COAGULANTS OF MORINGA OLEIFERA LAM. SEEDS PURIFICATION & CHARACTERISATION

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1 COAGULANTS OF MORINGA OLEIFERA LAM. SEEDS PURIFICATION & CHARACTERISATION THÈSE N O 3251 (2005) PRÉSENTÉE À LA FACULTÉ SCIENCES DE BASE Institut des sciences et ingénierie chimiques SECTION DE CHIMIE ET GÉNIE CHIMIQUE ÉCOLE POLYTECHNIQUE FÉDÉRALE DE LAUSANNE POUR L'OBTENTION DU GRADE DE DOCTEUR ÈS SCIENCES TECHNIQUES PAR Stephan Christos DÖRRIES Diplom-Biochemiker, Universität Hannover, Allemagne et de nationalité allemande acceptée sur proposition du jury: Dr I. W. Marison, directeur de thèse Prof. G. Folkard, rapporteur Dr C. Servis, rapporteur Prof. U. von Stockar, rapporteur Prof. C. Wandrey, rapporteur Lausanne, EPFL 2005

2 Table of Contents Coagulants of Moringa oleifera Lam. Seeds Global Water Supply Water Treatment using Primary Coagulants Commercial Flocculants Aim References Extraction of Flocculating Proteins from M.oleifera Seeds Abstract Introduction Materials and Methods Seed Extraction Multiple Salt Solution: Protein Extraction Buffer: M NaCl: Demineralised Water: Analytical Methods Chromatography SDS-PAGE / Western Blotting Results and Discussion Different Extraction procedures Evolution of Protein Content during Storage Effect of Thermo-Precipitation Conclusions References Purification of M.oleifera Seed Proteins for Sequence Determination Abstract Introduction Purification Strategies SDS-PAGE Modification of Proteins upon Extraction Materials and Methods Seed Extraction Chromatography Analytics SDS-PAGE Total Protein Mass Spectrometry (MS) Results and Discussion Semi-Preparative Reverse Phase HPLC iii

3 Preparative Reverse Phase Chromatography Influence of the Thermo-Precipitation Step Protein Extraction Buffer Pooling of Non-Reduced Main Protein Component Prior to Reduction Increased Theoretical Plate Number Conclusions Purification Strategy Protein Characteristics Protein Modification and Non-Proteinaceous Components References Characterisation of Non-Proteinaceous Components Present in M.oleifera Extracts Abstract Introduction Presence of Secondary Metabolites that Interfere with Protein Purification from Seeds Phenolic Compounds Glucosinolate derivatives Influence of Phenolics and Isothiocyanates on the Physicochemical Properties of Proteins Determination of Secondary Metabolites in M.oleifera seed extracts 71 Materials and Methods Chemicals Plant material Extraction and isolation of 4(-α-L-rhamnopyranosyloxy)- benzylglucosinolate (4RBGLS) Re-crystallization of 4RBGLS Characterization of isolated 4RBGLS Preparation and isolation of 4-(α-L-rhamnopyranosyloxy)- benzylisothiocyanate (4RBITC) Seed Extractions Modified Extraction Procedure for All Different Media Original Protein Extraction Procedures HPLC methods Quantification of secondary metabolites Results and Discussion Preparation of Standards for Glucosinolates and Isothiocyanates present in M.oleifera Qualitative Analysis of Different Extraction Media Quantification of Secondary Metabolites in Extracts of M.oleifera seeds Conclusions Importance of the Results in Regard to Protein Extraction Acknowledgments References iv

4 Measurement of Coagulating Activity of Different Components Present in M.oleifera Seed Extracts Abstract Introduction Testing of Coagulation Efficiency Influence of the Particle Concentration on the Coagulating Activity Choice of colloid material for coagulation experiments Measurement of the Coagulation Rate Definition of Coagulation and Flocculation Materials and Methods Chemicals M.oleifera Seed Extracts Coagulation Assay Results and Discussion Different Extraction Procedures Analysis of Fractions prepared by Chromatographic separation of Extracts Non-reduced extract Separation after Reduction of the Sample Flocculation of Suspensions of Glassbeads and Bentonite Conclusions Coagulating activity of M.oleifera extracts References Characterisation of M.oleifera Seed Proteins Abstract Introduction Seed Proteins Materials and Methods Chemicals Protein extraction and quantification Chromatographic Separation (prep. HPLC + Äkta) Tris-Tricine SDS PAGE / Western Blotting Non-Specific Protein Staining and Immuno Staining Specific Staining of Glycosylated Proteins MALDI/TOF-MS Peptide Mapping / Mass Mapping Results and Discussion Tag Sequences of Selected Protein Fractions Low molecular weight proteins present in oil body fractions Proteins Separated by Reverse Phase Chromatography after Sample Reduction Tryptic digest Chymotryptic Digest Immuno Detection of Proteins with Anti-Flo Antibodies v

5 Conclusions Difficulties of Purification of M.oleifera seed proteins Homology of Isolated Fragments to Mo Sequence Homologies of Mo2.1 with seed proteins References Scale-up of the Extraction Procedure Abstract Introduction Transfer from Laboratory to Pilot Scale Materials and Methods Chemicals Press Cake Extraction Batch Extraction Percolation Ultrafiltration and Diafiltration Laboratory Scale Pilot Scale Diafiltration Analytics Tris-Tricine SDS-PAGE Protein Determination (BCA & Bradford) Results and Discussion Solid-Liquid Separation Product Concentration Protein Extraction by Percolation Conclusions Solid-Liquid Separation Product Concentration Solid-Liquid Extraction References Coagulants of Moringa oleifera Lam. Seeds General Conclusions and Future Perspectives References Additional Figures and Tables Chapter 3 - Characterisation of Non-Proteinaceous Components Present in M.oleifera Extracts Direct Calculation of Secondary Metabolite Concentration from Signal Peak Areas Observed in Standard Protein HPLC Chromatograms 190 Chapter 4 - Measurement of Coagulating Activity of Different Components Present in M.oleifera Seed Extracts Absorption change during coagulation of a glass powder suspension Chapter 5 - Characterisation of M.oleifera Seed Proteins vi

6 Preparative HPLC of Extract prepared with Multiple Salt Solution from M.oleifera press cake Preparation of Fractions for Sequence Analysis by Reverse Phase HPLC of an Extract prepared with Multiple Salts Solution from M.oleifera Press Cake Chapter 6 - Scale-up of the Extraction Procedure Setup of the Diafiltration Device Reference Nomenclature Abbreviations Amino Acids (Single Letter Code) Curriculum Vitae Publications vii

7 SUMMARY Water has been declared a human right by the United Nations, thus urging countries who have ratified the International Covenant on Economic, Social and Cultural Rights to ensure supply with drinking water for their populations 1. However, supply of drinking water in developing countries underlies different constraints than those met in industrialized countries. An example is the dependency on treatment chemicals that may pose problems in these countries regarding importation and distribution 2. In pioneering work in the Darfur region (Sudan) S.A.A. Jahn has instructed rural people how to use local natural coagulants, that had been used traditionally by parts of the population, for the preparation of water for household use with special emphasis on the seeds of the Moringa oleifera tree 3. The implementation of water supply for the population on the other hand demands centralized water treatment for villages and cities and thus traditional techniques will reach their limitations. The present work tries to identify and characterise the coagulating principle present in the seeds and press-cake of M.oleifera, in order to produce quantities of a standardized coagulant that could be used in small treatment works. Therefore, different extraction methods, reported in the literature, are compared regarding extraction efficiency and composition. All extracts contained a large proteinaceous moiety, which differed from proteins described before as Mo 2.1 (flocculating proteins 4 ) in the way that two proteins were detected under reducing conditions, both not co-migrating with a synthetic protein having the amino acid sequence of Mo 2.1. Moreover a different group of proteins with higher molecular weight was only present during the first two days of storage of the liquid extract. An optimized extraction procedure is presented, resulting in a total protein yield of 40g/L. Separation of proteinaceous components present in the seed and press-cake extracts lead to the determination of relative molecular masses of two protein components present (4 and 8 kda) which form a 12 kda heterodimer under non-reducing conditions, such as those found in the raw extracts. Further evidence is presented that the number of different proteins present in the 1. UN, Substantive issues arising in the implementation of the international covenant on economic, social and cultural rights. 2002, United Nations, Economic and Social Council, Committee on Economic, Social and Cultural Rights. 2. Folkard, G. and J. Sutherland, Development of a naturally derived coagulant for water and wastewater treatment. Water Science and Technology: Water Supply, (5-6): p Jahn, S.A.A., Proper use of African natural coagulants for rural water supplies research in the Sudan and a guide for new projects. 1986, Eschborn: Deutsche Gesellschaft für Technische Zusammenarbeit. 541 S. 4. Gassenschmidt, U., et al., Isolation and Characterization of a Flocculating Protein from Moringa- Oleifera Lam. Biochimica Et Biophysica Acta-General Subjects, (3): p ix

8 Summary extracts using the reported techniques may be an artifact due to sample degradation, oxidation and reaction with non-proteinaceous substances present in the extracts. The presence of glucosinolates and of their derivatives that belong to the reactive group of isothiocyanates was show in the extracts of M.oleifera seeds and press-cake, thus increasing the risk of protein stability problems during storage of the potential product. Furthermore, a method for protein isolation which reduces interference of isothiocyanates has been developed for extraction from M.oleifera. In order to compare the coagulating activities of different components present in fractions after separation of the seed extracts a miniaturized assay has been developed. Highest coagulating activities were found for the 8 kda protein chain. Partial sequencing of different preparations of the 8 kda chain indicated the presence of several forms of the protein having slight sequence differences. The protein chain is very similar to the published Mo 2.1 protein, but appears to contain 70 amino acid residues instead of the 60 residues reported for Mo 2.1. These results clearly indicate that the original protein Mo 2.1 is only a fragment of the proteins present in extracts from M.oleifera seeds. Experiments for pilot scale production were conducted involving extraction of the coagulant from M.oleifera press cake. Different possible technologies for the preparation of coagulant from M.oleifera press-cake are compared. Difficulties were encountered in solid-liquid separation after initial extraction, due to high solid loads and very slow settling, and during concentration of extracts by ultrafiltration. Alternatively tests for percolation of press-cake were carried out, which resulted in high extraction efficiencies. Finally an extraction process using sequential percolation with two different solvents, in order to separate glucosinolates and proteins is proposed, followed by a precipitating concentration step of the product. x

9 ZUSAMMENFASSUNG Wasser ist von den Vereinten Nationen zu einem Menschenrecht erklärt worden, in der Absicht Staaten, die den Internationalen Pakt über wirtschaftliche, soziale und kulturelle Rechte ratifiziert haben, dazu zu bewegen, die Versorgung der Bevölkerung mit Trinkwasser zu gewährleisten 1. Bei der Umsetzung dieses Ziels ist jedoch zu bedenken, dass die Trinkwasserversorgung in den armen Ländern anderen Beschränkungen unterliegt, als die für industrialisierte Nationen gilt. Die Abhängigkeit von Chemikalien zur Wasseraufbereitung beispielsweise, welche aus anderen Ländern importiert werden müssen, birgt die Gefahr von Unregelmässigkeiten bei der Lieferung 2. S.A.A. Jahn versuchte, die Verwendung von natürlichen Flockungsmitteln (besonders der Samen des M.oleifera Baumes) zur Aufbereitung von Trinkwasser in der Landbevölkerung in der Region um Darfur (Sudan) mit einfachen Mitteln zu systematisieren 3. Dem gegenüber verlangt die Umsetzung der Wasserversorgung der Bevölkerung eine zentralisierte Wasseraufbereitung zur Versorgung ganzer Dörfer und Städte, sodaß traditionelle Methoden nicht mehr ausreichen. Die vorliegende Arbeit versucht, das die Koagulation verursachende Prinzip in den Samen und dem bei der Ölgewinnung anfallenden Filterkuchen von M.oleifera zu identifizieren und zu charakterisieren, um größere Mengen eines standardisierten Koagulationsmittels herstellen zu können, welches in kleinen bis mittleren Wasseraufbereitungsanlagen verwendet werden kann. Zu diesem Zweck wurden verschiedene, in der Literatur angegebene Extraktionsverfahren getestet und bezüglich der Extraktionsleistung sowie der Zusammensetzung des resultierenden Extraktes verglichen. Alle Extrakte zeigten einen hohen Proteingehalt, der jedoch charakteristische Unterschiede zu dem zuvor beschriebenen Protein Mo 2.1 (Flockungsaktives Protein 4 ) aufwies. Dies äußerte sich durch das Vorhandensein zweier Proteine, die beide in ihrem elektrophoretischen Migrationsverhalten von einer synthetisch hergestellten Probe des Mo 2.1 Proteins abwichen. Darüberhinaus konnte eine weitere Gruppe höhermolekularer 1. UN, Substantive issues arising in the implementation of the international covenant on economic, social and cultural rights. 2002, United Nations, Economic and Social Council, Committee on Economic, Social and Cultural Rights. 2. Folkard, G. and J. Sutherland, Development of a naturally derived coagulant for water and wastewater treatment. Water Science and Technology: Water Supply, (5-6): p Jahn, S.A.A., Proper use of African natural coagulants for rural water supplies research in the Sudan and a guide for new projects. 1986, Eschborn: Deutsche Gesellschaft für Technische Zusammenarbeit. 541 S. 4. Gassenschmidt, U., et al., Isolation and Characterization of a Flocculating Protein from Moringa- Oleifera Lam. Biochimica Et Biophysica Acta-General Subjects, (3): p xi

10 Zusammenfassung Proteine ausschlieslich während der ersten zwei Tage der Lagerung beobachtet werden. Eine maximale Protein Konzentration von 40 g/l wurde durch die Verwendung einer Lösung verschiedener Salze erhalten. Die Trennung verschiedener in den Extrakten von Samen und Filterkuchen vorhandener Proteine ermöglichte die Bestimmung der relativen Molmasse von zwei unterschiedlichen Komponenten (4 und 8 kda), die unter nicht reduzierenden Bedingungen, wie sie in den Extrakten vorliegen, ein 12 kda Heterodimer bilden. Weiterhin wurden Anhaltspunkte gefunden, daß die große Anzahl von Proteinen, die in den Extrakten gefunden wurden möglicherweise auf Artefakte, wie Zersetzung, Oxidation oder Reaktion mit nicht-proteinösen Substanzen, zurückzuführen ist. Das Vorhandensein von Glukosinolaten, und deren Derivaten, welche zur Gruppe der reaktiven Isothiocyanate gehören, erhöht die Gefahr der Instabilität für die Proteine in M.oleifera Samenund Filterkuchenextrakten. Aus diesem Grunde wurde eine weitere Methode zur Proteinisolation entwickelt, die den Einfluß von Isothiocyanaten minimiert. Zum Vergleich der koagulierenden Eigenschaften von verschiedenen Komponenten nach Fraktionierung der Extrakte wurde ein miniaturisierter Test erstellt. Die stärksten koagulierenden Effekte wurden für die 8 kda Kette des 12 kda Proteins gefunden. Teilweise Sequenzierung verschiedener Präparationen der 8 kda Kette ergab geringe Unterschiede in den Proteinsequenzen. Insgesamt ist die 8 kda Kette dem Mo 2.1 Protein sehr ähnlich, jedoch besteht sie aus 70 Aminosäureresten, anstelle von 60, wie es für Mo 2.1 berichtet wurde. Diese Ergebnisse zeigen deutlich, daß es sich bei dem Mo 2.1 um ein Fragment eines Proteines handelt, das in Extrakten von M.oleifera Samen gefunden wird. Versuche zur Produktion eines Koagulationsmittels aus M.oleifera Filterkuchen im Technikumsmaßstab wurden durchgeführt. Dabei wurden verschiedene Verfahren zur Herstellung des Koagulierungsmittels verglichen. Auf Schwierigkeiten stießen Versuche bei Fest-Flüssig Trennverfahren nach erfolgter Extraktion aufgrund hoher Feststoffgehalte und sehr schlechtem Sedimentationsverhalten. Weiterhin ließen sich Versuche zur Aufkonzentrierung der Lösungen mittels Ultrafiltration nur sehr schlecht reproduzieren. Alternativ wurden Versuche zur Perkolation des gemahlenen Filterkuchens durchgeführt, die zu hohen Extraktionsausbeuten führten. Abschließend wird ein Extraktionsprozeß mittels sequentieller Perkolation unter Verwendung zweier verschiedener Lösungsmittel, um eine Trennung von Glukosinolaten und Proteinen zu erreichen, gefolgt von einer Fällung der aktiven Proteine zur Produktkonzentrierung vorgeschlagen. xii