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Reactive extraction enables efficient and selective separation of fructose from glucosides (here: α-glucosyl glycerol) produced from sucrose by enzymatic transglycosylation.

Advanced biotransformation processes typically involve the upstream processing part performed continuously and interlinked tightly with the product isolation. Key in their development is a catalyst that is highly active, operationally robust, conveniently produced, and recyclable. A promising strategy to obtain such catalyst is to encapsulate enzymes as permeabilized whole cells in porous polymer materials. Here, we show immobilization of the sucrose phosphorylase from Bifidobacterium adolescentis (P134Q-variant) by encapsulating the corresponding E. coli cells into polyacrylamide. Applying the solid catalyst, we demonstrate continuous production of the commercial extremolyte 2-α-d-glucosyl-glycerol (2-GG) from sucrose and glycerol. The solid catalyst exhibited similar activity (≥70%) as the cell-free extract (~800 U g−1 cell wet weight) and showed excellent in-operando stability (40 °C) over 6 weeks in a packed-bed reactor. Systematic study of immobilization parameters related to catalyst activity led to the identification of cell loading and catalyst particle size as important factors of process optimization. Using glycerol in excess (1.8 M), we analyzed sucrose conversion dependent on space velocity (0.075–0.750 h−1) and revealed conditions for full conversion of up to 900 mM sucrose. The maximum 2-GG space-time yield reached was 45 g L−1 h−1 for a product concentration of 120 g L−1. Collectively, our study establishes a step-economic route towards a practical whole cell-derived solid catalyst of sucrose phosphorylase, enabling continuous production of glucosides from sucrose. This strengthens the current biomanufacturing of 2-GG, but also has significant replication potential for other sucrose-derived glucosides, promoting their industrial scale production using sucrose phosphorylase. Key points • Cells of sucrose phosphorylase fixed in polyacrylamide were highly active and stable. • Solid catalyst was integrated with continuous flow to reach high process efficiency. • Generic process technology to efficiently produce glucosides from sucrose is shown. Graphical abstract

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Advanced biomanufacturing builds on production processes that are both profitable and sustainable. Integrated design of process unit operations, geared to output efficiency and waste minimization and guided by a rigorous techno-economic assessment, is essential for development aligned to these central aims. Here, we demonstrate such a development for the biocatalytic production of the biological extremolyte 2- O -α- d -glucosyl-glycerol (2-GG) for functional ingredient application. The process was aligned in scale over all steps (∼180 g product; ∼2.5 L reaction mixture) and involved continuous enzymatic synthesis from sucrose and glycerol interlinked with reactive extraction and nanofiltration for product isolation (purity of ∼80 wt %) and side stream recovery. Glycerol used in ∼6-fold excess over sucrose was recycled, and hydrothermal conversion into 5-(hydroxymethyl)furfural was evaluated for the fructose by-product released from sucrose. Based on a process mass intensity (total mass input/mass product) of 146, ∼80% of the total mass input was utilized and an E -factor (mass waste/mass product) of 28 was obtained. EcoScale analysis revealed a penalty point score of 44, suggesting an acceptable process from a sustainability point of view. Process simulation for an annual production of 10 tons 2-GG was used for the techno-economic assessment with discounted cash flow analysis. The calculated operating costs involved 35 and 47% contributions from materials and labor, respectively. About 91% of the material costs were due to chemicals for the reactive extraction-acidic stripping step, emphasizing the importance of material reuse at this step. Glycerol recycling involved a trade-off between waste reduction and energy use for the removal of water. Collectively, the study identifies options and boundaries of a profitable 2-GG process. The minimum selling price for 2-GG was calculated as ∼240 € kg –1 or smaller. The framework of the methodology presented can be generally important in applied bio-catalysis: it facilitates closing of the gap between process design and implementation for accelerated development.