Department of Biotechnology, Dong-A University, Busan, South Korea.
Appl Biochem Biotechnol. 2013 Mar;169(6):1870-83. doi: 10.1007/s12010-013-0101-x. Epub 2013 Jan 24.
The enzyme from halophilic microorganisms often has unique properties such as organic-solvent-tolerance. In this study, a novel organic-solvent-tolerant α-amylase gene was cloned from the mild halophile Exiguobacterium sp. DAU5. The open reading frame (ORF) of the enzyme consisted of 1,545 bp and encoded 514 amino acids, the primary sequence revealed that it belongs to the glycoside hydrolase (GH) family 13. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed an AmyH monomer of 57 kDa. The enzyme exhibited maximal activity at 40 °C in pH 8.5 glycine-NaOH buffer, and the activity was strongly inhibited by Zn(2+), Cu(2+), and Fe(2+). The α-amylase AmyH exhibited high hydrolysis activity toward soluble starch, and the major hydrolysis products were maltose, maltotriose, and maltopentaose; the AmyH could not efficiently hydrolyze oligosaccharides smaller than maltoheptaose, nor could it act on the β-1,4 or α-1,6 glucosidic bonds in xylan or pullulan, respectively. In addition, the α-amylase exhibited better tolerance to organic solvents, as it was stable in the presence of dimethylsulfoxide (DMSO), methanol, ethanol, and acetone. Base on all of these results, the enzyme could be useful for practical application in the bakery industry and in biotechnological processes that occur in the presence of organic solvents.
嗜盐微生物来源的酶通常具有独特的性质，例如有机溶剂耐受性。在这项研究中，从中度嗜盐菌 Exiguobacterium sp. DAU5 中克隆到了一种新型的有机溶剂耐受性α-淀粉酶基因。该酶的开放阅读框（ORF）由 1545bp 组成，编码 514 个氨基酸，其一级序列表明它属于糖苷水解酶（GH）家族 13。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）显示 AmyH 单体为 57kDa。该酶在 pH8.5 甘氨酸-氢氧化钠缓冲液中于 40°C 时表现出最大活性，活性强烈受 Zn(2+)、Cu(2+)和 Fe(2+)抑制。α-淀粉酶 AmyH 对可溶性淀粉具有很高的水解活性，主要水解产物为麦芽糖、麦芽三糖和麦芽五糖；AmyH 不能有效地水解小于麦芽七糖的寡糖，也不能分别作用于木聚糖或普鲁兰中的β-1,4 或α-1,6 糖苷键。此外，该α-淀粉酶对有机溶剂具有更好的耐受性，因为它在二甲基亚砜（DMSO）、甲醇、乙醇和丙酮存在下稳定。基于所有这些结果，该酶在烘焙工业和在有机溶剂存在下发生的生物技术过程中可能具有实际应用价值。
