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標題: Effect of Adenosine Triphosphate on the Activity of N-Acetyl-D-Glucosamine 2-Epimerase
作者: Chen, Chuen-Fan
關鍵字: epimerase;ATP
出版社: 化學工程學系所
N-acetyl-D-manosamine, ManNAc, is the precursor for the biosynthesis of sialic acid, NeuAC. To reduce the cost of substrate, an two-enzyme process with N-acetyl-D-glucosamine 2-epimerase, GlcNAc2-epimerase, as the catalyst has been proposed, enabling the use of relatively inexpensive N-acetyl-D-glucosamine, as the starting reactant. Adenosine Triphosphate, ATP, is the activator for GlcNAc 2-epimerase for the alternative process. In this study, we search for the effect of ATP concentration on the activity of the free enzyme and the activity of the enzyme of co-immobilized with ATP. For the free enzyme,the activity will be the maximum as the concentration of ATP in the substrate reaches 1.0mM. For the study of enzyme co-immobilization with ATP, we regard the immobilized enzyme as the control group. The optimal concentration of ATP was 15 mM for the covalent bonding method and 12 mM for the cross-linking method, in these condition the relative activity was 74.2% and 83.3% higher than the control respectively. During the repetitive batch operation, the activity of immobilized enzyme which co-immobilized with ATP appears only at the first batch either covalent bonding or cross-linking method. There is no influence compared with the control after the second batch.

在酵素法製備唾液酸(sialic acid, NeuAc)的製程中,其主要原料為N-acetyl-D-mannosamine (ManNAc)。然而為了節省反應物成本,提出了先以價格低廉的N-acetyl -D-glucosamine (GlcNAc)經由GlcNAc 2-epimerase催化生成ManNAc的二步化酵素法。然而在此催化反應中,三磷酸腺&;#33527;(Adenosine Triphosphate, ATP)扮演了控制酵素活性的關鍵角色。本研究探討ATP濃度對自由態酵素活性之影響,以及添加ATP與酵素進行共固定化後對固定化酵素活性的影響。對自由態酵素而言,基質中ATP濃度為1.0 mM時酵素即發揮最大催化活性。在ATP共固定化的研究中,以未添加ATP共固定化做為控制組,共價鍵結法的最適ATP添加濃度為15 mM,其相對活性較控制組高74.2%;交聯鍵結法的最適ATP添加濃度為12 mM,其相對活性則是高出控制組83.3% 。在進行重複式批次操作下,無論是共價鍵結或是交聯鍵結法, ATP共固定化酵素僅有第一次批次有活性表現,第二次批次後相較於控制組幾無差別。
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