Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Capture and transformation: Participatory Irrigation management in Andhra Pradesh, India
Mollinga, P.P. ; Doraiswamy, R. ; Engbersen, K. - \ 2004
In: The Politics of Irrigation Reform: Contested Policy Formulation and Implementation in Asia, Africa and Latin America / Mollinga, P.P., Bolding, J.A., Hampshire : Ashgate Publishing Limited (Global environmental governance ) - ISBN 0754635155 - p. 240 - 262.
irrigatiesystemen - waterbeheer - participatie - boeren - india - irrigation systems - water management - participation - farmers
Interconnective host-guest complexation of b-cyclodextrin-calix[4]arene couples
Bügler, J. ; Sommerdijk, N.A.J.M. ; Visser, A.J.W.G. ; Hoek, A. van; Nolte, R.J.M. ; Engbersen, J.F.J. ; Reinhoudt, D.N. - \ 1999
Journal of the American Chemical Society 121 (1999). - ISSN 0002-7863 - p. 28 - 33.
Flexibility of enzymes suspended in organic solvents probed by time-resolved fluorescence anisotropy. Evidence that enzyme activity and enantioselectivity are directly related to enzyme flexibility.
Broos, J. ; Visser, A.J.W.G. ; Engbersen, J.F.J. ; Verboom, W. ; Hoek, A. van; Reinhoudt, D.N. - \ 1995
Journal of the American Chemical Society 117 (1995). - ISSN 0002-7863 - p. 12657 - 12663.
Steady-state fluorescence studies on lipase-vesicle interactions.
Mosmuller, E.W.J. ; Pap, E.H.W. ; Visser, A.J.W.G. ; Engbersen, J.F.J. - \ 1994
Biochimica et biophysica acta-protein structure and molecular enzymology 1189 (1994). - ISSN 0167-4838 - p. 45 - 51.
Studies on the stability of lipase from Candida cylindracea free and incorporated in polymerisable zwitterionic surfactant vesicles.
Mosmuller, E.W.J. ; Jongejan, H. ; Franssen, M.C.R. ; Engbersen, J.F.J. - \ 1993
Biocatalysis 8 (1993). - ISSN 0886-4454 - p. 173 - 190.
Studies on the incorporation of lipase in synthetic polymerisable vesicles
Mosmuller, E.W.J. - \ 1993
Agricultural University. Promotor(en): H.C. van der Plas; J.F.J. Engbersen. - S.l. : Mosmuller - ISBN 9789054851172 - 121
colloïden - dispersie - carboxyl ester hydrolasen - tannase - choline esterase - triacylglycerol lipase - synthese - organische verbindingen - colloids - dispersion - carboxylic ester hydrolases - tannase - cholinesterase - triacylglycerol lipase - synthesis - organic compounds

This thesis describes studies on the suitability of synthetic polymerisable vesicles for the incorporation and stabilisation of lipase for the bioconversion of organic chemical compounds.

In chapter 1 , some characteristics are reviewed of hydrolytic enzymes, and more specific those of lipases. In chapter 2 an overview is presented of the features and properties of surfactants and vesicles.

In chapter 3 , the incorporation is described of lipase from Candida cylindracea (CCL) into polymerisable positively charged dialkylammonium bromide surfactant vesicles.

Before incorporation the lipase has been purified and characterised. The enzyme has a molecular weight of 58.5 kD and an isoelectric point of 4.1; the pH optimum is broad, ranging from pH 4 to 6 and the optimal temperature is 45°C

The synthesis of several polymerisable surfactants and the preparation of nonpolymerised and polymerised vesicles from these surfactants are described. The vesicle systems were characterised in terms of morphology (electron microscopy) and stability. It appeared that polymerised vesicles are considerably more stable than their nonpolymerised analogues.

The enzyme was incorporated in the vesicle by the use of the dehydration- rehydration method or by incubation. In the latter case, trapping efficiencies are obtained of up to 100%. Activities of free and vesicle incorporated CCL are tested for three triglycerides: triacetin, tributyrin and tricaprylin and for 2,4-dinitrophenyl butyrate. Enzyme activity is lowest in homogeneous mixtures (triacetin and relatively low concentrations of tributyrin) and highest in heterogeneous mixtures (tricaprylin and relatively high concentrations of tributyrin and 2,4-dinitrophenyl butyrate). Incorporation of the enzyme in vesicular systems is advantageous for the activity, especially in homogeneous reaction mixtures, due to the presence of hydrophobic sites of the vesicles. Moreover, in the case of the production of insoluble fatty acid (caproate), inhibition by the acid is suppressed.

The influence of several surface active additives is tested on the activity of lipase. Vesicles have a positive influence on the activity, whereas positively charged surfactant addenda act as inhibitors. In the case of tricaprylin assays, the positively charged surfactant addenda increase enzymatic activity.

In addition, the sensitivity for tryptic digestion of free and incorporated CCL is compared. Free CCL is readily inactivated, whereas incorporated enzyme is protected from proteolytic degradation.

In chapter 4 the stability of vesicle incorporated Candida cylindracea lipase is described.
For this purpose, the enzyme was incorporated into vesicles of the polymerisable zwitterionic surfactant bis[2-(pentacosa-10,12-diynoyloxy)ethyll-2-aminoethanesulfonic acid WAS). Vesicle systems of BPAS were characterised
in terms of morphology (electron microscopy) and stability. Polymerisation of vesiculated BPAS surfactants does not alter the vesicle morphology. Polymeric vesicles are considerably more stable than the monomeric analogues. CCL incorporated into the vesicle membrane by the incubation method remains fully active; especially in homogeneous assay mixtures the vesicle incorporated enzyme shows an increased activity when compared to the free lipase. The stability of free and incorporated lipase was determined by measuring the residual activity of the various systems when mixed with ethanol (50% v/v) or 2-(n-butoxy)ethanol (37.5% v/v), at 50°C and 60°C and in the presence of the proteolytic enzyme trypsin. In all cases the vesicle incorporated enzyme displays an increased stability against denaturating conditions.

The interaction of lipase from Candida cylindracea with positively charged polymerisable surfactant vesicles was studied by the use of steady state fluorescence techniques. The results of these studies are described in chapter 5 .
The phase transition of vesicles composed of nonpolymerised and polymerised N- allylbis[2-(hexadecanoyloxy)ethyllmethylammonium bromide was determined by measuring the change in fluorescence anisotropy of the membrane probe diphenylhexatriene. The phase transition temperature for nonpolymerised vesicles is 49°C and for the polymerised analogues 45°C. Fluorescence anisotropy and energy transfer measurements were used to demonstrate that Candida cylindracea lipase is readily incorporated into the hydrophobic bilayer of the vesicle. By using an interfacial membrane probe (trimethylammonium diphenylhexatriene) and an internal membrane probe (diphenylhexatriene), it could be determined that the lipase is incorporated more efficiently into the nonpolymerised vesicles, and that the penetration of the enzyme into the bilayer is less deeply in the case of polymerised vesicles.

In chapter 6 , a rapid and sensitive assay for the detection of lipase activity is described. The method is based upon the increase in absorbance at 360 nm due to the formation of the 2,4-dinitrophenolate anion during the enzymatic hydrolysis of 2,4- dinitrophenyl esters. Several esters with different acyl chain length have been tested. 2,4-Dinitrophenyl butyrate proved to be a suitable standard substrate. This substrate can be used in homogeneous reaction systems and in emulsified form. In the latter case, a correction can be made for absorbance changes due to clearance of the emulsion during hydrolysis by using a diode array spectrophotometer with internal referencing. The small reaction volume and the high extinction coefficient of the product makes this method suitable for assay mixtures of low substrate and low enzyme concentration.

In chapter 7 the results from the preceding chapters are reviewed in a general discussion.

Metal-ion complexes of functionalised 1,10-Phenanthrolines as hydrolytic synzymes
Weijnen, J.G.J. - \ 1993
Agricultural University. Promotor(en): H.C. van der Plas; J.F.J. Engbersen. - S.l. : S.n. - ISBN 9789054851820 - 145
metaalionen - organominerale complexen - fenantreen - hydrolasen - metal ions - organomineral complexes - phenanthrene - hydrolases

In this thesis metal-ion complexes of functionalised 1,10-phenanthroline derivatives have been studied as model systems for hydrolytic metallo-enzymes. Amphiphilic metallo- complexes incorporated into micelles or vesicles and water-soluble complexes in pure aqueous buffer solutions, have been found catalytically active in the hydrolysis of activated (chiral) carboxylic and phosphate esters. The effect of changing the ligand structure and the metal ion on the activity and enantioselectivity of the complexes has been investigated.

After a general introduction and a description of the aim and contents of the thesis in chapter 1, an overview is given in chapter 2 of the catalytic roles that metal ions perform in hydrolytic reactions and of the application of micelles and vesicles as biomimetic systems, with emphasis on functionalised metallo-aggregates.

Mixed micellar systems containing Zn IIand Cu IIcomplexes of lipophilic ligands with the 1,10-phenanthroline or pyridine group as chelating moiety and a pendant Nτ-alkylated imidazole group (ligands 1 and 3, chapter 3), are efficient catalysts in the hydrolysis of p -nitrophenyl picolinate (PNPP) and diphenyl p -nitrophenyl phosphate (DPPNPP). The lipophilic 1,10-phenanthroline ligands 1 and 2 have a higher affinity for metal ions than the pyridine ligand 3. In the presence of one equivalent of metal ions, almost all 1,10-phenanthroline ligand sites are occupied by MU, whereas for the pyridine ligand this amounts to only about 50%. Kinetic studies of the hydrolysis of PNPP strongly indicate that catalysis proceeds by preliminary formation of a reactive ternary complex composed of metal ion, ligand, and substrate. These synzymes operate via a metal-hydroxide-ion catalysed mechanism and exhibit turn-over behaviour while retaining their full catalytic activity.

The catalytic role of a hydroxymethyl group covalently bound to the 1,10-phenanthroline ligand in the vicinity of the reaction centre and the effect of incorporation of the ligand into micelles are discussed in chapter 4. The Zn IIcomplex of the lipophilic ligand bearing the hydroxymethyl group (ligand 1) is 25 times more active in the hydrolysis of PNPP than the lipophilic metallo-complex lacking this group (ligand 3). Under turn-over conditions, the hydroxymethyl-containing metallo-catalyst displays a kinetically biphasic behaviour, characteristic for an acylation-deacylation mechanism. The acylation of the hydroxymethyl group is 133 times faster than the deacylation step. Lipophilic 1,10-phenanthroline derivatives in mixed micelles are able to form only 1 : 1 complexes with bivalent metal ions, whereas the water-soluble ligand 2 can both form 1 : 1 and 2: 1 (ligand : M II) complexes. Only 1 : 1 complexes appear to be catalytically active.

Metal-ion complexes of a lipophilic 1,10-phenanthroline ligand containing the (S)-2-(hydroxymethyl)pyrrolidine function at the α-position (ligand 1, chapter 5) are highly active and enantioselective in the hydrolysis of p -nitrophenyl esters of N-protected phenylalanine. The direction and magnitude of enantioselective catalysis are remarkably dependent on the nature of the metal ion and the co-surfactant. The Co IIcomplex in Brij 35 micelles exhibits the highest degree of enantioselectivity: kDa,obs / kLa,obs = 15 .3 toward the substrate D(L)-C 12 -Phe-PNP. In mixed micellar systems composed of the Zn IIcomplex and Brij 35 as co- surfactant, hydrolysis of the D-enantiomer predominates over that of the L-enantiomer ( kDa,obs / kLa,obs = 2 .4), whereas with CTABr as the co-surfactant an inversion of enantioselectivity is observed ( kDa,obs / kLa,obs = 0 .54). Enantioselective hydrolysis is an important effect of the hydrophobic interaction between substrate and catalyst, since water-soluble ligands containing a (S)-2-(hydroxymethyl)pyrrolidine group (ligands 5, 6, and 7) are less active and less stereoselective. Moreover, lipophilic 1,10-phenanthroline ligands with chiral ephedrine functions (ligands 3 and 4) show a lower activity and stereoselectivity.

Metal-ion complexes of functionalised 1,10-phenanthroline ligands having two long alkyl chains and a nucleophilic hydroxymethyl (ligand 1, chapter 6), (S)-2-(hydroxymethyl)pyrrolidine (ligand 2), or ephedrine group (ligands 3 and 4) at the a-position, incorporated in C 18 C 12 vesicles, are catalytically active toward PNPP and show activity and enantioselectivity toward p-nitrophenyl esters of N-protected leucine as the substrate. In mixed metallo-vesicles, the amphiphilic ligand is anchored in the core of the bilayer membrane by the alkyl chains, whereas the chelated headgroup protrudes into the aqueous interface. The metallo-complexes appear to be active in both the exo- and endovesicular side of the bilayer and the fluidity of the vesicle membrane has no influence on the enantioselectivity.

The catalytic activity of metal-ion complexes of 1,10-phenanthroline with two long alkyl chains at the 2 and 9 positions (C 12 Phen) in Brij 35 micelles toward various phosphate triesters, diesters, and monoesters is described in chapter 7. In the presence of Co IIand Zn IIcomplexes the rate of hydrolysis of DPPNPP is increased by factors of 600 and 240, respectively. The metallo-complexes exhibit turn-over behaviour without loss of activity. Saturation kinetics provide evidence for preliminary formation of ligand-M II-phosphate ester complexes, which decay to products. Kinetic studies indicate that phosphate triesters containing a metal-ion binding site in the leaving group are hydrolysed by the same mechanism as DPPNPP.

In chapter 8 an outline is given of the synthesis and enzymatic resolution of mono alanine a mides containing pyridine (5a) or 1,10-phenanthroline (5b) side chains and of bis-alanine amides with 1,4-phenyl (10a) or 2,9-(1,10-phenanthroline) (10b) linker moieties. Resolution of the bis-alanine derivatives using aminopeptidase from Pseudomonas putida requires an excess amount of enzyme due to substrate and product inhibition. The large amount of biocatalyst which, is necessary for the reaction, strongly hampers the work-up procedure, preventing the isolation of satisfactory amounts of enantiomerically pure product. In the cases of the mono-alanine derivatives, enzymatic resolution is successful and treatment of the racemic amino acid amides with the aminopeptidase yields the L-amino acid and the unchanged D-amino acid amide, which can easily be separated.

Lipase activity in vesicular systems: characterization of candida cylindracea lipase and its activity in polymerizable dialkylammonium surfactant vesicles
Mosmuller, E.W.J. ; Franssen, M.C.R. ; Engbersen, J.F.J. - \ 1993
Biotechnology and Bioengineering 42 (1993). - ISSN 0006-3592 - p. 196 - 204.
Hydrolysis of 4-nitrophenyl esters of picolinic acid and N-protected amino acids by metalloenzyme models in vesicular assemblies.
Weijnen, J.G.J. ; Koudijs, A. ; Tap, P.G.J.A. ; Engbersen, J.F.J. - \ 1993
Recueil des travaux chimiques des Pays-Bas = Journal of the Royal Netherlands Chemical Society 112 (1993). - ISSN 0165-0513 - p. 525 - 530.
Catalytic hydrolysis of phosphate esters by metallocomplexes of 1,10-phenanthroline derivatives in micellar solution.
Weijnen, J.G.J. ; Engbersen, J.F.J. - \ 1993
Recueil des travaux chimiques des Pays-Bas = Journal of the Royal Netherlands Chemical Society 112 (1993). - ISSN 0165-0513 - p. 351 - 357.
Activity and stability of lipase in synthetic polymerisable surfactant vesicles.
Mosmuller, E.W.J. ; Franssen, M.C.R. ; Jongejan, H. ; Engbersen, J.F.J. - \ 1992
In: Abstract Int. Symp. Stability and stabilization of enzymes, Maastricht - p. 79 - 79.
Inleiding in de bio-organische chemie, 5e geheel herz. dr.
Engbersen, J.F.J. ; Groot, Ae. de - \ 1992
Wageningen : Pudoc - ISBN 9789022010495 - 576 p.
organic compounds - biochemistry - organic chemistry
Metal ion and micellar triggering of enantioselectivity in the hydrolysis of N-protected amino acid esters.
Weijnen, J.G.J. ; Koudijs, A. ; Engbersen, J.F.J. - \ 1992
Journal of molecular catalysis 73 (1992). - ISSN 0304-5102 - p. L5 - L9.
Functionalised 1,10-phenanthroline metallocatalysts as models for hydrolytic metalloenzymes.
Weijnen, J.G.J. ; Koudijs, A. ; Schellekens, G.A. ; Engbersen, J.F.J. - \ 1992
Journal of Chemical Society, Perkin Transactions 2 (1992). - p. 829 - 834.
A new spectrophotometric method for the detection of lipase activity using 2,4-dinitrophenyl butyrate as a substrate.
Mosmuller, E.W.J. ; Heemst, J.D.H. van; Delden, C.J. van; Franssen, M.C.R. ; Engbersen, J.F.J. - \ 1992
Biocatalysis 5 (1992). - ISSN 0886-4454 - p. 279 - 287.
Synthesis of novel amphiphilic diacetylenes with amino or ammonium functionality.
Ohba, S. ; Engbersen, J.F.J. - \ 1991
Tetrahedron 47 (1991). - ISSN 0040-4020 - p. 9947 - 9952.
Carboxylix and phosphate ester hydrolysis catalysed by bivalent zinc and copper metallosurfactants.
Weijnen, G.J. ; Koudijs, A. ; Engbersen, J.F.J. - \ 1991
Journal of Chemical Society, Perkin Transactions 2 (1991). - p. 1121 - 1126.
Activity of lipase in synthetic surfactant vesicles.
Mosmuller, E.W.J. ; Engbersen, J.F.J. - \ 1990
In: Abstract Zomercongr. Kon. Ned. Chem. Vereniging, Leiden - p. 4 - 4.
Activity of enzymes entrapped in synthetic surfactant vesicles.
Mosmuller, E.W.J. ; Jongejan, H. ; Franssen, M.C.R. ; Engbersen, J.F.J. - \ 1990
In: Abstract 5th Eur. Congr. Biotechnology, Kopenhagen - p. 249 - 249.
Activity of enzymes entrapped in synthetic surfactant vesicles.
Mosmuller, E.W.J. ; Engbersen, J.F.J. - \ 1990
In: Proc. 3rd Neth. Biotechnol. Congr., Amsterdam, H. Breteler, R.F. Beudeker, K.Ch.A.M. Luyben (eds.). Neth. Biotechnol. Soc., Zeist. Part I - p. 115 - 120.
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