A gene coding endo-β-1,3(4)-glucanase(ENG16A)was cloned from Coprinopsis cinerea and heterologously expressed in Pichia pastoris.ENG16A only acts on substrates containingβ-1,3 glycosidic bonds but not on substrates containing onlyβ-1,4-orβ-1,6-glycosidic bonds.Interestingly,compared to the activity of this enzyme towards carboxymethyl(CM)-pachyman containing onlyβ-1,3-glycosidic bonds,its activity towards barleyβ-glucan containing bothβ-1,3-glycosidic andβ-1,4-glycosidic bonds was increased by 64.72%,its activity towards laminarin containing bothβ-1,3-glycosidic andβ-1,6-glycosidic bonds was decreased by 50.83%.In addition,ENG16A has a higher Km value and Vmax for barleyβ-glucan than laminarin,which may be related to the fact that barleyβ-glucan contains mainlyβ-1,4-glycosidic bonds mixed with a fewβ-1,3-glycosidic bonds,whereas laminarin mainly containsβ-1,3-glycosidic bonds with a fewβ-1,6-branched glucose residues.The adjacentβ-1,4-glycosidic bond promotes ENG16A to hydrolyzeβ-1,3-glycosidic bonds,leading to an increased Vmax;the nearbyβ-1,6-glycosidic bonds inhibited its hydrolysis ofβ-1,3-glycosidic bonds,resulting in a decreased Vmax,This property is suggested to be related to the mechanism that C.cinerea uses to degrade and utilize hemicellulose in straw medium and to protect its cell wall during the mycelium growth stage.