Spatially sequential co-immobilization of phosphorylases in tiny environments and its application in the synthesis of glucosyl glycerol.

2-O-(α- d -glucopyranosyl)-sn-glycerol (2-αGG) has been applied in the food industry due to its numerous physiological benefits. The synthesis of 2-αGG can be achieved through a cascade catalytic reaction involving sucrose phosphorylase (SP) and 2-O-α-glucosylglycerol phosphorylase (GGP). However, the low substrate transfer rates between free enzymes have hindered the efficiency of 2-αGG synthesis. To address this issue, a novel technology was developed to prepare sequential multi-enzyme nanoflowers via chemical crosslinking and protein assembly, thus overcoming diffusion limitations. Specifically, spatially sequential co-immobilized enzymes, referred to as SP-GGP@Cap, were created through the targeted assembly of Bifidobacterium adolescentis SP and Marinobacter adhaerens GGP on Ca2+. This assembly was facilitated by the spontaneous protein reaction between SpyTag and SpyCatcher. Compared to free SP-GGP, SP-GGP@Cap demonstrated improved thermal and pH stability. Moreover, SP-GGP@Cap enhanced the biosynthesis of 2-αGG, achieving a relative concentration of 98 %. Additionally, it retained the ability to catalyze the substrate to yield 61 % relative concentration of 2-αGG even after ten cycles of recycling. This study presents a strategy for the spatially sequential co-immobilization of multiple enzymes in a confined environment and provides an exceptional biocatalyst for the potential industrial production of 2-αGG. [Display omitted] • A spatially sequential multi-enzyme immobilization strategy was provided. • SP-GGP@Cap was constructed by SP and GGP via the above strategy. • SP-GGP@Cap showed excellent thermal and pH stability than free SP-GGP. • SP-GGP@Cap enabled the biosynthesis of 2-αGG, with a relative concentration of 98 %. • SP-GGP@Cap could catalyze the substrate to synthesize a 61 % relative concentration of 2-αGG after recycling ten times. [ABSTRACT FROM AUTHOR]