Michigan Biotechnology Institute, Lansing, Michigan 48909, and Departments of Biochemistry and Microbiology, Michigan State University, East Lansing, Michigan 48824.
Appl Environ Microbiol. 1990 Apr;56(4):881-6. doi: 10.1128/aem.56.4.881-886.1990.
A thermoanaerobe (Thermoanaerobacter sp.) grown in TYE-starch (0.5%) medium at 60 degrees C produced both extra- and intracellular pullulanase (1.90 U/ml) and amylase (1.19 U/ml) activities. Both activities were produced at high levels on a variety of carbon sources. The temperature and pH optima for both pullulanase and amylase activities were 75 degrees C and pH 5.0, respectively. Both the enzyme activities were stable up to 70 degrees C (without substrate) and at pH 4.5 to 5.0. The half-lives of both enzyme activities were 5 h at 70 degrees C and 45 min at 75 degrees C. The enzyme activities did not show any metal ion activity, and both activities were inhibited by beta- and gamma-cyclodextrins but not by alpha-cyclodextrin. A single amylolytic pullulanase responsible for both activities was purified to homogeneity by DEAE-Sepharose CL-6B column chromatography, gel filtration using high-pressure liquid chromatography, and pullulan-Sepharose affinity chromatography. It was a 450,000-molecular-weight glycoprotein composed of two equivalent subunits. The pullulanase cleaved pullulan in alpha1,6 linkages and produced multiple saccharides from cleavage of alpha-1,4 linkages in starch. The K(m)s for pullulan and soluble starch were 0.43 and 0.37 mg/ml, respectively.
在 60°C 的 TYE-淀粉（0.5%）培养基中生长的嗜热厌氧菌（Thermoanaerobacter sp.）产生了胞外和胞内普鲁兰酶（1.90 U/ml）和淀粉酶（1.19 U/ml）活性。这两种活性在多种碳源上都能高水平产生。普鲁兰酶和淀粉酶活性的最适温度和 pH 值分别为 75°C 和 pH5.0。两种酶活性在没有底物的情况下均可稳定至 70°C，在 pH4.5 至 5.0 下稳定。两种酶活性的半衰期在 70°C 下为 5 小时，在 75°C 下为 45 分钟。该酶活性不显示任何金属离子活性，并且两种活性均被β-和γ-环糊精抑制，但不受α-环糊精抑制。负责这两种活性的单一淀粉酶普鲁兰酶通过 DEAE-琼脂糖 CL-6B 柱色谱、使用高压液相色谱的凝胶过滤以及普鲁兰-Sepharose 亲和色谱被纯化为均一性。它是一种 450,000 分子量的糖蛋白，由两个相等的亚基组成。普鲁兰酶在α1,6 键上切割普鲁兰，并从淀粉的α-1,4 键切割产生多种糖。普鲁兰和可溶性淀粉的 K(m)分别为 0.43 和 0.37 mg/ml。
