-resistant, mesophilic endo-glucanase (EC 3.2.1.4) that hydrolyzed glucan-like substrates, generating predominantly G3 and G4 oligosaccharides, and which appeared to have glycosylation activity. These findings provided insights into the substrate specificity and product profiles of rumen-derived GH9 glucanases and contributed to the expanding knowledge of cellulolytic enzymes and novel herbivore rumen enzymes in general.
ABSTRACT
Amino acid balance is central to improving the efficiency of feed protein utilization and for reducing environmental pollution caused by intensive farming. In previous studies, supplementation with limiting amino acids has been shown to be an effective means of improving animal nutrient utilization and performance. In this experiment, the effects of methionine on the apparent digestibility of nutrients, antler nutrient composition, rumen fluid amino acid composition, fecal volatile fatty acids and intestinal bacteria in antler-growing sika deer were investigated by randomly adding different levels of methionine to the diets of three groups of four deer at 0 g/day (CON), 4 g/day (LMet) and 6 g/day (HMet). Methionine supplementation significantly increased the apparent digestibility of organic matter, neutral detergent fiber (NDF) and acid detergent fiber (ADF) in the LMet group (p < 0.05). The crude protein and collagen protein of antlers were significantly higher in the LMet and HMet groups compared to the CON group and also significantly higher in the HMet group compared to the LMet group, while the calcium content of antlers was significantly lower in the HMet group (p < 0.05). Ruminal fluid free amino acid composition was altered in the three groups of sika deer, with significant changes in aspartic acid, citrulline, valine, cysteine, methionine, histidine and proline. At the phylum level, Firmicutes and Bacteroidetes were highest in the rectal microflora. Unidentified bacterial abundance was significantly decreased in the HMet group compared to the CON group. Based on the results of principal coordinate analysis (PCoA) and Adonis analysis, there was a significant difference in the composition of the intestinal flora between the CON and HMet groups (p < 0.05). At the genus level, compared with the CON group, the abundance of Rikenellaceae_RC9_gut_group and Lachnospiraceae_UCG-010 in the LMet group increased significantly (p < 0.05), the abundance of dgA-11_gut_group in the HMet group decreased significantly (p < 0.05) and the abundance of Lachnospiraceae_UCG-010, Saccharofermentans and Lachnospiraceae_NK3A20_group increased significantly. Taken together, the results showed that methionine supplementation was beneficial in increasing the feed utilization efficiency and improving antler quality in sika deer, while affecting the composition of fecal bacteria.