title: Deduced amino acid sequence and possible catalytic residues of a thermostable, alkaline cellulase from an Alkaliphilic bacillus strain.
abstract: Alkaliphilic Bacillus sp. strain KSM-S237 (a relative of Bacillus pseudofirmus) produces a thermostable, alkaline endo-1,4-beta-glucanase (Egl). The entire gene for the enzyme harbored a 2,472-bp open reading frame (ORF) encoding 824 amino acids, including a 30-aminoacid signal peptide. The deduced amino acid sequence of the mature enzyme (794 amino acids, 88,284 Da) showed very high similarity to those of family 5 mesophilic, alkaline Egls from some alkaliphilic bacilli. The enzyme had a region similar to a novel cellulose binding domain proposed for an Egl (EngF) from Clostridium cellulovorans. Expression of the Bacillus Egl gene in Bacillus subtilis resulted in high carboxymethy cellulase activity (2.0 g/l) in the culture broth, concomitant with the appearance of a protein band on an SDS gel at 86 kDa. Site-directed mutagenesis delineated the importance of Arg111, His151, Glu190, His262, Tyr264, and Glu305 in catalysis and/or substrate binding of the enzyme.
authors.affiliation: Tochigi Research Laboratories of Kao Corporation, Haga, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Hakamada
authors.forename: Y
authors.initials: Y
authors.lastname: Hatada
authors.forename: K
authors.initials: K
authors.lastname: Koike
authors.forename: T
authors.initials: T
authors.lastname: Yoshimatsu
authors.forename: S
authors.initials: S
authors.lastname: Kawai
authors.forename: T
authors.initials: T
authors.lastname: Kobayashi
authors.forename: S
authors.initials: S
authors.lastname: Ito
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins; D001483:Base Sequence; D020134:Catalytic Domain; D002480:Cellulase / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial / Q000032:analysis; D004795:Enzyme Stability; D019143:Evolution, Molecular; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; D002480:Cellulase; C421179:Egl-237 protein, Bacillus