Kinetics of thermal inactivation of pea seed lipoxygenases and the effect of additives on their thermostability

Mature pea seeds contain two major lipoxygenases (LOX) isoenzymes designated LOX-2 and LOX-3. The thermal inactivation of crude pea LOX and the recombinant LOX (rLOX) were studied. Heat-inactivation plots for crude extracts of pea LOX were linear from which thermodynamic activation parameters, ΔH#, ΔS# sand ΔG# have been estimated. The enzymatic activity was relatively stable with a respective half-life (t1/2) at 60 °C of 54.2 min for LOX from pea (Pisum sativum L. cv. Birte) or 18.4 min for a mutant line lacking LOX-2. At 50°C the thermostability of LOX-3 present in crude extracts of the mutant strain (t1/2=66.8 min) was 90% greater than purified recombinant LOX-3 (rLOX-3; t1/2=34.6 min). However, rLOX-3 was more heat-stable than rLOX-2. Both rLOX-3 and pea mutant line lacking LOX-2 possessed considerable thermostability at 60°C (t1/2=16.5 min and 18.4 min, respectively). Even at the higher temperatures of 70°C the t1/2 values were 84 and 51, respectively. It is suggested that LOX in crude enzyme extracts was stabilised at 50°C due to protection by other constituents, possibly including starch and proteins. Separate tests at 70°C in the presence of additives (polyols, detergents and small ions) showed that sucrose was the most effective stabiliser and increased the stability of pea LOX by 400–600%.