A putative β-glucosidase gene, BglAc, was amplified from Acidilobus sp. through metagenome database sampling from a hot spring in Yellowstone National Park. BglAc is composed of 485 amino acid residues and bioinformatics analysis showed that it belongs to the GH1 family of β-glucosidases. The gene was successfully expressed in Escherichia coli with a molecular weight of approximately 55.3 kDa. The purified recombinant enzyme showed the maximum activity using p-nitrophenyl-β-D-glucopyranoside (pNPG) as the substrate at optimal pH 5.0 and 100 °C. BglAc exhibited extraordinary thermostability, and its half-life at 90 °C was 6 h. The specific activity, K_m, V_max, and K_cat/K_m of BglAc toward pNPG were 357.62 U mg⁻¹, 3.41 mM, 474.0 μmol min⁻¹·mg⁻¹, and 122.7 s⁻¹mM⁻¹. BglAc exhibited the characteristic of glucose tolerance, and the inhibition constant Ki was 180.0 mM. Furthermore, a significant ethanol tolerance was observed, retaining 96% relative activity at 10% ethanol, and even 78% at 20% ethanol, suggesting BglAc as a promising enzyme for cellulose saccharification. BglAc also had a strong ability to convert the major soybean isoflavone glycosides (daidzin, genistin, and glycitin) into their corresponding aglycones. Overall, BglAc was actually a new β-glucosidase with excellent thermostability, ethanol tolerance, and glycoside hydrolysis ability, indicating its wide prospects for applications in the food industry, animal feed, and lignocellulosic biomass degradation.

The study aimed to investigate the effects of different levels of added sucrose on the fermentation quality and microbial community of kudzu (Pueraria lobata) silage. The three sucrose supplementation levels utilized were 0, 0.5, and 1.0%, and kudzu was silaged for 15, 30, and 60 days. Sucrose supplementation significantly decreased the pH levels, acid detergent fiber, ammonia nitrogen content, and relative abundance of Pantoea in the silages (p < 0.05). The addition of 1% sucrose to kudzu silage at 60 days had the lowest acid detergent fiber content, the highest crude protein, and the highest relative feed value. Additionally, the sucrose-supplemented silage had a lower pH than that of the control group at each time point. The dominant genera in all groups were Klebsiella, Enterobacteriaceae, Lactobacillus, and Weissella, and the relative abundance of Enterobacteriaceae was lower in the 1% sucrose-supplemented group than in the control group. These results showed that sucrose addition could improve the quality of kudzu silage and increase its beneficial microbial community.

SUMOylation is a well-conserved post-translational modification that is essential for modulating plant adaptation to various abiotic stresses. Although the functions of small ubiquitin-like modifier (SUMO) genes have been reported in various plant species, systematic studies focusing on the SUMO gene family members in alfalfa remain limited. In this study, we identified 49 MsSUMO genes from the alfalfa genome using bioinformatics approaches, and conducted comprehensive analyses of their phylogenetic relationships, structural features, cis-regulatory elements, and expression patterns. Most MsSUMO genes were predicted to localize in the nucleus and cytoplasm, consistent with their roles in transcriptional regulation and protein modification. Phylogenetic analysis grouped MsSUMO, soybean and Arabidopsis SUMO genes into seven subfamilies, which exhibited both high homology and species-specific divergence, suggesting functional differentiation during evolution. Conserved motif and domain analyses revealed strong structural consistency among MsSUMO members, with relatively simple gene architectures. In total, 59 types of cis-elements were detected in the promoter regions, playing crucial roles in plant growth, light signaling, and responses to biotic and abiotic stresses. Abscisic acid-responsive elements (ABREs) were the most abundant, implying that this gene family may serve key functions in stress regulation via the abscisic acid (ABA) signal pathway. Protein interaction network analysis indicated that MsSUMO members cooperate with core enzymes to modulate downstream stress-responsive targets. Transcriptome and real-time quantitative polymerase chain reaction (RT-qPCR) results showed that eight MsSUMO genes exhibited significant expression responses to salt, drought, and waterlogging stresses. Remarkably, six genes consistently exhibited upregulation across all three stress conditions. This observation underscores their potential as pivotal players in abiotic stress tolerance and identifies them as promising candidates for subsequent functional characterization.