Construction of an Artificial In Vitro Synthetic Enzymatic Platform for Upgrading Low-Cost Starch to Value-Added Disaccharides

Abstract

Disaccharides are valuable oligosaccharides with an increasing demand in the food, cosmetic, and pharmaceutical industries. Disaccharides can be manufactured by extraction from the acid hydrolysate of plant-derived substrates, but this method has several issues, such as the difficulty in accessing natural substrates, laborious product separation processes, and troublesome wastewater treatment. A chemical synthesis using glucose was developed for producing disaccharides, but this approach suffers from a low product yield due to the low specificity and requires tedious protection and deprotection processes. In this study, we adopted an artificial strategy for producing a variety of value-added disaccharides from low-cost starch through the construction of an in vitro synthetic enzymatic platform: two enzymes worked in parallel to convert starch to glucose and glucose 1-phosphate, and these two intermediates were subsequently condensed together to a disaccharide by a disaccharide phosphorylase. Several disaccharides, such as laminaribiose, cellobiose, trehalose, and sophorose, were produced successfully from starch with the yields of more than 80% with the help of kinetic mathematical models to predict the optimal reaction conditions, exhibiting great potential in an industrial scale. This study provided a promising alternative to reform the mode of disaccharide manufacturing.

Thermodynamics-Driven Production of Value-Added d-Allulose from Inexpensive Starch by an In Vitro Enzymatic Synthetic Biosystem

“…In this study, the enzyme ratio was optimized by the method of titration experiment. Using the mathematical model based on the catalytic mechanisms and kinetic parameters of enzymes could provide a rapid and precise method to obtain the optimal reaction condition . However, the undesired reactions catalyzed by TtA6PE and CtA6PP bring difficulty and complexity to build a precise mathematical model to simulate the reaction process of the in vitro biosystem.…”

Recent advances in enzymatic synthesis of β-glucan and cellulose

“…d -cellobiose has been also a valuable and commercially attractive sugar with respect to zero-calorie sweeteners and as potential food additive. Although its synthesis from sucrose by SCP and CBP has been under implementation for industrial production [ 116 ], current research focussing on atom economy has been exploited via multi-enzyme one-pot biotransformation approaches with mathematical model assistance to predict optimal reaction conditions and enzyme loading ratio [ 117 , 118 ]. In this sense, d -cellobiose was synthesised from sucrose by three thermophilic enzymes with 60% yield within 10 h. The process involved phosphorolysis of sucrose by SCP ( Thermoanaerobacterium thermosaccharolyticum ) to form α-D-Glc-1P and fructose, which was isomerised by glucose isomerase ( Streptomyces murinus ) into D-Glc for further disaccharide synthesis by CBP ( Clostridium thermocellum ) ( Fig.…”

“…Amongst other valuable disaccharides synthesised in this study (laminaribiose, trehalose and sophorose), d -cellobiose was produced within 8 h with yield higher than 80% from low and high concentrations of starch. Since in vitro multi-enzymatic cascades have emerged as a promising biomanufacturing platform, this process proved to be robust and could represent an alternative approach to the current disaccharide production [ 118 ]. Although not covered in this review, manno-oligosaccharides are also of interest in the production of nutraceuticals and pharmaceuticals due to their prebiotic effects and many other biological properties [ 119 ].…”

“…Despite all the advantages of using sucrose as substrate and SCP as biocatalyst towards the synthesis of functional disaccharides, alternative multi-enzyme approaches have been also investigated using starch. Starch is cheaper than sucrose, the phosphorolysis produces more α-D-Glc-1P and the ratio α-D-Glc-1P/D-Glc can be tuned for optimisation [ 118 ] . In this sense, d -laminaribiose was successfully synthesised from starch and glucose by four thermophilic enzymes with 91% yield within 36 h. The process involved debranching of starch with isoamylase for conversion into α-D-Glc-1P by α-glucan phosphorylase and final catalysis by LBP ( Paenibacillus sp YM-1) in the presence of external D-Glc.…”

“…6 B). Some data indicated that D-Glc was slowly released from starch and remained at low concentration, thus minimising not only inhibition of the enzymes but also the effect of Maillard reaction during the process at high temperature [ 118 ].…”