Enzymatic properties, crystallization, and deduced amino acid sequence of an alkaline endoglucanase from Bacillus circulans

• Published in: Biochimica et Biophysica Acta (BBA) - General Subjects Vol. 1570; no. 3; pp. 174 - 180
• Main Authors: Hakamada, Yoshihiro, Endo, Keiji, Takizawa, Shuichi, Kobayashi, Tohru, Shirai, Tsuyoshi, Yamane, Takashi, Ito, Susumu
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15.04.2002; Elsevier BV
• Subjects: Amino Acid Sequence; Bacillus; Bacillus - enzymology; Base Sequence; Carboxymethylcellulose Sodium; Carboxymethylcellulose Sodium - metabolism; Cellulase; Cellulase - chemistry; Cellulase - genetics; Cloning; Crystallization; Crystallography, X-Ray; Family 8; Glycosyl hydrolase; Hydrogen-Ion Concentration; Hydrolysis; Isoelectric Point; Lichenase; Molecular Sequence Data; Molecular Weight; Open Reading Frames; Protein Conformation; Sequence Alignment; aa; Cloning; nt; Crystallization; Glycosyl hydrolase; PAGE; Egl; Cellulase; Family 8; Bacillus; Lichenase; CMC; p I; PCR
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/S0304-4165(02)00194-0

A high-isoelectric-point (p I), alkaline endo-1,4-β-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 °C. The molecular mass was 43 kDa, and the p I 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-β-glucanase. However, this enzyme was not active on p-nitrophenyl β- d-cellotrioside and p-nitrophenyl β- 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 Å resolution. It belongs to the space group P2 12 12 1 with unit cell parameters of a=62.5 Å, b=71.7 Å, and c=88.6 Å.