| Xylanase is a complex enzyme system that can degrade xylan to xylooligosaccharide or xylose,mainly including ?-1,4-endo xylanase,?-xylosidase,?-L-arabinfuranosidease,?-D-glucuronidase,acetyl xylanase and phenolic acid esterase,it can degrade the xylan-like hemicellulose that are abundant in nature.Therefore,xylanase can be widely used in industries and fields such as in the food,pharmaceutical,energy field,papermaking and feed industries.The streptomyces althioticus used in this study mainly produced ?-1,4-endo xylanase,which can randomly cleave the ?-1,4-glycosidic linkage of the xylan backbone skeleton to produce xylose and xylooligosaccharide or xylooligosaccharide with side chains.In this study,a strain with higher xylanase activity was screened by UV and sodium nitrite complex mutagenesis.Then the carbon source,nitrogen source and inorganic salt were screened out by single factor optimization.The Plackett-Burman design experiment,the steepest climbing experiment and the minimum addition experiment with insignificant factors were used to determine the composition in the medium.Finally,the optimal composition of fermentation medium was obtained through the Central Composite Design experiment.The separation and purification of xylanase were performed,the enzymatic properties of the xylanase were studied.Finally,the effect of synergistic action of the xylanase and cellulase on hydrolyzing the corn cob powder was studied.The main results were as follows.(1)A strain with the highest production of xylanase was screened by UV and sodium nitrite complex mutagenesis.The xylanase activity of the strain was determined to be 40.21 U/m L.The growth curve of the strain was determined by measuring the dry weight of the cells.0h-4h was the acclimation period,4h-28 h was the logarithmic growth period,and 28h-36 h was the stationary phase.Therefore,in the later fermentation,cells grown for 24 h were selected as seed culture medium to inoculate in the fermentation medium.(2)Single factor optimization method was used to determine the optimal fermentation conditions for producing xylanase from the strain,the results were 108 h fermentation period,40? fermentation temperature,4% inoculum,180r/min shaker speed,and 50 m L/250 m L liquid volume,the initial fermentation p H was 7.0.The Plackett-Burman design experiment was conducted to screen out the components that had significant effect on the xylanase activity of the strain.Then the steepest climbing experiment and the minimum addition amount experiment of insignificant factors were used to determine the addition of components in the medium.And finally the optimal composition of the fermentation medium obtained through Central Composite Design design experiment,respectively 12.77g/L for corncob powder,1.67g/L for potassium nitrate,and 1.45g/L for disodium hydrogen phosphate,other media components were dipotassium phosphate 0.4 g/L,sodium dihydrogen phosphate 0.4 g/L,and potassium dihydrogen phosphate 0.4 g/L.The predicted enzyme activity was 184.6 U/m L,and the experimentally measured enzyme activity was 178 U/m L.The difference of results were not significant,indicating that the experimental results obtained by the above optimization methods were reasonable and reliable.After optimization of fermentation conditions and media composition,The optimized xylanase activity of the strain was about 4.4 times than before the optimization(3)After purification by ammonium sulfate fractionation,Hi Prep 26/10 Desalting desalting,Hiprep DEAE FF 16/10 ion exchange chromatography and Sephadex G-100 gel filtration chromatography.The purification fold was 10.37.The specific activity of high purity sample was 952 U/mg,and the molecular weight was approximately 35.0 KDa as measured by SDS-PAGE.The enzymatic properties of the purified xylanase were studied.The optimum reaction temperature of xylanase was 55?.After incubation at 40?,45? and 50? for 4 hours,the residual xylanase was greater than 80%,the xylanase had good temperature stability in the temperature range of 40?to 50?.The optimum reaction p H of the xylanase was at p H5,and the incubation time was 4 h in the range of p H 4 to p H9.The residual enzyme activity remained 60%,the enzyme stability was good in the range of pH4 to pH9.(4)Optimizing the optimal conditions for xylanase,cellulase,xylanase and cellulase synergistic hydrolysis of corncob powder by single factor method.The optimum condition for hydrolysis of corncob with xylanase was p H6,the enzyme dose was 110 U,the hydrolysis time was 7h,and the amount of reducing sugar produced was 7.38mg;the optimum condition for hydrolysis of corncob with cellulase was p H5.5,the enzyme dose was 150 U,and the hydrolysis time was 28 h,the amount of reducing sugar was 39.69mg;the optimum condition for xylanase hydrolysis of corncob residues was p H 5.5,the enzyme dose was 110 U,the hydrolysis time was 7h,and the amount of reducing sugar produced was 4.08mg;the optimum condition for celluase hydrolysis corncob residues was p H 4.5 and the enzyme dose was 230 U,the amount of reducing sugar produced was 49.53 mg.At the same time,after the pretreatment of corncob with steam explosion,the hydrolysis of the corncobs by the four processes were separately investigated.The amount of reducing sugar produced by xylanase hydrolysis of corncob was 9.48 mg.The cellulase hydrolyzed corncob to produce 52.56 mg reducing sugar.The xylanase hydrolyzed corncob residues to produce 8.27 mg reducing sugar.The cellulase hydrolyzed corncob residues to produce 64.89 mg reducing sugar.The results showed that the pretreated corncob residues were hydrolyzed by cellulase to produce the highest amount of reducing sugars.In addition,the products from xylanase and cellulase hydrolysis of corncob were analyzed by high performance liquid chromatography(HPLC).The main products of the corncob hydrolyzed by cellulase were glucose,cellobiose and xylose.The main products of xylanase hydrolyzed corncob were xylose,xylobiose,and xylotriose,only a small amount of xylotetraose was present in xylanase hydrolyzed pretreated corncob.The surface of the enzyme hydrolyzed corncob was observed by scanning electron microscope(SEM).It was found that the corncob was hydrolyzed by enzymes,the surface of the corncob became rougher than the original sample and the diameter of the corncob became smaller,and some pores appeared on the surface.It further verified that the hydrolysis of corncob by cellulase and xylanase was obvious. |
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