Characterization of recombinant invertase expressed in methylotrophic yeasts

We studied, for the first time, characterization of the invertase expressed in the methylotrophic yeasts Hansenula polymorpha and Pichia pastoris in t erms of enzyme conformational stability and structural behaviour induced by temperature as a function of PH using enzymic assays, differential scannin g calorimetry, fluorescence and CD. The enzyme produced in both hosts was v ery stable over a broad range of pH values, keeping its enzymic activity an d structure above 60 degreesC, Thermal denaturation, as measured by differe ntial scanning calorimetry, war always irreversible. However, the fact that scanning rate had no effect on the calorimetric curves gave us the chance to analyse the data from a thermodynamic point of view. The conformational stabilities were essentially identical under the experimental conditions st udied, but stability was always slightly higher in the enzyme expressed in H. polymorpho. This fact indicates that the greater degree of glycosylation of this enzyme form contributed to its increased global stability. Reactiv ation upon heating at 80 degreesC depends on protein concentration, suggest ing that irreversibility could be associated with slow refolding kinetics a t high protein concentration.