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Enzyme Adsorption at Solid-Liquid Interfaces

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Enzyme Adsorption at Solid-Liquid Interfaces Enzyme adsorption at solid-liquid interfaces Ontvangen 1 5 OKT 1992 UB-OAHDEX CENTRALE LANOBOUWCATALOGUS 0000 0491 2420 Promotor: Dr. J. Lyklema, hoogleraar in de fysische chemie, met bijzondere aandacht voor de grensvlak- en kolloi'dchemie. Co-promotor: Dr. W. Norde, wetenschappelijk hoofdmedewerker in de fysische en kolloi'dchemie. S. Duinhoven Enzyme adsorption at solid-liquid interfaces Proefschrift ter verkrijging van de graad van doctor in de landbouw- en milieuwetenschappen op gezag van de rector magnificus, Dr. H.C. van der Plas, in het openbaar te verdedigen op woensdag 28 oktober 1992 des namiddags te vier uur in de aula van de Landbouwuniversiteit te Wageningen. { JTv-v - 5 k ^ \ I O BllJLlOl'HEfcR tANDBOUWUNIVERSIIEDI WAGENINGEM *vhff\''^'t te<JO , STELLINGEN behorende bij het proefschrift Enzyme adsorption at solid-liquid interfaces van Susan Duinhoven Opgedragen aan mijn ouders CONTENTS VOORWOORD CONTENTS CHAPTER 1 INTRODUCTION 1 CHAPTER 2 EXPERIMENTAL TECHNIQUES FOR SAVINASE ADSORPTION MEASUREMENT 19 CHAPTER 3 SAVINASE™ ADSORPTION ON POLYSTYRENE LATICES AND GLASS 57 CHAPTER 4 THE ADSORPTION OF SAVINASE VARIANTS ON POLYSTYRENE LATICES AND GLASS 83 CHAPTER 5 LIPOLASE™ ADSORPTION ON POLYSTYRENE LATICES AND GLASS 113 CHAPTER 6 SAVINASE™ ADSORPTION ON POLYESTER AND COTTON CLOTH 129 SUMMARY ENZYME ADSORPTION ON SOLID-LIQUID INTERFACES 151 SAMENVATTING ENZYM-ADSORPTIE VANUIT EEN OPLOSSING AAN EEN VASTE STOF 157 CURRICULUM VITAE 163 VOORWOORD Het onderzoek dat in deze dissertatie wordt beschreven, is uitgevoerd in het Unilever Research Laboratorium te Vlaardingen, binnen de sectie Fysische Chemie onder leiding van Dr. W.G.M. Agterof. Mijn promotieonderzoek was gericht op de werking van enzymen die in wasmiddelen worden gebruikt. Heti see n leerzame ervaring geweest tezien , welkuitgebrei d onderzoek ten grondslag ligt aan de productie en verbetering van alledaagse consumentenproducten als wasmiddelen, margarines, mayonaises en cosmetica. Hoe vanzelfsprekend deze producten inhe tdagelijks e gebruik ooklijken , vanuit natuurwetenschappelijk oogpuntzij n ze zeer complex. Vele van deze systemen bevatten tientallen componenten, die in interactie met elkaar functioneren onder steeds wisselende omstandigheden. Gaarne maak ik van de gelegenheid gebruik de directie van het Unilever Research Laboratorium te bedanken voor de geboden mogelijkheid mij binnen deze stimulerende omgeving aan onderzoek te wijden. Vierjaa r lang heb ik alle ondersteuning gekregen die nodig was voor de succesvolle afronding van mijn promotieonderzoek. Zo kon ik beschikken over zes Savinase-varianten, die ter beschikking werden gesteld door Novo- Nordisk, Denemarken en het Unilever Research Laboratorium Vlaardingen. Ik heb veel te danken aan de intensieve samenwerking met de verschillende afdelingen van de groep 'Detergents' o.l.v. Ir. H. Vonkeman, de groep 'Bio- and Nutrition Sciences' o.l.v. Dr. O. Korver, en de groep 'Physical and Analytical Sciences' o.l.v. Dr. L. de Galan. In het bijzonder heb ik gesteund op de experimentele kennis en vaardigheden van Ruud Poort en Gerrit van der Voet. Samen werkend hebben wij ons telkens weer verbaasd over de wondere wereld van de enzymen. Jullie interesse en volharding bij alle 'ups and downs' zijn voor mij een stimulans geweest. Ik wil jullie beiden bedanken voor je wezenlijke bijdrage aan de totstandkoming van dit proefschrift. Wim Agterof dank ik voor zijn niet aflatende ondersteuning van het project. Zijn brede onderzoekservaring en zijn aandacht voor de kwaliteit van industriele research garandeerden een gunstig werkklimaat. Zijn vertrouwen in de goede afloop van de onderneming heeft mijtege n menigeber g opgeholpen . Er was altijd tijd en aandacht voor overleg, zowel over detailproblemen als over de grote lijnen. Ik bewaar goede herinneringen aan onze uitvoerige, vaak verhitte discussies. Voorwoord Hans Lyklema, mijn promotor, wil ik bedanken voor zijn accurate en efficiente begeleiding. Zijn aanstekelijk enthousiasme voor wetenschappelijk onderzoek heeft mij geiinspireerd. Door zijn overzicht van het vakgebied en zijn vermogen het wezenlijke van de details te scheiden, heeft hij mijn onderzoek, ook bij tegenvallende resultaten, steeds met vastehan d op koers gehouden. Van meet af aan heeft hij benadrukt, dat de complexe problematiek allereerst vanuit modelsystemen moet worden benaderd, alvorens tot praktijkproeven over te gaan. De loop van het onderzoek heeft hem ten voile gelijk gegeven. Veel dank ben ik ook verschuldigd aan mijn co-promotor, Willem Norde. Ik heb zeer geprofiteerd van zijn grote experimentele ervaring op het gebied van eiwitadsorptie. Hij heeft me op een cruciaal moment methode en materialen kunnen leveren, die het onderzoek over een dood punt hebben heengetrokken. Dewetenschappelijk eachtergrondstudi e ten aanzien vanconsumentenproducte n doorloopt hethel e spectrum van denatuurwetenschappen , waardoor hetonderzoe k in een industrieel laboratorium multidisciplinair van aard is. Wie in zo'n omgeving werkt, kan veelzijdig onderzoek verrichten en daarbij profiteren van de grote diversiteit aan specialismen, die in het laboratorium aanwezig is. Gaarne noem ik degenen die vanuit hun specifieke deskundigheid hebben bijgedragen aan het ontstaan en deuitwerkin g van dit proefschrift. Met Maarten Egmond heb ik voor mij waardevolle discussies gevoerd over enzymen en eiwitten. Peter Ravestein en Willem Antheunissedan k ik de zuivering van alle benodigde enzympreparaten. Kest Hissink, Cora Engelen en Jan Bus hebben zich ingespannen te komen tot een goede labelingsmethode voor het Savinase. De berekeningen van de electrostatische eigenschappen van de Savinase-varianten zijn uitgevoerd door Hans Peters, Dennis de Jong en Jacob de Vlieg. Hans Meder en Petra Weisenborn hebben de CD-spectra opgenomen. Arend-Jan Adriaanse heeft tijdens zijn stage in de sectie Fysische Chemie de protease-varianten gekarakteriseerd met behulp van HPLC-scheidingstechnieken. De omslag van mijn dissertatie is ontworpen door Mari van der Giessen. I would also like to thank Jonathan Warr and Pete Hall for removing the 'steenkolen' English phrases. Any errors still occurring in the text are due to alterations subsequently made. Hen alien dank ik hartelijk voor hun bijdragen. II CONTENTS VOORWOORD CHAPTER 1 INTRODUCTION 1 1.1 Enzymes at interfaces 1 1.2 Savinase™ and Lipolase™ 5 1.2.1 The enzymes 5 1.2.2 Molecular charge of Savinase as a function of pH 5 1.3 Protein adsorption 9 1.4 The adsorption isotherm: experimental tool for investigation of the adsorption mechanism 11 1.5 References 14 CHAPTER 2 EXPERIMENTAL TECHNIQUES FOR SAVINASE ADSORPTION MEASUREMENT 19 Abstract 19 2.1 Introduction 19 2.2 Materials 21 2.2.1 Savinase 21 2.2.2 PMSF-inhibited Savinase 21 2.2.3 Polystyrene latices 22 2.2.4 Buffer solutions 23 2.3 Experimental methods 23 2.3.1 Adsorption measurement 23 2.3.2 Concentration determination by the Radio-active tracer technique 24 2.3.3 Concentration determination with UV 280 nm extinction detection 25 2.3.4 Concentration determination with the Lowry colouring method . 27 2.3.5 Concentration determination by measurement of proteolytic activity on AAPF 28 2.3.6 Determination of protein purity with Reversed Phase HPLC .... 29 III Contents 2.4 Results 30 2.4.1 RTT adsorption detection using "old labelled" PMS-Savinase ... 30 2.4.2 RTT adsorption detection using "new labelled" PMS-Savinase . 35 2.4.3 Adsorption measurement with UV 280 nm detection 40 2.4.4 Adsorption measurement with Lowry colouring method 41 2.4.5 Adsorption of active Savinase measured with RTT and Lowry colouring 43 2.4.6 Adsorption of active Savinase measured with the AAPF concentration determination method 44 2.5 Conclusions 46 2.6 References 47 APPENDIX 2.1 INHIBITION OF SAVINASE WITH PMSF 49 APPENDIX 2.2 RADIO-ACTIVE LABELLING OF SAVINASE AND PMS-SAVINASE 50 APPENDIX 2.3 PROCEDURE FOR PROTEIN DETERMINATION WITH LOWRY COLOURING METHOD 52 APPENDIX 2.4 PROCEDURE FOR THE DETECTION OF ENZYMATIC ACTIVITY ON AAPF 53 APPENDIX 2.5 REVERSED PHASE HPLC FOR THE DETERMINATION OF PROTEIN PURITY 54 APPENDIX 2.6 VARIATION CALCULATION OF ADSORPTION MEASUREMENT WITH THE LOWRY COLOURING METHOD 55 CHAPTER 3 SAVINASE™ ADSORPTION ON POLYSTYRENE LATICES AND GLASS 57 Abstract 57 3.1 Introduction 58 3.2 Experimental 58 3.2.1 Materials 58 3.2.2 Buffer solutions 60 IV Contents 3.2.3 Protein concentration determination 61 3.3 Results 61 3.3.1 Zeta-potentials of PS-latices 61 3.3.2 Comparison of the adsorption of active and PMSF-inhibited Savinase 62 3.3.3 Savinase adsorption on various surfaces 64 3.3.4 Variation of pH 66 3.3.5 Ionic strength dependence of PMS-Savinase adsorption 68 3.3.6 The influence of the type of salt ions on PMS-Savinase adsorption 69 3.3.7 Temperature dependence 71 3.3.8 Reversibility of PMS-Savinase adsorption 72 3.4 Discussion 74 3.4.1 The shape of the adsorption isotherm 74 3.4.2 The adsorption mechanism 76 3.5 Conclusions 80 3.6 References 80 CHAPTER 4 THE ADSORPTION OF SAVINASE VARIANTS ON POLYSTYRENE LATICES AND GLASS 83 Abstract 83 4.1 Introduction 84 4.2 Experimental 86 4.2.1 Adsorption measurement 86 4.2.2 Savinase variants 86 4.2.3 Calculation of variant characteristics 89 4.3 Results and Discussion 93 4.3.1 Characterisation of variants 93 4.3.2 The influence of stabilizing buffer on Savinase variants 97 4.3.3 Adsorption of variants with various isoelectric points 97 4.3.4 Adsorption of variants with varying number of charged residues . 100 4.3.5 Molecular cartography of the electrostatic potential on the protein surface 103 4.4 Conclusions 109 4.5 References 109 APPENDIX 4.1 POLARITY OF ATOMS IN AMINO-ACIDS 112 Contents CHAPTER 5 LIPOLASE™ ADSORPTION ON POLYSTYRENE LATICES AND GLASS 113 Abstract 113 5.1 Introduction 113 5.2 Experimental 115 5.2.1 Materials 115 5.2.2 Adsorption measurement 115 5.2.3 Experimental determination of titration curve 117 5.3 Results 117 5.3.1 Titration curves of Lipolase 117 5.3.2 Variation of surface characteristics 117 5.3.3 Variation of pH 118 5.3.4 Ionic strength dependence and the influence of Ca2+ ions 119 5.3.5 Temperature variation 121 5.3.6 Adsorption measurement with the Radio-active Tracer Technique .
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