Liebl W, Wagner B, Schellhase J
Lehrstuhl für Mikrobiologie, Technische Universität München, Germany.
Syst Appl Microbiol. 1998 Mar;21(1):1-11. doi: 10.1016/s0723-2020(98)80002-7.
Thermotoga maritima represents one of the few hyperthermophilic bacteria currently known. The chromosomal alpha-galactosidase gene of T. maritima strain MSB8 has been cloned and its nucleotide sequence was determined. The gene, designated galA, has coding capacity for a 552 residue polypeptide with a calculated molecular mass of 63,653 Da. GalA was found to be flanked by other genes probably involved in galactoside breakdown and utilization. The previously sequenced beta-galactosidase gene, lacZ, is localized immediately upstream of galA while two open reading frames that putatively encode enzymes of galactose catabolism, i.e. galactose-1-phosphate uridylytransferase (galT) and galactokinase (galK), were found downstream of galA. The identified genes are extremely close together or even overlap and have the same orientation, so they could all be part of one galactoside utilization operon of T. maritima MSB8. GalA displayed low-level amino acid sequence similarity with alpha-galactoside of glycosyl hydrolase family 36. However, GalA is smaller than the other members of this enzyme family. The galA gene was expressed in Escherichia coli and the recombinant alpha-galactosidase was purified and characterized. The molecular mass of the recombinant enzyme was estimated at about 62 kDa by denaturting gel electrophoresis. Maximal hydrolysis of the chromogenic substrate p-nitrophenyl-alpha-D-galactopyranoside was measured at pH 5.0-5.5 and 90-95 degrees C (5 min assay). Divalent cations were not required for activity. The enzyme released galactose from raffinose, melibiose and the synthetic substrates p-nitrophenyl-and omicron-nitrophenyl-alpha-D-galactopyranoside. The T. maritima alpha-galactosidase thus was highly specific for the galactose moiety and the alpha-anomeric configuration of the glycosidic linkage. Its extreme thermal stability (t 1/2 = 6.5 h at 85 degrees C) makes this enzyme an interesting candidate for biotechnological applications.
嗜热栖热菌是目前已知的少数几种超嗜热细菌之一。嗜热栖热菌MSB8菌株的染色体α-半乳糖苷酶基因已被克隆，并测定了其核苷酸序列。该基因命名为galA，编码一个由552个氨基酸残基组成的多肽，计算分子量为63,653道尔顿。发现GalA两侧还有其他可能参与半乳糖苷分解和利用的基因。先前测序的β-半乳糖苷酶基因lacZ位于galA的紧上游，而在galA下游发现了两个推测编码半乳糖分解代谢酶的开放阅读框，即半乳糖-1-磷酸尿苷酰转移酶（galT）和半乳糖激酶（galK）。所鉴定的基因彼此靠得极近甚至重叠，且方向相同，因此它们可能都是嗜热栖热菌MSB8半乳糖苷利用操纵子的一部分。GalA与糖基水解酶家族36的α-半乳糖苷显示出低水平的氨基酸序列相似性。然而，GalA比该酶家族的其他成员小。galA基因在大肠杆菌中表达，重组α-半乳糖苷酶被纯化并进行了特性分析。通过变性凝胶电泳估计重组酶的分子量约为62 kDa。在pH 5.0 - 5.5和90 - 95℃（5分钟测定）条件下，测定了生色底物对硝基苯基-α-D-吡喃半乳糖苷的最大水解率。该酶活性不需要二价阳离子。该酶从棉子糖、蜜二糖以及合成底物对硝基苯基-α-D-吡喃半乳糖苷和邻硝基苯基-α-D-吡喃半乳糖苷中释放出半乳糖。因此，嗜热栖热菌α-半乳糖苷酶对半乳糖部分和糖苷键的α-异头构型具有高度特异性。其极高的热稳定性（85℃下t1/2 = 6.5小时）使该酶成为生物技术应用中一个有趣的候选对象。
