Mutation Breeding And Fermentation Optimization Of Xylosidase Producing Strains,studying On Characterization And Application Of Xylosidase

The process of the degradation from xylan which is the main component of renewable hemicellulose to monomeric xylose requires the participation of a series of enzymes.The basic skeleton of this complex polysaccharide consists of D-1-glucopyranose linked by?-1,4 glycosidic bonds and contains different side chain substituents.The most common substituents are acetyl,arabinose and glucuronic acid.Due to the degree of plant xylan polymerization and the substitution of side chains,complete degradation requires a set of xylan hydrolase systems,mainly including?-1,4-xylanase and?-xylosidase.The Aspergillus niger used in this study can produce?-xylosidase and xylanase,wherein?-xylosidase hydrolyzes the non-reducing ends of soluble xylooligosaccharides and xylobiose to release xylose.In this study,the fermentation conditions and fermentation medium of xylosidase production from Aspergillus niger preserved in the laboratory were optimized.The optimum fermentation conditions for the production of xylosidase from the strain were determined by single factor optimization experiments.The optimum fermentation medium components for the production of xylosidase were determined by Plackett-Burman design experiment,steepest climbing experiment,minimum additive experiment with insignificant factors and Central Composite Design design experiment.Then,a high-yield xylosidase-producing strain was screened by UV mutagenesis,and the fermentation conditions and fermentation medium were optimized twice.Finally,the enzymatic properties of the isolated and purified xylosidase and the effect of the enzyme and xylanase on the hydrolysis of xylanwere studied.The main results are as follows.?1?The laboratory-preserved strain Aspergillus niger was determined to be the starting strain.The growth curve of the strain was determined by measuring the dry weight of the cells,and the seed culture time was selected for 36 hours.The optimum fermentation conditions for the production of xylosidase by the Plackett-Burman design experiment and Central Composite Design experimental experiment were as follows:fermentation period 144h,fermentation temperature 34°C,inoculum volume 7%,shaker speed 180r/min,the liquid loading 110 mL/300 mL,and the initial fermentation pH 3.5.The optimal fermentation medium consisted of corncob powder 31.55 g/L,yeast powder8.00 g/L,peptone 5.48 g/L,MgSO4 0.70 g/L,NaCl 1.00 g/L,and CaCl2 1.50 g/L.The predicted enzyme activity?15.04 U/mL?and the measured enzyme activity?14.87U/mL?were similar,which indicat that the experimental results obtained by the above optimization method are reasonable and credible.After optimizing,the xylosidase enzyme activity was 8.89 times that before optimization.?2?The original strain CAN was subjected to UV mutagenesis,and the optimal time for UV irradiation was 1.5 min.The UV mutagen mutant CANT was obtained with good genetic stability.The xylosidase activity was 25.12 U/mL by shake flask fermentation,which was about 69%higher than CAN.The growth curve was measured and 40 h was selected as the seed culture time.?3?The optimum fermentation conditions for the production of xylosidase by the single factor experiment,Plackett-Burman design experiment and Central Composite Design experimental experiment were as follows:fermentation period 156 h,fermentation temperature 34?,inoculum volume 5%,shaker speed 160 r/min,the liquid loading110 mL/300 mL,and the initial fermentation pH 4.0.The optimal fermentation medium consisted of corncob powder 40.00 g/L,yeast powder 5.00 g/L,peptone 22.41g/L,KH₂PO₄ 0.50 g/L,K₂HPO₄ 0.50 g/L,MgSO₄ 0.50 g/L,NaCl 1.00 g/L,and CaCl₂ 1.50g/L.The predicted enzyme activity?47.02 U/mL?and the measured enzyme activity?46.77 U/mL?were similar,which indicat that the experimental results obtained by the above optimization method are reasonable and credible.After optimizing,the xylosidase enzyme activity was 8.89 times that before and 27.97 times than CAN.?5?The separation and purification steps were carried out by one-step salting out and three-step chromatography,and finally a high-purity enzyme protein having a purification ratio of 33.68 and an enzyme activity of 1954.23 U/mg was obtained,and the molecular weight was about 121.0 KDa by SDS-PAGE.The electrophoretic pure xylosidase was characterized.The optimum reaction temperature of the xylosidase was70°C,and the residual enzyme activity was still 75%after 4 h at 30°C⁶0°C.The optimum pH was 4.0.The relative residual enzyme activity was still 70%after 4 h in the range of pH 4^pH 10.The synergistic effect of xylosidase and xylanase on the degradation of corncob powder was studied.The amount of reducing sugar produced by the two enzymes together with corncob powder was 347.1%of the amount of reducing sugar produced by single xylanase.The products of two enzyme-degraded corncob powders were analyzed.It was found that the main products of single xylan-degraded corncob powder were xylo-oligosaccharides,xylotriose and xylo-oligosaccharides,and the main product of the corncob powder degraded by the two enzymes together has an increased xylose content compared with the degradation product of a single xylanase,and the content of xylooligosaccharides such as xylobiose,xylotriose and xylotetraose is reduced.