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 Mg²⁺ . 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.