Ozaki K, Ito S
Tochigi Research Laboratories, Kao Corporation, Japan.
J Gen Microbiol. 1991 Jan;137(1):41-8. doi: 10.1099/00221287-137-1-41.
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).
从土壤中分离得到的芽孢杆菌属KSM - 330培养物中发现了一种新型酸性纤维素酶（内切 - 1,4 - β - 葡聚糖酶，EC 3.2.1.4）。在CM - Bio - Gel A柱上进行一步层析得到了一种均一的酶，这通过十二烷基硫酸钠（SDS）凝胶和非变性凝胶的银染得以确定。通过SDS - 聚丙烯酰胺凝胶电泳测定，该酶的分子量为42 kDa。其等电点高于pH 10。该酶的N端氨基酸序列为Val - Ala - Lys - Glu - Met - Lys - Pro - Phe - Pro - Gln - Gln - Val - Asn - Tyr - Ser - Gly - Ile - Leu - Lys - Pro。这种酶的最适pH值为5.2，在极其狭窄的pH值范围内（4.2至6.9）有活性；在这些pH值以下和以上均未检测到活性。在pH 5.2时的最适温度约为45℃。该酶能有效水解羧甲基纤维素（CMC）和地衣多糖，但对结晶度更高的纤维素、凝胶多糖、海带多糖、4 - 硝基苯基 - β - D - 吡喃葡萄糖苷和4 - 硝基苯基 - β - D - 纤维二糖苷几乎不水解。酶活性受到Hg2 + 的抑制，但不受其他巯基酶抑制剂（如4 - 氯汞苯甲酸、N - 乙基马来酰亚胺和碘乙酸）的影响。N - 溴代琥珀酰亚胺使酶活性丧失，而CMC可保护该酶不被这种色氨酸特异性氧化剂灭活。这表明一个或多个色氨酸残基参与了芽孢杆菌纤维素酶的作用机制，并且Hg2 + 对酶活性的抑制归因于与色氨酸残基的相互作用而非巯基。
