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

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.