Novel route to mono- and diglycerides synthesis in miniemulsion catalyzed by lipases [Elektronische Ressource] / vorgelegt von Jean-Baptiste Doucet

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Informations

• Publié par: universitat_ulm
• Publié le: 01 janvier 2007
• Nombre de lectures: 25
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Novel route to mono- and diglycerides
synthesis in miniemulsion catalyzed by
lipases



Dissertation
Zur Erlangung des Doktorgrades Dr. rer. nat.
der Fakultät für Naturwissenschaften
der Universität Ulm

vorgelegt von
Jean-Baptiste Doucet
geboren am 07.10.1976 in Marseille, Frankreich
Ulm, Dezember 2007

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Amtierender Dekan: Prof. Dr.
1. Gutachter: Prof. Dr. Katharina Landfester
2. Gutachter: Prof. Dr. Nicola Hüsing
Universität Ulm, Fakultät für Naturwissenschaften, 2008
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- 4 -TABLE OF CONTENT

1. INTRODUCTION............................................................................................................ 7
2. THEORETICAL SECTION ........................................................................................... 9
2.1. Emulsions .................................................................................................................. 9
2.2. Miniemulsion .......................................................................................................... 11
2.3. Preparation and homogenization of miniemulsions................................................. 14
2.4. Description of lipases and of its properties – Attractive applications .................... 16
2.4.1. Structure and mechanism ....................................................................................... 16
2.4.2. Occurrence and preparation of lipases ................................................................... 23
2.4.3. Kinetics of lipase catalysis ..................................................................................... 25
2.4.4. Applications of lipases ........................................................................................... 33
2.4.4-i. Lipases as biocatalysts in organic synthesis .................................................... 33
2.4.4-ii. Lipases in oleochemistry ................................................................................ 33
2.4.4-ii.a) Soaps and fatty acids ............................................................................... 33
2.4.4-ii.b) Transesterified triglycerides .................................................................... 33
2.4.4-ii.c) Monoglycerides ....................................................................................... 34
2.4.4-ii.d) Lipolysis and glycerolysis....................................................................... 34
3. RELEVANT METHODS FOR CHARACTERIZATION............................................. 39
3.1. Dynamic light scattering (DLS) ................................................................................. 39
3.2. Nuclear magnetic resonance 41
4. RESULTS AND DISCUSSION......................................................................................... 60
4.1. Analysis of different products .................................................................................... 60
14.1.1. H-NMR spectroscopy of acylglycerols of tricaprylin........................................... 60
4.1.2. Determination of caprylic acid content by acid-base titration ............................... 67
4.2. Kinetics of lipolysis of tricaprylin by lipase in miniemulsion.................................. 68
4.2.1. Typical exploitation of the results obtained by the Pseudomonas Cepacia lipase-
catalyzed lipolysis of tricaprylin in miniemulsion at 35 °C............................................. 69
4.2.2. Description of kinetics ........................................................................................... 77
4.2.2-i. Description of kinetics of tricaprylin lipolysis in miniemulsion carried out with
the lipases RML, RAL and lipase PS at respective different optimal temperatures .... 77
4.2.2-ii. Influence of the different type of lipases ........................................................ 82
4.2.2-iii. Influence of the miniemulsion homogeneity................................................. 89
4.2.2-iv. Influence of the droplet size........................................................................... 95
4.2.2-v. Glycerolysis versus lipolysis......................................................................... 102
- 5 -4.2.2-vi. Hydrolysis in miniemulsion......................................................................... 108
5. SUMMARY AND CONCLUSIONS............................................................................... 112
6.ZUSAMMENFASSUNG .................................................................................................. 115
7. EXPERIMENTAL PART ............................................................................................... 118
7.1. Hydrolysis in miniemulsion...................................................................................... 118
7.2. Enzymatic lipolysis in miniemulsion ....................................................................... 118
7.3. Enzymatic lipolysis in miniemulsion – Kinetics ..................................................... 119
st7.3.1. Preparation of the miniemulsion – 1 Method 119
nd7.3.2. Preparation of the miniemulsion – 2 Method .................................................... 119
7.4. Enzymatic glycerolysis in miniemulsion – Kinetics ............................................... 120
7.5. Methods...................................................................................................................... 121
8. REFERENCES ................................................................................................................. 123
9. APPENDIX ....................................................................................................................... 129
9.1. Abbreviations............................................................................................................. 129
9.2. Symbols 130
9.3. Chemicals ................................................................................................................... 131
9.4. Enzymes 132
19.5. H-NMR spectra of acylglycerols at 400 MHz in CDCl ....................................... 132 3
10. DANKSAGUNG 135
11. LEBENSLAUF ............................................................................................................... 137
12. ERKLÄRUNG................................................................................................................ 139













- 6 -1. INTRODUCTION

Lipids are key elements in the chemistry of life. Most organisms use the supramolecular
chemistry inherent to phospholipids to form their exterior and compartimental membranes.
Many plants and animals store chemical energy in the form of triglycerides, which are
sparingly soluble in water. For the metabolic turnover of these and other biochemicals, they
produce esterases, enzymes which can hydrolyze bonds of water-soluble esters. Esterases
which can hydrolyze triglycerides at the water/oil boundary are termed lipases or more
systematically triacylglycerol hydrolases, and those which attack phospholipids are termed
[1] [2-13]phospholipases. Both types of enzymes have recently received considerable attention.
Whereas phospholipases are involved in key metabolic events such as membrane turnover and
signal transduction, lipases have diverse functions in the degradation of food and fat. They
have qualified as valuable drugs against digestive disorders and diseases of the pancreas.
They also find applications in biotechnology (in particular as detergent additives) and as
catalysts for the manufacture of specialty (oleo) chemicals and for organic synthesis. Their
broad synthetic potential is large due to the fact that lipases, in contrast to most other
enzymes, accept a wide range of substrates. They are quite stable in organic solvents, and
thus, depending on the solvent system used, can be applied to hydrolysis reactions or ester
synthesis. One of those applications is the synthesis of monoglycerides (MG) and diglycerides
(DG), which are used in food industry and are used as emulsifiers in cosmetics and
[14]pharmaceuticals for the controlled-release preparations. The most frequently used method
to produce MG and DG is the glycerolysis. In principle, there are two possibilities of
glycerolysis, one where free fatty acid (FFA) and glycerol are mixed, and another one where
triglyceride (TG) and glycerol are mixed together in a 1:2 ratio.
In 1958 Sarda and Desnuelle defined lipases