Preparation of crosslinked enzyme aggregates of lipoxygenase and its use in the synthesis of various quinones

In this study, lipoxygenase (LOX) partially purified from soybean seeds (Glycine max (L.) Merr. Cv. Atakişi) was immobilized as crosslinked enzyme aggregates (LOX-CLEA). The optimal glutaraldehyde and ammonium sulfate concentrations and immobilization time for the immobilization were determined as 20% (w/v), 55% (w/v) and 10 h, respectively by using response surface methodology (RSM). The optimum pH and temperature of free LOX and LOX-CLEA were determined as 8.5 and 25 ºC, respectively for the free LOX and 8.5 and 60 ºC, respectively for LOX-CLEA. Under the predetermined optimal pH and temperature values, Km and Vmax values of the free LOX were 28 mM and 1593 nmol/min mg protein, respectively towards linoleic acid. The corresponding Km and Vmax values of the LOX-CLEA were 4.4 mM ve 1046 nmol/min g CLEA. After 24 h preincubation time, the free LOX protected 85 and 47% of its initial activity at 35 and 60 °C, respectively whereas LOX-CLEA protected 90 and 73% of its initial activity, respectively under the same conditions. The hydroperoxidase activities of free lipoxygenase and LOX-CLEA were investigated for the synthesis of 1,4-benzoquinone and thymoquinone. After 15 min reaction time the highest 1,4-benzoquinone yield was obtained as 24 and 33%, respectively for the free LOX and LOX-CLEA whereas the highest thymoquinone yields were obtained as 12 and 15%, respectively for the free LOX and LOX-CLEA. After 15 reuses, LOX-CLEA protected 39% of its initial activity for the synthesis of 1,4-benzoquinone in a batch type reactor.