Plant A R, Clemens R M, Morgan H W, Daniel R M
Microbial Biochemistry and Biotechnology Unit, School of Science, University of Waikato, Private Bag, Hamilton, New Zealand.
Biochem J. 1987 Sep 1;246(2):537-41. doi: 10.1042/bj2460537.
Thermoanaerobium Tok6-B1 pullulanase (EC 3.2.1.41) was active on alpha 1-6-glucosidic linkages of pullulan, amylopectin and glycogen and the alpha 1-4 linkages of amylose, amylopectin and glycogen but not of pullulan. Hydrolysis of short-chain-length malto-oligosaccharides (seven or fewer glucose residues) yielded maltose as product. Pullulan hydrolysis was pH-dependent and a plot of log(V/Km) versus pH implied a carboxy group with pKa 4.3 at the active site. Modification with 1-(3-dimethylaminopropyl)-3-ethylcarbodi-imide (EDAC) confirmed this view, and analysis of the order of reaction and inactivation kinetics suggested the presence of a single carboxy group at a catalytic centre of the active site. EDAC-mediated inhibition of pullulan alpha 1-6-bond hydrolysis was relieved by amylose or pullulan. Similarly both pullulan and amylose protected the activity directed at alpha 1-4 bonds of amylose from EDAC inhibition. When both amylose and pullulan were simultaneously present, the observed rate of product formation closely fitted a kinetic model in which both substrates were hydrolysed at the same active site.
嗜热厌氧菌Tok6-B1支链淀粉酶（EC 3.2.1.41）对支链淀粉、支链淀粉和糖原的α-1,6-糖苷键以及直链淀粉、支链淀粉和糖原的α-1,4键有活性，但对支链淀粉的α-1,4键无活性。短链麦芽寡糖（七个或更少葡萄糖残基）水解产生麦芽糖作为产物。支链淀粉水解依赖于pH值，log(V/Km)对pH值的作图表明活性位点存在pKa为4.3的羧基。用1-(3-二甲基氨基丙基)-3-乙基碳二亚胺（EDAC）进行修饰证实了这一观点，反应顺序和失活动力学分析表明活性位点的催化中心存在一个单一的羧基。直链淀粉或支链淀粉可解除EDAC介导的对支链淀粉α-1,6-键水解的抑制。同样，支链淀粉和直链淀粉都能保护针对直链淀粉α-1,4键的活性免受EDAC抑制。当直链淀粉和支链淀粉同时存在时，观察到的产物形成速率紧密符合一个动力学模型，即两种底物在同一活性位点水解。
