Abstract
BACKGROUND: Cellobiose (4-O-β-D-Glucopyranosyl-D-glucopyranose) is an important disaccharide utilized, for example in food and cosmetics. It can be enzymatically synthesized involving two steps from sucrose and glucose, where first the sucrose undergoes phosphorolysis by sucrose phosphorylase, yielding glucose 1-phosphate and fructose. Glucose 1-phosphate is then combined with glucose into cellobiose, releasing phosphate in a reaction catalyzed by cellobiose phosphorylase. To better control and reuse the enzymes in the two main reaction steps, immobilization on Bacillus subtilis spores is a promising approach due to the ease of production and recyclability. RESULTS: Here we describe the display of a sucrose phosphorylase and a cellobiose phosphorylase on B. subtilis spores through fusion with the crust protein CotY, to our knowledge marking the first use of multiple enzymes directly displayed on the spore surface during sporulation in a reaction cascade. While immobilization had no effect on thermostability, we demonstrate the recyclability of the individual spore variants over four reaction cycles at 45 °C with sucrose phosphorylase maintaining 35% of its initial activity and cellobiose phosphorylase maintaining 65%. Both spore variants were used together to catalyse a reaction cascade in a separated two-pot, as well as in a one-pot reaction. The one-pot reaction achieved a 90% yield with respect to the initially available 40 mM of glucose. The one-pot cascade maintained activity after being recycled five times over the course of 120 hours. Furthermore, we report on improving the reaction yield in the two-pot reaction from 60% to 80% by using calcium to precipitate excess phosphate. CONCLUSION: In this study we demonstrate that spores are a suitable immobilization platform for multistage reaction cascades. The spores displaying biocatalysts can be recovered and reused over multiple reaction cycles. The immobilization of glycosylic enzymes on spores enables cost-effective, scalable enzyme production on a temperature-resistant carrier that facilitates purification. The potential modularity of this approach adds to the adaptability of the system to different requirements in terms of substrate and product. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-026-02943-w.