Identification And Characterization Of A Bacterium Strain With High Yield Of Cellulase

Cellulose is the most abundant regenerative resources in the nature. Cellulase, being the hydrolase of cellulose, breaks down cellulose through cooperative hydrolysis into oligosaccharide, cellobiose, and eventually the simple monosaccharide, glucose. Glucose has been broadly applied in chemical industry, food industry, animal husbandry, pharmaceutical industry and other industries. However, there still remains a challenge for the mass production of cellulase, as a becoming focus of current(?)search in both domestic and international labor(?)ries. But more and more researchers pay attention to bacterial because they growth speed, the structure is relatively simple, and the enzyme is single.Here we report the identification of a strain with especially higher productivity of cellulase, selected by CMC-Na culture medium from Yellow Mountain rotten leaves in the woods. After morphological observation, investigation of its cultivating characteristics, molecular biological identification, and construction of its phylogenic trees, we found this strain shares over95%similarity with Bacillus subtilis, and concluded its identification as Bacteria; Firmicutes; Bacillales; Bacillaceae Bacillus. Name Bacillus TZT-01.We performed composite mutation using physical and chemical mutagenesis on TZT-01protoplast, and obtained TZT-02strain with even higher productivity than the original strain. After10generations of genetic stability test on TZT-02strain, the stability of its product cellulase has increased39%compared to the original. Next, in optimization of the culture medium condition, we find that adding0.2%～0.4%glucose helps promote the cellulase production in TZT-02while addition of too much glucose would cause inhibition due to the glucose effect, which also indicates that its cellulase production is inducible. The final optimized condition for cellulase production is0.30%nitrogen source,0.10%Na2HPO4and initial pH7.0, with fluid amount of60mL/250mL fermentation time36h, and4%inoculation. In this condition, the best enzyme activity of cellulase is586.7U/mL,13%higher than that before optimization.Finally v(?)e cloned the endoglucanase gene from TZT-02genome and obtained the DNA of1470bp, which shares high homology with the published endoglucanase from bacillus subtilis. The protein product of this gene com(?)s487amino acides, with molecular weight o(?).84kD and theory isoelectric point pI7.60. Based on the domain conservation analysis, this endoglucanase can be divided into two functional groups: GH-5and CBD-Ⅲ. GH-5is mainly catalytic while CBD-Ⅲ interacts with cellulose. There is a short peptide connecting these two domains, making it possible to catalyze cellulose hydrolysis simultaneously with substrate binding. Software analysis found that its function domain structure is not always same. Upload sequence to Genbank, get a registration number JQ864356.In conlusion, after screening, identification, optimization, biochemical function and molecular biology analysis on the high-celluose-yield bacteria strain, it provides us a much clearer and more in-depth understanding about its production of cellulose. Meanwhile, facing the current status that fungi is being most broadly used as microorganisms for cellulase production, our research will undoubtedly bring a great breakthrough in and promote the area of prokaryotic cellulase production. In the future, the alkaline cellulase producing Bacillus subtilis strain can be optimized to apply in the large-scale production to improve the industrial efficiency. Moreover, further studies of the cellulase endoglucanase genes will reveal more information on the structure ar’ activity center of the protein encoded by the gene, facilitating the development of genetic transformation and optimization in the molecular biology level, for even gr(?)er improvements in the future production.