Improving the thermostability of horseradish peroxidase by incorporating into water-immiscible coacervates

Abstract

Looking for novel matrix materials for encapsulation of enzymes based on water immiscible coacervates prepared by reaction of negatively charged hyaluronic acid and a positively charged recombinant mussel adhesive protein containing tyrosine residues was the subject of the investigation of this work. The results of experimental study of the thermostability of horseradish peroxidase (HRP) by means its encapsulation in these coacervates at temperature from 30 to 95^oC is presented in this paper. The Michaelis-Menten equation was applied to analyze of the enzymatic activity of HRP. The kinetic parameters were interpreted using a Lineweaver-Burk plot. According to the data obtained, Michaelis-Menten parameters, K_M and K_Cat , interpreted from the Lineweaver- Burk plots, were 0.271 mM and 2265 s^-1 for the free HRP and 0.325 mM and 2158 s^-1 for the rMAP/HA coacervate, containing HRP, respectively, which indicate that the enzyme did not lose its activity during the coacervate formation. It was founded that the free enzyme began to lose activity above 40^oC, while the encapsulated HRP remained stable to 85^oC. The encapsulated HRP lost only 18% and 25% of activity at temperature of 90 and 95^oC, respectively, while as free HRP loses all its initial activity, although they show similar activity at room temperature.

Keywords: encapsulation, hyaluronic acid, coacervate, recombinant mussel adhesive protein