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

Abstract

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.

Three‐Enzyme Phosphorylase Cascade for Integrated Production of Short‐Chain Cellodextrins

“…The three‐enzyme phosphorylase cascade in Figure A was promising for cellodextrin synthesis. Here, cellobiose phosphorylase (CbP) from Cellulomonas uda ( Cu CbP) and CdP from Clostridium cellulosi ( Cc CdP) had been identified for cellodextrin biosynthesis in a recent study . Sucrose phosphorylase (ScP) from Bifidobacterium adolescentis ( Ba ScP) was known from literature for the efficient conversion of sucrose .…”

“…Sucrose phosphorylase (ScP) from Bifidobacterium adolescentis ( Ba ScP) was known from literature for the efficient conversion of sucrose . The three enzymes showed suitable overlap in their pH and temperature profiles of activity, which is critical when their reactions are to be telescoped in one pot. At the conditions selected for the overall conversion (pH 7.0; 45 °C), the enzymes also display useful stability, each with half‐life of 2 days or more.…”

“…We recently demonstrated a linear cascade of Cu CbP and Cc CdP for synthesis of soluble cellodextrins from αGlc1‐ P and glucose . To prepare the cellodextrins from sucrose, we first considered a strictly modular approach in which the formation of αGlc1‐ P from sucrose was uncoupled from the cellodextrin synthesis.…”

“…The approach entails a phosphate concentration equal to, or higher than, that of sucrose and limits the phosphate/αGlc1‐ P shuttle to only one single operational cycle. However, the approach enabled us to directly apply the conditions previously established for the linear cascade . In particular, we had shown in the earlier study that precipitation as magnesium salt of the phosphate released from αGlc1‐ P was an efficient means of pulling the Cu CbP‐ Cc CdP reaction toward completion.…”

“…Chemical and enzymatic methods are known for both types of route . Control of the product DP is essential and remains a challenge . Robust process technology fit for the bulk production of soluble cellodextrins is currently lacking.…”