De diervoederhygiëne verordening als opmaat voor 'toezicht op controle' in de diervoederketen

Per 1 januari 2006 werd de diervoederhygiëne verordening EG Nr. 183/2005 van kracht. In deze verordening staan traceerbaarheid, aansprakelijkheid, risicobewust handelen en HACCP principes centraal. Doel van dit project was om praktijkkennis en ervaringen te gebruiken om de naleving en het draagvlak voor de diervoederhygiëne verordening gericht te kunnen bevorderen. De volgende onderzoeksvragen stonden centraal: voldoen bedrijven die GMP+-gecertificeerd zijn ook aan diervoederhygiëne verordening?; hoe is de situatie voor bedrijven die nu niet aan een kwaliteitssysteem meedoen?; welke knelpunten zijn er t.a.v. de diervoederhygiëne verordening?; waar moet rekening mee worden gehouden bij implementatie van de diervoederhygiëne verordening?; hoe kunnen bovengenoemde knelpunten opgelost worden?; welke acties kunnen / moeten LNV, VWA, PDV, Nevedi en anderen op zich nemen

Exploring the reductive capacity of Pyrococcus furiosus : the reduction of carboxylic acids and pyridine nucleotides

This Ph.D. project started in 1997 and its main goal was to obtain insight in the reductive capacity of the hyperthermophilic archaeon Pyrococcus furiosus . The research was focused on the biocatalytic reduction of carboxylic acids.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. furiosus is an anaerobic hyperthermophilic organism growing optimally at temperatures ranging from 90 to 100°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, P. furiosus is an archaeon and these organisms are non-pathogenic to men. These features and the fact that P. furiosus produced dihydrogen during growth make P. furiosus a very useful organism to study the reduction of acids.First, an inventory was made of which carboxylic acids can be reduced by P. furiosus to the corresponding aldehydes and subsequently to the alcohols (chapter 2). Both aliphatic and aromatic acids were tested and P. furiosus 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.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 P. furiosus cells and dihydrogen are essential to reduce carboxylic acids.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.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°C. The reduction rate, however, was optimal at pH 7.0, 10 mM substrate and 90°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.Dihydrogen showed to be essential for the reduction of carboxylic acids and P. furiosus 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.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.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.The enzyme showed an operational stability of at least 37 days (hydrogen oxidation activity) and proved to be active even at lower temperatures (20°C).One of the most important results of the NADPH production and regeneration experiments was that hydrogenase was not inhibited by high concentrations NADP +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.Chapters 2 and 3 described that P. furiosus 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.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 P. furiosus 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.The results described in this thesis showed that P. furiosus is well able to catalyze several reductive reactions. Carboxylic acids as well as NADP +can be reduced and provide a potential alternative for chemical reductions. As yet, the production and regeneration of NADPH by P. furiosus hydrogenase seem to have the best chances to a successful commercial application.</font

Waterberging en veehouderijen: dier- en plantgezondheid, voedselveiligheid en bedrijfsvoering, kennis uit wetenschap en praktijk

Waterberging of inundatie speelt een grote rol in de waterhuishouding van oppervlaktewater voor de veehouderij in Nederland. Waterberging kan voor de veehouderij effect hebben op plant- en diergezondheid, bedrijfsvoering en voedselveiligheid. Dit rapport beoogt enerzijds op basis van bestaande kennis een analyse te geven van de risico's van waterberging op bedrijfsvoering, dier- en plantziekten en voedselveiligheid in dierlijke productieketens. Hoofdstuk 2 geeft een literatuurstudie: effecten van waterberging op veehouderijen (via water en gras). Daarnaast zijn verschillende betrokken partijen zoals provincies, waterschappen, LTO en veehouders benaderd om de belangrijkste kennishiaten, knelpunten en kansen voor deze betrokkenen te benoemen (hoofdstuk 3 en 4). Tenslotte wordt een kort verslag gegeven van de discussie van het minisymposium "Kortdurende inundaties in natuur- en landbouwgebieden: kansen en bedreigingen" (hoofdstuk 5

Practical applications of hydrogenase I from Pyrococcus furiosus for NADPH generation and regeneration

The soluble hydrogenase I (H-2:NADP(+) oxidoreductase, EC 1.18.99.1) from the marine hyperthermophilic strain of the archaeon Pyrococcus furiosus was partially purified by anion-exchange chromatography. This P furiosus hydrogenase I preparation (PF H(2)ase I) has been used as biocatalyst in the enzymatic production and regeneration of beta-1,4-nicotinamide adenindinucleotide phosphate, reduced form (NADPH), utilizing cheap molecular hydrogen and forming protons as the only side-product. Any excess of dihydrogen can be removed easily. It could be demonstrated, that this hyperthermophilic hydrogenase exhibits a high stability under reaction conditions. Generation as well as regeneration of NADPH were performed in batch and repetitive batch experiments with recyclisation of the biocatalyst. In two repetitive batch-series 6.2 g l(-1) NADPH could be produced with a total turnover number (ttn: mol produced NADPH/mol consumed enzyme) of 10,000. Utilizing the thermophilic NADPH-dependent alcohol dehydrogenase from Thermoanaerobium spec. (ADH M) coupled to the PF H(2)ase I in situ NADPH-regenerating system, two prochiral model substrates, acetophenone and (2S)-hydroxy-1-phenyl-propanone (HPP), were quantitatively reduced to the corresponding (S)-alcohol and (1R,2S)-diol. An e.e. >99.5% and d.e. >98%, respectively, with total turnover numbers (ttn: mol product/mol consumed cofactor NADP(+)) of 100 and 160 could be reached. (C) 2003 Elsevier B.V. All rights reserved

Bioreduction of carboxylic acids by Pyrococcus furiosus in batch cultures

The reduction of aromatic and aliphatic (di)carboxylic acids to their corresponding aldehydes and alcohols by the hyperthermophilic organism Pyrococcus furiosus was investigated. The reduction was performed with P. furiosus cells growing in the presence of 1 mM acid with starch as a carbon and energy source at 90oC. The aromatic acids t-cinnamic and 3-phenylpropionic acid were reduced to their corresponding alcohols with the highest yields in the described batch cultures: 67 and 69%, respectively. The aliphatic acid reduced with the highest yield was hexanoic acid (yield: 38%). No aldehydes were detected during the reduction of acids, indicating that the reduction of aldehydes to alcohols is faster than the reduction of acids to aldehydes. Some aldehydes were both reduced to the corresponding alcohol and oxidized to the corresponding acid. Besides reduction of the unsaturated t-cinnamaldehyde to t-cinnamyl alcohol (63%), the double bond of t-cinnamaldehyde was also reduced by P. furiosus

De diervoederhygiëne verordening als opmaat voor 'toezicht op controle' in de diervoederketen

Per 1 januari 2006 werd de diervoederhygiëne verordening EG Nr. 183/2005 van kracht. In deze verordening staan traceerbaarheid, aansprakelijkheid, risicobewust handelen en HACCP principes centraal. Doel van dit project was om praktijkkennis en ervaringen te gebruiken om de naleving en het draagvlak voor de diervoederhygiëne verordening gericht te kunnen bevorderen. De volgende onderzoeksvragen stonden centraal: voldoen bedrijven die GMP+-gecertificeerd zijn ook aan diervoederhygiëne verordening?; hoe is de situatie voor bedrijven die nu niet aan een kwaliteitssysteem meedoen?; welke knelpunten zijn er t.a.v. de diervoederhygiëne verordening?; waar moet rekening mee worden gehouden bij implementatie van de diervoederhygiëne verordening?; hoe kunnen bovengenoemde knelpunten opgelost worden?; welke acties kunnen / moeten LNV, VWA, PDV, Nevedi en anderen op zich nemen

Enzymes of hydrogen metabolism in Pyrococcus furiosus

The genome of Pyrococcus furiosus contains the putative mbhABCDEFGHIJKLMN operon for a 14-subunit transmembrane complex associated with a Ni-Fe hydrogenase. Ten ORFs (mbhA-I and mbhM) encode hydrophobic, membrane-spanning subunits. Four ORFs (mbhJKL and mbhN) encode putative soluble proteins. Two of these correspond to the canonical small and large subunit of Ni-Fe hydrogenase, however, the small subunit can coordinate only a single iron-sulfur cluster, corresponding to the proximal [4Fe-4S] cubane. The structural genes for the small and the large subunits, mbhJ and mbhL, are separated in the genome by a third ORF, mbhK, encoding a protein of unknown function without Fe/S binding. The fourth ORF, mbhN, encodes a 2[4Fe-4S] protein. With P. furiosus soluble [4Fe-4S] ferredoxin as the electron donor the membranes produce H2, and this activity is retained in an extracted core complex of the mbh operon when solubilized and partially purified under mild conditions. The properties of this membrane-bound hydrogenase are unique. It is rather resistant to inhibition by carbon monoxide. It also exhibits an extremely high ratio of H2 evolution to H2 uptake activity compared with other hydrogenases. The activity is sensitive to inhibition by dicyclohexylcarbodiimide, an inhibitor of NADH dehydrogenase (complex I). EPR of the reduced core complex is characteristic for interacting iron-sulfur clusters with Em 0.33 V. The genome contains a second putative operon, mbxABCDFGHH'MJKLN, for a multisubunit transmembrane complex with strong homology to the mbh operon, however, with a highly unusual putative binding motif for the Ni-Fe-cluster in the large hydrogenase subunit. Kinetic studies of membrane-bound hydrogenase, soluble hydrogenase and sulfide dehydrogenase activities allow the formulation of a comprehensive working hypothesis of H2 metabolism in P. furiosus in terms of three pools of reducing equivalents (ferredoxin, NADPH, H2) connected by devices for transduction, transfer, recovery and safety-valving of energ

Enzymes of hydrogen metabolism in Pyrococcus furiosus

The genome of Pyrococcus furiosus contains the putative mbhABCDEFGHIJKLMN operon for a 14-subunit transmembrane complex associated with a Ni-Fe hydrogenase. Ten ORFs (mbhA-I and mbhM) encode hydrophobic, membrane-spanning subunits. Four ORFs (mbhJKL and mbhN) encode putative soluble proteins. Two of these correspond to the canonical small and large subunit of Ni-Fe hydrogenase, however, the small subunit can coordinate only a single iron-sulfur cluster, corresponding to the proximal [4Fe-4S] cubane. The structural genes for the small and the large subunits, mbhJ and mbhL, are separated in the genome by a third ORF, mbhK, encoding a protein of unknown function without Fe/S binding. The fourth ORF, mbhN, encodes a 2[4Fe-4S] protein. With P. furiosus soluble [4Fe-4S] ferredoxin as the electron donor the membranes produce H2, and this activity is retained in an extracted core complex of the mbh operon when solubilized and partially purified under mild conditions. The properties of this membrane-bound hydrogenase are unique. It is rather resistant to inhibition by carbon monoxide. It also exhibits an extremely high ratio of H2 evolution to H2 uptake activity compared with other hydrogenases. The activity is sensitive to inhibition by dicyclohexylcarbodiimide, an inhibitor of NADH dehydrogenase (complex I). EPR of the reduced core complex is characteristic for interacting iron-sulfur clusters with Em 0.33 V. The genome contains a second putative operon, mbxABCDFGHH'MJKLN, for a multisubunit transmembrane complex with strong homology to the mbh operon, however, with a highly unusual putative binding motif for the Ni-Fe-cluster in the large hydrogenase subunit. Kinetic studies of membrane-bound hydrogenase, soluble hydrogenase and sulfide dehydrogenase activities allow the formulation of a comprehensive working hypothesis of H2 metabolism in P. furiosus in terms of three pools of reducing equivalents (ferredoxin, NADPH, H2) connected by devices for transduction, transfer, recovery and safety-valving of energ