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The Adsorption of Lysozyme and Chymosin Onto Emulsion Droplets and Their Association with Caseins

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The Adsorption of Lysozyme and Chymosin Onto Emulsion Droplets and Their Association with Caseins Theadsorptio n oflysozym ean d chymosin ontoemulsio n dropletsan d their association with caseins Promoter: dr. ir. P. Walstra emeritus-hoogleraar ind ezuivelkund e A.L. de Roos Theadsorptio n ofchymosi nan dlysozym e onto emulsion droplets andthen - association withcasein . Proefschrift ter verkrijging van degraa d van doctor op gezag van rector magnificus van deLandbouwuniversitei t Wageningen, dr.C.M. Karssen, inhe t openbaar te verdedigen op vrijdag 12maar t 1999 desnamiddag st e vieruu r ind eAul a OVi ISBN 90-5808-033-1 Cover :Andr e de Roos Theresearc h described inthi sthesi swa sfinance d byGist-Brocade s BV. STELLINGEN K)W>f2QlJ'Zj59Z. 1.D eretenti eva n chymosine inwronge lword tveroorzaak t door associatie vanchymosin e metpara-K-casei"ne . Ditproefschrift: hoofdstuk5,6 en 7 2. Deinaktiverin gva n enzymente n gevolgeva n adsorptie aanee ngrensvla k wordt niet noodzakelijkerwijs veroorzaakt door ontvouwinge nspreidin gva n de peptideketen. Ditproefschrift: hoofdstuk3 3. Olie-in-water emulsieskunne n fungeren alsee nvloeibaa r enfood-grad e dragermateriaal voor immobiliseringva nbiologisc h actieve eiwitten metbehou d van aktiviteit. Ditproefschrift: hoofdstuk4 en 5 4.He t bepalenva n associatieconstanten van gezuiverde caseinesi noplossin g geeft geen infor- matieove r het associatiegedrag van casei'nesi nmelk . Ditproefschrift: hoofdstuk 7 5.D e associatie van chymosineme t geadsorbeerde caseinesword t primair bepaald door de aard vanhe t adsorbens. Ditproefschrift en K.I. Larsson &A. Andren. Int. DairyJ. (1997), 7, 615. 6. Deberekenin g doorKumosinsk i et al.va nd edrie-dimensional estructuu r van eencaseine - submicelme tbehul pva n "energy minimizedtechniques "heef t eenlag evoorspellend e waarde. T.F. Kumosinski, G. Kingand KM. Farrelljr., J.Protein Chemistry, 13,681 (1994). 7.D einaktiverin gva nlysozy mi nkaa sword t nietveroorzaak t door associatieva n lysozym met caseines, maardoo rzou t afkomstig uit depekel . Ditproefschrift: hoofdstuk4 8.D e invloedva n adsorptie op de drie-dimensionale conformatie vanbiologisc h actieve eiwit­ ten isgoe d te constateren door veranderingva nhu n activiteit vanwege dieadsorptie . 9.He t verhogen van de stabiliteit van een enzymverhoog t niet altijd zijn commercielewaarde . 10.D e neigingva n industrien omonderzoek ,behoren d bijhu nkerncompetentie , uitt ebeste - denka nbedreigen d zijn voor het voortbestaan van dieIndustrie . 11. De oer-Hollandsegewoont e vanhe t prakken van het etenka ngezie nworde n alsee n emstigevor mva n fysisch voedselbederf door vereveningva n de samenstelling. 12. De evolutiemaakt ed evrou w niet alleenmee rmulti-taskin g maarvoora l ook multi-asking. 13. De stromingCobr a in de schilderkunstbegin t eerder bijJea nDubuffe t danbi jKare lAppel . Stellingenbehorend ebi jhe t proefschrift "Theadsorptio n oflysozym e andchymosi n onto emulsion droplets andthei r associationwit hcaseines"doo r A.L. deRoos . Wageningen, 12maar t 1999. Contents Abstract 1 GENERAL INTRODUCTION 1 Abstract 2 1.1 Immobilization of biologically active proteins 3 1.2 The aim and outline of the research 4 1.3 References 6 2 THEORETICAL ASPECTS 7 2.1 Interfacial phenomena 8 2.2 Protein adsorption 9 2.3 Protein conformation and its change on adsorption 12 2.4 Adsorption of biologically active proteins 13 2.5 Protein adsorption onto emulsion droplets 15 2.6 Milk proteins 18 2.6.1 Casein-casein interactions and micelle structure 21 2.7 Chymosin 24 2.8 Lysozyme 28 2.9 References 30 3 LOSS OF ENZYME ACTIVITY DUE TO ADSORPTION ONTO 33 EMULSION DROPLETS Abstract 34 3.1 Introduction 35 3.2 Materials and methods 35 3.3 Results and discussion 37 3.3.1 Influence of homogenization 37 3.3.2 Enzymes as surfactants 38 3.3.3 Enzymes added to soya-oil/BSA emulsions 41 3.3.4 Emulsification of soya-oil using BSA and the enzyme simultaneously 42 3.4 Conclusions 43 3.5 References 44 4 THE ASSOCIATION OF LYSOZYME WITH CASEIN 47 Abstract 48 4.1 Introduction 49 4.2 Materials and methods 49 4.2.1 Caseins in solution 49 4.2.2 Caseinated emulsions 50 4.2.3 Artificial Micelle Milk (AMM) 51 4.3 Results and discussion 52 4.3.1 Association of lysozyme with caseins in solution 52 4.3.2 Association of lysozyme with casein micelles 55 4.3.3 Association of lysozyme with adsorbed caseins 57 4.4 Concluding remarks 60 Contents 4.5 References 61 5 THEASSOCIATIO N OF (THYMOSIN WITH ADSORBED CASEINS 63 Abstract 64 5.1 Introduction 65 5.2 Materials and methods 65 5.3 Results and discussion 66 5.4 References 72 6 THEASSOCIATIO N OF CHYMOSIN WITH CASEINS IN SOLUTION 73 Abstract 74 6.1 Introduction 75 6.2 Material and methods 75 6.3 Results and discussion 77 6.3.1 Contact time 77 6.3.2 Casein composition and casein concentration 78 6.3.3 Temperature 81 6.3.4 Ionic strength and pH 84 6.4 References 85 7 THEASSOCIATIO N OF CHYMOSIN WITH CASEIN MICELLES 87 Abstract 88 7.1 Introduction 89 7.2 Materials and methods 89 7.3 Results and discussion 90 7.3.1 Casein composition 90 7.3.2 pH 92 7.3.3 Casein concentration 95 7.3.4 Contact time 95 7.3.5 Competitive association 98 7 4 Discussion 98 7.5 Concluding remarks 103 7.6 References 103 SUMMARY 105 SAMENVATTING 111 NAWOORD 115 CURRICULUM VITAE 116 Abstract De Roos, A.L. (1999) The Adsorption of Lysozyme and Chymosin onto emulsion droplets and their association with caseins. Ph.D. thesis, Wageningen Agricultural University, Wageningen, The Netherlands, (pp.116, English and Dutch summaries). Keywords: enzyme immobilisation, emulsion droplet, casein micelle, chymosin, lysozyme, adsorption, association. Abstract: In this study the enzymes chymosin and lysozyme were immobilised on soya-oil emulsion droplets by means of direct adsorption onto the oil droplets'surface during homogenisation. This emulsion was stabilised not only using the enzymes as the only emulsifier, but also in combination with bovine serum albumin in various ratios of concentration, simultaneously and successively. In every system the adsorbed enzyme lost all activity insitu, and also after desorption from the oil/water interface by means of a detergent. Soya-oil emulsion droplets stabilized with casein proved to be a good adsorbant for the enzymesstudied . Chymosinboun d onlywit h emulsion droplets stabilized withK-casei nan d was strongly dependent on pH and ionic strength. The extents of association are given in adsorption isotherms. The enzyme immobilized in this way showed in situ activity. Lysozyme associated with every type of casein-stabilized emulsion droplet in the order as -casein > P-casein > K-casein. The association is less dependent on pH and ionic strength within the ranges tested. Lysozyme also proved to be fully active while associated, indicating that the active sitei sno t involved inth e association. The characteristics of association of the enzymes with casein were worked out using casein micelles of various casein composition and the individual caseins in solution. Lysozyme only associates with ou- and p-casein and not with K-casein, the latter in contrast with the binding capacity of adsorbed K-casein. Consequently, casein micelles of high K-casein content bind less lysozyme. The extent of association of lysozyme with ou- and P-casein is about equimolar. Theassociatio n didno t result inlos so factivity . As with adsorbed caseins chymosin only associates with K-casein. The characteristics for association are more or less the same. The association with K-casein in solution, however, decreases strongly with contact time, which is not the case with adsorbed K-casein. This dissociation seemed to be very much related to the proteolytic activity ofth e enzyme and thus related to temperature, pH and extent of association. The association of chymosin with K-casein is one in competition with the other caseins for K-casein and is determined by the association constants for casein-casein and chymosin-K-casein interactions. Akineti c model for competitive association further explains the effect of casein micelle compostion and the low extent of chymosin association with mixtures of caseinsan d casein micelles in solution. GENERAL INTRODUCTION Chapter 1 Chapter 1 Abstract Immobilization of biologically active proteins is widely used in industry. The main advantage of immobilization is that the proteins can be easily regained after having played their role in the biochemical process. Immobilization is mostly carried out with various solid carriers. These carriers as well as the chemicals used in the immobilization procedure can mostly not be considered as food grade. For this reason, application of immobilized biologically active proteins to play a role in the realization of food stuffs or in pharmaceutics may not be allowed. The aim of this study is to immobilize enzymes, while retaining activity, on carriers that have a food grade status. We used soya-oil droplets and protein aggregates (casein micelles) as the carrier particles, and chymosin and lysozyme as the enzymes. general introduction 1.1 Immobilization ofbiologicall y active proteins. Immobilization of enzymes can be defined as the transfer of enzymes from a water-soluble, mobile state into awater-insoluble , immobile state. Immobilization deprives the enzymes of their diffusional mobility and facilitates recycling by simple means such as centrifugation or filtration. Moreover immobilization often increases the conformational stability of the enzyme molecule to protect them against influences of pH, temperature, ionic strength and shear forces. The food processing industry is the largest consumer of industrial enzymes and many applications of immobilized enzymes can be found in this area [1]. Also in pharmaceutical and personal care products, biologically active agents are immobilized by encapsulation (e.g. inliposomes )t o bring about specific targetting and controlled release. Various techniques of immobilization are applied: adsorption, covalent binding, ionic binding, cross-linking, entrapment and encapsulation.
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