Efficient isoprimeverose production using an enzyme cocktail from engineered Aspergillus oryzae and yeast-assisted purification.

Aspergillus oryzae is a filamentous fungus that possesses various types of carbohydrate -degrading enzymes. Among these, isoprimeverose-producing enzyme (IpeA), acts on a key component of the plant cell wall structure, xyloglucan, to catalyze the release of isoprimeverose - a rare disaccharide that is expected to possess valuable prebiotics properties. Despite these expectations, however, a process for the effective production of isoprimeverose from the xyloglucan still requires further development for commercial-level application. A complicating factor for the lack of such a valuable process is that plant-derived xyloglucan is often modified with other sugars such as galactose and arabinose. Therefore, the effective production of isoprimeverose requires a cooperative form of degradation that must utilize different enzymes. In this study, we genetically engineered two A. oryzae strains - one produces IpeA and the other produces endoglucanase. The two strains were cultivated separately, and an enzyme cocktail was prepared using their respective culture supernatants. This enzyme cocktail successfully produced isoprimeverose from tamarind xyloglucan and tamarind seed gum. Approximately 14 g/L of isoprimeverose was obtained, which corresponds to a theoretical conversion rate of over 90 %. Although glucose and galactose remained in the reaction solution after enzymatic degradation, these by-products could be easily removed via treatment with Saccharomyces cerevisiae. Our developed process, which mimics traditional Japanese sake fermentation using A. oryzae and S. cerevisiae, has enabled efficient production of isoprimeverose.