Production Optimization And Enzymatic Properties Of Thermostable Xylanase Produced By Thermoascus Aurantiacus

Xylanases hydrolyze the main chain of xylan to release xylooligosaccharides and xylose,and have been applied widely in food,feed and textile industries.Thermophilic xylanases present the advantages such as their higher catalytic efficiency,better thermal stability and lower energy consumption compared to other mesophilic xylanases.Generally,the thermophilic fungi are the main producers of thermophilic xylanases.Thus,the present study mainly aimed to screen and indentify a thermophilic fungus from soil samples collected from high temperature compositing and/or hot spring,optimized its culture conditions and well investigate enzymatic characteristics of the produced xylanase.The main conclusions are as follows:?1?Ten thermostable strains numbered from M1 to M10 were isolated from the high temperature compositing and hot spring by dilution plating,congo red staining and extracellular enzyme activity testing.The selected strain M2 with highest xylanase?34.58U/m L?was identified as Thermoascus aurantiacus by morphological observation,ITS sequence homology and phylogenetic tree and patent-preserved in China General Microbiological Culture Collection Center?CGMCC?with the number of 11334.The T.aurantiacus CGMCC11334 produced xylanase reached to the maximum level of 39.07 U/mL after 8 d fermentation at 45 oC.?2?The comprehensive hydrolysis ability index?XCHI?was developed by using principal component analysis.XCHI was proved to represent the hydroysis ability of xylanase by regressively analyzing the relation of XCHI and sugar yields with XCHI as independent variable and reducing sugar yield as dependent variable.Using XCHI as response,the culture compositions were optimized by using orthogonal experimental design as?g/L?: wheat bran 16,microcrystalline cellulose 7,beef extract 4,corn syrup 3,wheat peptone 4,KH₂PO₄ 1.0,MgSO4 2.0,CuSO4 0.15,FeSO4 0.05,VB1 0.15.Under the optimal condition,the Xyn,CMC,FPA and XCHI were 135.12 U/mL,1.95 U/mL,1.68 U/mL and1649.44,respectively.?3?The xylanase produced by T.aurantiacus CGMCC11334 was purificed 5.27 fold with a yield of 36.5% by ammonium sulphate,DEAE-Sepharose Fast Flow and Sephadex G-75 chromatography.The molecular mass was approximately 31.0 kDa and structure characteristics of xylanase was predicted by SDS-PAGE and MALDI-TOF-MS analysis.The xylanase produced by T.aurantiacus CGMCC11334 had better thermal and acid stability with the optimal temperature?75 oC?and pH?5.0?by enzymatic properties analysis.Within30⁸0 oC and pH 2.0¹0.0,the residual activity was above 80% and 70%,respectively.Mn²⁺and Ag+enhanced reletive activity to 120.0% and 119.6%.Co²⁺,Cu²⁺and Mn²⁺inhibited its catalytic reaction with reletive activity dropped to 48.3%,96.5% and 20.7%,respectively.The Km and V_max of the xylanase from T.aurantiacus CGMCC11334 for beechwood xylan were 4.81 mg/mL and 476.2 mg/?min·mL?,respectively.From the relative molecular mass,main hydrolysis product of xylobiose and three-dimensional structure,the xylanase of T.aurantiacus CGMCC11334 belongs to family 10 and endo-?-1,4 xylanases.