| Sporocytophaga is a kind of gliding bacteria that can decompose the cellulose efficiently.But the cellulolytic degradation mechanism has not been revealed till now.During the work,we mainly chose Sporocytophaga sp. JL-01 as research object.We studied the cellulolytic degradation efficiency of Sporocytophaga sp. JL-01 through solid and liquid cultivation;the variation of pH and reducing sugar content during the procession of liquid cultivation;the method of enzyme activity determination;the optimization of enzyme-producing and the FPase purification.So we further clarified the cellulolytic degradation mechanism of Sporocytophaga sp. JL-01.The result are illustratie as follows:1.During the solid cultivation,we found that Sporocytophaga sp. JL-01 could totally decompose filter paper within 168 hours and cellulose powder within 192 hours. During the liquid cultivation, Sporocytophaga sp. JL-01 could totally decompose filter paper within 144 hours.Sporocytophaga sp. JL-01 can decompose cellulose more efficient than Sporocytophaga sp. SD-02 whether during solid cultivation or liquid cultivation.So we chose Sporocytophaga sp. JL-01 as research object.2.when we processed liquid cultivation with filter paper as sole carbon source,we found that the pH and total carbohydrate value increased,and no reducing sugar was detected.When using glucose as sole carbon source,the contrary results obtained, the pH value decreased.The results illustrated that the metabolic pathways might be different with different carbon sources used.As carbon sources varied, the enzyme system changed.3.We studied the influnces to FPase enzyme activity determination,such as crude enzyme-buffer ratio,reaction time, pH value and the treatment of filter paper.The FPase enzyme activity obtained when reaction conditions optimized. Reaction conditions as follows:crude enzyme-buffer ratio 1:1,reaction time 2 hours, pH value 7.0, filter paper treated with NaOH.4.Because of the low FPase enzyme activity assayed,we studied the optimization of enzyme-producing by means of orthogonal test.We take thease factors into consideration,such as cultivation time, inoculation amount,temperature, pH value and rotating speed. The results of orthogonal experiment show that the optimal operation conditions are as follows: cultivation time 120 hours, inoculation amount 5% ,temperature 30℃, pH value 7.2 and rotating speed 120r/min. Repeated experiments done as optimized conditiongs and showed better results.5.During the procession of FPase purification,in order to remove the impurity protein and mucopolysaccharide,we processed the ultrafiltration, ammonium sulfate fractionation,CTAB and Sepharose CL-6B preparative column. The FPase was purified by the Sephacryl s-100 and DEAE-Sephadex G-50 sequently. The purified cellulose assayed by SDS-PAGE showed one protein band and the molecular weights of this enzyme is about55.4KD. The optimal degrading conditions for FPase were obtained: pH7.2 and temperature 50℃. The enzyme is stable between pH 6.8 and 7.4, and temperature between 30℃-50℃. The enzyme activity is activitied by Zn2+ and Co2+,inhibited by Fe3+, Ca2+,Mg2+and Na+ have no significant effect on its activity.We studied the substrate specificity of FPase, FPase can also hydrolysis CMC,and the activity of CMCase is 10 times higher than that of FPase.So we can conclude that FPase is a bifunctional enzyme,also the domain of FPase may have multifunctional areas,the endoglucanase and CBH functional area act synergistically to effect the extensive hydrolysis of crystalline cellulose. So we further clarified the cellulolytic degradation mechanism of Sporocytophaga sp. JL-01.
|
PDF Full Text Request