Cellodextrins are non-digestible oligosaccharides that have attracted interest from the food industry as potential prebiotics. They are typically produced through the partial hydrolysis of cellulose, resulting in a complex mixture of oligosaccharides with a varying degree of polymerisation (DP). Here, we explore the defined synthesis of cellotriose as product since this oligosaccharide is believed to be the most potent prebiotic in the mixture. To that end, the cellobiose phosphorylase (CBP) from Cellulomonas uda and the cellodextrin phosphorylase (CDP) from Clostridium cellulosi were evaluated as biocatalysts, starting from cellobiose and α-D-glucose 1-phosphate as acceptor and donor substrate, respectively. The CDP enzyme was shown to rapidly elongate the chains towards higher DPs, even after extensive mutagenesis. In contrast, an optimised variant of CBP was found to convert cellobiose to cellotriose with a molar yield of 73%. The share of cellotriose within the final soluble cellodextrin mixture (DP2-5) was 82%, resulting in a cellotriose product with the highest purity reported to date. Interestingly, the reaction could even be initiated from glucose as acceptor substrate, which should further decrease the production costs.Key points• Cellobiose phosphorylase is engineered for the production of cellotriose.• Cellotriose is synthesised with the highest purity and yield to date.• Both cellobiose and glucose can be used as acceptor for cellotriose production.

中文翻译：

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纤维二糖磷酸化酶的工程化，用于确定合成纤维三糖。

纤维糊精是不可消化的低聚糖，作为潜在的益生元已引起食品工业的兴趣。它们通常是通过纤维素的部分水解产生的，从而产生具有不同聚合度 (DP) 的低聚糖的复杂混合物。在这里，我们探索作为产品的纤维三糖的明确合成，因为这种低聚糖被认为是混合物中最有效的益生元。为此，来自纤维单胞菌的纤维二糖磷酸化酶 (CBP) 和来自纤维素梭菌的纤维糊精磷酸化酶 (CDP) 被评估为生物催化剂，分别以纤维二糖和 α-D-葡萄糖 1-磷酸作为受体和供体底物。即使经过广泛的诱变，CDP 酶也能迅速将链拉长到更高的 DP。相比之下，发现 CBP 的优化变体以 73% 的摩尔产率将纤维二糖转化为纤维三糖。纤维三糖在最终可溶性纤维糊精混合物 (DP2-5) 中的份额为 82%，产生迄今为止报告的最高纯度的纤维三糖产品。有趣的是，该反应甚至可以从作为受体底物的葡萄糖开始，这将进一步降低生产成本。关键点• 纤维二糖磷酸化酶是为生产纤维三糖而设计的。• 纤维三糖的合成纯度和产率是迄今为止最高的。• 两者纤维二糖和葡萄糖可用作纤维三糖生产的受体。产生了迄今为止报告的最高纯度的纤维三糖产品。有趣的是，该反应甚至可以从作为受体底物的葡萄糖开始，这将进一步降低生产成本。关键点• 纤维二糖磷酸化酶是为生产纤维三糖而设计的。• 纤维三糖的合成纯度和产率是迄今为止最高的。• 两者纤维二糖和葡萄糖可用作纤维三糖生产的受体。产生了迄今为止报告的最高纯度的纤维三糖产品。有趣的是，该反应甚至可以从作为受体底物的葡萄糖开始，这将进一步降低生产成本。关键点• 纤维二糖磷酸化酶专为生产纤维三糖而设计。• 纤维三糖的合成纯度和产率是迄今为止最高的。• 两者纤维二糖和葡萄糖可用作纤维三糖生产的受体。