Isolation Of A Microbial Community Producing Cellulase And The Analysis Of The Enzymatic Characteristics

A microbial community was isolated through three different culture conditions,including aerobic, anaerobic and facultative anaerobic. Three fast and effective communities in facultative anaerobic condition were obtained finally. The MO of these three communities have maximal CMCase activity up to 1.36 IU.The characteristics of cellulase of the MO community were researched. The optimal reaction temperature and pH of MO's CMCase were 60℃and pH 7.0 respectively. CMCase activity was not reduced when the MO enzyme liquid was kept below 60℃or under pH 3-8.for 30 minutes. CMCase activity was inhibited by cobalt, zinc and copper and 47% of which could be reduced. Sodium and potassium ions enhanced the enzyme activity by 7% and 2% respectively. CMCase activity was 2.13 IU under this optimal reaction condition (peptone as the source of nitrogen).Carbon source, for exemple, microcrystalline cellulose (MCC), fermented saccharine sorghum stalk or crushed saccharine sorghum stalk could induce the production cellulase, while it was inhibited by CMC, crushed corn stalk and vinasse. Compared to peptone, yeast extract and beef extract as nitrogen source could enhance the cellulase activity by 2.42 and 2.37 times, respectively. While inorganic nitrogen source urea, NH4Cl and (NH4)2SO4, were harmful to cellulase production.Dynamic variation of pH, CMCase and filter paper degradation ratio during MO's culture period was studied and the relationships between them were also analyzed. The results indicated that each function strains played a different dominant role in the different growing phases. Extracellular proteins of MO were separated by native-PAGE, and were reacted with agarose gel containing CMC in-situ for 30 mins. This agarose gel was later stained by Congo red and seven activity protein bands were observed. PCR-DGGE results indicated the change of the function strain composition of MO during the cultivation.Five function bacteria were isolated on the medium with cellulose as sole carbon source. Three of them were most related to Ureibacillus thermosphaericus, Brevibacillus borstelensis and Bacillus sp. 50LAy-3 respectively according to the 16S rDNA sequences, with the similarity of 99%, 100% and 99%.