Study On Isolation And Purification Of Cellulase Production By Mixed Culture Fermentation And Enzymatic Properties

Separation and purification of cellulase that was mix fermented with three cellulase producing strains(Aspergillus niger D2-1,Bacillus subtilis S3-7,Candida lipolytica) was researched in this paper.The properties of separated cellulose were also researched.Two kinds of endoglucanase components was separated from the cellulase with these methods respectively:fractional precipitation of ammonium sulphate,Gel column chromatography,Ultrafiltration,Weak anion exchange column chromatography.The main results are as following:1.Comparing ammonium sulfate saturationon effect on salting out effect of β-glucosidase,endo-glucanase and exo-glucanase,we found the optimum range of ammonium sulfate saturation of saltin-g out effect was20%-60%.2.1n the experiment of Sephadex G-100column chromatography,it was concluded that the optimum separation conditions were:gel column length70cm,sample volume2mL,speed22r/min.The optimum eluent was Na2HPO4-citric acid buffer solution.Then peak3that mainly contains endoglucanase was appeared.3.In the ultrafiltration process,through the experiment of single factor and center composite design,it showed that the optimum separation conditions were:pH5.09,speed4567r/min,time32.19min,sample volume940.98mL.The specific activity was6.58.4.After the separation of DEAE FF ion exchange chromatography,two peaks of endoglucanase were got. The specific activity of endoglucanase1increasd by14.41times.The en2yme was purified16.95times, and the reclaim ratio of the activity was22.72%.The specific activity of endoglucanase2increased by14.78times.The enzyme was purified17.38times, and the reclaim ratio of the activity was only0.13%.5.Through the analysis of SDS-PAGE electrophoresis,it was showed that the endo-glucanase co m-ponents was electrophoretic purity.The molecular weight of them were43KD and38KD.6.The study on properties of the endo-glucanase showed that the optimum conditions of43KD endo-glucanase were as following:temperature50℃,pH5.5.It has good stability at temperature of30-50℃and pH of4.5-6.0.Zn2+and Ba2+could promote endo-glucanase activities while K+has no obvious effect on the activities.Mg2+、Ca2+、Na+could inhibite endo-glucanase activities partly while Cu2+could inhibite the activities completely.The optimum conditions of38KD endo-glucanase were as following:temperature40℃,pH5.0.It had good stability at temperature of30～50℃and pH of5.0-6.0.Zn2+and Ba2+could promote endo-glucanase activities while Fe2+had no obvious effect on the activities.K+、Mg2+、Cu2+、Ca2+、 Na+could inhibite endo-glucanase activities partly.