Product solubility control in cellooligosaccharide production by coupled cellobiose and cellodextrin phosphorylase

Soluble cellodextrins (linear β‐1,4‐d‐gluco‐oligosaccharides) have interesting applications as ingredients for human and animal nutrition. Their bottom‐up synthesis from glucose is promising for bulk production, but to ensure a completely water‐soluble product via degree of polymerization (DP) control (DP ≤ 6) is challenging. Here, we show biocatalytic production of cellodextrins with DP centered at 3 to 6 (~96 wt.% of total product) using coupled cellobiose and cellodextrin phosphorylase. The cascade reaction, wherein glucose was elongated sequentially from α‐d‐glucose 1‐phosphate (αGlc1‐P), required optimization and control at two main points. First, kinetic and thermodynamic restrictions upon αGlc1‐P utilization (200 mM; 45°C, pH 7.0) were effectively overcome (53% → ≥90% conversion after 10 hrs of reaction) by in situ removal of the phosphate released via precipitation with Mg(2+). Second, the product DP was controlled by the molar ratio of glucose/αGlc1‐P (∼0.25; 50 mM glucose) used in the reaction. In optimized conversion, soluble cellodextrins in a total product concentration of 36 g/L were obtained through efficient utilization of the substrates used (glucose: 98%; αGlc1‐P: ∼80%) after 1 hr of reaction. We also showed that, by keeping the glucose concentration low (i.e., 1–10 mM; 200 mM αGlc1‐P), the reaction was shifted completely towards insoluble product formation (DP ∼9–10). In summary, this study provides the basis for an efficient and product DP‐controlled biocatalytic synthesis of cellodextrins from expedient substrates.