Introduction

We computationally designed and experimentally validated two thermostable Amadoriase mutants featuring significantly improved thermal resistance properties relative to the wild type enzyme. These stabilized Amadoriase variants can be directly applied to improve currently available biosensors used in the detection and monitoring of diabetes or as additive in detergents for stain removal. The thermostabilization design strategy can be applied on other enzyme targets.

Technical features

Amadoriase enzymes are used as biosensor for the detection of glycated haemoglobin (HbA1c) and Glycated Albumin (GA) in the diagnosis and monitoring of diabetes. We designed and characterized two thermostable Amadoriase mutants featuring significantly improved thermal resistance properties relative to the wild type enzyme. As the enzyme represents the most sensitive component of the HbA1c and GA sensing systems, our engineered enzyme  increase the stability of the biosensor during transport and storage. Other potential applications include the reduction of undesidered glycation (browning) of food proteins during thermal treatments (e.g., milk UHT treatment), or as additive in detergent for stain removal. Finally, the thermostabilization design strategy can be applied on other enzyme targets. Two different engineered forms of the enzyme have been already produced in small (mg) quantities. We are currently fully characterizing their stability and enzymatic activity profiles. They show a remarkable increase in their thermostability, as they are still active at around 80°C when the wild type enzyme shows activity only up to 50°C. This has been confirmed by standard enzymatic assays produced in triplicates.

Possible Applications

• HbA1c monitoring in diabetes;
• Glycated albumin monitoring in diabetes;
• Reduce glycation in food products;
• Additive in detergents (stain removal);
• Design of thermostable enzymes.

Advantages

• Increased shelf life, increased stability during storage and transportation;
• Increased activity at high temperatures;
• Increase yield in protein production.