Self‐assembling protein scaffold‐mediated enzymes' immobilization enhances in vitrod‐tagatose production from lactose

Abstract

As a rare low-calorie sugar with special medicinal value, D-tagatose is widely used in the field of food, beverages, medicine, and cosmetics. However, enzymatic D-tagatose production in vitro is commonly limited to low conversion efficiency and poor thermo-stability. Herein, taking advantage of the self-assembling property of protein scaffold EutM (ethanolamine bacterial microcompartments), Spy and Snoop peptide pairs was used to drive the linkage between the EutM and cargo proteins, βgalactosidase (BagB), and L-arabinose isomerase (TMAI) to construct a dual-enzymes cascade and realize the D-tagatose production from lactose. The optimal conditions of the cascade were shown to be pH of 8.0, temperature of 60°C, 100 g/L lactose as substrate with supplementing 5 mM Mn 2+ . When the ratio of immobilized enzymes to EutM scaffold reached 1:6, the D-tagatose productivity of the dual-enzymes cascade could reach 1.03 g/L/h, which was 1.24-fold higher than free enzymes. In addition, the EutM-based scaffold could efficiently improve the stability of immobilized enzymes, in which 45% of the activity remained after 12 h, 2.14-fold higher than the free one. Overall, an attractive EutM-based selfassembling platform immobilizing BagB and TMAI was developed, showing enhanced catalysis efficiency and enzyme thermo-stability for D-tagatose production.

Theoretical and Practical Aspects of Multienzyme Organization and Encapsulation

“…Activity enhancement varied depending on which EutM homolog was used. In addition, two two-enzyme pathways producing either trehalose from soluble starch or tagatose from lactose were scaffolded on EutM (11,64). Two EutM constructs were used to simultaneously scaffold two proteins, with each containing either the SpyCatcher or SnoopCatcher tag to match the enzyme with the corresponding tag.…”

Marine chitin upcycling with immobilized chitinolytic enzymes: current state and prospects

“…Living cellular and also non-living protein and peptide bioscaffolds 167–178 were previously promoted as capable alternatives for enzyme immobilization. One of their greatest advantages is that they can be genetically modified for high performance, can be simply produced in large quantities through cellular expression, and can be coupled to the biocatalyst targets by the integration of biomolecular recognition and binding domains.…”

“…One of their greatest advantages is that they can be genetically modified for high performance, can be simply produced in large quantities through cellular expression, and can be coupled to the biocatalyst targets by the integration of biomolecular recognition and binding domains. Examples of uses of this promising tactic are the involvement of preparations of synthetic cellulosomes, the multienzyme complexes used by anaerobic cellulolytic bacteria to degrade lignocellulosic biomass, for hydrolytic degradation of softwood pulp, 170 the design of self-assembled enzyme/bacterial microcompartment conjugates for enhanced biocatalytic d -tagotose production from lactose, 175 and the use of chemically cross-linked enzyme aggregates for lignocellulosic waste processing 179 For in-depth practical details of the technology behind protein- and peptide-mediated enzyme assembly for assorted biotechnology uses the interested reader is referred to the cited topical reviews and examples of original articles in the literature databases.…”