In vitro assembly of the trehalose bi-enzyme complex with artificial scaffold protein

Abstract

Introduction: Trehalose is a significant rare sugar known for its stable properties and ability to protect biomolecules from environmental factors.Methods: In this study, we present a novel approach utilizing a scaffold protein-mediated assembly method for the formation of a trehalose bi-enzyme complex. This complex consists of maltooligosyltrehalose synthase (MTSase) and maltooligosyltrehalose trehalohydrolase (MTHase), which work in tandem to catalyze the substrate and enhance the overall catalytic efficiency. Utilizing the specific interaction between cohesin and dockerin, this study presents the implementation of an assembly, an analysis of its efficiency, and an exploration of strategies to enhance enzyme utilization through the construction of a bi-enzyme complex under optimal conditions in vitro.Results and Discussion: The bi-enzyme complex demonstrated a trehalose production level 1.5 times higher than that of the free enzyme mixture at 40 h, with a sustained upward trend. Compared to free enzyme mixtures, the adoption of a scaffold protein-mediated bi-enzyme complex may improve cascade reactions and catalytic effects, thus presenting promising prospects.

Research progress of multi-enzyme complexes based on the design of scaffold protein

“…When the substrate level was low, the channelization of the substrate was more significant (You, 2012 ). Some articles have highlighted the latest progress of multi-enzyme reactions, mainly focusing on the substrate channel strategy based on DNA and protein scaffold (Schoffelen and Hest 2013 ; Wheeldon 2016 ; Buddingh' BC 2017 ; Nakata et al 2019 ; Tsitkov 2019 ; Wang et al 2023b ). The method by which synthetic multi-enzyme complexes increase product synthesis has long been debated.…”

“…4 B) Another research group has demonstrated that the multi-enzyme complex based on the SpyTag/SpyCatcher system improves catalytic efficiency (Fig. 4 C) (Wang et al 2023a , b ). The stereoselective carbonyl reductase (CpCR) and glucose dehydrogenase (GDH) successfully fused with SpyCatcher and SpyTag to form two double enzyme self-assembled clusters, named CpCR–SpyCatcher–SpyTag–GDH and GDH–SpyCatcher–SpyTag–CpCR.…”

“…The stereoselective carbonyl reductase (CpCR) and glucose dehydrogenase (GDH) successfully fused with SpyCatcher and SpyTag to form two double enzyme self-assembled clusters, named CpCR–SpyCatcher–SpyTag–GDH and GDH–SpyCatcher–SpyTag–CpCR. CpCR–SpyCatcher–SpyTag–GDH showed better activity and efficiently converted ethyl 2-oxo-4-phenylbutyrate (OPBE) to ethyl(R)2-hydroxy-4-phenylbutanoate ((R)-HPBE), while regenerating NADPH with a unique structure and excellent catalytic activity, improving the catalytic efficiency of the enzyme (Wang et al 2023a , b ). Overall, these examples demonstrate the practicality of the SpyTag/SpyCatcher system in biotechnology.…”

“…B Preparation of the oil synthesis with TLL–CvFAP (Zhong et al, 2022a). C Regenerating NADPH with a special structure and excellent catalytic activity with CpCR–SpyCatcher–SpyTag–GDH (Wang et al 2023a , b ). D Assembly of the TIM, ALD, and FBP fused dockerin in the mini-scaffoldin (You, 2012).…”