Introduction

A combined computational and experimental approach was used to obtain an engineered Amadoriase with increased stability with respect to the wild-type one. Using a computational approach, pairs of amino acids that can be mutated to cysteines were identified in order to introduce one or two disulphide bonds (i.e., cysteine-cysteine covalent bond) in the structure of Amadoriase I.

Technical features

The measurement of glycated haemoglobin (HbA1c ) in the blood is a very powerful method for monitoring the insurgence and development of diabetes. A method for HbA1c detection exploits the deglycating properties of Amadoriases enzyme, which can be included in a fast, easy and cost-effective HbA1c monitoring system, but the enzymes should be able to sustain the thermal treatments without losing activity. Experimentally, for the patent, several mutants were tested and those where the introduction of the disulfide bond(s) has proved effective in increasing the thermal stability of the enzyme without affecting the activity were identified and samples of the engineered enzyme were obtained and experimentally tested.

Possible Applications

• Measurement of glycated hemoglobin for diabetes detection/monitoring;
• Food industry: in controlling and preventing protein glycation in food products, but the enzymes should be able to sustain the thermal treatments without losing activity;
• Reduce undesired Maillard Reaction in food processing (for example during pasteurization or sterilization);

Advantages

• The engineered enzyme has a high thermal stability, enabling the use of the engineered form of the enzyme to prevent aminoacid glycation glycation in application where it is important the long-term stability, stability against temperature variations and/or the use of high temperature.
• Increased thermal stability likely involves longer stability at milder conditions (i.e. longer storage and expiry date).