Glucosylglycerate (R-2-O-α-D-glucopyranosyl-glycerate, GG) is a negatively charged compatible solution with versatile functions. Here, an artificial in vitro enzymatic cascade was designed to feasibly and sustainably produce GG from affordable starch and glycerol. First, glucosylglycerate phosphorylase (GGP) was carefully selected because of its excellent heterologous expression, specific activity, and thermostability. The optimized two-enzyme cascade, consisting of alpha-glucan phosphorylase (αGP) and GGP, achieved a remarkable 81 % conversion rate from maltodextrin and D-glycerate. Scaling up this cascade resulted in a practical concentration of 58 g/L GG with a 62 % conversion rate based on the added D-glycerate. Additionally, the production of GG from inexpensive starch and glycerol in one-pot using artificial four-enzyme cascade was successfully implemented, which integrates alditol oxidase and catalase with αGP and GGP. Collectively, this sustainable enzymatic cascade demonstrates the feasibility of the practical synthesis of GG and has the potential to produce other glycosides using the phosphorylase-and-phosphorylase paradigm.

中文翻译：

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通过人工体外酶级联从淀粉和甘油一锅可持续合成葡萄糖基甘油酯

葡萄糖基甘油酸酯（R-2-O-α-D-吡喃葡萄糖基甘油酸酯，GG）是一种带负电荷的兼容溶液，具有多种功能。在此，设计了一种人工体外酶级联，以从负担得起的淀粉和甘油中可行且可持续地生产 GG。首先，精心选择葡萄糖基甘油酸磷酸化酶（GGP），因为它具有优异的异源表达、比活性和热稳定性。优化的双酶级联由 α-葡聚糖磷酸化酶 (αGP) 和 GGP 组成，麦芽糖糊精和 D-甘油酸的转化率高达 81%。放大该级联导致实际浓度为 58 g/L GG，基于添加的 D-甘油酸，转化率为 62%。此外，还成功实现了利用人工四酶级联将廉价淀粉和甘油一锅法生产GG，该级联将糖醇氧化酶和过氧化氢酶与αGP和GGP整合在一起。总的来说，这种可持续的酶级联证明了 GG 实际合成的可行性，并有可能使用磷酸化酶和磷酸化酶范式生产其他糖苷。