Purification and Characterization of LOX Isoenzymes from Germinating Barley : Biotransformation of Complex Lipids

Transcription

1 Purification and Characterization of LOX Isoenzymes from Germinating Barley : Biotransformation of Complex Lipids vorgelegt von M.Sc. Roberto Barbosa de Almeida aus Lorena, Sao Paulo, Brasilien von der Fakultat III - Prozesswissenschaften der Technischen Universitat Berlin zur Eriangung des akademischen Grades Doktor der Ingenieurwissenschaften - Dr.-lng. - genehmigte Dissertation Promotionsausschuss: Vorsitzender: Prof. Dr.-lng. Dr. e.h. Friedrich Meuser Berichter: Prof. Dr. rer. nat. Roland Tressl Berichter: Prof. em. Dr.-lng. Karl Wackerbauer Tag der wissenschaftiichen Aussprache : 11. August 2005 Berlin 2005 D83

2 INDEX 1 - Introduction Flavour stability Lipids in the brewing process Oxidation of fatty acids Photooxidation of fatty acids Autoxidation of fatty acids Enzymatic oxidation of fatty acids Lipoxygenases LOX in the malting / beer process Lipoxygenase - mechanism LOX - substrate acceptance Hydroxy fatty acids in the malting / beer process Hydroperoxy fatty acid metabolizing enzymes (HPME) Aims of the thesis Materials and Methods Chemicals Raw material Synthesis Synthesis of (S)-9-hydroperoxy-(E,Z)-10,12-octadecadienoic acid First step - extraction of lipoxygenase from tomato Second step - enzymatic transformation of linoleic acid Synthesis of (S)-9-hydroxy-(E,Z)-10,12-octadecadienoic acid Synthesis of 9-keto-(E,Z)-10,12-octadecadienoic acid Synthesis of racemic 9-hydroxy-(E,Z)-10,12-octadecadienoic acid Synthesis of (S)-13-hydroperoxy-(Z,E)-9,11-octadecadienoic acid Synthesis of (S)-13-hydroxy-(Z,E)-9,11-octadecadienoic acid Synthesis of 13-keto-(Z,E)-9,11-octadecadienoic acid 28

3 Synthesis of racemic 13-hydroxy-(Z,E)-9,11-octadecadienoic acid Enzyme assays Lipoxygenase assay Hydroperoxy - metabolizing enzymes (HPME) assay Lipoxygenase isoenzymes - extraction and partial purification / separation Lipoxygenases - extraction and partial purification Lipoxygenases - isoenzymes partial purification / separation Hydroxylapatite chromatography Isoelectric focussing (IEF) LOX isoenzymes - characterization SDS-PAGE ' ph optimum Kinetic parameters Influence of temperature on the enzyme activity Protein concentration Extraction of malt lipids - fatty acid composition Extraction of non-polar lipids Extraction of polar lipids Determination of the fatty acid composition of the extracted malt lipids Preparation of stock emulsions Substrate stock solution of free linoleic acid Substrate stock emulsions of esterified derivatives of linoleic acid "Substrate stock emulsions of extracted lipids Preparative enzymatic transformations Enzymatic transformations using free linoleic acid as substrate Hydroxylapatite chromatography partial purified LOX Isoelectric focussing partial purified LOX Crude extract LOX 37 IV

4 Enzymatic transformations using esterified derivatives of linoleic acid as substrate Isoelectric focussing partial purified LOX Crude extract LOX Enzymatic transformations using extracted lipids as substrate Isoelectric focussing partial purified LOX Product analysis of the enzymatic transformations Determination of the amount of the regioisomers 9- and 13-HPODE and the remained linoleic acid Sample preparation GC- MS analysis Determination of'the stereoisomer ratios from 9-HPODE and 13-HPODE Sample preparation HPLC-analysis Determination of the LOXs relative activities Statistic methodology Purification, separation and characterization of LOX isoenzymes using free linoleic acid Introduction Results Analysis of 9- and 13-HPODE Purification / separation methods Hydroxylapatite chromatography Isoelectric focussing Regiqselectivity of LOX isoenzymes with free linoleic acid as substrate Hydroxylapatite chromatography Isoelectric focussing Total amount of HPODE after enzymatic transformations of linoleic acid with IEF separated LOX Hydroperoxide-metabolising enzymes Stereoselectivity of partially purified LOX isoenzymes Formation of (E.E)-isomers 62

5 Kinetic parameters Electrophoresis pH optimum Influence of temperature Discussion Summary Substrate-, regio- and stereospecificity of LOX isoenzymes with complex lipids and extracted malt lipids Introduction Results Analysis of extracted lipids Transformation of trilinolein with crude enzyme extract Enzymatic transformation of esterified fatty acids by LOX1 and LOX Relative activities of LOX isoenzymes with free and esterified linoleic acids Regioselectivity of LOX isoenzymes with esterified linoleic acid Stereoselectivity of LOX isoenzymes with free and esterified derivatives of linoleic acid Fatty acid composition of polar lipids and non-polar lipids extracted from malt Regioselectivity of LOX isoenzymes with malt lipid extracts Stereoselectivity of LOX isoenzymes with malt lipid extracts Discussion Biotransformation of esterified linoleic acids with LOX Biotransformation of esterified linoleic acids with LOX Biotransformation of trilinolein with crude enzyme extract Biotransformation of extracted polar lipids with LOX1 and LOX Biotransformation of extracted non-polar lipids with LOX1 and LOX Summary VI

6 6 - Conclusion References 99 8-Appendix 116 VII