| Xylanase mainly including the β-1,4-D-of cut xylosidase and β-1,4-D-of Endo xylanase constitute, it can degrade the xylan which including a group of enzymes that xylose and wood oligosaccharides, and was extracted from the microbial fermentation product. In this paper, Rhizopus stolonifer TP-02 mutagenesis used ARTP mutagenesis. After screening a strain of genetically stable high yield of wood poly xylanase strain TZD-01; response surface method was used to optimize Rhizopus stolonifer TZD-01 fermentation medium; then on Rhizopus stolonifer TZD-01 liquid fermentation production of xylanase batch fermentation kinetics; at the end of fermentation production of xylanase was isolated and purified, and its enzymatic characteristics was studied.Rhizopus stolonifer TP-02 as starting strain, in ventilation 10L/min, 100W, mutagenesis time 240s, plasma emission end and spore suspension distance under the condition of 2mm ARTP mutagenesis treatmentRhizopus stolonifer, eventually bred mutant strain TZD-01. The wood xylanase ability is 2.7 times of the starting strain. The ten times passage fermentation experiment and Strains enzyme activity basically remain unchanged,showing good genetic stability of Rhizopus stolonifer TZD-01.On the basis of single factor experiment, Plackett Burman experiment and the steepest ascent experiment of liquid fermentation were used,screening three of the most significant factors:pineapple peel, peptone, KH2PO4,and then through the response surface method was used to optimize the culture medium which included pineapple skin 4.5%, protein peptone 0.45%, KH2PO4 0.35%,0.1% MgSO4, tween - 800.04%, CaCl2,0.3%, under the condition of optimization Rhizopus stolonifer TZD-01 xylanase to 350.8U/ml, which increased 24.9% than before optimization.Using logistic and luedeking piret model to liquid state fermentation kinetics analysis, Rhizopus stolonifer TZD-01 cell growth, wood xylanase synthesis and substrate consumption kinetics model are studied, the model parameters are obtained by using the nonlinear least squares fitting method, establishing dynamics model can be used to predict the xylanase batch fermentation process of Rhizopus stolonifer fermentation producing, the average relative error of cell growth, xylanase synthesis and substrate consumption variable experiment value and simulation value were 3.89%,2.59% and4.22%.The single factor and response surface experiments were used to optimize the culture medium production of xylanase by solid state fermentation of Rhizopus stolonifer TZD-01. The optimized culture medium composition included bran as 8g, (NH4)2SO42.3%, water quantity 1.5g/g, inoculation amount of 7.5%, MgSO4-7H2O 1%, KH2PO4 1%, CaCl2 2%, Tween 800.025%(all accounted for the mass fraction of solid matrix). Under the optimized conditions, the measuring enzyme activity achieved 693.24U/g. More than that before optimization with an increase of 23.5%.The crude enzyme liquid of Liquid fermentation was precipitated by ammonium sulfate first; then through DEAE weak anion exchange chromatography and Sephadex G-75 column chromatography separation of target protein, which was analyzed by SDS-PAGE, target protein showed a single band with, wood xylanase I molecular weight of 45kda, xylan molecular weight xylanase Ⅱ 26kDa. Through the properties of enzyme, xylanase I the optimum temperature of 50℃, the optimum pH value was 6, Ca2+,Mn2+ activation is better, CO2+, Hg2+ inhibition effect is very obvious, to an almost complete inhibition of Hg2+; xylan enzyme Ⅱ of the optimum temperature of 60℃, the optimal pH value is 5, Mg2+ to promote effect is good, CO2+, Fe2+, Hg2+ on xylanase Ⅱ inhibition effect are obvious, Hg2+almost completely inhibited. |
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