低聚木糖(XOS)被认为是一种有前途的益生元,可用于食品,饲料,和保健产品。木聚糖酶在由木聚糖生产XOS中起关键作用。在这项研究中,我们对饲喂不同类型饲料的奶牛的粪便微生物群进行了宏基因组分析。尽管他们的饮食多样化,在所有粪便微生物群中观察到的主要门是厚壁菌和拟杆菌。在属一级,一组饲喂含有益生菌发酵草药混合物的饲料的奶牛显示出甲烷的丰度降低和有益的Akkermansia细菌的生长增加。此外,该组表现出高度的微生物丰富度和多样性。通过我们的分析,我们获得了一个包含超过280,000个碳水化合物活性酶基因的综合数据集。其中,我们鉴定出了163个潜在的木聚糖酶基因,随后在大肠杆菌中表达了其中的34个。在34个表达的基因中,获得了两种具有优异的温度稳定性和pH耐受性的碱性木聚糖酶。值得注意的是,CDW-xyl-8表现出96.1±7.5U/mg蛋白质的木聚糖酶活性,最佳工作温度为55℃,最佳pH值为8.0。CDW-xyl-16的活性为427.3±9.1U/mg蛋白,最适pH为8.5,最适温度为40℃。生物信息学分析和结构模型表明,CDW-xyl-8属于GH10家族木聚糖酶,CDW-xyl-16是GH11家族木聚糖酶。两种酶都具有水解山毛虫木聚糖并产生XOS的能力。总之,这项宏基因组研究为饲喂不同饲料类型的奶牛的粪便微生物组成提供了有价值的见解,揭示了主要的微生物群体,并证明了木聚糖酶的丰度。此外,两种新型木聚糖酶的表征突出了它们在XOS生产中的潜在应用。
Xylo-oligosaccharides (XOS) are considered as a promising type of prebiotics that can be used in foods, feeds, and healthcare products. Xylanases play a key role in the production of XOS from xylan. In this study, we conducted a metagenomic analysis of the fecal microbiota from dairy cows fed with different types of fodders. Despite the diversity in their diets, the main phyla observed in all fecal microbiota were Firmicutes and Bacteroidetes. At the genus level, one group of dairy cows that were fed probiotic fermented herbal mixture-containing fodders displayed decreased abundance of Methanobrevibacter and increased growth of beneficial Akkermansia bacteria. Additionally, this group exhibited a high microbial richness and diversity. Through our analysis, we obtained a comprehensive dataset comprising over 280,000 carbohydrate-active enzyme genes. Among these, we identified a total of 163 potential xylanase genes and subsequently expressed 34 of them in Escherichia coli. Out of the 34 expressed genes, two alkaline xylanases with excellent temperature stability and pH tolerance were obtained. Notably, CDW-xyl-8 exhibited xylanase activity of 96.1 ± 7.5 U/mg protein, with an optimal working temperature of 55 ℃ and optimal pH of 8.0. CDW-xyl-16 displayed an activity of 427.3 ± 9.1 U/mg protein with an optimal pH of 8.5 and an optimal temperature at 40 ℃. Bioinformatic analyses and structural modeling suggest that CDW-xyl-8 belongs to GH10 family xylanase, and CDW-xyl-16 is a GH11 family xylanase. Both enzymes have the ability to hydrolyze beechwood xylan and produce XOS. In conclusion, this metagenomic study provides valuable insights into the fecal microbiota composition of dairy cows fed different fodder types, revealing main microbial groups and demonstrating the abundance of xylanases. Furthermore, the characterization of two novel xylanases highlights their potential application in XOS production.