嗜热脂肪芽孢杆菌中葡萄糖激酶的纯化与特性分析
The purification and characterization of glucokinase from the thermophile Bacillus stearothermophilus.
作者信息
Goward C R, Hartwell R, Atkinson T, Scawen M D
出版信息
Biochem J. 1986 Jul 15;237(2):415-20. doi: 10.1042/bj2370415.
Abstract
Homogeneous glucokinase (EC 2.7.1.2) from the thermophile Bacillus stearothermophilus was isolated on the large scale by using four major steps: precipitation of extraneous material at pH 5.5, ion-exchange chromatography on DEAE-Sepharose, pseudo-affinity chromatography on Procion Brown H-3R-Sepharose 4B and gel filtration on Ultrogel AcA 34. The purified enzyme had a specific activity of about 330 units/mg of protein and was shown to exist as a dimer of subunit Mr 33,000. Kinetic parameters for the enzyme were determined with a variety of substrates. The glucokinase was highly specific for alpha-D-glucose, and the only other sugar substrate utilized was N-acetyl-alpha-D-glucosamine. The enzyme shows Michaelis-Menten kinetics, with a Km value of 150 microM for alpha-D-glucose. The glucokinase was maximally active at pH 9.0.
摘要
嗜热脂肪芽孢杆菌的均一葡萄糖激酶（EC 2.7.1.2）通过四个主要步骤进行大规模分离：在pH 5.5下沉淀杂质、在DEAE-琼脂糖上进行离子交换色谱、在Procion Brown H-3R-琼脂糖4B上进行假亲和色谱以及在Ultrogel AcA 34上进行凝胶过滤。纯化后的酶比活性约为330单位/毫克蛋白质，显示以Mr 33,000亚基的二聚体形式存在。用多种底物测定了该酶的动力学参数。该葡萄糖激酶对α-D-葡萄糖具有高度特异性，唯一利用的其他糖类底物是N-乙酰-α-D-葡萄糖胺。该酶呈现米氏动力学，对α-D-葡萄糖的Km值为150微摩尔。葡萄糖激酶在pH 9.0时活性最高。
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