Kim Dae Hoon, Morimoto Naoki, Saburi Wataru, Mukai Atsushi, Imoto Koji, Takehana Toshihiko, Koike Seiji, Mori Haruhide, Matsui Hirokazu
Research Faculty of Agriculture, Hokkaido University, Sapporo, Japan.
Biosci Biotechnol Biochem. 2012;76(7):1378-83. doi: 10.1271/bbb.120164. Epub 2012 Jul 7.
α-Amylase (EC 3.2.1.1) hydrolyzes an internal α-1,4-glucosidic linkage of starch and related glucans. Alkalophilic liquefying enzymes from Bacillus species are utilized as additives in dishwashing and laundry detergents. In this study, we found that Bacillus sp. AAH-31, isolated from soil, produced an alkalophilic liquefying α-amylase with high thermostability. Extracellular α-amylase from Bacillus sp. AAH-31 (AmyL) was purified in seven steps. The purified enzyme showed a single band of 91 kDa on SDS-PAGE. Its specific activity of hydrolysis of 0.5% soluble starch was 16.7 U/mg. Its optimum pH and temperature were 8.5 and 70 °C respectively. It was stable in a pH range of 6.4-10.3 and below 60 °C. The calcium ion did not affect its thermostability, unlike typical α-amylases. It showed 84.9% of residual activity after incubation in the presence of 0.1% w/v of EDTA at 60 °C for 1 h. Other chelating reagents (nitrilotriacetic acid and tripolyphosphate) did not affect the activity at all. AmyL was fully stable in 1% w/v of Tween 20, Tween 80, and Triton X-100, and 0.1% w/v of SDS and commercial detergents. It showed higher activity towards amylose than towards amylopectin or glycogen. Its hydrolytic activity towards γ-cyclodextin was as high as towards short-chain amylose. Maltotriose was its minimum substrate, and maltose and maltotriose accumulated in the hydrolysis of maltooligosaccharides longer than maltotriose and soluble starch.
α-淀粉酶（EC 3.2.1.1）可水解淀粉及相关葡聚糖内部的α-1,4-糖苷键。芽孢杆菌属的嗜碱液化酶被用作洗碗和洗衣洗涤剂的添加剂。在本研究中，我们发现从土壤中分离出的芽孢杆菌属AAH-31菌株产生了一种具有高热稳定性的嗜碱液化α-淀粉酶。芽孢杆菌属AAH-31菌株（AmyL）的胞外α-淀粉酶经过七步纯化。纯化后的酶在SDS-PAGE上显示出一条91 kDa的单条带。其水解0.5%可溶性淀粉的比活性为16.7 U/mg。其最适pH和温度分别为8.5和70℃。它在pH 6.4 - 10.3范围内以及60℃以下稳定。与典型的α-淀粉酶不同，钙离子不影响其热稳定性。在60℃下于0.1% w/v的EDTA存在下孵育1小时后，它显示出84.9%的残余活性。其他螯合剂（次氮基三乙酸和三聚磷酸钠）对活性完全没有影响。AmyL在1% w/v的吐温20、吐温80和Triton X-100以及0.1% w/v的SDS和市售洗涤剂中完全稳定。它对直链淀粉的活性高于对支链淀粉或糖原的活性。其对γ-环糊精的水解活性与对短链直链淀粉的水解活性一样高。麦芽三糖是其最小底物，在水解比麦芽三糖和可溶性淀粉更长的麦芽寡糖时会积累麦芽糖和麦芽三糖。
