title: Enzymatic properties, crystallization, and deduced amino acid sequence of an alkaline endoglucanase from Bacillus circulans.
abstract: A high-isoelectric-point (pI), alkaline endo-1,4-beta-glucanase (Egl-257) of Bacillus circulans KSM-N257 was purified to homogeneity and crystallized. The purified enzyme hydrolyzed carboxymethyl cellulose (CMC) with optima of pH 8.5 and 55 degrees C. The molecular mass was 43 kDa, and the pI was pH 9.3. The structural gene contained a single open reading frame of 1221 bp, corresponding to 407 amino acids (aa), including a 30-aa signal peptide (377 aa and 41,680 Da for the mature enzyme). Egl-257 hydrolyzed lichenan and showed 76.3% aa identity to a lichenase from B. circulans WL-12 belonging to glycosyl hydrolase family 8 but did not hydrolyze laminarin, curdran, and xylan at all. This indicates that Egl-257 is a true endo-1,4-beta-glucanase. However, this enzyme was not active on p-nitrophenyl beta-D-cellotrioside and p-nitrophenyl beta-D-cellotetraoside. It was crystallized by the hanging-drop vapor-diffusion method with phosphate plus CdCl(2) as precipitant. Pyramid-like crystals were formed, and they diffracted X-rays beyond 2.2 A resolution. It belongs to the space group P2(1)2(1)2(1) with unit cell parameters of a=62.5 A, b=71.7 A, and c=88.6 A.
authors.affiliation: Tochigi Research Laboratories of Kao Corporation, 2606, Akabane, Ichikai, Haga, Tochigi 321-3497, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Hakamada
authors.forename: Keiji
authors.initials: K
authors.lastname: Endo
authors.forename: Shuichi
authors.initials: S
authors.lastname: Takizawa
authors.forename: Tohru
authors.initials: T
authors.lastname: Kobayashi
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Shirai
authors.forename: Takashi
authors.initials: T
authors.lastname: Yamane
authors.forename: Susumu
authors.initials: S
authors.lastname: Ito
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001483:Base Sequence; D002266:Carboxymethylcellulose Sodium / Q000378:metabolism; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics; D018360:Crystallography, X-Ray; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D011487:Protein Conformation; D016415:Sequence Alignment
chemical_list: D002480:Cellulase; D002266:Carboxymethylcellulose Sodium