Thermal stabilization of the deglycating enzyme Amadoriase I by rational design

Amadoriases are a class of FAD-dependent enzymes that are found in fungi, yeast and bacteria and that are able to hydrolyze glycated amino acids, cleaving the sugar moiety from the amino acidic portion. So far, engineered Amadoriases have mostly found practical application in the measurement of the concentration of glycated albumin in blood samples. However, these engineered forms of Amadoriases show relatively low absolute activity and stability levels, which affect their conditions of use. Therefore, enzyme stabilization is desirable prior to function-altering molecular engineering. In this work, we describe a rational design strategy based on a computational screening method to evaluate a library of potentially stabilizing disulfide bonds. Our approach allowed the identification of two thermostable Amadoriase I mutants (SS03 and SS17) featuring a significantly higher T-50 (55.3 degrees C and 60.6 degrees C, respectively) compared to the wild-type enzyme (52.4 degrees C). Moreover, SS17 shows clear hyperstabilization, with residual activity up to 95 degrees C, whereas the wild-type enzyme is fully inactive at 55 degrees C. Our computational screening method can therefore be considered as a promising approach to expedite the design of thermostable enzymes.