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 designate d LOX-2 and LOX-3. The thermal inactivation of crude pea LOX and the recomb inant LOX (rLOX) were studied. Heat-inactivation plots for crude extracts o f pea LOX were linear from which thermodynamic activation parameters, Delta H-#, Delta S-# sand Delta G(#) have been estimated. The enzymatic activity was relatively stable with a respective half-life (t(1/2)) at 60 degrees 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 degrees C the thermostability of LOX-3 p resent in crude extracts of the mutant strain (t(1/2) = 66.8 min) was 90% g reater than purified recombinant LOX-3 (rLOX-3; t(1/2) = 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 degrees C (t(1/2 ) = 16.5 min and 18.4 min, respectively). Even at the higher temperatures o f 70 degrees C the t(1/2) values were 84 and 51, respectively. It is sugges ted that LOX in crude enzyme extracts was stabilised at 50 degrees C due to protection by other constituents, possibly including starch and proteins. Separate tests at 70 degrees C in the presence of additives (polyols, deter gents and small ions) showed that sucrose was the most effective stabiliser and increased the stability of pea LOX by 400-600%. (C) 1999 Elsevier Scie nce Ltd. All rights reserved.