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

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. Th...

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Vydané v:	Biochimica et Biophysica Acta (BBA) - General Subjects Ročník 1570; číslo 3; s. 174 - 180
Hlavní autori:	Hakamada, Yoshihiro, Endo, Keiji, Takizawa, Shuichi, Kobayashi, Tohru, Shirai, Tsuyoshi, Yamane, Takashi, Ito, Susumu
Médium:	Journal Article
Jazyk:	English
Vydavateľské údaje:	Netherlands Elsevier B.V 15.04.2002 Elsevier BV
Predmet:	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
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Shrnutí:	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.
Bibliografia:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0304-4165 0006-3002 1872-8006
DOI:	10.1016/S0304-4165(02)00194-0