Glycogen branching enzymes (GBEs; 1,4-α-glucan branching enzyme; E.C. 2.4.1.18) have so far been described to be capable of both α-1,6-transglycosylation (branching) and α-1,4-hydrolytic activity. The aim of the present study was to elucidate the mode of action of three distantly related GBEs from the glycoside hydrolase family 13 by in depth analysis of the activity on a well-defined substrate. For this purpose, the GBEs from R. marinus (RmGBE), P. mobilis (PmGBE1), and B. fibrisolvens (BfGBE) were incubated with a highly pure fraction of a linear substrate of 18 anhydroglucose units. A well-known and characterized branching enzyme from E. coli (EcGBE) was also taken along. Analysis of the chain length distribution over time revealed that, next to hydrolytic and branching activity, all three GBEs were capable of generating chains longer than the substrate, clearly showing α-1,4-transglycosylation activity. Furthermore, the GBEs used those elongated chains for further branching. The sequential activity of elongation and branching enabled the GBEs to modify the substrate to a far larger extent than would have been possible with branching activity alone. Overall, the three GBEs acted ambiguous on the defined substrate. RmGBE appeared to have a strong preference towards transferring chains of nine anhydroglucose units, even during elongation, with a comparably low activity. BfGBE generated an array of elongated chains before using the chains for introducing branches while PmGBE1 exhibited a behaviour intermediate of the other two enzymes. On the basis of the mode of action revealed in this research, an updated model of the mechanism of GBEs was proposed now including the α-1,4-transglycosylation activity.

糖原分支酶（GBE；1,4-α-葡聚糖分支酶；EC 2.4.1.18）迄今已被描述为具有 α-1,6-转糖基化（分支）和 α-1,4-水解活性. 本研究的目的是通过对明确定义的底物的活性进行深入分析，阐明来自糖苷水解酶家族 13 的三个远缘 GBE 的作用模式。为此，将来自R. marinus (RmGBE)、P. mobilis (PmGBE1) 和B. fibrisolvens (BfGBE) 的 GBE 与 18 个脱水葡萄糖单位的线性底物的高纯度级分一起孵育。来自大肠杆菌的众所周知的特征性分支酶(EcGBE) 也被带走。随着时间的推移对链长分布的分析表明，除了水解和支化活性之外，所有三种 GBE 都能够产生比底物更长的链，清楚地显示出 α-1,4-转糖基化活性。此外，GBE 使用这些拉长的链进一步分支。延伸和分支的连续活动使 GBE 能够在更大程度上修饰底物，而不是单独使用分支活动。总体而言，三个 GBE 在定义的基材上表现不明确。RmGBE 似乎对转移 9 个脱水葡萄糖单元的链具有强烈的偏好，即使在伸长期间也是如此，但活性相对较低。BfGBE 在使用链引入分支之前生成了一系列细长链，而 PmGBE1 表现出其他两种酶的行为中间体。根据本研究揭示的作用模式，现在提出了一种更新的 GBE 机制模型，包括 α-1,4-转糖基化活性。