IN-VITRO CHARACTERIZATION OF ASTAXANTHIN BIOSYNTHETIC-ENZYMES

Escherichia coli strains expressing the marine bacteria (Agrobacterium aurantiacum and Alcaligenes sp. strain PC-1) astaxanthin biosynthetic genes (crtZ and W), Haenatococcus pluvialis bht, and Erwinia uredovor a crtZ genes were used for in, vitro characterization of the respectiv e enzymes. Specific enzyme assays indicated that all of the enzymes ar e bifunctional, in that the CrtZ enzymes formed zeaxanthin from beta-c arotene via beta-cryptoxanthin, as well as astaxanthin from canthaxant hin via phoenicoxanthin (adonirubin). The BKT/CrtW enzymes synthesized canthaxanthin via echinenone from beta-carotene and 4-ketozeaxanthin (adonixanthin) with trace amounts of astaxanthin from zeaxanthin. Comp arison of maximum catalytic activities as well. as selectivity experim ents carried out in the presence of both utilizable substrates indicat ed that the CrtZ enzymes from marine bacteria converted canthaxanthin to astaxanthin preferentially, whereas the Erwirnia CrtZ possessed a f avorability to the formation of zeaxanthin from beta-carotene. The Crt W/BKT enzymes were not so defined in their substrate preference, respo nding readily to fluctuations in substrate levels. Other properties ob tained indicated that the enzymes were strictly oxygen-requiring; and a cofactor mixture of a oxoglutarate, ascorbic acid, and Fe2+ was bene ficial to activity. Based on enzymological data, a predicted pathway f or astaxanthin biosynthesis is described, and it is proposed that CrtZ -like enzymes be termed carotenoid 3,(3')-beta-ionone ring hydroxylase and CrtW/BKT carotenoid 4,(4')-beta-ionone ring oxygenase.