Screening Xylanase Production Strain And Optimising Fermentation Processes And Studying On Characterization Of Xylanase

This paper studies on using of agricultural wastes to produce xylanase with microorganism, and the research of purification and characterization of xylanase. The concrete research content is as follows:（1）Transparent circle method was used to screen a strain that produced xylanase, which called LMZY was identified as Streptomyces althioticus. 26 h was chosen for inoculation fermentation time.（2）Using single factor experiment, the Plackett-Burman experiment design, central composite design method for LMZY fermentation was optimized, and the optimal culture conditions was as follows: temperature 40℃, the period of 120 h, the initial p H 5.0, speed of rotary shaker 160 r/min, inoculation amount 6%, loaded liquid 50 m L/250 m L.The optimal fermentation medium formula（g/L） as follows: corn cob powder 24.4, soybean 16, KNO₃8, K₂HPO₄0.4, Na₂HPO₄0.754, Mg SO₄ ? 7H₂ O 0.4. The predictive value was 36.65 U/m L, and the actual value was 36.45 U/m L, which was coinciding well with the predicted values.（3） The original strains LMZY was found using the ultraviolet mutagenic had best effect. The ultraviolet illuminate time was 1.0 min. By shake flask fermentation, determining enzyme activity was 46.61 U/m L, which was about 27% higher than that of LMZY, with well genetic stability. 26 h was chosen for inoculation fermentation time.（4）Using single factor experiment, the Plackett-Burman experiment design, central composite design method for LMZY fermentation was optimized, and the optimal culture conditions was as follows: temperature 40℃, the period of 108 h, the initial p H 5.5, speed of rotary shaker 160 r/min, inoculation amount 8%, speed of rotary shaker 160 r/min, loaded liquid 50 m L/250 m L.The optimal fermentation medium formula（g/L） as follows: corncob powder 27.85, soybean 16.25, yeast extract powder 3.46, Na₂HPO₄ 0.4 g, K₂HPO₄ 0.6 and Mg SO₄ 1.176. The predictive value was 117.804 U/m L, and the actual value was 116.63 U/m L, which was coinciding well with the predicted values. And it was 1.52 times higher than the enzyme activity of LMZM.（5）The xylanase was purified by ammonium sulfate fractionation, desalination, ion exchange chromatography, and gel chromatography. The molecular mass is 31.75 k D.（6）The optimum temperature for the enzymatic reaction was 60℃. The optimum p H for the enzyme activity was 8.0. It was indicate that K+ and Na+ had non-significant effect on xylanase enzymatic reaction; Cu²⁺ had stronge effect on the xylanase enzyme reactions, weaker by Ca²⁺, Zn²⁺ and Mg²⁺; Mn²⁺ was not significant with low concentration, but had obvious restraining with high concentration effect; Hg²⁺ had strong inhibition on enzymatic reaction,weaker by Co²⁺, Ba²⁺, Fe²⁺ and Fe³⁺. At the same time, the β- mercaptoethanol and EDTA had non- significant effects on enzymatic reaction. SDS, DEPC, EDC, NBS and L-tryptophan had a certain inhibition on enzymatic reaction. Triton X-100 and DTT have acceleration effect on the enzymatic reaction, urea and L-cysteine played significant role in enzymatic reaction.The Michaelis constant is 43.03 mg/m L, and the maximum reaction rate is 312.5μmol/（m L.min）.And the analysis results of its hydrolysate of thin-layer chromatography chromatography showed that the enzyme was endo xylanase. In addition, the substrate specificity was strong, and the weaker with other substrates. The brightness of kraft pulp was improved on some degree.