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KMID : 0525720150200040267
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Journal of Chitin and Chitosan
2015 Volume.20 No. 4 p.267 ~ p.272
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Characterization of Cellulase from Bacillus subtilis KB1 Isolated from Environmental Soil and Determination of Its Gene
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Kwon Eun-Ryeng
Park Chang-Su
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Abstract
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We isolated a strain producing celluase from environmental soil. The strain was formed activity zone on the LB agar plate containing carboxymethylcellulose (CM-cellulose) stained with trypan blue as substrate with its cellulase activity. In the analysis of the 16S rRNA gene sequence, it was identified as Bacillus subtilis and was named as Bacillus subtilis KB1. The B. subtilis KB1 cellulase showed the maximum activity at pH 5.0 and 50°C. The half-lives of the enzyme at 30, 40, 50, 60 and 70°C were 5,404, 133, 37, 24, and 6 min, respectively. In the investigation of substrate specificity, B. subtilis KB1 cellulase showed the most high enzyme activity for the CM-cellulose substrate. To clone the cellulase gene from B. subtilis KB1, the cellulase gene was amplified from genomic DNA of the B. subtilis KB1 with designed primers based on the cellulase gene from Bacillus subtilis (GenBank: AGN52749) by PCR. In the analysis of the cellulase gene sequence, it was confirmed that the cloned cellulase gene was consisted of a 1,500 bp open reading frame (ORF) encoding a 499 amino acid protein with calculated molecular mass of 55,251 Da. The deduced amino acid sequences from cellulase gene showed high identity with glycosyl hydrolases family (GH) 1.
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KEYWORD
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Bacillus subtilis, Screening, Cellulase, Characterization, Cloning
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