Study On Thermomyces Lanuginosusproducinghighproduction Xylanase And Preparation Of Xylooligosaccharides By Enzymes Hydrolyzing

Xylooligosaccharides are oligasaccharides composed of 2-7 xylose units linked byβ-1,4-xylosidete bonds. Among many methods of oligosaccharide prepared, the enzyme hydrolysis is the method, which is most convenient, effective, well targeted and controllable. Strain selection and process optimization are considered to be a key factor in the method. In this paper, a xylanase production fungi Thermomyces lanuginosus DSM10635 was used as the original strain for mutation. Firstly, protoplast was dealt with UV and nitrous acid separately, in order to screen a high-producing and stable xylanase mutant. Then liquid fermentation process was optimized and the basic characterizations of xylanase were investigated. Finally, the optimum conditions of enzymatic hydrolysis were determined.1.The best conditions for protoplast preparation were identified, which were as follows: with the mixture solution of 0.01g/mL cellulase and 0.01g/mL snailase (volume ratio 1:1),the mycelia incubated for 58h were most suitable for protoplast release. The suitable time and enzyme temperature were 2.5h and 30℃³4℃,respectively. The most effective osmotic stabilizer was 0.6mol/L mannitol. The number of protoplasts was up to 4.47×10⁶ / mL, and the regeneration rate was 4.74%. The best condition of UV mutagenesis was 30cm department, with a 15W UV light, and then protoplast was irradated for 5min. A name for the T-U-03 mutant was screened. The protoplast of T-U-03 was disposed with 120s by nitrite, and then, a high yield of xylanase T-U-03-10 strain was bred, at last. The xylanase production by T-U-03-10 increased from 1011U/mL to1510U/mL, increasing by 49.36% comparing to xylanase production by start strains.2. Firstly, the optimal carbon, nitrogen sources and surfactants were determined by sheet factor experiment, which were corn cob, urea and Tween-80.The best medium components were determined by PB experimental design and response surface experiment, with SAS-JMP software in the application of the conditions. Medium included the following components: corn cob 4.570%, Urea1.797%, KH2PO4 0.497%, ZnSO₄ 0.2%, MgSO₄·7H₂O 0.03%, CaCl₂ 0.03%, FeSO₄·7H₂O 0.03%, and Tween-80 0.03%.The optimum fermentation condition was determined by a single factor test. As were included: spore inoculation medium for amounting to about 105¹06 /80mL, culture time 7d,medium initial pH6.5⁷.0, fermentation temperature 50⁵2℃, speed180²00r/min,and liquid volume 90mL/250mL.The producing enzyme activity of strain can reach to 1726.80U/mL, comparing with the optimization of the former increased by 14.36%.3. The basic enzyme properties of mutant were discussed. The results showed that specificity of the enzyme was strong, most temperature of the reaction was 65℃, and the stable range should be not higher than 65℃. The optimal pH was 6.8, and under this condition, the stability of the enzyme was better, with the survival rate still 85.20%. Mg²⁺, Mn²⁺ at low concentrations and Fe²⁺, Zn²⁺ at high concentrations had a significant role in the activation of the enzyme, but Hg²⁺ could inhibit the enzyme significantly, with even at high concentrations completely being inactivated. Cu²⁺, Co²⁺ also had a greater inhibitory effect, the inactivation rates of which were 10.40% and 19.46%, respectively. The enzyme solution stored at room was instability, but it should be stored under 4℃. The michaelis constant of xylanase was about 38.71mg/mL, and the maximum reaction rate was about 277.78μmol / (mL.min).4. Xylooligosaccharides were made of xylanase by Thermomyces lanuginosus, and the main conditions were discussed. The best hydrolysis technology was determined by single factor and orthogonal tests for preparation of oligosaccharide. They were hydrolysis time 2.5h, substrate concentration 4%, and enzyme addition level of enzyme per gram for substrate 120U.The solution purified by carbon column filtration was analyzed by the HPLC. The main components of liquid glucose included wood two sugar and wood three-four sugar, of which accounted for about 74.56% among oligosaccharide components.