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

我们已经开发出一种简单而有效的方法，以粪霉细胞为催化剂，以L-同型半胱氨酸和相应的核苷为原料生产同型半胱氨酸和相关核苷，但在与粪霉细胞的反应体系中引入一种酶活性来催化D-同型半胱氨酸和L-同型半胱氨酸异构体的相互转化，似乎是解决这一问题的一种非常有希望的方法。基于这一思想，我们再次筛选出既能利用D-同型半胱氨酸异构体又能利用L同型半胱氨酸异构体的微生物。作为筛选的结果，恶臭假单胞菌IFO 12996和其他几个细菌菌株被发现对腺苷和D-同型半胱氨酸转化为L式的ADOhcy起到催化剂作用。但在实际应用中，粪假单胞菌仍优于恶臭假单胞菌，因为它们表现出较高的ADHcy水解酶活性和较低的腺苷脱氨酶活性。因此，我们只使用恶臭假单胞菌作为消旋酶的来源。通过将腺苷和D，L-同型半胱氨酸与粪便假丝酵母细胞和恶臭假丝酵母细胞在磷酸二氢钾黄油中混合，基本上获得了100%的产率。以类似的方式，相应的腺苷类似物和L-同型半胱氨酸可以有效地合成几种新的S-核苷基同型半胱氨酸。K-卡拉胶包裹的粪肠弯曲杆菌细胞在重复反应中表现出较高的稳定性，而游离细胞则迅速失活。这些新型的S-核苷基同型半胱氨酸在药物和化疗领域也有很好的应用前景。

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.