Vihinen M, Mäntsälä P
Department of Biochemistry, University of Turku, Finland.
Biotechnol Appl Biochem. 1990 Aug;12(4):427-35.
Liquefying-type Bacillus stearothermophilus alpha-amylase was characterized. The coding gene was cloned in Bacillus subtilis and the enzyme was produced in three different host organisms: B. stearothermophilus, B. subtilis, and Escherichia coli. Properties of the purified enzyme were similar irrespective of the host. Temperature optimum was at 70-80 degrees C and pH optimum at 5.0-6.0. The enzyme was stable for 1 h in the pH range 6.0-7.5 at 80 degrees C. The enzyme was stabilized by Ca2+, Na+, and bovine serum albumin. About 50% of the activity remained after heating at 70 degrees C for 5 days or 45 min at 90 degrees C. Metal ions Cd2+, Cu2+, Hg2+, Pb2+, and Zn2+ were inhibitory, whereas EDTA, ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid, and Tendamistat were without effect. The enzyme was fully active after treatment in acetone or ethanol at 55 or 70 degrees C, respectively, for 30 min. Sodium dodecyl sulfate (1%) did not affect stability, whereas 6 M urea denatured totally at 70 degrees C. The Km value for soluble starch was 14 mg/ml. Mr is 59,000 and pI 8.8. The only difference between the enzymes produced in different hosts was in signal peptide processing.
对液化型嗜热脂肪芽孢杆菌α-淀粉酶进行了特性鉴定。将编码基因克隆到枯草芽孢杆菌中，并在三种不同的宿主生物中生产该酶：嗜热脂肪芽孢杆菌、枯草芽孢杆菌和大肠杆菌。无论宿主如何，纯化酶的性质相似。最适温度为70-80℃，最适pH为5.0-6.0。该酶在80℃下于pH 6.0-7.5范围内稳定1小时。Ca2+、Na+和牛血清白蛋白可使该酶稳定。在70℃加热5天或在90℃加热45分钟后，仍保留约50%的活性。金属离子Cd2+、Cu2+、Hg2+、Pb2+和Zn2+具有抑制作用，而EDTA、乙二醇双（β-氨基乙醚）N,N,N',N'-四乙酸和抑肽酶则无影响。该酶分别在55℃或70℃下于丙酮或乙醇中处理30分钟后仍具有完全活性。1%的十二烷基硫酸钠不影响稳定性，而6 M尿素在70℃下完全变性。可溶性淀粉的Km值为14 mg/ml。分子量为59,000，等电点为8.8。不同宿主产生的酶之间的唯一差异在于信号肽加工。
