The Study On Solid-state Fermentation Of Sheep Gastric Contents To Produce Xylanase By Trichoderma M-112

Xylanase(β-1,4-D-endoxylanase, EC 3.2.1.8)is an important class of biological enzyme preparation, which can degrade xylan to oligosaccharide and a small amount of xylose.Currently, xylanase has been shown a high application potential in many areas.The effects of single factor,such as medium ratio,SSR,salt,inoculum and culture time on the solid fermentation of sheep gastric contents by Trichoderma M-112 is studied,and the solid-state fermentation condition is optimized by orthogonal experimental design.The results showed that the solid-state fermentation of sheep gastric contents by Trichoderma M-112 to produce xylanase is feasible,and the most important factor affecting enzyme production is inoculation. Optimization of culture condition is 50% of stomach contents, corn / wheat bran = 5:5, CaCl2 0.5%, the initial pH 5.0, solid to liquid ratio of 1:3, medium dry inoculum per 10g 2ml (spore 1.2～1.6×10⁸cfu/ml), fermentaton time of 72h, the fermentation temperature is 30℃. After optimization of the activity levels reached 138.504 IU/ml. The activity Increased to three times compared with the reported maximum fermentation activity by the same specie of Trichoderma.Secondly, the xylanase was separated and purified from a culture filtrate of Trichoderma M-112 in Solid-state Fermentation of Sheep Gastric Contents through ammonium sulfate precipitation,Sephadex G-25 column chromatography and Sephadex G-150→Sephadex G-100 successive column chromatography.The purified xylanase was proved by SDS-PAGE and Native PAGE to be a single composition with molecular mass of 67.4 KDa.The recovery rate is 5.59%.Purification fold is 10.43 and the specific activity is 253.36 IU/mg.By study of characterization,The results show that its Km is 11.20g/L,and Vmax is 0.70μmol/min; Optimum temperature is 65℃, the optimum pH is 6.0;This xylanase have good stability at medium temperature.After 65℃for 120 min, residual activity remained above 60%;It also have good stability at pH 3.0～8.0. After 65℃for 120 min, activity remained above 58%.Different metal ions have different impacts on xylanase activity. Fe²⁺,Fe³⁺,Ba²⁺,Al³⁺are inhibitors to xylanase,while Ca²⁺,Mg²⁺,Cu²⁺,Zn²⁺,Mn²⁺ are strong stimulators.5～15mmol/L Ca²⁺ has the strongest activation,and 15mmol/L Al³⁺ the strongest inhibition. K⁺,Na⁺,Co²⁺ have less impacts on xylanase activity.The data of this study provided some reliable scientific basis for potential industrial development application of biological waste-based renewable resources.