Purification And Characterization Of Xylanases From The White-rot Fungus Trametes Gallica

Xylan is the main important component of plant hemicelluloses,which accounts for one third of the total sugar in plants.Next to cellulose,xylan is the most abundant reproducible biomass resource.Xylanases can hydrolyze xylan into xylooligosaccharides,xylopentose,xylobiose and xylose,which is widely distributed in eukaryotes and prokaryotes.These enzymes has great prospect for many applications such as in paper-making,feed,food and medicine industry. White-rot basidiomycete Trametes gallica is one of the main bio-degraders of poplar in China.Previous study has proved that it has strong capacity to degrade lignocellulose.Using T.gallica as a donor strain,studies were carded out on purification and characterization of the three isoenzyme xylanases produced by this strain when grown on wheat straw powder medium.Trametes gallica was cultivated on wheat straw powder for 40 days.The fractions showing xylanase activity was separated and purified from the soaking fluid of solid culture by ultrafilration concentration,(NH₄)₂SO₄ fractionation, Phenyl Sepharose CL-4B,Sepharose-Q fast folw and Sephadex G-150 chromatography.The recovery rate and purification fold were 1.45%and 15.6, respectively.Partially purified xylanases were luther recovered by native-polyacrylamide gel electrophoresis(PAGE)and three isoenzyme xylanases (â…©â… ,â…©â…¡andâ…©â…¢) of SDS-PAGE purity were finally obtained.SDS-PAGE and isoelectfic focusing(IEF) were used to evaluated molecular weight and isoelectric point(pI) of the three isoenzymes,respectively.The components had similar molecular weights(approximately 19.0KDa),but exhibited different isolectfic points(5.6 forâ…©â… ,4.7 forâ…©â…¡and 4.0 forâ…©â…¢) and carbohydrate content(0.25%forâ…©â… ,0.63%forâ…©â…¡and 3.4%forâ…©â…¢), respectively.The results showed that isoenzymeâ…©â… can degrade both xylan and cellulose,meaning it is a difuntional enzyme,whileâ…©â…¡andâ…©â…¢can only degrade xylan.The optimal temperature and pH forâ…©â…¡were 45℃and 5.0, respectively and its activity was increased by Mg²⁺ and Fe²⁺,while inhibited by Mn²⁺ and Co²⁺.Its K_m and V_max,were 0.75mg/mL and 5000mmoL/(min.mg), respectively.