CLONING AND SEQUENCING OF CELLULASE-ENCODING GENE (cel 2) FROM LOCALLY ISOLATED BACILLUS SP. STRAIN A4

Abstract

A Bacillus bacterium, Bacillus sp. strain A-4 was isolated from the Egyptian soil and
identified according to the methods in Bergey’s Manual of Systematic Bacteriology. The
bacterial isolate, Bacillus sp. strain A-4 was a facultative anaerobic, mesophilic, sporeforming,
Gram-positive, motile, rod-shaped organism and produced catalase. Bacillus sp.
strain A-4 can degrade cellulose, hemicellulose, amylose and other forms of carbon
sources. A DNA genomic library constructed from Bacillus sp. Strain A-4 and screened
for cellulase (CMCase) activity. Recombinant β-glycosyl hydrolase activity was detected
on the basis of the clearing of hallos around Escherichia coli colons grown on a substrate
containing carboxymethylcellulose (CMC). The nucleotide sequence of the cellulase gene
(cel 2), corresponded to complete open reading frame 1167 bp that codes for a 389 amino
acid and a protein with a molecular mass of 4249Da. From sequence analysis, cel 2
belongs to glycosyl hydrolase family 5 and exhibit high similarity to engD and engO from
Clostridium cellulovorans and celB from Ruminococcus albus. The crude enzyme
preparation from Bacillus sp. A-4 exhibits activity over a broad range of pH5 to 6 and has
good stability across temperatures 50°C and pH6.