Research On Cellulases Fermentation、Separation And Enzymatic Characterization

Cellulase was a complex enzyme, which can hydrolyze cellulose into simplesugars, it mainly includes cellobiohydrolase, endoglucanase and β-glucosidase.Cellulase was widely used in the food process, feed, light industry and bioenergyprocess. It was the one of main objectives for researchers to get high enzymaticactivty and complete cellulase composition, and control the cost of cellulaseproduction. In this paper, the main contents and results showed as followings:(1) Optimization of cellulase-producing conditions by three strains mixedsolid-state fermentation. In the solid culture media consisted of rice straw powderand wheat bran, the cellulases production by solid state fermentation with White rotfungi NS75, Aspergillus Niger NS83and Saccharomyces cerevisiae SP5three strainsmixing have been investigated. The results showed that the activities of cellulasesproduced reached the peak value at the seventh day of fermentation process, inwhich the yeast Saccharomyces cerevisiae SP5was inoculated while the White rotfungi NS75and Aspergillus Niger NS83had grown2days in the solid culture media.The β-glucosidase (β-G) and carboxymethyl cellulase (CMCase) activities producedby three strains mixed fermentation have increased143.3%and68.2%, respectively,than that produced by two strains mixed fermentation. The single factor andorthogonal experiment results revealed that, under the optimum fermentationconditions: dosage ratio of rice straw powder and bran powder8:2(w/w), feed-waterratio1:2(w/v), inoculation volume ratio of White-rot fungi NS75、Aspergillus nigerNS83and Saccharomyces cerevisiae SP51:2:1.5（v/v/v）,30℃culture7d, the β-Gand CMCase activities produced reached62305U/g and30241U/g, respectively.(2) Optimization of cellulase extraction conditions. Single factor and ResponseSurface Methodology were employed to optimize the cellulase extraction conditions,which used CMCase activity as index. The optimal conditions showed as following:dosage of citric acid buffer10.42mL/g, pH of citric acid buffer5.22, leaching time2.00h. The CMCase activity extracted reached30782U/g, and had increased1.79% than that of the original conditions. Compared with the predicted value, the relativeerror is0.12%. It means that the response value is coincided well with the predictedvalue of the regression equation.(3) Cellulase pellets were developed. Through the hollow fiber ultrafiltrationmembrane ultrafiltration, the concentrated enzyme liquid had been gotten, then itwas adsorbed by solid medium fermented, and dried. The suitable conditions ofdeveloping cellulase pellets showed as following: concentrated enzyme liquidactivity was12635U/mL, the ratio of liquid enzyme to solid adsorbent was3:1(v/w),dried at30℃, did repeat three times, the CMCase activity of pellets reached127835U/g, which had increased4.16times than that to the original enzyme pellet.(4) Enzymatic characterization. The factors that affect the activity and stabilityof cellulase, such as reaction temperature, pH, glucose, cellobiose, xylose andglycerol, were investigated. The results indicated that the cellulase presented thestrongest cellulase activity under50℃, and pH5.0. The glucose, cellobiose, xyloseand glycerol can cause the inhibition of normal CMCase enzyme and β-G enzymeactivity, and the inhibition effect became stronger with the increasing of sugarconcentration. However, the inhibition effect of cellobiose on CMCase enzyme wasstronger, but on β-G enzyme was not obvious when a small amount existed.