Clostridium thermocellum 來源重組纖維二糖磷酸化酶之受質特異性

Abstract

醣基修飾是影響多種化合物物理化學性質與生理功能的關鍵因素。纖維二糖磷酸化酶 (Cellobiose phosphorylases, CBPs) 是一種具醣基修飾潛力的生物催化劑，可以透過磷酸解產生葡萄糖磷酸，此類醣磷酸在醣基合成上，相較於醣基轉移酶所需的核苷活化醣是更容易取得的受質，在細胞工廠的建立上，相較於直接使用葡萄糖，醣磷酸也可以節省生物體代謝過程中的 ATP 消耗。因此，深入探討 CBP 的受質特異性，就有機會產生出不同的醣磷酸，並應用在更多元的醣基修飾上。然而，大多數文獻均著重於逆磷酸解方向的討論，目前針對不同 CBP 在磷酸解方向之受質特異性與產物分布的系統性比較仍相當有限。本研究選擇 Cellulomonas uda (Cu) 與 Clostridium thermocellum (Ct) 來源的 CBP，系統性比較其在磷酸解方向反應之受質特異性與產物分布。根據 30 種不同受質的篩選結果，CuCBP 和 CtCBP 對於醣苷鍵的鍵結位置、異頭構型及聚合度皆為高度保守。然而，本研究發現 CtCBP 相較於 CuCBP 表現出更廣泛的受質接受性，特別是其 −1 亞位點對半乳糖基的耐受性甚至高於葡萄糖基。除了葡萄糖基之外，CtCBP 在 +1 亞位點也可以接受苯基及對硝基苯基等平面結構之取代基，相對纖維二糖之比活性分別為 2.3% 與 3.8%，相較之下，含有官能基修飾或多環結構的苯基衍生物則不具反應活性。此外，本研究首次釐清了乳糖磷酸解反應的整體產物分布，並發現 CtCBP 在反應過程中產生 6–12% 的 β1→6 異乳糖 (Allolactose)，此結果不同於在雙醣受質特性的結果中觀察到的鍵結保守性。另一方面，在半乳糖基衍生受質中也觀察到了非預期產物半乳糖的生成，推測反應中可能涉及磷酸解外的潛在反應途徑。整體而言，本研究為 CBP 在磷酸解方向之受質特異性提供了新的見解，並為未來醣基修飾策略的發展奠定基礎。

Glycosyl modification is a key determinant of the physicochemical properties and biological functions of diverse molecules. Cellobiose phosphorylases (CBPs) are promising biocatalysts for producing glycosyl phosphates, such as glucose 1-phosphate, through phosphorolysis. These phosphorylated sugars are valuable for precise enzymatic glycosyl synthesis and cell factory construction because they serve as more accessible glycosyl donors than nucleotide-activated sugars required by glycosyltransferases and reduce ATP consumption relative to free glucose in cellular metabolism. Understanding the substrate specificity of CBPs may therefore provide a basis for developing diverse glycosyl modification strategies. However, systematic comparisons of phosphorolytic substrate specificity and product distribution among CBPs remain limited. In this study, CBPs from Cellulomonas uda (CuCBP) and Clostridium thermocellum (CtCBP) were selected to compare their phosphorolytic substrate specificity and product distribution. Screening of 30 substrates showed that both CBPs exhibited conserved specificity for glycosidic linkage position, anomeric configuration, and degree of polymerization. CtCBP showed broader substrate acceptance than CuCBP, with greater tolerance toward galactosyl moieties at the −1 subsite. At the +1 subsite, CtCBP also accepted planar substituents, including phenyl and p-nitrophenyl groups, with relative specific activities of 2.3% and 3.8%, respectively, whereas phenyl derivatives containing additional functional groups or multi-ring structures were not accepted. The product distribution of lactose phosphorolysis was also clarified for the first time. CtCBP synthesized 6–12% β1→6 allolactose during the reaction, a result inconsistent with the strict linkage specificity observed in disaccharide phosphorolysis. The unexpected formation of galactose from galactosyl-derived substrates further suggested the involvement of a potential alternative reaction pathway. Overall, this study provides new insights into the phosphorolytic substrate specificity of CBPs and establishes a basis for future glycosyl modification strategies.