Characterization of UDP-glucose 4-epimerase from Pyrococcus horikoshii: Regeneration of UDP to produce UDP-galactose using two-enzyme system with trehalose

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• 作者：Seung-Kyung Chung¹ ; Soo-In Ryu¹ ; Soo-Bok Lee ; ^{soobok@yonsei.ac.kr}
• 关键词：Pyrococcus horikoshii ; UDP-glucose 4-epimerase ; Trehalose synthase ; Trehalose ; Regeneration of UDP
• 刊名：Bioresource Technology
• 出版年：2012
• 期刊代码：11_09608524
• 类别：ce
• 出版时间：April, 2012
• 卷：110
• 期：Complete
• 页码：423-429
• 文件大小：418 K

摘要

A gene encoding a putative UDP-glucose 4-epimerase (pGALE) in Pyrococcus horikoshii was cloned and expressed in Escherichia coli. The purified enzyme could reversibly catalyze both the synthesis of UDP-Gal and UDP-Glc but preferred the binding of UDP-Gal by approximately 10-fold. The optimum pH and temperature were 6.5 and 65 掳C. The enzyme acted effectively without the addition of nicotinamide adenine dinucleotide (NAD⁺), possibly due to the presence of tightly bound NAD⁺. In particular, pGALE could be coupled with trehalose synthase (TreT) from P. horikoshii to regenerate UDP-Gal from UDP. The possible byproduct of glycosyltransferase, UDP, was capable of being converted to UDP-Glc with trehalose by TreT, and UDP-Glc was simultaneously converted to UDP-Gal by pGALE. Conclusively, the results suggest that pGALE and TreT with trehalose is an effective one-pot two-enzyme system for the regeneration of UDP-Gal, a high-cost substrate of galactosyltransferase, to complete a sugar nucleotide cycle.