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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Assessment of the environmental status of the coastal and marine aquatic environment in Europe: A plea for adaptive management
Laane, R.W.P.M. ; Slijkerman, D.M.E. ; Vethaak, A.D. ; Schobben, J.H.M. - \ 2012
Estuarine Coastal and Shelf Science 96 (2012)1. - ISSN 0272-7714 - p. 31 - 38.
estrogenic endocrine disruption - flounder platichthys-flesus - ecological risk-assessment - critical load concept - precautionary principle - north-sea - water-quality - pelagic fish - chemicals - exposure
Policymakers and managers have a very different philosophy and approach to achieving healthy coastal and marine ecosystems than scientists. In this paper we discuss the evolution of the assessment of the chemical status in the aquatic environment and the growing rift between the political intention (precautionary principle) and scientific developments (adaptive and evidence-based management) in the context of the pitfalls and practicalities confronting the current Marine Strategy Framework Directive (MSFD). The conclusion is that policymakers and water managers should move with the times and take on board new techniques that scientists are using to assess chemical status and apply new scientific developments in assessment studies of the chemical status. These new techniques, such as bioassays, are cheaper than the classic approach of checking whether concentrations of certain individual priority compounds comply with permissible thresholds. Additionally, they give more insight into the real impacts of chemical compounds
Monitoring en Evalautie Pilot Zandmotor Fase 2 - Plan van Aanpak Kwaliteitsborging
Baptist, M.J. ; Laane, R.W.P.M. - \ 2011
Den Burg [etc.] : IMARES en Deltares (Rapport / IMARES C168/11 - 1205045-000-ZKS-0037) - 13
zandsuppletie - kustbeheer - natuurbeheer - natuurontwikkeling - monitoring - zuid-holland - sand suppletion - coastal management - nature management - nature development
Dit Plan van Aanpak Kwaliteitsborging beschrijft het proces voor de borging van de inhoudelijke kwaliteit van de tijdens het project op te leveren producten, en de rollen en verantwoordelijkheden van het reviewteam in het geheel van de projectkwaliteit.
Chemicals & energy; building a sustainable bio-based economy for the Netherlands: 2020 & beyond
Wielen, L.A.M. van der; Arends, I. ; Heijnen, S. ; Sanders, J.P.M. ; Scott, E.L. - \ 2009
In: Partners in de polder; A vision for the life sciences in the Netherlands and the role of public-private partnership / Laane, Colja, Besteman, Koen, S.n. - ISBN 9789080735774 - p. 136 - 157.
onderzoek - samenwerking - innovaties - levenswetenschappen - publiek-private samenwerking - toekomst - Nederland - research - cooperation - innovations - life sciences - public-private cooperation - future - Netherlands
Partners in the Polder is a book in two parts. Part I is called Unity, and presents the field’s joint vision of the future of the life sciences in the Netherlands, and the past, present and future role of public-private partnerships. Part II, Diversity, looks at (the future of) the life sciences and PPPs from the perspectives of the sectors involved (food, health, chemicals & energy and agriculture) and the disciplines linking them (technology, education, valorization and social aspects).The two parts can be read separately, but together illustrate a field united in diversity. Each chapter in Part II can be read on its own.
Horseradish peroxidase-catalyzed cross-linking of feruloylated arabinoxylans with ß-casein
Boeriu, C.G. ; Oudgenoeg, G. ; Spekking, W.T.J. ; Berendsen, L.B.J.M. ; Vancon, L. ; Boumans, H. ; Gruppen, H. ; Berkel, W.J.H. van; Laane, N.C.M. ; Voragen, A.G.J. - \ 2004
Journal of Agricultural and Food Chemistry 52 (2004)21. - ISSN 0021-8561 - p. 6633 - 6639.
ferulic acid - fungal laccase - proteins - polysaccharides - conjugation - pentosans - bran
Heterologous conjugates of wheat arabinoxylan and beta-casein were prepared via enzymatic cross-linking, using sequential addition of the arabinoxylan to a mixture of beta-casein, peroxidase, and hydrogen peroxide. The maximal formation of adducts between the beta-casein and the feruloylated arabinoxylan was reached at a protein-to-arabinoxylan ratio of 10:1, in combination with a molar ratio hydrogen peroxide to substrate of 2:1 and a molar protein-to-enzyme ratio between 10(2) and 10(4). The protein-arabinoxylan adducts were separated from the arabinoxylan homopolymers by size exclusion and anion exchange chromatography. The molar ratio protein:arabinoxylan in the purified conjugates varied between 0.1 and 5.6. This is the first report on the large-scale enzymatic preparation of heterologous protein-arabinoxylan conjugates.
Peroxidase catalyzed conjugation of peptides, proteins and polysaccharides via endogenous and exogenous phenols.
Oudgenoeg, G. - \ 2004
Wageningen University. Promotor(en): N.C.M. Laane; Fons Voragen, co-promotor(en): Willem van Berkel; Harry Gruppen. - [S.l.] : S.n. - ISBN 9058089657 - 192
peroxidasen - ferulazuur - pyrocatechol - katalyse - peroxidases - ferulic acid - catalysis
Peroxidase mediated cross-linking of proteins
Oudgenoeg, G. ; Gruppen, H. ; Hilhorst, M.H. ; Berkel, W.J.H. van; Boumans, H. ; Piersma, S.R. ; Boeriu, C.G. ; Voragen, A.G.J. ; Laane, N.C.M. - \ 2003
Industrial Proteins 11 (2003)2. - ISSN 1381-0022 - p. 14 - 17.
Comparison of content in phenolic compounds, polyphenol oxidase and peroxidase in grains of fifty sorghum cultivars from Burkina Faso.
Dicko, M.H. ; Hilhorst, M.H. ; Gruppen, H. ; Traore, A.S. ; Laane, N.C.M. ; Berkel, W.J.H. van; Voragen, A.G.J. - \ 2002
Journal of Agricultural and Food Chemistry 50 (2002)13. - ISSN 0021-8561 - p. 3780 - 3788.
oxidative-enzymes - ferulic acid - wheat - monophenolase - localization - sensitivity - vegetables - resistance - products - vulgare
Analysis of fifty sorghum [Sorghum bicolor (L.) Moench] varieties used in Burkina Faso showed that they have different contents of phenolic compounds, peroxidase (POX), and polyphenol oxidase (PPO). Most of the varieties (82%) had a tannin content less than 0.25% (w/w). POX specific activity was higher than the monophenolase and o-diphenolase specific activities of PPO. For POX, there was a diversity of isoforms among varieties. No clear correlation could be made between the quantitative composition of the grain in phenolics, PPO, and POX, and resistance of plant to pathogens. In general, varieties good for a thick porridge preparation (¿tô¿) had low phenolic compounds content and a medium POX activity. From the red varieties, those used for local beer (¿dolo¿) had a high content in phenolic compounds and PPO, and a low POX activity. The variety considered good for couscous had a low POX content. The characteristics might be useful as selection markers for breeding for specific applications.
Flavoenzyme-catalyzed oxygenations and oxidations of phenolic compounds
Moonen, M.J.H. ; Fraaije, M.W. ; Rietjens, I.M.C.M. ; Laane, C. ; Berkel, W.J.H. van - \ 2002
Advanced Synthesis and Catalysis 344 (2002). - ISSN 1615-4150 - p. 1023 - 1035.
Flavin-dependent monooxygenases and oxidases play an important role in the mineralization of phenolic compounds. Because of their exquisite regioselectivity and stereoselectivity, these enzymes are of interest for the biocatalytic production of fine chemicals and food ingredients. In our group, we have characterized several flavoenzymes that act on phenolic compounds, including 4-hydroxybenzoate 3-hydroxylase, 3-hydroxyphenylacetate 6-hydroxylase, 4-hydroxybenzoate 1-hydroxylase (decarboxylating), hydroquinone hydroxylase, 2-hydroxybiphenyl 3-monooxygenase, phenol hydroxylase, 4-hydroxyacetophenone monooxygenase and vanillyl-alcohol oxidase. The catalytic properties of these enzymes are reviewed here, together with insights obtained from site-directed and random mutagenesis.
Zymography of monophenolase and o-Diphenolase activities of polyphenol oxidase
Dicko, M.H. ; Hilhorst, R. ; Gruppen, H. ; Laane, C. ; Berkel, W.J.H. van; Voragen, A.G.J. - \ 2002
Analytical Biochemistry 306 (2002). - ISSN 0003-2697 - p. 336 - 339.
Recombinant gelatin and collagen from methylotrophic yeasts
Bruin, E.C. de - \ 2002
Wageningen University. Promotor(en): N.C.M. Laane; Frits de Wolf. - S.l. : S.n. - ISBN 9789058085832 - 110
collageen - gelatine - recombinant dna - hansenula - Hansenula polymorpha - Pichia pastoris - collagen - gelatin - Methylotrophic yeast - Fermentation - Secretion - Recombinant expression
<font size="3"><p>Based on its structural role and compatibility within the human body, collagen is a commonly used biomaterial in medical applications, such as cosmetic surgery, wound treatment and tissue engineering. Gelatin is in essence denatured and partly degraded collagen and is, as a result of its unique functional and chemical properties, also used in many medical and pharmaceutical products. Collagen and gelatin are traditionally extracted from animal tissues. The quality and the characteristics of the proteins are not very reproducible in today's batch-to-batch production processes and recently, potential contamination of collagen and gelatin with viruses and prions (causing BSE) became a matter of concern. BSE is thought to cause a new variety of the brain- wasting Creutzfeldt-Jacob disease in humans.</p><p>Recombinant DNA technology may provide safe collagen and gelatins from which the quality and characteristics can precisely be controlled and reproduced and, in addition, opens up possibilities for novel functional "tailor-made" proteins.For the heterologous production of animal proteins yeasts are frequently used. Since yeasts are eukaryotes, most translational modification, needed for functionality and stability of recombinant animal proteins, normally occur. However prolyl 4-hydroxylation, essential for gelling properties of recombinant gelatin and thermal stability of recombinant collagen, is generally considered to be absent in yeast systems.</p><p>In this study we explored the methylotrophic yeasts <em>Hansenula polymorpha</em> and <em>Pichia pastoris</em> for their use as recombinant production systems of natural and "tailor-made" gelatins and human collagen.We found that both yeasts are well able to cope with the repetitive gene sequences encoding animal gelatin and human collagen and showed that <em>P. pastoris</em> can produce synthetic gelatins with highly hydrophilic properties at high levels. Furthermore, it was discovered that <em>H. polymorpha</em> unexpectedly produced endogenous collagen-like proteins with 4-hydroxyproline amino acid residues. This finding indicated that the yeast <em>H. polymorpha</em> , in contract to what was generally believed, must contain intrinsic proly 4-hydroxyalse activity. Indeed, expression of murine gelatin in <em>H. polymorpha</em> yielded a secreted and hydroxylated product. We also investigated if <em>H. polymorpha</em> could be used for the production of recombinant human collagen. Intract human collagen trimers were obtained but they were not stable at temperatures higher than 15 °C, indicating that hydroxylation in the product was poor.</p><p>In the course of this study we found putative prolyl 4-hydroxylase genes in different eukaryotic microbial systems. In the future these genes may be used to further develop yeasts into cell factories for the production of animal gelatins and thermally stable human collagen.
Exploring conformational dynamics of flavoenzymes with flavin fluorescence relaxation spectroscopy
Berg, P.A.W. van den - \ 2002
Wageningen University. Promotor(en): A.J.W.G Visser; N.C.M. Laane. - S.l. : S.n. - ISBN 9789058086945 - 223
amine oxidoreductasen - fluorescentie-emissiespectroscopie - moleculaire structuur - amine oxidoreductases - fluorescence emission spectroscopy - molecular conformation
<p>Research described in this thesis was aimed at gaining more insight into the active-site dynamics of dimeric flavoproteins by means of fluorescence relaxation spectroscopy. Three flavoproteins for which crystallographic data have suggested different types of functionally important motions were chosen as central systems; <em>E. coli</em> glutathione reductase, which displays a local conformational change in the protein environment; <em>E. coli</em> thioredoxin reductase, for which a major domain rotation was proposed to be essential for catalysis; and <em>P. fluorescens p-</em> hydroxybenzoate hydroxylase, in which the isoalloxazine ring of the flavin cofactor itself is mobile during catalysis. For interpretation of fluorescence data in terms of dynamic events in the proteins, explicit attention was paid to the photophysical and dynamic characteristics of the flavin cofactor.</p><strong><em><p>Chapter 1</strong></em> provides a general introduction into the enzyme systems and into the principles and mechanisms of conformational dynamics and fluorescence relaxation spectroscopy.</p><p>In <strong><em>Chapter 2,</strong></em> the dynamic properties of wild-type <em>E. coli</em> glutathione reductase (GR) are studied in comparison with those of the mutant enzymes GR Y177F and GR Y177G. Emphasis is laid on the relations between fluorescence lifetime patterns, protein dynamics and the mechanisms for fluorescence quenching in proteins. Experimental evidence is provided for the multiple quenching sites model.</p><p>The implications of the comparative study on the gluthatione reductase enzymes for the interpretation of time-resolved fluorescence anisotropy decays are described in <strong><em>Chapter 3</strong></em> , where a new mechanism for flavin fluorescence depolarization is proposed.</p><strong><em><p>Chapter 4</strong></em> focuses on the conformational dynamics of <em>E. coli</em> thioredoxin reductase (TrxR) and the mutant enzyme TrxR C138S. Two catalytically important conformational states of the enzyme are detected and characterized by (sub)picosecond time-resolved and spectrally resolved fluorescence techniques. Flavin fluorescence relaxation experiments are combined with steady-state optical techniques to gain insight into the dynamic properties of the enzyme and the conformational equilibrium. The importance of enlarging the time window for the fluorescence detection of dynamic events is discussed.</p><p>The mobile flavin in <em>p-</em> hydroxybenzoate hydroxylase (PHBH) is subject of a time-resolved fluorescence investigation in <strong><em>Chapter 5</strong></em> . Different binary (mutant) enzyme/substrate (analogue) complexes are used to direct the conformation of the cofactor. The chapter reflects on possibilities and limitations of ensemble fluorescence lifetime data for studying protein dynamics.</p><p>In <strong><em>Chapter 6</strong></em> , a link is created between time-resolved fluorescence data of ensembles of molecules and the molecular dynamics of single molecules as retrieved from molecular dynamics (MD) simulations. Hereto, the system of investigation is simplified to the FAD cofactor, which can exist in both 'open' and 'closed' conformations. MD simulations provide insight into the dynamic behaviour of the free cofactor and into pathways for conformational transitions.</p><strong><em><p>Chapter 7</strong></em> describes the first steps into the world of single-molecule detection through natural flavin fluorescence. Fluorescence Correlation Spectroscopy studies on FAD, FMN and lipoamide dehydrogenase provide a first glance into the future perspectives of detecting single flavoproteins and give an understanding of the specific obstacles that need to be overcome.</p><p>The thesis is concluded by a summarizing discussion reflecting on the research described in this thesis in relation to developments in the field.
Peroxidase mediated conjugation of proteins and carbohydrates to tailor their functional properties
Oudgenoeg, G. ; Berkel, W.J.H. van; Boumans, H. ; Boeriu, C.G. ; Gruppen, H. ; Laane, C. ; Voragen, A.G.J. - \ 2002
In: Biocatalysis in the Food and Drink Industries, London, 2002
Method of enzymatically cross-linking proteins and phenolic polymers
Oudgenoeg, G. ; Boeriu, C.G. ; Hilhorst, H.M. ; Gruppen, H. ; Laane, N.C.M. ; Voragen, A.G.J. - \ 2002
Octrooinummer: EP1169922, verleend: 2002-01-09.
The present invention relates to a method of cross-linking a protein or peptide and a phenolic polymer or oligomer having substituents derived from carboxylic acids containing hydroxyl substituted phenyl groups by means of an enzyme and an oxidizing agent suitable for the enzyme in a solvent, which method comprises reacting a mixture of protein or peptide, oxidizing agent, enzyme and polymer or oligomer in the solvent, wherein the method is controlled such that in the mixture the ratio of target amino groups in the protein or peptide to hydroxyl substituted phenyl groups in the polymer or oligomer is more than equimolar. With the present method predominantly hetero-cross-linking of protein and polymer occurs.
Horseradish peroxidase-catalyzed oligomerization of ferulic acid on a template of a tyrosine-containing tripeptide
Oudgenoeg, G. ; Dirksen, E. ; Ingemann, S. ; Hilhorst, R. ; Gruppen, H. ; Boeriu, C.G. ; Piersma, S.R. ; Berkel, W.J.H. van; Laane, C. ; Voragen, A.G.J. - \ 2002
Journal of Biological Chemistry 277 (2002)24. - ISSN 0021-9258 - p. 21332 - 21340.
Ferulic acid (FA) is an abundantly present phenolic constituent of plant cell walls. Kinetically controlled incubation of FA and the tripeptide Gly-Tyr-Gly (GYG) with horseradish peroxidase and H2O2 yielded a range of new cross-linked products. Two predominant series of hetero-oligomers of FA linked by dehydrogenation to the peptidyl tyrosine were characterized by electrospray ionization (tandem) mass spectrometry. One series comprises GYG coupled with 4-7 FA moieties linked by dehydrogenation, of which one is decarboxylated. In the second series 4-9 FA moieties linked by dehydrogenation, of which two are decarboxylated, are coupled to the tripeptide. A third series comprises three hetero-oligomers in which the peptidyl tyrosine is linked to 1-3 FA moieties of which none is decarboxylated. Two mechanisms for the formation of the FA-Tyr oligomers that result from the dualistic, concentration-dependent chemistry of FA and their possible role in the regulation of plant cell wall tissue growth are presented.
Endogenous prolyl 4-hydroxylation in Hansenula polymorpha and its use for the production of hydroxylated recombinant gelatin
Bruin, E.C. de; Werten, M.W.T. ; Laane, C. ; Wolf, F.A. de - \ 2002
FEMS Yeast Research 1 (2002)4. - ISSN 1567-1356 - p. 291 - 298.
Several yeast systems have recently been developed for the recombinant production of gelatin and collagen. Amino acid sequence-specific prolyl 4-hydroxylation is essential for the gel-forming capacity of gelatin and for the proper folding of (pro)collagen. This post-translational modification is generally considered to be absent in microbial eukaryotic systems and therefore co-expression of heterologous (human or animal) prolyl 4-hydroxylase would be required. However, we found that the well-known protein expression host Hansenula polymorpha unexpectedly does have the endogenous capacity for prolyl 4-hydroxylation. Without co-expression of a heterologous prolyl 4-hydroxylase, both an endogenous collagen-like protein and a heterologously expressed collagen fragment were found to be sequence-specifically hydroxylated.
Effects of Xylanase and peroxidase on soluble and insoluble arabinoxylans in wheat bread dough
Hilhorst, R. ; Gruppen, H. ; Orsel, R. ; Laane, C. ; Schols, H.A. ; Voragen, A.G.J. - \ 2002
Journal of Food Science 67 (2002). - ISSN 0022-1147 - p. 497 - 506.
Bread doughs supplemented with xylanase and xylanase plus peroxidase were fractionated into 4 insoluble and 3 soluble fractions. Chemical analysis and high-performance size-exclusion chromatography analysis of apparent molecular weight distribution indicated that xylanase acts on both cold-water-extractable arabinoxylans and on those that can be solubilized from cell wall fragments by hot water extraction. Peroxidase action increased the amount of insoluble small cell wall fragments, notably the amount of protein and arabinoxylan. Arabinoxylans were retained in the small cell wall fragments because cross-linking of arabinoxylans through ferulic acid residues to other arabinoxylans rendered them insoluble. Peroxidase did not affect the composition of gluten, nor was evidence obtained for peroxidase-catalyzed cross-linking of arabinoxylans to protein in the gluten and other fractions.
Peroxidase-mediated cross-linking of a tyrosine-containing peptide with ferulic acid
Oudgenoeg, G. ; Hilhorst, H. ; Piersma, S.R. ; Boeriu, C.G. ; Gruppen, H. ; Voragen, A.G.J. ; Laane, C. - \ 2001
Journal of Agricultural and Food Chemistry 49 (2001). - ISSN 0021-8561 - p. 2503 - 2510.
The tyrosine-containing peptide Gly-Tyr-Gly (GYG) was oxidatively cross-linked by horseradish peroxidase in the presence of hydrogen peroxide. As products, covalently coupled di- to pentamers of the peptide were identified by LC-MS. Oxidative cross-linking of ferulic acid with horseradish peroxidase and hydrogen peroxide resulted in the formation of dehydrodimers. Kinetic studies of conversion rates of either the peptide or ferulic acid revealed conditions that allow formation of heteroadducts of GYG and ferulic acid. To a GYG-containing incubation mixture was added ferulic acid in small aliquots, therewith keeping the molar ratio of the substrates favorable for hetero-cross-linking. This resulted in a predominant product consisting of two ferulic acid molecules dehydrogenatively linked to a single peptide and, furthermore, two ferulic acids linked to peptide oligomers, ranging from dimers to pentamers. Also, mono- and dimers of the peptide were linked to one molecule of ferulic acid. A mechanism explaining the formation of all these products is proposed.
Exploring the limits of vanillyl-alcohol oxidase
Heuvel, R.H.H. van den - \ 2001
Wageningen University. Promotor(en): N.C.M. Laane; Willem van Berkel. - S.l. : S.n. - ISBN 9789058083722 - 146
vanilline - biosynthese - alcohol oxidoreductasen - vanillin - biosynthesis - alcohol oxidoreductases
<p>Vanillin is the world's principal flavoring compound, extensively used in food and personal products. The curing process of vanilla beans is labor-intensive and the <em>Vanilla</em> plant only grows in a few territories over the world, making synthetically produced vanillin far cheaper than natural vanillin. Nowadays, only 0.5% of the total market is met by extraction of <em>Vanilla</em> beans. The remaining 99.5% of the vanillin produced is of synthetic origin. However, with the increasing interest in natural products alternative methods are being developed to produce natural vanillin from sources other than <em>Vanilla planifolia</em> . Cell cultures, microorganisms, and isolated enzymes form potentially alternative sources for the production of vanillin from natural feedstock.</p><p>The Ph. D. project described here was initiated in the framework of the Innovation Oriented Research Program (IOP) Catalysis of the Dutch Ministry of Economy Affairs. In the Enzymatic Oxidation cluster of this research program the catalytic potential of oxidative enzymes for the production of valuable compounds was investigated. Enzymes are an almost unlimited source for the production of these compounds as they can produce natural products and are often highly regio- and/or stereospecific. Furthermore, biocatalytic (enzymatic) processes are in general environmentally friendlier than chemical processes. In this project, we aimed to enlarge the catalytic potential of the flavin-containing enzyme vanillyl-alcohol oxidase (VAO). To that end, the VAO-mediated production of natural vanillin and optically pure aromatic alcohols was addressed. Two different methods were used to direct the reactions to the most favorable product. In the first method we controlled the reaction medium and in the second method we introduced a few subtle changes in the enzyme. For these studies insight in the protein-flavin and protein-protein interactions were of crucial importance.</p><h3>Enzymatic production of natural vanillin and optically pure alcohols</h3><p><strong>Chapter 2</strong> describes the VAO-catalyzed conversion of creosol and vanillylamine to vanillin. The enzymatic conversion of creosol proceeds via a two-step process in which the initially formed vanillyl alcohol is further oxidized to vanillin. The production of vanillin is not optimal due to the competitive binding of creosol and vanillyl alcohol in the enzyme active site and the fact that creosol forms a non-reactive covalent adduct with the flavin cofactor.</p><p>The oxidation of vanillylamine to vanillin proceeds readily at pH 10. However, as vanillylamine is too expensive for industrial use, we searched for a natural precursor compound. Capsaicin from red pepper is rather cheap and can be hydrolyzed enzymically to vanillylamine by a carboxylesterase from liver or chemically at basic pH values. Therefore, the use of capsaicin as feedstock for the production of vanillin is very promising.</p><p>VAO is active with a wide range of 4-alkylphenols bearing aliphatic side chains up to seven carbon atoms. In <strong>Chapter 3</strong> , we describe the enzymatic conversion of short-chain 4-alkylphenols to optically pure aromatic alcohols and the conversion of medium-chain 4-alkylphenols to aromatic alkenes. The VAO-mediated hydroxylation of 4-alkylphenols is highly stereospecific (enantiomeric excess = 94%), and the enantiomeric excess of the <em>R</em> -product is even increased by the VAO-mediated oxidation of the ( <em>S</em> )-isomer of the alcohol. The enzymatic dehydrogenation of medium-chain 4-alkylphenols is also stereospecific, suggesting that the <em>p</em> -quinone methide intermediate products are bound in a fixed orientation in the enzyme active site. Some medium-chain 4-alkylphenols are dehydrogenated to the <em>cis</em> -isomer and others to the <em>trans</em> -isomer of the alkene product. Thus, the specificity of the VAO-mediated conversions is dictated by the intrinsic reactivity, water accessibility, and orientation of the enzyme-bound <em>p</em> -quinone methide intermediate.</p><h3>Tuning the product specificity</h3><p>In the following chapters (4-7), we studied the possibilities to direct the VAO-mediated conversion of 4-alkylphenols into the most favorable direction using two different strategies. First, we varied the medium in which the reaction was performed and second, we modified the protein by rationale mutagenesis. In <strong>Chapter 4</strong> , we investigated the reactivity of VAO with 4-alkylphenols in the hydrophobic solvent toluene and the hydrophilic solvent acetonitrile. In both solvents the efficiency of substrate hydroxylation decreased compared to aqueous conditions. This effect on the hydroxylation efficiency was dependent on the water activity, but independent on the solvent used. This shows that the availability of water determines the efficiency of the hydroxylation reaction. A similar result was obtained by the addition of the monovalent anions chloride, bromide, or thiocyanate. The binding of these ions near the flavin prosthetic group inhibited the attack of water to the enzyme-bound quinone methide, providing a similar effect as lowering the water activity.</p><p>Protein engineering of VAO by site-directed mutagenesis proved to be another method to tune the reactivity of VAO with 4-alkylphenols ( <strong>Chapter 5 and 6</strong> ). The catalytic center of VAO harbors an acidic residue (Asp170), which is located in the proximity of the flavin N5-atom (3.6 Å) and the substrate Cα-atom (3.0 Å). The location of this residue is intriguing as in most flavin-dependent oxidoreductases of known structure the flavin N5-atom contacts a hydrogen bond donor rather than an acceptor. Asp170 appeared to be crucial for the activity of VAO, the efficiency of hydroxylation of 4-alkylphenols, and the covalent binding of the flavin. Studies from site-directed mutagenesis and protein crystallography showed that Asp170 raises the oxidative power of the flavin cofactor and, therefore, the activity of the enzyme. Replacement of Asp170 by Ser or Ala resulted in a better hydroxylation efficiency of VAO, whereas the Asp170Glu replacement decreased the hydroxylation efficiency. These changes in product specificity are caused by steric effects. The small side chains of Ser170 and Ala170 increase the accessibility of water to the enzyme-bound <em>p</em> -quinone methide intermediate, whereas the more bulky side chain of Glu170 protects the quinone methide from water attack.</p><p>In <strong>Chapter 7</strong> , we describe the inversion of the stereospecificity of VAO by protein redesign. The active site residue Asp170, involved in water activation, was transferred to the opposite face of the substrate binding pocket (Thr457Glu mutation). As a result, the double mutants D170S/T457E and D170A/T457E hydroxylated 4-ethylphenol to the inverse enantiomer of the aromatic alcohol. This change in stereospecificity is caused by the activation of a water molecule, attacking the <em>p</em> -quinone methide, positioned at the opposite face of the substrate compared to wild type VAO. Crystallographic data confirmed that the distinctive properties of the redesigned mutants are caused by the selective mutations and not by structural changes within the protein. This is the first example of the inversion of the stereospecificity of an enzyme using a rationale redesign strategy.</p><h3>Rationale of covalent flavin binding</h3><p>The reason of covalent flavin binding in flavoenzymes is still a matter of debate. It has been suggested that the covalent interaction might a) increase the protein stability, b) enhance the enzyme activity, c) prevent flavin dissociation, and d) improve the resistance against proteolysis.</p><p>In <strong>Chapter 8</strong> , the role of the covalent protein-flavin interaction was studied by changing the residue to which the flavin is linked. The non-covalent VAO mutant H422A firmly binds the FAD cofactor, but the activity of the enzyme is decreased ten-fold. The lower enzymatic activity is not caused by structural changes but can be fully attributed to the decreased redox potential of the flavin cofactor. Thus, the covalent flavin bond is essential for the high oxidative power of the enzyme.</p><h3>Oligomeric structure of VAO</h3><p>At neutral pH, VAO predominantly forms homooctamers. The crystal structure of VAO has revealed that the octamer can be described as a tetramer of dimers in which each dimer is stabilized by extensive intersubunit interactions. Because some dimers are present at neutral pH and low ionic strength, it was of interest to study the stability of the protein assembly as a function of pH by electrospray ionization mass spectrometry ( <strong>Chapter 9</strong> ). At low pH values, the octamer-dimer equilibrium shifts to the dimeric form, whereas at neutral pH the enzyme is mainly present in the octameric form. Interestingly, also higher oligimerization assemblies of VAO were observed, indicating that weak interactions between the octamers exist. This information about the oligomeric structure of VAO is very useful for further studies, directed towards the stability of VAO under operational conditions. It is the first time that the mass of such a large molecule (larger than 1 million Da) is determined using this technique.</p><h3>Conclusions</h3><p>This research project was performed within the framework of the Enzymatic Oxidation cluster of the Innovation Oriented Research Program (IOP) Catalysis, funded by the Ministry of Economy Affairs. The aim of this cluster was to develop processes for the production of pharmaceuticals, fine-chemicals, and flavors and fragrances using oxidative enzymes, like heme peroxidases, vanadium peroxidases, and flavin-dependent oxidases.</p><p>In this thesis work, we focussed on the catalytic potential of the flavoprotein vanillyl-alcohol oxidase (VAO). VAO is active with a wide range of phenolic compounds and can produce a variety of industrially relevant products like vanillin and optically pure aromatic alcohols. We have demonstrated that the reactivity and selectivity of VAO can be modulated by medium engineering and protein engineering.</p><p>The principal component of red pepper, capsaicin, proved to be a promising candidate to produce natural vanillin using a bi-enzyme system, consisting of VAO and a hydrolase. By combining these two enzymes a one-pot conversion from capsaicin to vanillin can be realized. This production method yields natural vanillin, which is more valuable than synthetic vanillin. Moreover, the enzymatic production has, in general, environmental advantages compared to the traditional synthetic vanillin production.</p><p>VAO produces optically pure aromatic alcohols from 4-alkylphenols. The efficiency of substrate hydroxylation can be tuned by varying the availability of water in the catalytic center or by substituting a single amino acid residue (Asp170) in the enzyme. Furthermore, we were able to invert the stereospecificity of VAO by relocation of the active site base. This demonstrates that protein engineering is a powerful tool to introduce new enzyme characteristics. A major goal for further research would be to enlarge the substrate scope of VAO and to improve the catalytic performance of VAO variants. Interesting target compounds are creosol and capsaicin, as being precursors of vanillin, and epinephrine analogs. Here, random mutagenesis and/or gene shuffling are attractive approaches, since the required changes in VAO are not easy to predict.</p><p>VAO is active over a wide pH range, but the protein assembly falls apart under extreme conditions. For possible future applications of the enzyme it is important to study the relationship between the conformational stability and oligomeric structure of VAO. In this aspect, the influence of the covalent flavin linkage is of importance as well. An interesting option to obtain a protein with improved stability properties would be gene shuffling between VAO and a homolog from a thermophilic organism. Another possibility would be a combination of directed evolution methods.</p>
Structural studies on metal-containing enzymes: T4 endonuclease VII and D. gigas formate dehydrogenase
Raaijmakers, H.C.A. - \ 2001
Wageningen University. Promotor(en): N.C.M. Laane; D. Suck. - S.l. : S.n. - ISBN 9789058084125 - 85
enzymen - röntgenkristallografie - desulfovibrio - oxidoreductasen - wolfraam - enzymes - x ray crystallography - oxidoreductases - tungsten
<p>Many biological processes require metal ions, and many of these metal-ion functions involve metalloproteins. The metal ions in metalloproteins are often critical to the protein's function, structure, or stability. This thesis focuses on two of these proteins, bacteriophage T4 endonuclease VII (EndoVII) and D. gigas fonnate dehydrogenase, which are studied by X-ray crystallography. The structure of EndoVII reveals how a magnesium or calcium ion is used to cleave several kinds of irregular but flexible DNA, while a zinc ion maintains the structural integrity of this DNase.</p><p>The formate dehydrogenase contains a tungsten ion and a seleno-cysteine at the active site, that catalyses the oxidation of formate to carbon dioxide. The two released electrons are transferred through four [4Fe-4S] clusters before they can be handed over to another protein. Two of the [4Fe-4S] and the selenium have been overlooked by other techniques, but could be localised and identified by crystallography.</p><p><strong>Chapter 1</strong> gives a general introduction on metals in biological systems, X-ray crystallography and also describes the biological background of both proteins.</p><p><strong>Chapter 2</strong> presents the structure of the four-way DNA-junction resolving enzyme T4 endonuclease VII, and that of the inactive N62D mutant. The betterexpressed mutant was solved first, using seleno-methionine, mercury and gold derivatives. These mercury and gold derivatives bind to the sulphurs that also ligand the zinc. The wild-type was solved with help of a single mercury derivative since molecular replacement with the mutant structure failed.</p><p>On its own, the EndoVII monomer would not represent a stable fold, as it exposes many hydrophobic residues to the solvent. But two monomers intertwine to form a dimer without this problem. In this dimer, the monomers are aligned head-to-tail; the N-terminus of one monomer interacts with the C-terminus of the other monomer and <em>vice versa</em> . The major dimerization element, unique to EndoVII, is the "four-helix-cross" domain, which consists of helix-2 and helix-3 from each monomer. It contains an extended hydrophobic core.</p><p>Another feature is the "beta-finger", residues 38-56. Its stability depends critically on the zinc. This zinc ion is tetrahedrally co-ordinated to four cysteines, linking helix- I through residues C23 and C26 firmly to the N-terminal part of helix-2 (C58, C61). Indeed, interfering mutations inactivate the protein. Finally, the calcium ion, which marks the active site, is liganded to aspartate-40 and asparagine-62. Mutation studies show that these amino acids are essential for activity: The N62D mutant is completely inactive.</p><p>The EndoVII structure has been docked to a "stacked-X" four-way DNA junction, one of its many substrates. This model is not refined, since both the DNA and the protein are known to be flexible and might undergo conformational changes. However, its overall features confirm experimental data: 1) The EndoVII dimer binds to the minor groove side of the four-way junction; 11) Basic residues on helix-2 can interact with phosphates on the exchanging strands and those on the C-terminal domain can interact with phosphates in the continuous strands, consistent with observed foot-printing patterns; 111) The C-terminus binds up to nine base pairs away from the junction, confirming the minimal length of two arms of the substrate; IV). The active sites do not cleave both the scissile phosphates simultaneously.</p><p>Surprisingly, the N62D mutant shows a major rearrangement in the "four-helixcross" domain, when compared to the wild-type: helices-2 are translated by half a turn each, in opposite direction and the opening of the "bays", between each helix-2 and betafinger, is wider. These differences might be attributed to the point-mutation, which introduces an extra charge in the active site, to differences in crystallisation conditions, to the different pH employed, to crystal contacts or perhaps they are simply a sign of the intrinsic flexibility of EndoVII.</p><p>This dilemma is partly solved in <strong>chapter 3, which</strong> presents the crystal structure of wild-type EndoVII in a different space group, which contains less solvent. It crystallised in the same drop, so that differences observed between the two wild-type structures cannot be attributed to the mutation, pH or salt concentrations. Since the helical-cross region of this second structure is very similar to that of the mutant, rearrangements in this region must be seen as a consequence of intrinsic flexibility of EndoVII. The widening of the "bays", however, might still be a consequence of the mutation, different pH, absence of Ca <sup>2+</sup> or crystal packing. An investigation of the flexibility of EndoVII with TLS- refinement, i.e. anisotropic refinement of rigid bodies, provides only limited insight. However, it confirms that rotations along the axes of the helices 2 and 4 and along the beta-finger are a main source of flexibility and also that the C-terminus, helix-4, 5 and 6, behave as a rigid body.</p><p>The high-resolution structure of the N62D mutant brings more clarity towards the reaction mechanism of the nuclease. This model contains important water molecules and reveals the position and orientation of 14 sulphate ions, which may indicate favoured phosphate (DNA) binding sites. Supported by new mutation data (Birkenbihl, unpublished), these sulphate and water positions, combined with the Ca <sup>2+</sup> positions in the wild-type structures, suggest a reaction-mechanism similar to those proposed for some other magnesium dependent nucleases.</p><p>2 Asparagine-62, glutamate-65 and aspartate-40 are important to position Mg <sup>2+</sup> or Ca <sup>2+</sup> next to the scissile phosphate of the DNA substrate. Histidine-41 activates a water molecule, which in turn executes a nucleophilic attack on the phosphor atom. Histidine-43 stabilises this phosphate directly through a hydrogen bond. Unfortunately it is still unclear why the N62D shows no DNase activity at all; an aspartate would also be able to ligand/position a divalent cation. The extra charge that this mutation introduces in the active site might distort the geometry of the active site, and repel the DNA. A more attractive, albeit more speculative hypothesis, assumes that the amino group of asparagine-62 donates a hydrogen-bond to the phosphate, which would also stabilise the transition state.</p><p>At present, there are no known proteins with significant sequence homology to EndoVII, though nucleases with structural similarities do exist. One group consists of magnesium-dependent nucleases, which have a similar geometry of liganding sidechains around the magnesium (or calcium) ion in the active site; e.g. the E. coli proteins RuvC (Ariyosi et al., 1994) and RNase H (Katayanagi et al., 1990). However, these nucleases have no resembling fold. Most likely, this just shows that magnesium-dependent nucleases need a certain geometry to function.</p><p>A more interesting group shares a folding motif similar to the beta finger and helix-2: Serratia Nuclease (Miller et al., 1994), Ppol (Flick et al., 1998) and perhaps even Colicin E9 (Kleanthous et al., 1999). Asparagine-62 and histidine-41 are conserved between Serratia nuclease, Ppol and T4 endonuclease VII. Ppol has also been crystallised in complex with DNA. If one superimposes this with the EndoVII structure, it turns out that the Ca <sup>2+</sup> in EndoVII is buried deeper within the protein, but small rotations (10-20 degrees) along helix 2 and the beta-finger suffice to superimpose them. These two nucleases act on different substrates, and maybe the larger DNA junctions of EndoVII need a wider and deeper binding groove than the double stranded DNA of Ppol. However, it could also be the source of EndoVII's specificity; flexible DNA might impose this conformational change of EndoVII upon binding, readying the enzyme for cleavage, while the magnesium or calcium ion might be too far away if EndoVII approaches more rigid DNA. A structure of EndoVII in complex with DNA would solve these questions.</p><p><strong>Chapter 4</strong> presents the major part of the determination of the 3D structure of the tungsten-containing formate dehydrogenasc (W-FDH) from Desulfovibrio gigas, one of the first tungsten-containing enzymes isolated from a mesophile. The large subunit (92 kDa) is structurally related to several tungsten- and molybdenumcontaining enzymes and X-ray structures have been determined for two of them. One of these, the periplasmic nitrate reductase (Dias et al, 1999), could be used to obtain a molecular replacement solution. But the quality of phasing was not sufficient to generate a clear, interpretable electron density map. Furthermore, the amino acid sequence of W-FDH has not yet been determined, what makes model building complicated. Multiple wavelength diffraction (MAD) measurements were undertaken at the absorption edges of W and Fe to define unambiguously the number, positions and identity of these anomalous scatterers and to improve the X-ray phases. The MAD-analysis revealed one W-atom with a Se-cys ligand and one [4Fe-4S] cluster bound to the large subunit, and three [4Fe-4S] clusters in the small subunit. The four [4Fe-4S] clusters are ca. 10 Å apart, creating a feasible electron transfer pathway, which connects the exterior of the protein to the W/Se site in the large subunit. Two of the four iron-sulphur clusters had not been predicted before by spectroscopic techniques (Almendra et al., 1999). A reinvestigation of the spectroscopic data was performed, but gave the same results as before. If these data were correct, this means that the [4Fe-4S] clusters are instable, and that only protein with fully occupied clusters crystallises.</p><p>The formate dehydrogenase H (FDH-H) from E. coli catalyses the same reaction as W-FDH, but uses a molybdenum instead of tungsten. Both are liganded to two molybdopterin-cofactors and to a seleno-cysteine, so the question remains why W-FDH prefers tungsten to the more common molybdenum. The full structure will allow a comparison of the two enzymes in atomic detail, and perhaps, it will shed some light on this phenomenon.</p><p>X-ray crystallography has been used to characterise the nature of metal-centres in proteins, their coordination geometry and even their identity. Sometimes, the way metal ions are bound to the protein already clarifies its role in the protein. In other cases it has to be supplemented with other studies before the role can be fully understood. Either way, crystallography provides a powerful tool for the study of metalloproteins.</p><dl><dt><em>References</em></dt><dd>Almendra, M.J., Brondino, C.D., Gavel, 0., Pereira, A.S., Tavares, P., Bursakov, S., Duarte, R., Caldeira, J., Moura, J.J.G., Moura, 1. (1999) <em>Biochemistry, 38</em> , 16366-16372</dd><dd>Ariyosi, M., Vassylyev, D., Iwasaki, H., Shinagawa, H. and Morikawa, K. (1994) <em>Cell, 78</em> , 1063-1072.</dd><dd>Flick, K.E., Jurica, M.S., Monnart Jr, R.J. and Stoddard, B.L. (1998), <em>Nature, 394</em> , 96-101.</dd><dd>Katayanagi, M., Miyagawa, M., Matsushima, M., Ishikawa, M., Kanaya, S., Ikehara, M., Matsuzaki, M. and Morikawa, K. (1990) <em>Nature, 347</em> , 306-309.</dd><dd>Kleanthous, C., Kuhlmann, U.C., Pornmer, A.J., Ferguson, N., Radford, S.E., Moore, G.R., James, R. and Hemmings, A.M. (1999) <em>Nature Struct. Biol., 6</em> , 243-252.</dd><dd>Miller, M.D., Tanner, J., Alpaugh, M., Benedik, M.J. and Krause, K.L. (1994) <em>Nature Struct Biol., 1</em> , 461-468.</dd></dl>
Exploring the reductive capacity of Pyrococcus furiosus : the reduction of carboxylic acids and pyridine nucleotides
Ban, E.C.D. van den - \ 2001
Wageningen University. Promotor(en): N.C.M. Laane. - S.l. : S.n. - ISBN 9789058085085 - 141
carbonzuren - pyridinenucleotiden - redoxpotentiaal - thermofiele micro-organismen - pyrococcus furiosus - carboxylic acids - pyridine nucleotides - redox potential - thermophilic microorganisms
<FONT FACE="Book Antiqua"><p>This Ph.D. project started in 1997 and its main goal was to obtain insight in the reductive capacity of the hyperthermophilic archaeon <em>Pyrococcus furiosus</em> . The research was focused on the biocatalytic reduction of carboxylic acids.</p><p>Reductions of carboxylic acids are interesting reactions, since the generated products, aldehydes and alcohols, are potentially applicable in the fine-chemical industry. However, the reduction of carboxylic acids to the corresponding aldehydes is a thermodynamically difficult reaction, both chemically and biologically, because the reaction needs strong reducing agents. Nevertheless, there are several microorganisms able to catalyze the reduction of acids. For some microorganisms, the mechanism catalyzing the acid reduction has been elucidated. Two of the proposed mechanisms proceed via activation of the acid to an acyl-AMP or acyl-CoA intermediate followed by reduction of the activated acid to the corresponding aldehyde. The third one is reduction of the acid without activation.</p><em><p>P. furiosus</em> is an anaerobic hyperthermophilic organism growing optimally at temperatures ranging from 90 to 100<FONT FACE="Symbol">°</font>C. The advantage of these high temperatures is that infectious mesophilic contaminations during culturing of this organism do not occur. Other advantages of this organism are the relatively fast growth and the usage of inexpensive carbon and energy sources like (potato)-starch. Next to that, <em>P. furiosus</em> is an archaeon and these organisms are non-pathogenic to men. These features and the fact that <em>P. furiosus</em> produced dihydrogen during growth make <em>P. furiosus</em> a very useful organism to study the reduction of acids.</p><p>First, an inventory was made of which carboxylic acids can be reduced by <em>P. furiosus</em> to the corresponding aldehydes and subsequently to the alcohols (chapter 2). Both aliphatic and aromatic acids were tested and <em>P. furiosus</em> was able to catalyze the reduction of compounds from both groups. The best results were obtained with aromatic compounds, of which the reduction of 3-phenylpropionic acid resulted in the highest yield: 69% of the acid was reduced to the corresponding alcohol.</p><p>The aldehyde, intermediate in this reduction, was not detected. This could be explained by the thermodynamically unfavorable reduction of the acid to the aldehyde followed by the thermodynamically favorable reduction of the aldehyde to the alcohol. It means that the 'difficult-to-generate' aldehyde is immediately converted into the corresponding alcohol. The experiments also showed that growing <em>P. furiosus</em> cells and dihydrogen are essential to reduce carboxylic acids.</p><p>Based on the results described in chapter 2, it was investigated which factors influenced the bioreduction and how the reduction rate as well as the yield of the alcohol could be optimized (chapter 3). Five factors were studied: 1) pH, 2) partial dihydrogen pressure, 3) substrate concentration, 4) carbon and energy concentration, and 5) temperature. To study the effects of these factors on the reduction of acids, experiments were performed according to the principle of 'factorial design'. 3-Phenylpropionic acid was used as model substrate.</p><p>The yield of the alcohol was found to be optimal at pH 6.3, a substrate concentration of 1 mM, and a temperature of 80<FONT FACE="Symbol">°</font>C. The reduction rate, however, was optimal at pH 7.0, 10 mM substrate and 90<FONT FACE="Symbol">°</font>C. Both the yield and the reduction rate showed to be dependent on the same variables, but the dependencies were opposite. As a result, it was not possible to maximize both terms at the same time within the limits of these experiments.</p><p>Dihydrogen showed to be essential for the reduction of carboxylic acids and <em>P. furiosus</em> is able to produce this gas. Chapter 4 describes the functions of several hydrogenases. It was demonstrated that previous hypotheses do not sustain, since particularly sulfhydrogenase has insufficient capacity to dispose off the reducing equivalents (reduced ferredoxin and NADPH), generated during starch fermentation, as dihydrogen. However, a membrane-bound hydrogenase was able to catalyze the disposal of catabolically generated reducing equivalents as dihydrogen. This membrane-bound hydrogenase was partially purified. The complex consists of 14 subunits and the N-terminal sequences of two subunits were determined.</p><p>Based on the results, four functions were proposed for the dihydrogen metabolism: 1) the generation of dihydrogen coupled to a proton pump, 2) the production of NADPH for biosynthetic purposes, 3) an additional route to generate NADPH, and 4) a safety valve to dispose off a surplus of reducing equivalents.</p><p>One of the functions of the, in chapter 4 mentioned, cytosolic hydrogenase is the regeneration of NADPH. This reaction is very interesting from a commercial point of view, since NADPH is an expensive cofactor applied in numerous biosynthetic reactions. The potential applications of this hydrogenase for the production and regeneration of NADPH are described in chapter 5.</p><p>The enzyme showed an operational stability of at least 37 days (hydrogen oxidation activity) and proved to be active even at lower temperatures (20<FONT FACE="Symbol">°</font>C).</p><p>One of the most important results of the NADPH production and regeneration experiments was that hydrogenase was not inhibited by high concentrations NADP <sup>+</SUP>and NADPH. Moreover, substrates and products present in the NADPH-dependent reduction of the model substrates 2-ketoglutarate, 2-pentanone, or butyraldehyde did not inhibit NADPH regeneration either. Furthermore, hydrogenase catalyzed the production of 100 mg/ml NADPD in a relative short period of time (4 hours). Finally, the NADPH-regeneration experiments resulted in 'total turnover' numbers of 500.</p><p>Chapters 2 and 3 described that <em>P. furiosus</em> was able to catalyze the reduction of carboxylic acids during growth. These experiments, however, did not provide indications by which mechanism the reduction was catalyzed. The research to elucidate this is described in chapter 6.</p><p>The most obvious route, the reduction of the acid to the aldehyde catalyzed by ferredoxin:aldehyde oxidoreductase, could not be demonstrated by the used electrochemical and enzymatic methods. Yet, it could be shown that the reduction was dependent on ATP, dihydrogen, and partially dependent on NADPH. Based on these results it was proposed that the reduction of carboxylic acids in <em>P. furiosus</em> proceeds by the activation of the acid with ATP to form an acyl-AMP intermediate and that the reduction of the acyl-AMP intermediate is NADPH-dependent. It is remarkable that NADPH was not essential, but that it could be replaced by an unknown protein factor, possibly ferredoxin.</p><p>The results described in this thesis showed that <em>P. furiosus</em> is well able to catalyze several reductive reactions. Carboxylic acids as well as NADP <sup>+</SUP>can be reduced and provide a potential alternative for chemical reductions. As yet, the production and regeneration of NADPH by <em>P. furiosus</em> hydrogenase seem to have the best chances to a successful commercial application.</p></font>
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