Screening Endoglucanase-producing Bacillus C-36 And Cloning, Sequence Analysis Of Its Gene

One strain of endoglucanase-producing Bucillus which can degrade sodium carbox-ymethylcellulose was isolated on the selected medium using CMC-Na as only carbon source from the mud. The strain had high cellulase producing ability proved by the congored dying method on the culture plates. C-36 is Gram positive after it was cultured twenty hours under the microscope. The young cell is most double grow or chain shape. The cell is straight not bend, (2.2~3.0)um long and (0.8~0.9)um tall. The cell and can form middle, closer middle spore. The spore is egg-round, (1.1~1.4)um long and (0.8~0.9)um tall, doesn't make sporangium big. In CMC-Na plate, the colonies are ivory, dense and mass, wet and bright, grow rapidly, can see distinctly transparent circle after 24h. It is identified as Bacillus subilius according to its morphological, physiological and biochemiscal characteristics. It is revealed by the experiments that C-36 grows in a broad range of pH.,while the optimum pH for its growing is pH7.0, and it also grows well in more alkaline circumstances. Results in shaking flask culture showed that high cellulase activity (CMCase) could be detected in the condition of pH6.8, and the cellulase activity is 79.2U/mL, More than sixty-five percent of the full activity remains at pH9.0. The optimal condition for the enzymatic reactions is 50℃, and pH value is about 6.8.According to the Bacilius endoglucanase gene sequence downloaded from GenBank, a pair of primer was designed to amplify the full sequence of endoglucanase gene from B.subtilis C-36 by the polymersase chain reaction(PCR). The PCR product was cloned and sequenced. The sequence analysis results showed the full gene was 1500bp in length, and it coded a 499 amino acid multi-peptide. The gene was accepted by GenBank with accession number DQ782954.Comparison of this sequence with the endoglucanase gene of the others B.subtilis (Genbank Accession: Z29076.1, X04689.1, AY044252.1, X67044.1, AY766381.1), shows that the nucleotide homology is as high as 90%~93%, and the amino acid (Genbank Acc -ession: CAA82317, CAA97610,AAK94871,AAZ22322, AAN07019) homology comes up...