Rapid, real-time sucrase characterization: Showcasing the feasibility of a one-pot activity assay

Abstract

Sucrases can modify numerous carbohydrates, and short-chain oligosaccharides produced by the unique transfructosylation activity of levansucrases are promising candidates for the growing sugar substitute market. These compounds could counteract the increasing number of diseases associated with the consumption of high-calorie sugars. Thus, there is great interest in the characterization of novel levansucrases. The commonly used method for sucrase activity determination is to quantify d-glucose released in the sucrose-splitting reaction. This is usually done in a discontinuous mode, i.e., several samples taken from the sucrase reaction are applied to a separately performed d-glucose determination (e.g., GOPOD assay). Employing the newly isolated levansucrase LevSKK21 from Pseudomonas sp. KK21, the feasibility of a one-pot sucrase characterization was investigated by combining sucrase reaction and GOPOD-based d-glucose determination into a single, continuous assay (Real-time GOPOD). The enzyme was characterized with respect to kinetic parameters, ion dependency, pH value, and reaction temperature in a comparative approach employing Real-time GOPOD and HPLC. High data consistency for all investigated enzyme parameters demonstrated that current processes for sucrase characterization can be considerably accelerated by the continuous assay while maintaining data validity. However, the assay was not applicable at acidic pH, as decolorization of the quinoneimine dye formed during the GOPOD reaction was observed. Overall, the study presents valuable data on the potentials of real-time sucrase activity assessment for an accelerated discovery and characterization of interesting enzymes such as the hereby introduced levansucrase LevSKK21. Progress in sucrase discovery will finally foster the development of health-promoting sucrose substitutes.