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XOS is from 2 to 7 D-xylose, β-1, 4 - xylosidase bond of oligomeric functional polysaccharide. XOS prepared a variety of methods, enzyme hydrolysis method is the most convenient and effective, well targeted, controllable strong xylanase produced by strain selection and optimization of process conditions can be utilized the method key factors. Thermophilic fungus Thermomyces lanuginosus DSM10635 as the original strain, protoplast UV and nitrous acid compound mutagenesis method, the final screening to yield a stable xylanase mutant strains (TU-03-10), in this based on mutant strain liquid fermentation xylanase produced by process optimization, as well as the production of xylanase preparation XOS the optimal enzymatic process conditions. 1 this test to determine the optimum conditions for this strain Protoplast: enzymes and enzyme concentration of 0.01 g cellulase 0.01 g helicase (volume ratio of 1:1), strain age for 58h, hydrolysis time of 2.5h, hydrolysis temperature of 30 ℃ to 34 ℃ osmotic stabilizer 0.6mol / L mannitol; protoplast number up to 4.47 × 10 6 a / mL regeneration rate of 4.74%. Using a 15W UV lamp at 30cm irradiated protoplasts 5min, screened a named TU-03 mutant, and then to nitrous acid treatment TU-03 protoplasm body 120s, screening and shake the bottle through a transparent circle rescreening last breeding a xylanase yield bacteria of TU-03-10, produced enzyme up to 1510U/mL, a 49.36% increase compared with the original strain enzyme activity 1011U/mL. First through the single factor experiment to determine the most suitable carbon and nitrogen sources and surfactant, respectively, for the corn cob, urea, Tween-80; recycling SAS-JMP software PB experiment, the main component of the medium experiment and response surface, determine the best medium group divided into: the corncob 4.570%, urea 1.797% KH 2 PO 4 0.497%, 0.2% of ZnSO4 the MgSO 4 · 7H O 0.03%, the CaCl 2 0.03%, FeSO 4 · 7H 2 O 0.03% Tween-80 0.03% single factor approach to determine the optimum fermentation conditions: inoculum size the medium spore count of approximately 10 5 10 6 / 80mL, incubation time 7d medium initial pH6.5 ~ 7.0, fermentation temperature 50 to 52 ℃, speed 180 ~ 200r/min medium volume 90mL/250mL. Under these conditions, the strain producing enzyme high 1726.80U/mL, than before optimization enzyme activity increased by 14.36%. Explore the enzymatic properties of the mutant enzyme production results: strong enzyme substrate specificity, optimum reaction temperature of 65 ° C, the thermal stability range no higher than 65 ° C, the stability of the enzyme is preferred. Enzymatic reaction optimum pH value of 6.8, and better stability of the enzyme under this condition, the survival rate at 85.20%. The the low concentrations Mg 2 , Mn, 2 and high concentrations of Fe 2 Zn 2 have the enzyme apparent activation; Hg 2 great inhibitory effect on the enzyme, even at a high concentration so that complete inactivation of the enzyme, while the Cu 2 , Co 2 also have a larger extent, inactivation rates were 10.40% and 19.46%. The enzyme was stored at room temperature instability, should be placed at 4 ℃ save. Determination of xylanase Michaelis constant of about 38.71mg/mL maximum reaction rate is about 277.78μmol / (mL.min). 4 using the the thermophilic fungus producing the xylanase preparation XOS, and discussed mainly on the enzymatic hydrolysis process conditions. By single factor and orthogonal experiment to determine the optimum conditions of XOS Enzymatic Preparation: hydrolysis time of 2.5h, substrate concentration of 4%, the enzyme is added per gram of substrate and enzyme amount of 120U . Enzymatic hydrolyzate is purified by filtration through a carbon column, the main component of the sugar was measured by HPLC analysis as xylobiose, xylotriose and wood tetrasaccharide XOS component content of about 74.56%.
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