Kinetic characterization of amino acid oxidase from different sources and their application in the oxidation of l-methionine

Oksidaze L-aminokiselina (L-AAO) su poznate po svojoj specifičnosti prema nizu različitih supstrata. U ovom radu su kinetički karakterizirani enzimi oksidaze L-aminokiselina iz triju različitih izvora. Korišteni su enzimi iz zmijskog otrova kobre roda Crotalus adamanteus, zmijskog otrova kobre Crotalus atrox i novi enzim iz bakterije Rhodococcus opacus. Kao supstrat je korišten L-metionin koji se u prisustvu enzima L-AAO oksidativno deaminira u odgovarajuću α-keto kiselinu uz nastanak vodikovog peroksida i amonijaka kao sporednih produkata. Prisustvo vodikovog peroksida je nepoželjno u reakcijskoj otopini, a za njegovo uklanjanje je korištena katalaza porijeklom iz goveđe jetre. Iz eksperimentalno određene ovisnosti specifične aktivnosti enzima o početnoj koncentraciji supstrata L-metionina, odnosno produkta, dobivenih metodom početnih brzina, nelinearnom regresijom su procijenjeni parametri Michaelis-Menteničinog izraza sva tri enzima, Vm i Km, kao i konstanta inhibicije produktom Ki. Biotransformacija L-metionina katalizirana oksidazama L-aminokiselina je provedena u kotlastom reaktoru bez dodatka katalaze te u ponovljenom kotlastom reaktoru s dodatkom katalaze. Postavljen je i validiran matematički model procesa biotransformacije L-metionina kataliziranog oksidazama L-aminokiselina.L-amino acid oxidases (L-AAO) are well known for their broad substrate specifity. In this work, three L-AAOs from different sources were kinetically characterized: two enzmyes from the snake venom of Crotalus adamanteus and Crotalus atrox, and a new enzyme from bacteria Rodococcus opacus. L-methionine was used as substrate. In the presence of L-AAO, L-methionine oxidizes to corresponding α-keto acid with production of hydrogen peroxide and ammonia as by-products. The presence of hydrogen peroxide in the reaction solution was unwanted and for its decomposition catalase from beef liver was used. Parameters of Michaelis-Mentens’s equation, Vm, Km and product inhibition constant, Ki were determined by non-linear regression from the experimentally observed dependence of the specific activity of enzyme on the initial concentration of substrate L-methionine and product (method of initial rates). Biotransformation of L-methionine catalysed by L-AAOs was carried out in batch reactor without the presence of catalase and in repetitive batch reactor with the addition of catalase. A mathematical model of L-methionine biotransformation process was developed and validated