Abstract
Several alkalophilic Bacillus spp. strains were selected for their capacity to produce alkaline cellulases. Culture supernatants of these strains showed optimal cellulase activities between pH 8 and 9 and they were stable from pH 6 to pH 12. A cellulase gene (celB1) from the alkalophilic Bacillus sp. strain N186-1 was cloned in Escherichia coli using polymerase chain reaction techniques. The cloned gene was present in a 2.539-bp HindIII fragment and its nucleotide sequence was determined. The coding sequence showed an open-reading frame encoding 389 amino acids. The amino acid sequence, deduced from the nucleotide sequence, permitted us to include it in family 5 (or A) of the glycosyl hydrolases. The complete open-reading frame of celB1 was cloned in the plasmid pET-11d and expressed in E. coli BL21 (DE3), in which a protein of 39 kDa was obtained in the cytoplasm; however, no endoglucanase activity was detected. A second construction in pET-12a allowed the production of a 39-kDa protein located in the periplasmic space of E. coli that had endoglucanase activity. The protein produced has optimal activity at pH 7 and 50°C and it retains more than 70% of its activity after incubation for 1 h at pH 12.
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Received: 27 December 1995/Received revision: 14 March 1996/Accepted: 25 March 1996
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Sánchez-Torres, J., Pérez, P. & Santamaría, R. A cellulase gene from a new alkalophilic Bacillus sp. (strain N186-1). Its cloning, nucleotide sequence and expression in Escherichia coli . Appl Microbiol Biotechnol 46, 149–155 (1996). https://doi.org/10.1007/s002530050797
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DOI: https://doi.org/10.1007/s002530050797