Bioproduct Engineering, Engineering and Technology Institute Groningen, University of Groningen, Nijenborgh 4, Groningen, 9747 AG, the Netherlands.
Bioproduct Engineering, Engineering and Technology Institute Groningen, University of Groningen, Nijenborgh 4, Groningen, 9747 AG, the Netherlands.
Enzyme Microb Technol. 2021 Oct;150:109882. doi: 10.1016/j.enzmictec.2021.109882. Epub 2021 Jul 31.
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）迄今为止被描述为能够同时进行α-1,6-转糖苷（分支）和α-1,4-水解活性。本研究的目的是通过深入分析在明确的底物上的活性，阐明糖苷水解酶家族 13 中三种亲缘关系较远的 GBE 的作用模式。为此，使用来自 R. marinus (RmGBE)、P. mobilis (PmGBE1) 和 B. fibrisolvens (BfGBE) 的 GBE 与 18 个无水葡萄糖单位的高度纯分数的线性底物孵育。还同时使用了一种来自大肠杆菌的已知和特征分支酶（EcGBE）。随着时间的推移分析链长分布表明，除了水解和分支活性外，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-转糖苷活性。此外，GBE 还使用那些延长的链进行进一步的分支。延伸和分支的连续活性使 GBE 能够比仅分支活性更有效地修饰底物。总的来说，三种 GBE 在定义的底物上表现出模糊的作用。RmGBE 似乎强烈倾向于转移九个无水葡萄糖单位的链，即使在延伸过程中也是如此，其活性相对较低。BfGBE 在使用链引入分支之前会生成一系列延长的链，而 PmGBE1 表现出两种酶之间的中间行为。基于本研究中揭示的作用模式，现在提出了 GBE 机制的更新模型，包括α-1,4-转糖苷活性。
