Studies On Fermentation Production,enzymatic Properties And Immobilization Of Xylanase From Streptomyces Sp.t7

Xylanase are hydrolytic enzymes that degrades xylan,which is widely used for medicine,food,feed and other industries.In this article,a strain,numbered T7,producing high-yield xylanase was isolated from soil samples.Strain T7 had the typical morphological and chemotaxonomic characteristics of members of the genus Streptomyces.The 16s r RNA gene sequence analysis of strain T7 showed high sequence indentity to Streptomyces.The fermentation conditions of strain T7 were optimized to improve the yield of xylanase by one-single factor experiment and response surface methodology.The optimal data from one-single factor experiment are listed as follows:inoculum size1%,liquid volume 100 m L,the culture temperature 30℃and the culture time 96 h.Under the above conditions,the total enzyme activity of the fermentation broth of strain T7 increased from 30 to 150 U/m L.Based on one-single factor experiment,the optimal nitrogen source of cell growth and enzyme production was NH₄NO₃,the optimal carbon source was oat,and the optimal inorganic salts were K₂HPO₄,Na₂HPO₄and Mg SO₄·7H₂O.Furthermore,the results of the Plackett-Burman design showed that the significant variables were oat,NH₄NO₃and K₂HPO₄.Then the steepest ascent search experiment,the Box-Behnken design,and ANOVA analysis was performed,the results showed that the optimal medium consisting of NH₄NO₃15.94 g/L,oat bran 1.70 g/L,and K₂HPO₄1.14 g/L,which produced the predicted total enzyme activity of 405.187 U/m L,while the total enzyme activity from verified experiment was 430.15 U/m L that was 14 times higher than before optimization.The purification of the T7 xylanase was carried out by ammonium sulfate precipitation,ultrafiltration,Sephadex ^TMG-100 coloum chromatography,etc.T7xylanase exhibited maximum enzyme activity at 60℃,and it was stable below 60℃,which could maintain more than 90%of the highest activity at 60℃for 2 h.T7xylanase exhibited maximum enzyme activity at p H 6.0,and it was stable in the solution over a broad p H range of 5.0～8.0.The metal ions including Mn²⁺,K⁺,Ca²⁺,Mg²⁺,Co²⁺could enhance enzyme activity,while Fe²⁺,Fe³⁺,SDS and EDTA inhibited enzyme activity.The K_mof the enzyme for xylan from corn was 1.05 mg/m L,and the V_maxwas 85.40μmol·min^-1·m L^-1.The results of the substrate specificity showed that the enzyme specifically hydrolyzed xylan,which displayed endoglucanase activity.The immobilization of T7 xylanase was performed by sodium alginate embedding method,chitosan-glutaraldehyde cross-linking method and membrane cross-linking method（PVDF membrane,nylon membrane and mixed cellulose membrane）.Among these methods,the nylon membrane immobilization had the highest recovery rate of enzyme activity,whose optimal conditions were as follows:0.5%glutaraldehyde concentration,30%enzyme concentration,immobilization time of 18 h,immobilization temperature of 40℃and p H 7.5.The optimal temperature of immobilized enzyme was 60℃,which was the same as that of free enzyme.But the thermal stability of immobilized enzymes was better than the free enzyme,which was able to maintain more than 75%of enzyme activity at 80℃for 1 h.The optimal p H was 8.0,which was more stable in the alkaline reaction solution than the free enzyme.Moreover,the mmobilizated enzyme could retain over 80%of enzyme activity at 4℃at p H 11.0 for 24 h.It still maintained more than 60%of enzyme activity after for bacths reaction,and the resulting XOS has the average yield of more than 100 mg/g.