Biocatalytic Synthesis of the Rare Sugar Kojibiose: Process Scale-Up and Application Testing

Abstract

Cost-efficient (bio)chemical production processes are essential to evaluate the commercial and industrial applications of promising carbohydrates and also are essential to ensure economically viable production processes. Here, the synthesis of the naturally occurring disaccharide kojibiose (2-O-α-d-glucopyranosyl-d-glucopyranoside) was evaluated using different Bifidobacterium adolescentis sucrose phosphorylase variants. Variant L341I_Q345S was found to efficiently synthesize kojibiose while remaining fully active after 1 week of incubation at 55 °C. Process optimization allowed kojibiose production at the kilogram scale, and simple but efficient downstream processing, using a yeast treatment and crystallization, resulted in more than 3 kg of highly pure crystalline kojibiose (99.8%). These amounts allowed a deeper characterization of its potential in food applications. It was found to have possible beneficial health effects, including delayed glucose release and potential to trigger SCFA production. Finally, we compared the bulk functionality of highly pure kojibiose to that of sucrose, hereby mapping its potential as a new sweetener in confectionery products.

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

“…The yeast however utilizes all other sugars present in the reaction (fructose, sucrose, glucose) for growth . Generally, product purification via selective microbial conversion of the accompanying sugars (ideally into biomass, CO 2 and H 2 O) is an effective strategy for the downstream processing of carbohydrate‐based reaction mixtures . The S. cerevisiae conversion are typically performed at carbohydrate concentrations not exceeding 200 g L −1 in total .…”

On the donor substrate dependence of group‐transfer reactions by hydrolytic enzymes: Insight from kinetic analysis of sucrose phosphorylase‐catalyzed transglycosylation

“…The molecular interactions involved in the proposed complex are not known in detail. However, it suffices to emphasize that the (acceptor) binding pocket of the E-Glc form of BaSucP is wide and flexible (Mirza et al, 2006;Sprogøe et al, 2004) to accommodate a range of bulky acceptors (Dirks-Hofmeister, Verhaeghe, De Winter, & Desmet, 2015;Goedl et al, 2010;Seibel et al, 2006), including glucose in multiple orientations (Beerens et al, 2017;Kraus, Görl, Timm, & Seibel, 2016;Verhaeghe et al, 2016). The kinetic significance of the (E-Glc •• G1P) complex, in terms of the amount formed at steady state and the rate of breakdown, have strong implications on canonical (apparent) enzyme kinetic parameters obtained from standard experiments (e.g., app V X , app K, TC; see Table 1).…”

Unravelling the Specificity of Laminaribiose Phosphorylase from Paenibacillus sp. YM‐1 towards Donor Substrates Glucose/Mannose 1‐Phosphate by Using X‐ray Crystallography and Saturation Transfer Difference NMR Spectroscopy

“…Substrates for GP‐catalysed glycosylations are more readily available, in comparison to those for GT‐catalysed reactions; this makes GPs attractive biocatalysts for carbohydrate syntheses. The use of GP biocatalysts has been demonstrated in academic research, such as in the synthesis of homogeneous crystalline cellulose, self‐assembled structures of alkylated cellulose, cellulose nanoribbons with primary amino groups, and the formation of oligo(ethylene glycol)‐bearing cellulose hydrogels, and more widely at an industrial scale, such as for the synthesis of 2‐ O ‐(α‐ d ‐glucopyranosyl)‐ sn ‐glycerol, a cosmetic ingredient, by sucrose phosphorylase; kilogram‐scale synthesis of lacto‐ N ‐biose, a prebiotic made with lacto‐ N ‐biose phosphorylase; and the synthesis of disaccharide sweetener kojibiose, produced with a sucrose phosphorylase variant from Bifidobacterium adolescentis …”