Study On Screening And Its Enzymatic Characteristics Of Cellulolytic Strains From Rumen

Cellulose is the most abundant renewable source in the natural environment, using microbial degradation on the fiber resources, and transforming them into raw materials which can be used for human. Meanwhile cellulase is a low-carbon and green protein applied to pharmaceutical field etc. based on biological. Nearly half a century, Cellulase had been researched extensively by domestic and foreign researchers, particularly on the metabolism regulation of cellulase protein, the interaction mechanisms of enzyme proreins, how to improve the cellulase activity and application of cellulase etc, and achieved good results. These were mainly focus on the fungi Trichoderma as the representative, while research on cellulase produced by bacterium was few. In this paper, microorganisms of degradation cellulose were isolated from cattle rumen liquid, and a bacterium with high cellulase activity had been screened. Main study results as following:1.11 wide strains capable of producing cellulase were isolated from rumen liquid. To improve the traditional selective medium, the modified medium composition was 0.5% CMC, 0.2% NaNO3,0.1% K2HPO4,0.05% KC1,0.05% MgSO4-7H2O,0.0001% FeSO4,1.6% agar, which could reduce the photocopying process. The size of the hydrolysis cycle could be changed by adjusting the CMC concentration in medium.2. Strain N07 was identified as genus Cellulomonas on the basis of 16S rDNA sequence comparisons in combination with morphological, physiological and biochemical experiments, named Cellulomonas uda NJ0807.3. The fermentation conditions producing cellulase were optimized by strain Cellulomonas uda NJ0807. The BBD (Box-Behnken) Response Surface experiment was applied to optimize fermentation conditions for producing cellulase based on the single factor experiment. Under the condition: medium composition of 1% peptone,0.5% sucrose,1% CMC,0.3% NaCl,0.1% K2HPO4,0.04% MgSO4-7H2O, the initial pH 7.7,5% inoculation,23℃,180 r-min-1 for 53 hours, the cellulase activity achieved 1.35 U·mL"1.4. Cellulase produced from the strain Cellulomonas uda NJ0807 was purified by anion exchange chromatography and gel filtration chromatography based on FPLC system, obtaining three endoglucanse components. The molecular weight was estimated by SDS-PAGE, it was approximately 66 KDa,40 KDa and 31 KDa, respectively. The specific activity of purified endoglucanase components was 11.76 U-mg-1,36.4 U-mg-1 and 72.4 U·mg-1, respectively. 5. The purified 40 KDa cellulase hydrolysising CMC had an optimum temperature with 50℃and pH at 6.0. the Km value was 11.4 mg-mL-1 and Vmaxwas 224.2μmol·L-1·min-1. The enzyme was highly stable in the temperature between 40℃and 50℃, also in the pH range 5-9.