Isolation, Fermentation Conditions And Characterization Of CMCase Of Penicillium Sp.DS-04

A new strain of cellulolytic fungus is isolated from soil, when grown on cellulose doubleplate, the velocity of growth is rapid and the result of degradation of cellulose is good. Themorphological and physiological properties of this strain are studied. According to these, thestrain is identified as Penicillium frequentans Westling. In this base, the medium componentsof the optimal liquid state fermentation, the purification of cellulase from this strain and thephysiological propertise of the purified cellulase are studied.The results are illustrated as follows:1. A new strain of cellulolytic fungus is isolated from soil by cellulose double plate, theresult of degradation of cellulose powder CF-11 is good. After 4 days, the greencolonies appear when this strain is inoculated on cellulose double plate in 28 ℃. After 10 days,the visible clearance zone around colonies is seen by three points inoculation, cellulose iscompletely degraded. To see the characterization of the colonies, the mycelia and conidia, thisstrain is inoculated on the Czapek medium after isolation. According to Manual ofDeterminative Fungus and related bibliography, the strain is identified as Penicilliumfrequentans Westling DS-04.2. Penicillium sp. DS-04 is inoculated in 5 different liquid state culture mediums. CMCase,FPase in each medium and the time of cultivation are compared so that the optimal originalliquid state fermentation medium is decided. In the base of the original fermentation medium, the optimum culture conditions isstudied. The culture conditions include carbon source, nitrogen source, initial pH of medium,inoculation amount, velocity of shaker and amount of CaCO3. The results show that theoptimum conditions of producing cellulase are as follows:The temperature is 32℃-34℃, the initial pH is 4.5-5.0, the shaking speed is 130r/min, theinoculation amount is 7%, meanwhile 0.1%-0.3% CaCO3 facilitates cellulase production. Thehighest cellulase activities are obtained by using 1% cellulose powder and 1% wheat bran ascarbon sources and 0.25% NaNO3 as nitrogen source.Under the conditions mentioned above, the activities of CMCase and Fpase in thecultivation broth after growing 72h are 187.46U/mL and 75.95U/mL, respectively.3. The crude cellulase broth of Penicillium sp. DS-04 is purified twice by ammoniumsulfate fractionation, dialysis and Sephadex G-100 column chromatography to obtain apurified component, and the CMCase activity of which is higher. The purified cellulaseassayed by SDS-PAGE shows one protein band which shows that the purified cellulase ishaplo-component.Meanwhile, this shows molecular weights of this enzyme about 64,000Da. The optimaldegrading conditions for cellulose obtained are pH3.6 and 50℃. The enzyme is stablebetween pH3.6 and 6.0, and at a temperature below 60℃. The enzyme activity is distinctivelyinhibited by metal ions such as Fe3+, but enhanced by Zn2+. Na+,Mg2+,Ca2+ have nosignificant effect.Key words:Penicillium sp.;Degradation of cellulose;Screening of stain;Fermentation Conditions;Properties of cellulase...