Functional Identification Of Three Glycosyl Hydrolases From Sphingomonas Elodea ATCC 31461

Xylanase and glucanase are glycosyl hydrolases and both of them are belong to hemicellulases,which have been widely used in brewing,feed,washing and biomass energy.In this study,three genes xyn10A,eg5A,and eg16A which are from Sphingomonas elodea ATCC 31461 were cloned and expressed in the E.coli,the specific substrates of the three crude enzymes showed that xyn10A encodes aβ-xylanase of the GH10 family,eg5A encodes aβ-1,3-1,4-glucanase of the GH5family,and eg16A encodes aβ-1,3（4）-glucanase of the GH16 family.Xyn10A,EG5A and EG16A were purified by nickel affinity chromatography,and the enzymatic properties of these three glycoside hydrolases were studied in detail.At pH7.0 and 37℃,Xyn10A can hydrolyze beechwood xylan,birchwood xylan,corncob xylan,wheat arabinoxylan,oat spelt xylan,bagasse xylan,and p NPC.Xyn10A showed the highest hydrolytic activity against beechwood xylan.Using beechwood xylan as a substrate,the optimal reaction pH of Xyn10A was6.5 which can be stable between pH4.5-10.5 and the optimal reaction temperature was 45℃ which was stable between 25℃-40℃.The Km and Vmax values of Xyn10A were 13.26±1.813 g/L and 138.3±8.648μmol*min^-1*mg^-1,respectively.The organic reagents such as alcohols inhibited the activity of Xyn10A to varying degrees.Xyn10A hydrolyzes xylooligosaccharides and xylan substrates to finally produce xylose and xylodiose.Xyn10A was detected to have a weak transglycosidic activity.At pH7.0 and 37℃,EG5A can hydrolyze barley glucan,oat glucan,lichenan,avicel,CMC,beechwood xylan,and p NPC.EG5A showed the highest hydrolytic activity against barley glucan.Using barley glucan as a substrate,the optimal reaction pH of EG5A was 6.0 which can be stable between pH4.5-10.5and the optimal reaction temperature was 45℃ which was stable between45℃-50℃.The Km and Vmax values of EG5A were 2.034±0.189 g/L and515.3±17.79μmol*min^-1*mg^-1,respectively.The organic reagents such as alcohols also inhibited the activity of EG5A to varying degrees.The hydrolysis products of barley glucan,oat glucan and lichenan by EG5A are the same,which are glucose,cellobiose,3-O-cellobiosyl-D-glucose（G4G3G）,3-O-cellotriosyl-D-glucose（G4G4G3G）.With avicel and CMC as the substrates,the final products are mainly glucose,cellobiose,and cellotriose.Using beechwood xylan as the substrate,the final products are mainly xylobiose,xylotriose,xylotetraose.At the same time,EG5A is capable of hydrolyzing cellooligosaccharides.At pH7.0 and 37℃,EG16A can hydrolyze barley glucan,oat glucan,lichenan,laminarin,yeast glucan,andβ-1,3-glucan.EG16A showed the highest hydrolytic activity against barley glucan.Using barley glucan as a substrate,the optimal reaction pH of EG16A was 7.0 which can be stable between pH3.0-7.0and the optimal reaction temperature was 40℃ which was stable between25℃-40℃.The Km and Vmax values were 4.363±0.230 g/L and 15.94±0.320μmol*min^-1*mg^-1,respectively.The organic reagents such as alcohols also inhibited the activity of EG16A to varying degrees.The end products of EG16A hydrolysis of barley glucan,oat glucan,and lichenan are glucose,laminaribiose,4-O-laminaribiosyl-glucose（G3G4G）,4-O-laminaribiosyl-cellobiose（G3G4G4G）.With laminarin and yeast glucan as the substrates,the final products are mainly trisaccharide and tetrasaccharide.