Optimization Of Culture Conditions And Characteristics Of Xylanase By Pichia Stipitis In Solid State Fermentation

With the advent of the global food crisis, the energy crisis and environmental problems,people are gradually paying attention to the most abundant and cheapest lignocelluloseresource on the earth. The natural lignocellulose has complex structure, and it is difficult todecompose. However solid-state-fermentation technology can make good use oflignocellulosic materials and break them up into metabolites such as xylanase and ethanol bymicrobial fermentation. The main contents of the paper were as follows: taking theagricultural waste such as corn cobs and wheat bran as the substrate of solid-statefermentation, and studying the influence of the type of substrate, the initial moisture contentand fermentation time on xylanase activity and ethanol production, and also confirming theoptimum process conditions of solid state fermentation, and concurrently studying theenzymatic properties purified xylanase.Using Pichia stipitis m4as fermentation strains, the impact of different fermentationconditions on production of xylanase was studied. According to the experimental results, theoptimal medium of xylanase production by solid state fermentation was as follows: the ratioof corncob and wheat bran1:1, initial moisture content80%, the range of medium densityfrom0.7g/mL to0.9g/ml, cultivation temperature28℃for9days and then the highestxylanase activity reached to5536U/g.The optimal medium of ethanol production by solid state fermentation was as follows:the ratio of corncob and wheat bran0:1，initial moisture content74%，the range of mediumdensity from0.7g/mL to0.9g/ml, cultivation temperature28℃for5days, and the highestethanol content was0.261g/g dry matter.The xylanase produced by solid state fermentation of Pichia stipitis was separated andpurified. After the ammonium sulfate fractionation, the yield of xylanase activity was88.2%and its purification fold was11.7times. An electrophoresis pure xylanase was obtained withits yield of enzyme activity of5.7%and its purification fold of38.7times after SepharoseCL-6B gel filtration chromatography and Q Sepharose Fast Flow ion exchangechromatography. Measured by SDS-PAGE, the relative molecular mass of the purifiedxylanase was31.6kDa.Enzymatic properties of purified xylanase were also studied in this paper. The optimum pH value was pH6.0, and the optimum temperature was50℃. According to theLineweaver-Burk plot method, the Km value was1.42mg/mL, and the Vmax value was9.17μmol/（min·mL）. Moreover, Cu²⁺and K+had no significant influence on xylanase activity,while the inhibit effects of different metal ions on the enzyme activity were as follows: Hg²⁺>Co²⁺, Pb²⁺> Mn²⁺, Fe3+> Ag+, Fe²⁺> SDS, Na+>EDTA, Mg²⁺> Zn²⁺, Ca²⁺> Cu²⁺, K+.Hydrolysates of purified xylanase analyzed by HPLC mainly were xylotriose and xylobiose,as well as a small amount of xylose.