Continuous process technology for glucoside production from sucrose using a whole cell-derived solid catalyst of sucrose phosphorylase

Abstract

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

Biocatalytic Production of 2-α-d-Glucosyl-glycerol for Functional Ingredient Use: Integrated Process Design and Techno-Economic Assessment

“…The column was connected to a circulating water bath for temperature control at 40 °C. 16 Feed was from an Azura P4.1S pump (Knauer, Berlin, Germany). The packed bed was washed with water and substrate solution (0.35 M sucrose, 1.9 M glycerol) at 1 mL min –1 .…”

“…For direct application, the 2-GG content in the retentate is too low (∼5 wt %). Therefore, an evaporation step is necessary to remove the surplus of water which can be recycled to the diafiltration step (16). The concentrated retentate is ultrafiltered and gives the final product stream (17) with a 2-GG content of ≥50 wt %.…”

“…Flow synthesis in a packed-bed reactor achieves high productivity while it still meets the high demand in sucrose conversion. 16 Process technologies that accomplish individual tasks of the downstream processing have been proposed. Glycerol is removed by multistep discontinuous diafiltration.…”

“… a Hydrolysis is a minor side reaction when glycerol is used in excess. The figure was adapted from Kruschitz et al 2021 …”

“…Escherichia coli cells that express the recombinant enzyme in high amounts are immobilized through entrapment in the polyacrylamide (PAM) matrix. Flow synthesis in a packed-bed reactor achieves high productivity while it still meets the high demand in sucrose conversion . Process technologies that accomplish individual tasks of the downstream processing have been proposed.…”