Inactivation Kinetics and Structural Changes of High Pressure Treated Actinidin

Z.Alexandrakis, G.Katsaros, Ph.Stavros, G.Nounesis, P. Taoukis

Abstract


Plant sulfydryl proteases such as actinidin are mainly used as meat tenderisers, milk clotting agents for the development of novel dairy products and proteolytic agents in biotechnological procedures. The main issue with regards their application is the method for the control of their enzymatic activity after the desired extent of proteolysis, considering that thermal treatment is not applicable. High Pressure (HP) processing could be applied for the enzyme activity regulation. Actinidin, extracted from kiwi fruit (Hayward var., A. chinensis), was purified using anion-exchange and gel filtration chromatography. After purification procedure, only one single peak was finally received for this enzyme corresponding to a protein of molecular mass of 24 kDa with an isoelectric point equal to or lower than 3.5. The effect of HP (500–900MPa and 50- 70°C) on the remaining activity of purified actinidin in phosphate buffer solution (pH 6.0) was studied. Inactivation of purified actinidin was described by a first-order kinetic model both for thermal and HP treatment at the studied processing conditions. Aiming at an optimal process design, a composite mathematical model, which describes the actinidin inactivation rate as a function of pressure and temperature conditions, taking into account the dependence of both activation energy and activation volume on the applied pressure and temperature respectively, was used. HP-induced conformational changes of the purified actinidin were also investigated using Circular Dichroism spectroscopy (CD). A direct comparison of the CD results for the treated and the untreated enzymes reveals that reversible changes were depicted in the far- and near-UV. It is suggested that the exposure to HP may affect the linkage between thiolate ion of cysteine group and positively charged nitrogen of the imidazole group of the active site of actinidin leading to the reduction of the enzyme activity.

Keywords


Purified Actinidin; High Pressure Inactivation; Circular Dichroism; Structural Alterations

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