A Glycosyl Hydrolase 30 Family Xylanase from the Rumen Metagenome and Its Effects on In Vitro Ruminal Fermentation of Wheat Straw

The challenge of wheat straw as a ruminant feed is its low ruminal digestibility. This study investigated the impact of a xylanase called RuXyn, derived from the rumen metagenome of beef cattle, on the in vitro ruminal fermentation of wheat straw. RuXyn encoded 505 amino acids and was categorized within subfamily 8 of the glycosyl hydrolase 30 family. RuXyn was heterologously expressed in Escherichia coli and displayed its highest level of activity at pH 6.0 and 40 &deg:C. RuXyn primarily hydrolyzed xylan, while it did not show any noticeable activity towards other substrates, including carboxymethylcellulose and Avicel. At concentrations of 5 mM, Mn2+ and dithiothreitol significantly enhanced RuXyn&rsquo:s activity by 73% and 20%, respectively. RuXyn&rsquo:s activity was almost or completely inactivated in the presence of Cu2+, even at low concentrations. The main hydrolysis products of corncob xylan by RuXyn were xylopentose, xylotriose, and xylotetraose. RuXyn hydrolyzed wheat straw and rice straw more effectively than it did other agricultural by-products. A remarkable synergistic effect was observed between RuXyn and a cellulase cocktail on wheat straw hydrolysis. Supplementation with RuXyn increased dry matter digestibility: acetate, propionate, valerate, and total volatile fatty acid yields: NH3-N concentration, and total bacterial number during in vitro fermentation of wheat straw relative to the control. RuXyn&rsquo:s inactivity at 60 &deg:C and 70 &deg:C was remedied by mutating proline 151 to phenylalanine and aspartic acid 204 to leucine, boosting activity to 20.3% and 21.8% of the maximum activity at the respective temperatures. As an exogenous enzyme preparation, RuXyn exhibits considerable potential to improve ruminal digestion and the utilization of wheat straw in ruminants. As far as we know, this is the first study on a GH30 xylanase promoting the ruminal fermentation of agricultural straws. The findings demonstrate that the utilization of RuXyn can significantly enhance the ruminal digestibility of wheat straw by approximately 10 percentage points. This outcome signifies the emergence of a novel and highly efficient enzyme preparation that holds promise for the effective utilization of wheat straw, a by-product of crop production, in ruminants.