Screening Of Cellulase-producing Strain And Studies On Optimization Of Fermentation Condition And Characteristics Of Enzyme

Cellulose is a renewable, abundant and inexpensive resource for the bio-conversion to biofueland bio-products, with the world on the verge of running of fossil energy, it is becominginternational issues. The bio-degradation of cellulose by cellulase has incomparable advantages, thekey problem is to screen cellulase-producing strain which has excellent performance.In the thesis, samples collected from rotten wood, paper mill waste and humus. R2A mediumwas selected for microbial enrichment,and180strains were separated and purified through platestreaking method, of which44strains were cellulase-producing strains, the preliminary screeningresults was obtained. Then, the44strains were used for fermentation,and the total cellulaseactivities were measured for secondary screening. The result showed that J1-3-1had highest totalcellulase activities3.56u/ml, and J1-3-1was selected for further experiments.In the experiment, the fermentation conditions of the strain J1-3-1were optimized, optimumschemes were established by a series of single-factor experiments and orthogonal test. CMC-Na wasthe best carbon source, and beef extract (0.6%) was the optimal nitrogen source.(1) For the FPase,the most suitable fermentation conditions: loaded125ml liquid fermentation medium into250mltriangular flask, the initial medium pH was7.5, the addition of Tween-80was0.5%, the inoculumconcentration was6%, and then fermented for3d at28℃and150rpm.(2) For the CMCase, the mostsuitable fermentation conditions: loaded75ml liquid fermentation medium into250ml triangularflask, the initial medium pH was6.5, the addition of Tween-80was0.6%, the inoculumconcentration was6%, and then fermented for3d at28℃and150rpm.(3) For the β-glucosidase, themost suitable fermentation conditions: loaded75ml liquid fermentation medium into250mltriangular flask, the initial medium pH was7.5, the addition of Tween-80was0.8%, the inoculumconcentration was6%, and then fermented for4d at30℃and150rpm. Under the optimalconditions,the highest enzyme production was obtained, FPase,CMCase, β-glucosidase was11.05U/ml,28.61U/ml and11.28U/ml, respectively.In the test, the extracting condition of crude enzyme, the stabilization of cellulase and theenzymatic reaction conditions were studied. The results showed that:(1) the speed6000rpm andcentrifuge time of10min, the reaction time was60min, FPase was highest;(2) the optimal conditionof FPase and β-glucosidase was at pH5.5,50℃, and CMCase was at pH6.5,60℃;(3) Themaximum reaction rate and Km of CMCase were obtained, Vmax=0.49mg/(min·ml) andKm=10.64g/L;(4) within the range of pH6.5~7.5, the pH on stabilization of FPase was relativelyhigher; about pH7.5, the pH on stabilization of CMCase and β-glucosidase were relatively higher;(5) within the range of40℃~50℃,the thermal stability of FPase was relatively higher, that is to say,lower temperature is favorable for cellulase;(6) the activity of FPase was inhibited by cellobiose, butthe activity of β-glucosidase was enhanced within a certain rang;(7) the effects of mental ions to theenzymatic reaction were obvious, the activity of CMCase was improved dramatically by1.0mmol/LMg2+, by contrast, Cu2+and Mn2+were inhibition;(8) for β-glucosidase, Mg2+,Na+and K+wereenergetically, conversely, Cu2+was suppressed.