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Studies on the enzymatic production of nucleosides in the activated methyl cycle

活化甲基循环中酶促生产核苷的研究

基本信息

批准号：
62470123
负责人：
YAMADA Hideaki
金额：
$ 4.29万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

项目摘要

We have developed a simple and efficient method for the production of AdoHcy and related nucleosides from L-homocysteine and corresponding nucleosides with A. faecalis cells as catalysts.However, the involvement of an enzyme activity that can catalyze the interconversion of the D- and L-homocysteine isomers in the reaction system with A. faecalis cells seems very promising as a means of solving this problem. Based on this idea, we again screened microorganisms capable of utilizing both the D- and L-homocysteine isomers as substrater. As a result of this screening, Pseudomonas putida IFO 12996 and several other bacterial strains were found to act as catalysts for the conversion of adenosine and D-homocysteine to the L-form of Adohcy. For practical purposes,though, A. faecalis cells were still superior to P. putida cells because they showed high AdoHcy hydrolase activity and low adenosine deaminase activity. Therefore, we used P. putida cells only as a source of the racemase. By mixing adenosine and D,L-homocysteine with a mixture of A. faecalis cells and P. putida cells in potassium phosphate butter, we essentially obtained a 100% yield.In a similar manner, several novel S-nucleosidylhomocysteines could be synthesized efficiently from the corresponding adenosine analogues and L-homocysteine. The K-carrageean entrapped cells of A. faecalis showed high stability on repeated reactions, whereas free cells were inactivated rapidly. These novel S-nucleosidylhomocysteines alsocould be promising for application in the pharmaceutical and chemotherapeutic fields.

我们建立了一种简单有效的方法，以L-同型半胱氨酸和相应的核苷为原料，用A.然而，可以催化D-和L-同型半胱氨酸异构体在与A.粪便细胞似乎很有希望作为解决这个问题的手段。基于这个想法，我们再次筛选能够利用D-和L-同型半胱氨酸异构体作为底物的微生物。作为筛选的结果，发现恶臭假单胞菌IFO 12996和几种其他细菌菌株作为催化剂用于腺苷和D-高半胱氨酸转化为L-型腺苷。但实际上，A。粪肠球菌细胞仍然上级恶臭假单胞菌细胞，因为它们显示出高的Hcy水解酶活性和低的腺苷脱氨酶活性。因此，我们仅使用恶臭假单胞菌细胞作为消旋酶的来源。通过将腺苷和D，L-高半胱氨酸与A.在磷酸钾缓冲液中，利用腺苷类似物和L-同型半胱氨酸合成了几种新的S-核苷酰同型半胱氨酸。K-卡拉胶包埋A.粪肠球菌在重复反应中表现出高稳定性，而游离细胞迅速失活。这些新的S-核苷酰同型半胱氨酸在药物和化学治疗领域也有很好的应用前景。