Thermostabilized Amadoriase I from Aspergillus Fumigatus
Data di pubblicazione
Data di priorità
International PCT patent application
Politecnico di Milano, IIT
Department of Electronics, Information and Bioengineering
Alfonso Gautieri, Emilio Parisini, Federica Rigoldi, Stefano Donini, Alberto Redaelli
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. Such enzyme-based system has been recently proposed and commercialized. However, one of the issues of these enzyme sensors is their unsatisfactory absolute activity and stability. This issue affects storage, stability against temperature changes, which in turn limits the applicability of enzymatic HbA1c monitoring systems based on enzymes.
Moreover, glycation of food proteins is a drawback effect of several thermal treatment (e.g., milk UHT treatment), which results in alteration of the sensory and nutritional profile of the products. Amadoriase enzymes have a potential use in 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.
Combined computational and experimental approach was used to obtain an engineered Amadoriase enzyme 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.
Experimentally, 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.
Campo di applicazione
This engineered enzyme can be used for measurement of glycated hemoglobin for diabetes detection/monitoring and to reduce undesired Maillard Reaction in food processing (for example during pasteurization or sterilization).
The engineered enzyme has a high thermal stability; the activity of this enzyme towards its natural substrate (fructosyl lysine) is retained up to a temperature of 80°C, whereas the wild-type enzyme is active only at T < 50°C.
Importantly, increased thermal stability likely involves longer stability at milder conditions (i.e. longer storage and expiry date).
The remarkable increase of thermal stability of the engineered enzymes allows envisioning the use of our engineered form of the enzyme to prevent aminoacid glycation in application where it is important the long-term stability, stability against temperature variations and/or the use of high temperature.
Stadio di sviluppo
Samples of the engineered enzyme were obtained and experimentally tested.