Purification and properties of an acid endo-1,4-beta-glucanase from Bacillus sp. KSM-330.
A novel acid cellulase (endo-1,4-beta-glucanase, EC 3.2.1.4) was found in a culture of Bacillus sp. KSM-330 isolated from soil. One-step chromatography on a column of CM-Bio-Gel A yielded a homogeneous enzyme, as determined by silver staining of both sodium dodecyl sulphate (SDS) and nondenaturing gels. The enzyme had a molecular mass of 42 kDa, as determined by SDS-polyacrylamide gel electrophoresis. The isoelectric point was higher than pH 10. The N-terminal amino acid sequence of the enzyme was Val-Ala-Lys-Glu-Met-Lys-Pro-Phe-Pro-Gln-Gln-Val-Asn-Tyr-Ser-Gly-Ile-Leu- Lys-Pro . This enzyme had an optimum pH for activity of 5.2, being active over an extremely narrow range of pH values, from 4.2 to 6.9; below and above these pH values no activity was detectable. The optimum temperature at pH 5.2 was around 45 degrees C. The enzyme efficiently hydrolysed carboxymethylcellulose (CMC) and lichenan, but more crystalline forms of cellulose, curdlan, laminarin, 4-nitrophenyl-beta-D-glucopyranoside and 4-nitrophenyl-beta-D-cellobioside were barely hydrolysed. The enzymic activity was inhibited by Hg2+ but was not affected by other inhibitors of thiol enzymes, such as 4-chloromercuribenzoate. N-ethylmaleimide and monoiodoacetate. N-Bromosuccinimide abolished the enzymic activity, and CMC protected the enzyme from inactivation by this tryptophan-specific oxidant. It is suggested that a tryptophan residue(s) is involved in the mechanism of action of the Bacillus cellulase and that the inhibition of enzymic activity by Hg2+ is ascribable to interactions with the tryptophan residue(s) rather than with thiol group(s).
Ozaki
K
K
Tochigi Research Laboratories, Kao Corporation, Japan.
Ito
S
S
D000595:Amino Acid Sequence; D001407:Bacillus / Q000145:classification / Q000187:drug effects / Q000201:enzymology*; D001974:Bromosuccinimide / Q000494:pharmacology; D002266:Carboxymethylcellulose Sodium / Q000378:metabolism; D002480:Cellulase / Q000737:chemistry* / Q000302:isolation & purification*; D002729:Chloromercuribenzoates / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000187:drug effects; D005033:Ethylmaleimide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013379:Substrate Specificity; D013696:Temperature; D020245:p-Chloromercuribenzoic Acid
D002729:Chloromercuribenzoates; D007461:Iodoacetates; D020245:p-Chloromercuribenzoic Acid; C089185:endoglucanase K; D002480:Cellulase; D002266:Carboxymethylcellulose Sodium; D001974:Bromosuccinimide; D005033:Ethylmaleimide; D019807:Iodoacetic Acid