Studies on Distribution of Marine Macro-algae in Europe Which Produce Novel Useful Enzymes and Their Structure-Function

海藻作为一种未开发的遗传资源，其生物化学研究很少，主要是由于技术上的困难。本研究的目的是从日本和欧洲收集的海洋巨藻中寻找新的酶。此外，我们旨在阐明一种独特的钒依赖性盐过氧化物酶（VHPO）的结构和功能。在本研究期间，我们收集了英国埃克塞特鸟取县和荷兰阿姆斯特丹附近的海藻，并测定了海藻的无细胞提取物的各种酶活性。我们发现了一种具有高磷酸酶活性的藻类。该酶的最适pH为8左右，最适温度为60 ~ 70℃，60℃孵育30 min， pH为3.5 ~ 9.0，稳定性为60% ~ 80%。此外，该酶对果糖1,6-二磷酸（53%）、葡萄糖6-磷酸（19%）、甘油醛3-磷酸（16%）和对硝基苯基磷酸（100%）表现出底物特异性。研究了毛菌VHPO的热稳定性和有机溶剂耐受性的影响因素。结果表明，holo-VHPO对四氢呋喃和乙腈以及乙醇和丙酮均有耐受性，而apo-VHPO仅对丙酮有耐受性。从VHPO的x射线分析中，我们发现了VHPO分子中的钙原子。钙原子和钒酸盐（酶的假体基）对酶的稳定性起重要作用。

Little has been done about biochemical studies on marine algae as an undeveloped genetic resource mainly because of technical difficulty. The purpose of this study is to find novel enzymes from marine macro-algae collected in Japan and Europe. Moreover we aimed to elucidate the structure and function of a unique vanadium-dependent haloperoxidase (VHPO) from a marine alga Corallina sp.During the period of this study, we collected marine algae around Tottori, Exeter, U.K., and Amsterdam, the Netherlands, and then determined various enzyme activities of cell-free extracts of the algae. We found an alga with a high phosphatase activity. The enzyme activity showed an optimal pH of about 8, optimal temperature of 60-70℃, and the 60% and 80% stabilities after the incubation at 60℃ for 30 min and at pH 3.5-9.0, respectively. Furthermore, the enzyme showed the substrate specificity toward fructose 1,6-bisphosphate (53%), glucose 6-phosphate (19%), and glyceraldehyde 3-phosphate (16%) as well as p-nitrophenylphosphate (100%).We examined the factors for thermostability and organic solvent tolerance of VHPO of C.pilulifera. As a result, holo-VHPO showed tolerance against tetrahydrofuran and acetonitrile as well as ethanol and acetone, whereas apo-VHPO showed tolerance only against acetone.From the X-ray analyses of VHPO, we found the calcium atoms in the VHPO molecule. The Ca atom and vanadate, the prosthetic group of the enzyme, were found to play an important part of the enzyme stabilities.