Igarashi K, Hatada Y, Hagihara H, Saeki K, Takaiwa M, Uemura T, Ara K, Ozaki K, Kawai S, Kobayashi T, Ito S
Tochigi Research Laboratories of Kao Corporation, Ichikai, Haga, Tochigi 321-3497, Japan.
Appl Environ Microbiol. 1998 Sep;64(9):3282-9. doi: 10.1128/AEM.64.9.3282-3289.1998.
A novel liquefying alpha-amylase (LAMY) was found in cultures of an alkaliphilic Bacillus isolate, KSM-1378. The specific activity of purified LAMY was approximately 5,000 U mg of protein-1, a value two- to fivefold greater between pH 5 and 10 than that of an industrial, thermostable Bacillus licheniformis enzyme. The enzyme had a pH optimum of 8.0 to 8.5 and displayed maximum activity at 55 degreesC. The molecular mass deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approximately 53 kDa, and the apparent isoelectric point was around pH 9. This enzyme efficiently hydrolyzed various carbohydrates to yield maltotriose, maltopentaose, maltohexaose, and maltose as major end products after completion of the reaction. Maltooligosaccharides in the maltose-to-maltopentaose range were unhydrolyzable by the enzyme. The structural gene for LAMY contained a single open reading frame 1, 548 bp in length, corresponding to 516 amino acids that included a signal peptide of 31 amino acids. The calculated molecular mass of the extracellular mature enzyme was 55,391 Da. LAMY exhibited relatively low amino acid identity to other liquefying amylases, such as the enzymes from B. licheniformis (68.9%), Bacillus amyloliquefaciens (66.7%), and Bacillus stearothermophilus (68.6%). The four conserved regions, designated I, II, III, and IV, and the putative catalytic triad were found in the deduced amino acid sequence of LAMY. Essentially, the sequence of LAMY was consistent with the tertiary structures of reported amylolytic enzymes, which are composed of domains A, B, and C and which include the well-known (alpha/beta)8 barrel motif in domain A.
在嗜碱芽孢杆菌分离株KSM - 1378的培养物中发现了一种新型液化α -淀粉酶（LAMY）。纯化后的LAMY比活性约为5000 U mg蛋白-1，在pH 5至10之间，该数值比工业用嗜热栖热芽孢杆菌的耐热酶高两到五倍。该酶的最适pH为8.0至8.5，在55℃时表现出最大活性。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳推导的分子量约为53 kDa，表观等电点约为pH 9。该酶能有效水解各种碳水化合物，反应完成后产生麦芽三糖、麦芽五糖、麦芽六糖和麦芽糖作为主要终产物。麦芽糖至麦芽五糖范围内的麦芽寡糖不能被该酶水解。LAMY的结构基因包含一个长度为1548 bp的单一开放阅读框，对应于516个氨基酸，其中包括一个31个氨基酸的信号肽。计算得出的细胞外成熟酶分子量为55391 Da。LAMY与其他液化淀粉酶，如地衣芽孢杆菌（68.9%）、解淀粉芽孢杆菌（66.7%）和嗜热栖热芽孢杆菌（68.6%）的酶相比，氨基酸同一性相对较低。在LAMY的推导氨基酸序列中发现了四个保守区域，分别命名为I、II、III和IV，以及推定的催化三联体。本质上，LAMY的序列与已报道的淀粉分解酶的三级结构一致，这些结构由结构域A、B和C组成，并且在结构域A中包括著名的(α/β)8桶基序。
