Regio- and Stereoselectivity of Malt Lipoxygenases LOX1 and LOX2
Roberto Barbosa de Almeida1, Leif-Alexander Garbe1,2, Renate Nagel1, Karl Wackerbauer1,3, and Roland Tressl1
1 Technische Universität Berlin, Institut für Biotechnologie, Fachgebiet Molekularanalytik, Seestrasse 13, D-13353 Berlin.
2 Corresponding author. E-mail: Leif-A. Garbe@TU-Berlin.de
3 Professor Emeritus Dr.-Ing. Karl Wackerbauer - Formerly Technische Universität Berlin, Institut für Biotechnologie, Fachgebiet Brauwesen.
J. Inst. Brew. 111(3), 265-274, 2005 | VIEW ARTICLE
ABSTRACT
The characterisation of lipoxygenases LOX1 and LOX2 and hydroperoxyoctadecadienoic acid (HPODE) degrading enzymes from barley green malt is reported. Hydroxylapatite chromatography (HAC) and isoelectric focussing (IEF) were performed to separate and purify LOX isoenzymes. The regio- and stereoselectivity of LOX1 and LOX2 towards linoleic acid as substrate was characterised. HAC purified isoenzyme LOX1 showed a 9-HPODE:13-HPODE ratio of 75:25 and LOX2 a ratio of 39:61. IEF separated LOX1 and LOX2 transformed linoleic acid to 9-:13-HPODE ratios of 90:10, and 13:87, respectively. 9- HPODE stereoisomers from LOX1 exhibited a S:R ratio of 93:7 and 13-HPODE from LOX2 a S:R ratio of 89:11. However, the minor regioisomers were analysed with S:R = 48:52 (LOX1, 13-HPODE) and 40:60 (LOX2, 9-HPODE). These results indicate a complete LOX isoenzyme separation by IEF. Hydroperoxide- metabolising enzymes, which were investigated in the IEF fractions, did not interfere with the dual position specificities of LOX isoenzymes.
Key words:
Green malt, HPODE, lipoxygenase, LOX1, LOX2, regioselectivity, stereoselectivity.
Publication no. G-2005-1012-297 ©2005 The Institute & Guild of Brewing