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

Abstract

Cellodextrins are linear β‐1,4‐gluco‐oligosaccharides that are soluble in water up to a degree of polymerization (DP) of ≈6. Soluble cellodextrins have promising applications as nutritional ingredients. A DP‐controlled, bottom‐up synthesis from expedient substrates is desired for their bulk production. Here, a three‐enzyme glycoside phosphorylase cascade is developed for the conversion of sucrose and glucose into short‐chain (soluble) cellodextrins (DP range 3–6). The cascade reaction involves iterative β‐1,4‐glucosylation of glucose from α‐glucose 1‐phosphate (αGlc1‐P) donor that is formed in situ from sucrose and phosphate. With final concentration and yield of the soluble cellodextrins set as targets for biocatalytic synthesis, three major factors of reaction efficiency are identified and partly optimized: the ratio of enzyme activity, the ratio of sucrose and glucose, and the phosphate concentration used. The efficient use of the phosphate/αGlc1‐P shuttle for cellodextrin production is demonstrated and the soluble product at 40 g L−1 is obtained under near‐complete utilization of the donor substrate offered (88 mol% from 200 mm sucrose). The productivity is 16 g (L h)−1. Through a simple two‐step route, the soluble cellodextrins are recovered from the reaction mixture in ≥95% purity and ≈92% yield. Overall, this study provides the basis for their integrated production.

Three‐Enzyme Phosphorylase Cascade Immobilized on Solid Support for Biocatalytic Synthesis of Cello−oligosaccharides

“…Besides the considerations of overall immobilization efficiency of each individual enzyme (Figure 2), we took into account that the DP-controlled synthesis of celloÀ oligosaccharides necessitates the three phosphorylases to be present in a balanced ratio of activities. In previous studies of the cascade reaction in solution, [30] we identified a favorable ratio 10 : 2 : 3 for the activities of ScP, CbP and CdP. The activity of the immobilized enzymes was 234 U/g (ZÀ BlScP), 49 U/g (ZÀ CuCbP) and 74 U/g (ZÀ CcCdP), thus well in line with the required ratio of activities for efficient production.…”

“…We used the conditions previously established for the enzymatic cascade reaction in solution: 200 mM sucrose, 65 mM glucose and 50 mM phosphate. [30] The volumetric activity of the immobilized enzymes in suspension was 9 U/mL for ZÀ BlScP, 2 U/mL for ZÀ CuCbP and 3 U/mL for ZÀ CcCdP. As shown in Figure 5B,~90 % of the initial sucrose was converted within 3 hrs and celloÀ oligosaccharides were released concomitantly.…”

“…We summarize the results in Figure 2, showing the immobilization yield, activity, and catalytic effectiveness of the immobilized enzyme dependent on the enzyme loading. In anticipation of an enzyme co-immobilization in which the activity of ZÀ BlScP should be present in excess over the activities of ZÀ CuCbP and CcCdP (see later), [30] we examined a larger range of enzyme loadings for the ZÀ BlScP than for the other two enzymes. Although all the enzymes were adsorbed to the carrier in useful amount (� 50 U/g carrier), details of their immobilization behavior were yet enzyme-specific.…”

“…Using co-immobilized enzymes, product precipitation occurred at a higher rate than the study of celloÀ oligosaccharide synthesis with the soluble enzymes had suggested. [30] Difference in behavior of soluble and immobilized enzyme cascades is not straightforward to explain and certainly warrants further mechanistic inquiry. The comparably high volumetric reaction rates in the confinement of the porous particle might be a relevant factor.…”

“…[29] Previous study of the three-enzyme cascade in solution shows, that shuttling of the glucosyl residues to and from phosphate constitutes an important factor of conversion efficiency. [30] To avoid celloÀ oligosaccharide precipitation during reaction in solution, tight control of DP in product is additionally required. [29,31] Without such control, the CdP would elongate the growing oligosaccharide chains until they become largely insoluble.…”