摘要Background Lignocellulose biomass is the most abundant and renewable material in nature.The objectives of this study were to characterize twoendoglucanases TrepCel3 andTrepCel4,and determine the effect of the combination of them(1.2 mgTrepCel3,0.8 mgTrepCel4)on in vitro rumen fermentation characteristics.In this study,three nature lignocellulosic substrates(rice straw,RS;wheat straw,WS;leymus chinensis,LC)were evaluated for their in vitro digest-ibility,gas,NH3-N and volatile fatty acid(VFA)production,and microbial protein(MCP)synthesis by adding enzymatic combination.Methods Two endoglucanases'genes were successfully expressed in Escherichia coli(E.coli)BL21(DE3),and enzy-matic characteristics were further characterized.The combination ofTrepCel3 andTrepCel4 was incubated with lignocellulosic substrates to evaluate its hydrolysis ability.Results The maximum enzymatic activity ofTrepCel3 was determined at pH 5.0 and 40℃,whileTrepCel4 was at pH 6.0 and 50℃.They were stable over the temperature range of 30 to 60℃,and active within the pH range of 4.0 to 9.0.TheTrepCel3 andTrepCel4 had the highest activity in lichenan 436.9±8.30 and 377.6±6.80 U/mg,respectively.The combination of TrepCel3 andTrepCel4 exhibited the highest efficiency at the ratio of 60:40.Compared to maximum hydrolysis ofTrepCel3 orTrepCel4 separately,this combination was shown to have a superior ability to maximize the saccharification yield from lignocellulosic substrates up to 188.4％for RS,236.7％for wheat straw WS,222.4％for LC and 131.1％for sugar beet pulp(SBP).Supplemental this combination enhanced the dry matter digestion(DMD),gas,NH3-N and VFA production,and MCP synthesis during in vitro rumen fermentation.Conclusions The TrepCel3 and TrepCel4 exhibited the synergistic relationship(60∶40)and significantly increased the saccharification yield of lignocellulosic substrates.The combination of them stimulated in vitro rumen fermentation of lignocellulosic substrates.This combination has the potential to be a feed additive to improve agricultural residues utilization in ruminants.If possible,in the future,experiments in vivo should be carried out to fully evaluate its effect.