Study On Mutagenesis Strain For Producing Xylanase And Property Of Xylanase

The xylanase is one kind of enzyme which may degrade the xylan. The xylan is polysaccharide which varies molecular structure in a wide range, its main chain was composed of D-xylose withβ-1, 4-glycoside linkage, and the chain is composed of many short substituents, such as L-arabinose sugar residues. 4-methyl-D-glucuronic acid residues, O-acetyl and so on. The xylan is an important renewable resource as second only to natural cellulose type of a polysaccharide. The xylanase which is the use of xylan important tool in paper, food, fodder, biomass conversion, as well as fruit juices, wines, coffee, spices and pigment production have a wide range of potential applications. Aspergillus oryzae in the traditional fermented food application for long history, has been used for industrial enzyme production, which is recognized safety bacteria by FDA.Aspergillus oryzae Cohn(AS 3.4382) was chosed as the most suitable species mutation with the initial screening for the genetic stability of the mutants and the higher production of xylanase. The producing condition of mutant strain was optimized. The use of aqueous two-phase for xylanase extraction and purification was studied. The strain's produced model was identified as synchronize type and established by analyzing the curves of enzyme production and the growth of cells. The results are as follows :1. Mutation of Aspergillus oryzae. UV induced mutagenesis and nitrous acid induced mutagenesis were used for mutagenesis of Aspergillus oryzae. With Congo Red staining plate screening mutants, mortality rates determined the optimum conditions: bacteria suspension concentration was 1.0×10⁶, the time of nitrite mutagenesis was 60s, the time of UV irradiation was 10s. The mutant strain with higher activity and genetic stable performance was obtained by treatment.2. The optimization for enzyme production. The single factor (one time one factor) experiment was to determine the optimal conditions for enzyme production: Ages 72 hours, the inoculum size 5%, liquid loading 50ml/250ml, speed 120r/min, temperature 28℃, initial pH of 5.0, the fermentation period three days. Plackett-burman design and surface Response methodology(RSM) were applied for optimizing the medium: bran, 2%; yeast extract, 1%; Tween 80,0.25ml; K₂HPO₄, 2.6%; calcium chloride, 0.5%; magnesium sulfate, 1%; ferrous sulfate, 0.016%; zinc sulfate, 0.006%.3. The extraction of xylanase with aqueous two-phase. The aqueous two-phase system was applied for extraction of xylanase, and the optimum conditions for the purification: PEG 8000, PEG 8000 concentration of 19% (w/w), K₂HPO₄ concentration of 13% (w/w). NaCl concentration of 0 (w/w), xylanase yield amounted to 97.58%.4. Xylanase enzyme properties. The optimum reaction temperature of xylanase was 50℃, the optimal pH was 5.0, between 4.0-6.5 the activities can be maintained at the highest activity of 80%. This enzyme in acidic conditions was relatively stable. The enzyme was strongly activated by Co²⁺ and Fe²⁺ and normally activated by Fe³⁺ and Ba²⁺. In addition to Ca²⁺ and Zn²⁺ was a strong inhibition, Pb²⁺, Mg²⁺, Cu²⁺, Mn²⁺, Li²⁺ were all have different levels of inhibition, but the residual activity was above 50%. The enzyme was measured in various temperature for the half-life: 50℃for 105 min. 55℃for 45min, 60℃15min,4℃for about 33 days.5. The growth model of Aspergillus oryzae. The growth model of Aspergillus oryzae was identified as producing synchronize type by comparison the curve of growth and enzyme production, as well as the growth model of mutant was established to mathematical model : N= 139.3388/(1+28.7922e-^0.0593t.This model was fit better with the experimental data. The limit value of bacterial was 1.39 x 106/ml, the specific growth rate was 0.0593h^-1, the inflection point for the time was 56.661h.