The Screening And Identification Of Cellulose-degrading Strain And Its Properties Of Cellulase

Tibetan pig, the only plateau type and grazing type miniature pig in China, hold a richerand more complex gut microflora compared with domestic Duroc pig and Chinese domesticpig. These characteristics make the research towards cellulase-producing microorganismsfrom gastrointestinal tract of Tibetan pig possessing more scientific values and practicalsignificances in development of grain saving swine industry. The aims of this study were thescreening of a cellulase-producing bacterium from gastrointestinal tract（GI） of Tibetan pig,purification and characterization of the cellulase. The main conclusions are as follows:1. A cellulose-degrading bacterium originally isolated from gastrointestinal (GI) tract ofTibetan pig was identified as Bacillus subtilis BY-4by morphological feature and16S rDNAanalysis.2. Strain BY-4was found competent in utilizing wheat bran, wheat straw, and corn stoveras energy sources and inducer to multiply and produce cellulase. The highest CMCase activityof strain BY-4was achieved in the present of wheat bran up to8.6U/mL, followed by wheatstraw (7.1U/mL) after48h cultivation. The cellulase had relaxed substrate specificityespecially wheat bran and wheat straw, with the relative activities of108.1±14.2、90.3±5.3.41.76%of relative activity was also found from avicel. Scanning electron microscopy (SEM)certificated the structural and morphological changes occurred during the hydrolysis ofcellulase against wheat straw and wheat bran.3. Cellulase was purified through HiTrap Phenyl HP hydrophobic chromatography andHiPrep16/60Sephacryl S-100gel filtration chromatography, generated8.9fold purifiedcellulase with23.7%recovery. The specific activity increased from0.95U/mL to14.7U/mL.The molecular weight of the purified cellulase was estimated to be55kDa by SDS-PAGE andzymogram analysis.4. The enzymatic properties revealed that the optimal values of pH and temperature on cellulase response were4.5and60℃respectively. The pH stability of the cellulase showedthat the enzyme was stable under acidic condition and remained90%of its original activityover pH range between4and6.5. The cellulase can be activated by Mg2+and Zn2+, whileinhibited by Mn2+and Co2+. Notably, intensive inhibitory action against Staphylococcusaureus was observed in crude enzyme with inhibition zone of15.24±0.15mm.5. A cellulase gene, showed a GH5-CBM3modular organization, was successfullycloned from the strain BY-4. The open reading frame (ORF) of the gene was1500bp,encoding499amino acid redidues with a molecular weight of55.21kDa.This research is very important for the revelation of mechanisms of roughage utilizationby pigs and can contribute to the development of grain saving swine industry.