Huang H S, Ito K, Yin C H, Kabashima T, Yoshimoto T
School of Pharmaceutical Sciences, Nagasaki University, Japan.
Biosci Biotechnol Biochem. 1998 Dec;62(12):2375-81. doi: 10.1271/bbb.62.2375.
Glycerol kinase (EC 2.7.1.30) is a key enzyme of glycerol uptake and metabolism in bacteria. Using PCR, we amplified and cloned a glycerol kinase gene, glpK, from Thermus aquaticus. The complete gene has 1488 base pairs, coding for a protein of 496 amino acids with a predicted molecular weight of 54,814. The amino acid sequence deduced from T. aquaticus glpK was found to have identities of 97 and 81%, respectively, with those of Thermus flavus and Bacillus subtilis glpK genes. After overproduction in Escherichia coli, the expressed enzyme was easily purified to homogeneity by DEAE-Toyopearl chromatography. The purified enzyme has been crystallized by the hanging drop vapor diffusion method at 22 degrees C. Comparison of the amino acid sequence with that of the B. subtilis enzyme showed that Ser and Lys are replaced by Ala and Arg, as was seen in mesophile and thermophile enzymes.
甘油激酶（EC 2.7.1.30）是细菌中甘油摄取和代谢的关键酶。我们利用聚合酶链式反应（PCR）从嗜热水栖菌中扩增并克隆了一个甘油激酶基因glpK。该完整基因有1488个碱基对，编码一个由496个氨基酸组成的蛋白质，预测分子量为54814。结果发现，嗜热水栖菌glpK推导的氨基酸序列与嗜热栖热菌和枯草芽孢杆菌的glpK基因的氨基酸序列分别具有97%和81%的同源性。在大肠杆菌中过量表达后，通过DEAE - Toyopearl层析法可轻松将表达的酶纯化至同质。纯化后的酶已通过悬滴气相扩散法在22℃下结晶。将该氨基酸序列与枯草芽孢杆菌的酶进行比较，结果表明，如同在嗜温酶和嗜热酶中所见，丝氨酸和赖氨酸分别被丙氨酸和精氨酸取代。
