title: A cellulase gene from a new alkalophilic Bacillus sp. (strain N186-1). Its cloning, nucleotide sequence and expression in Escherichia coli.
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 degrees C and it retains more than 70% of its activity after incubation for 1 h at pH 12.
authors.affiliation: Departamento de Microbiología y Genética, Consejo Superior de Investigaciones Cientificas/Universidad de Salamanca, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Sánchez-Torres
authors.forename: P
authors.initials: P
authors.lastname: Pérez
authors.forename: R I
authors.initials: RI
authors.lastname: Santamaría
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001483:Base Sequence; D002480:Cellulase / Q000235:genetics*; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D017386:Sequence Homology, Amino Acid
chemical_list: D004269:DNA, Bacterial; D002480:Cellulase