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Met enzymen
Berkel, W.J.H. van - \ 2011
Wageningen : Wageningen University - ISBN 9789085858935 - 28
enzymologie - enzymen - moleculaire interacties - enzymology - enzymes - molecular interactions
Characterization and redesign of galactonolactone dehydrogenase, a flavoprotein producing vitamin C
Leferink, N.G.H. - \ 2009
Wageningen University. Promotor(en): Sacco de Vries, co-promotor(en): Willem van Berkel. - [S.l.] : S.n. - ISBN 9789085853527 - 168
oxidoreductasen - arabidopsis thaliana - enzymologie - oxidoreductases - enzymology
Keywords: aldonolactone oxidoreductases, Arabidopsis thaliana, flavoprotein, galactonolactone dehydrogenase, molecular gatekeeper, oxidase, protein engineering, vanillyl-alcohol oxidase family, vitamin C

Redox enzymes are attractive biocatalysts because of their intrinsic (enantio-)selectivity and catalytic efficiency, which are often difficult to achieve by conventional chemical approaches. The discovery of new redox enzymes together with novel insights into their catalytic mechanism will increase the biocatalytic potential for application of these enzymes. Carbohydrate oxidases are valuable enzymes that can be applied in diagnostics and the food industry. Such enzymes often contain a flavin cofactor as redox-active group. Most carbohydrate oxidase are up to now isolated from fungi, but an extensive genome analysis revealed that also plants are a rich source of these enzymes. Carbohydrate oxidases are, for example, involved in maintenance of the plant cell wall, de protection against pathogens, and the production of vitamin C.
In this research the vitamin C producing enzyme galactonolactone dehydrogenase from the model plant Arabidopsis thaliana was studied. This enzyme is a so-called aldonolactone oxidoreductases that belongs to the vanillyl-alcohol oxidase family of flavoproteins. Most members of this family are oxidases with a covalently bound FAD cofactor. Galactonolactone dehydrogenase differs in some crucial properties from aldonolactone oxidoreductases from animals, yeasts and fungi. The plant enzyme contains a non-covalently bound FAD cofactor, has a different substrate specificity and hardly reacts with molecular oxygen. Several critical amino acid residues involved in cofactor and substrate binding were identified and the enzyme was redesigned into variants with altered substrate and electron acceptor specificities. One of the major results was the identification of a gatekeeper residue in galactonolactone dehydrogenase that prevents molecular oxygen from reacting with the flavin cofactor. Removal of this residue resulted in a catalytically competent galactonolactone oxidase that can efficiently react with oxygen.
The knowledge obtained with this research provides a firm basis for the design of suitable biocatalysts that can be used for the biotechnological production of vitamin C or related carbohydrates, as an alternative for the currently applied chemical methods.

Molecular characterization of hydrolytic enzymes from hyperthermophilic archaea
Voorhorst, W.G.B. - \ 1998
Agricultural University. Promotor(en): W.M. de Vos. - S.l. : Voorhorst - ISBN 9789054857969 - 136
iso-enyzmen - enzymologie - moleculaire genetica - translatie - eiwitsynthese - isoenzymes - enzymology - molecular genetics - translation - protein synthesis
<p>Hyperthermophiles are recently discovered microorganisms which are able to grow optimally above 85 °C. Most hyperthermophiles belong to the <em>Archaea,</em> the third domain of life. One of the main interests in hyperthermophiles to deepen the insight in the way their proteins are stabilized and how to apply this knowledge to improve the stability of biotechnologically relevant enzymes. In this thesis attention has been focused on hydrolytic enzymes from hyperthermophilic archaea to provide insight in the way these microorganisms stabilize their proteins and are able to perform catalysis around the normal boiling point of water as well as to functionally produce these enzymes in a mesophilic heterologous host. Additionally, the organization and expression of a number of genes in a hyperthermophilic archaeon were studied. Members of two different classes of hydrolytic enzymes, that represent key enzymes in the metabolism of hyperthermophilic archaea, have been characterized at the molecular level: (i) glycosyl hydrolases that are required for growth on beta-linked sugars, and (ii) serine proteases that are involved in the growth on proteins and peptides. The most extensively studied hyperthermophilic archaeon <em>Pyrococcus furiosus</em> was used as model organism for the work described in this thesis.</p><p>Chapter 1 gives a brief introduction into different aspects of hyperthermophilic archaea, including an overview of the metabolism of polymeric substrates and the hydrolytic enzymes involved, a listing of the mechanisms by which hydrolases and other enzymes from hyperthermophiles are stabilized, and the main characteristics of their molecular biology. The main part of the thesis deals with the <em>celB</em> locus (Fig. 1) of <em>P.furiosus</em> and serine proteases of <em>P.furiosus</em> and the related <em>Thermococcus stetteri.</em></p><p><CENTER><img src="/wda/abstracts/i2422_1.gif" width="494" height="120"/><br/><strong>Figure 1</strong> : Genetic and transcriptional organization of the <em>P.furiosus celB</em> locus.</CENTER><p>In chapter 2 the isolation and characterization of the extremely thermostable beta-glucosidase (half-life of 3 days at 100 °C) and its <em>celB</em> gene of <em>P.furiosus</em> is described. The transcriptional organization of the <em>celB</em> gene was analyzed and indicated a single transcriptional unit, which was sustained by Northern blot analysis (see below) (Fig. 1). The deduced amino sequence of the beta-glucosidase showed that it is a member of the glycosyl hydrolase family 1. The pyrococcal <em>celB</em> gene was overexpressed in the mesophilic host <em>Escherichia coli</em> using the <em>tac</em> promoter, which resulted in a high production level of beta-glucosidase (up to 20% of the total soluble proteins). The <em>P.furiosus</em> beta-glucosidase was produced in an active form by <em>E.coli</em> with kinetic and stability characteristics identical to that of the native pyrococcal enzyme. The production of the hyperthermostable beta-glucosidase in a mesophilic host allowed for a simple purification procedure consisting of a heat-treatment of the cell-extracts followed by a single column chromatography. The high production level of the beta-glucosidase in the genetically well-accessible host <em>E.coli</em> allowed for protein engineering to gain insight in structure-stability and function relations. Mutational analysis of the active site of the beta-glucosidase from <em>P.furiosus</em> showed that the mechanisms for catalysis near the boiling point of water does not differ from that used by homologous enzymes from the family 1 of glycosyl hydrolases optimally active at lower temperatures.</p><p>Over the last years, the potential of the extremely thermostable <em>P.furiosus</em> beta-glucosidase as a biocatalyst has been studied. Due to its broad substrate specificity and high chemical and thermal stability a wide variety of products could be in glucoconjugation and transglycosylation reactions in water and organic solvents at temperatures in between 75 to 95 °C (Fischer <em>et al.,</em> 1996; Trincone <em>et al.,</em> 1997). The beta-glucosidase from <em>P.furiosus</em> has also been analysed for its potential as biocatalyst in the production of beta-galacto-oligosaccharides from lactose (Jansen <em>et al.,</em> 1997). Furthermore, the <em>celB</em> gene has been developed into a genetic marker to study plant-bacterium interactions and in competition experiments with differently marked <em>Rhizobium</em> strains (Sessitsch <em>et al</em> ., 1996). The applicability of this system is exemplified by the development of the CelB Gene Marking Kit (FAO/IAEA).</p><p>Due to the high production level of the pyrococcal beta-glucosidase in <em>E.coli</em> and its simple purification procedure, sufficient pure protein has been generated to initiate crystalization experiments in collaboration with the group of Prof. G.E. Schulz (University of Freiburg) and resulted in crystals that diffract to 3.5 Ångstrom resolution (Schulz, 1997). Efforts are under way to elucidate a high resolution three-dimensional structure of beta-glucosidase from <em>P.furiosus</em> guided by the recently determined structure of the homologous <em>Sulfolobus solfataricus</em> beta-glycosidase LacS in conjunction with a mutational approach to improve the packaging of the pyrococcal crystals and resolve uncertain regions.</p><p>Analysis of the genomic region preceding the <em>celB</em> gene of <em>P.furiosus</em> , revealed the presence of two tandem genes, oppositely orientated of <em>celB</em> (Fig. 1). These have been designated <em>adhA</em> and <em>adhB</em> , since their predicted products showed high homology to short-chain and iron-containing NADP(H)-dependent alcohol dehydrogenases (ADHs), respectively. In chapter 3 these two distinct types of ADHs are studied. The AdhA of <em>P.furiosus</em> showed a high degree of conservation in its primary structure with bacterial and eucaryal short-chain ADHs, suggesting that a short-chain ADH was present in the last common ancestor. The AdhB is a member of the group of iron-containing ADHs that is characterized by the presence of four conserved histidine residues. The <em>adhA</em> and <em>adhB</em> genes were overexpressed in <em>E.coli</em> resulting in functional proteins.</p><p>AdhB showed NADP(H)-dependent activity with methanol as substrate, but this activity was rapidly lost preventing a detailed characterization. The purified AdhA showed a stable NADP(H)-dependent activity towards a broad range of primary and secondary alcohols. AdhA revealed an optimum activity for substrates with a carbon chain of 5 residues, with a preference for the oxidation of secondary over primary alcohols. A higher affinity for aldehydes than for alcohols was observed for AdhA and the pH optimum of this reaction is near the optimum pH for growth of <em>P.furiosus</em> . Therefore, it is most likely that the physiological role of AdhA is the reduction of the aldehyde to the corresponding alcohol thereby removing reduction equivalents and regenerating the cofactor. Given the localization of the <em>adhA</em> and <em>adhB</em> genes, in the vicinity of the <em>lamA</em> and <em>celB</em> genes, it tempting to speculate that AdhA and AdhB both have a function in the metabolism of beta-linked glucose polymers.</p><p>In chapter 4 the molecular characterization of an endo-beta-1,3-glucanase and its <em>lamA</em> gene from <em>P.furiosus</em> is described. The <em>lamA</em> gene was found to be located downstream of the tandem <em>adhA-adhB</em> genes within the <em>celB</em> locus (Fig. 1). The endo-beta-1,3-glucanase is the first archaeal member of the glycosyl hydrolases family 16 that is composed of endo-beta-1,3 and endo-beta-1,3-1,4-glucanases. The <em>lamA</em> gene, without the coding sequence for its N-terminal leader, was cloned behind the T7-promoter in <em>E.coli</em> . Using this expression system a functional and extremely stable endo-beta-1,3-glucanase was produced in <em>E.coli</em> up to 15 % of the total soluble protein. The purified endoglucanase showed the highest activity on the beta-1,3-glucose polymer laminarin, but has also activity on the beta-1,3-1,4-glucose polymers lichenan and beta-glucan. The pyrococcal endoglucanase showed optimal activity at pH 6-6.5 and the temperature for maximum activity was 100-105 °C, while at 100 °C it has a half-life of 19 h. Amino acid sequence alignment of the glycosyl hydrolases of family 16 showed two subgroups; one with endo-beta-1,3-1,4-glucanase activity and another with predominantly endo-beta-1,3-glucanase activity. The latter group is characterized by an additional methionine residue in the predicted active site. Removal of this methionine in the pyrococcal endo-beta-1,3-glucanase by protein engineering did not alter its substrate specificity, but only the catalytic activity.</p><p>It was found that <em>P.furiosus</em> was able to grown on laminarin and that the endoglucanase <em>in vitro</em> hydrolysed laminarin into oligomers and glucose. However, the hydrolysis proceeded more efficiently in combination with the beta-glucosidase of <em>P.furiosus</em> . These observations suggest a key role for the extracellular endoglucanase as well as the intracellular beta-glucosidase in the utilization of beta-1,3-linked glucose polymer laminarin.</p><p>Chapter 5 describes the regulation of transcription of the divergent <em>celB</em> gene and the <em>adhA-adhB-lamA</em> gene cluster in <em>P.furiosus</em> . Northern blot showed that the <em>adhA-adhB-lamA</em> gene cluster form a 2.8-kb operon, designated the <em>lamA</em> operon. This operon is flanked upstream by the <em>celB</em> gene in opposite orientation and downstream by the <em>birA</em> gene, with the <em>celB</em> gene being transcribed as monocistronic messengers (Fig. 1). The expression of the enzymes encoded by the <em>celB</em> gene and the <em>lamA</em> operon of <em>P.furiosus</em> was found to be largely dependent on the carbon source present and was highest when the pyrococcal cells were grown on the beta-linked glucose polymers cellobiose and laminarin. The <em>celB</em> gene and the divergently orientated <em>lamA</em> operon were found to be controlled at the transcriptional level. Moreover, the transcripts were co-regulated and induced by growth on beta-linked glucose polymers. The transcription initiation sites of the <em>celB</em> gene and the <em>lamA</em> operon were found to be separated by a relatively small intergenic spacer of 142 nucleotides that included the back-to-back promoters, that showed a high degree of conservation, including identical archaeal TATA-box sequences. Transcriptional analysis using an <em>in vitro</em> transcription system for <em>P.furiosus</em> revealed that both transcripts are initiated from their <em>in vivo</em> initiation site. However, the efficiency of transcription initiation was significantly lower than that of the <em>gdh</em> gene of <em>P.furiosus</em> , suggesting that a positive regulator may contribute to increase the efficiency of transcription of the divergent <em>celB</em> gene and <em>lamA</em> operon in <em>P.furiosus</em> . If so, this would be a bacterial type of regulation. A putative regulatory binding-site could be identified upstream of the promoters of the <em>celB</em> gene and <em>lamA</em> operon. These findings open the way use the cell-free transcription system of <em>P.furiosus</em> for the identification of the regulator that is likely to be involved in regulation of the divergently orientated genes of the <em>celB</em> locus. Alternatively, <em>in vivo</em> analysis of the transcriptional control of the <em>celB</em> locus may become feasible with the recent development of a transformation system in <em>P.furiosus</em> (Aagaard <em>et al.,</em> 1996).</p><p>A combination of the current knowledge and ideas of the utilization of beta-linked glucose polymers and the transcriptional regulation in the hyperthermophilic archaeon <em>P.furiosus</em> led to the following working model (Fig. 2). Large polymeric substrates containing beta-1,3-linked glucose residues, such as laminarin are depolymerized to smaller fragments by the extracellular endo-beta-1,3-glucanase (LamA). After uptake these fragments can be degraded by the intracellular beta-glucosidase activity of CelB. The complementary activity of these two hydrolases has been shown <em>in vitro</em> . The co-regulation of transcription of the genes encoding these hydrolases support their concerted action in polymere degradation.</p><p><CENTER><img src="/wda/abstracts/i2422_2.gif" width="302" height="410"/><br/><strong>Figure 2.</strong> : Model for the utilization of beta-linked glucose polymers, like laminarin. 1, the extracellular endo-beta-1,3-glucanase (LamA); 2, unknown uptake system for oligosaccharides; 3, beta-glucosidase (CelB); 4, ADP-dependent hexokinase.</CENTER><p>The two remaining experimental chapters deal with the characterization and molecular modelling of proteases involved in the growth on proteinaceous substrates. In chapter 6 the isolation and characterization of the hyperthermostable serine protease, pyrolysin, and its gene from <em>P.furiosus</em> are presented. The extracellular pyrolysin was found to be associated to the cell membrane of <em>P.furiosus</em> . The major purification step for pyrolysin was obtained via an autoincubation of the membrane fraction in 6 M urea at 95 °C, during which a 100-fold purification was obtained. The purification procedure resulted in two proteolytically activity fractions, HMW and LMW pyrolysin that were found to have identical NH <sub>2</sub> -termini and were glycosylated to a similar extent. Additionally, autoincubation of the HMW pyrolysin resulted in a proteolytically active fraction with the size of LMW pyrolysin. Together, these data indicate that the LMW pyrolysin is generated from the HMW pyrolysin by the autoproteolytic removal of its COOH-terminal part.</p><p>Via reversed genetics the <em>pls</em> gene, coding for the pyrolysin of <em>P.furiosus</em> , was cloned and characterized. The deduced pyrolysin amino acid sequence consists of 1398 residues and is synthesized as prepro-enzyme, with the mature part of 1249 residues that showed the highest homology with eucaryal tripeptidyl-peptidases, that form a distinct subgroup of the subtilisin-like serine proteases (also referred to as subtilases). The NH <sub>2</sub> -terminal catalytic domain (approximately 500 residues) showed considerable homology to subtilisin-like serine protease and contained a large insert of more than 150 residues between the aspartate and histidine active site residues. The COOH-terminal domain of pyrolysin together with the large insert in the catalytic domain contains almost all (29 of 32) of the possible <em>N</em> -glycosylation sites present in pyrolysin. Substrate specificity studies indicated that pyrolysin is a true endopeptidase with a rather broad substrate specificity and may autocatalytically remove its own propeptide.</p><p>The amino acid sequence homology in the catalytic domain of pyrolysin with other subtilases was sufficiently high to allow for homology modelling to gain insight in how the structure of the enzyme is stabilized. However, the identification of features as being important for protein stabilization based on only a single enzyme may lead to misinterpretations. Therefore, we screened a number of hyperthermophiles for the presence of subtilases using a PCR approach. Degenerated oligonucleotides were deduced based on the amino acid sequence of pyrolysin as well as on that surrounding the active site residues which are highly conserved among most of the subtilisin-like serine proteases. Chapter 7 describes the results of such a PCR approach that led to the identification of a DNA fragment in the genome of the extreme thermophilic archaeon <em>Thermococcus stetteri</em> , which could encode a serine protease, designated stetterlysin. The deduced sequence of stetterlysin showed high homology with pyrolysin and contained a similar sized insert between the aspartate and histidine active site residues. The high homology between the two subtilases, stetterlysin and pyrolysin, and subtilases with an identified three dimensional structure allowed for homology modelling.</p><p>Three-dimensional structure models for stetterlysin and pyrolysin were constructed based on the crystal structures of subtilases from mesophilic and thermophilic origin, respectively subtilisin BPN' and thermitase, and on the sequence alignment of the core residues of stetterlysin and pyrolysin with subtilisin and thermitase. The predicted model of subtilisin-S41 from psychrophilic origin was also included in the comparisons. The alignment and the predicted three-dimensional models were used to the analyze amino acid composition and structural features of the catalytic domain that could be related to thermostability. The higher thermostability of the subtilases, especially stetterlysin and pyrolysin, was found to be correlated with an increased number of residues involved in pairs and networks of charge-charge and aromatic-aromatic interactions. For stetterlysin and pyrolysin, most of the aromatic residues were located on the surface of the catalytic core and present in inserts within this domain, suggesting that for the overall structure of the proteases aromatic-aromatic interactions may have a even larger impact on the stability of the structure.</p><p>Analysis of the location of N-glycosylation sites in highly thermostable subtilases, including stetterlysin and pyrolysin, showed that most of these sites are located in surface loops. The modelling of the substrate binding region with known substrates was in good agreement with the observed broad substrate range for pyrolysin and the proposed autocatalytic activation (chapter 6).</p><p>The PCR approach with deduced oligonucleotides for subtilisin-like serine proteases also resulted in products with the expected size with DNA from the hyperthermophilic archaeon <em>Pyrodictium abyssi</em> and the hyperthermophilic bacterium <em>Fervidobacterium pennavorans</em> , that hybridized with a pyrolysin-derived probe and had the expected size. The PCR products were cloned and characterized and their deduced amino acid sequences showed significant homology with the catalytic domain of subtilases. However, unlike pyrolysin and stetterlysin, they did not contain the large inserts between the first two active site residues. Using this approach a <em>F.pennavorans</em> gene for a subtilisin-like serine has been isolated and is currently being characterized.</p><p>The hydrolases described in this thesis have been used as models to study different aspects of enzymes from hyperthermophiles, also referred to as thermozymes. Industry has screened hyperthermophiles for enzymes that can be applied as industrial biocatalysts to replace less stable homologous in current processes or to initiate new processes. The industrial use of thermozymes is hampered by the cost-ineffective fermentation properties of the hyperthermophilic organisms, in which they reside. Therefore, functional overproduction of thermozymes in heterologous hosts may have considerable impact on the development of these enzymes as biocatalyst. This thesis describes the functional overproduction of number of thermozymes including a beta-glucosidase an short-chain alcohol dehydrogenase and an endo-beta-1,3-glucanase. Moreover, this work has contributed to the insight hydrolytic enzymes from hyperthermophilic archaea in the relation between their structure-function and structure-stability.</p>
Physico - chemical stability of tomato products
Ouden, F.W.C. den - \ 1995
Agricultural University. Promotor(en): A.G.J. Voragen; T. van Vliet. - S.l. : Den Ouden - ISBN 9789054853985 - 113
vloeistofmechanica - reologie - visco-elasticiteit - celwanden - enzymen - enzymologie - fermentatie - voedselindustrie - voedseltechnologie - solanum lycopersicum - tomaten - fluid mechanics - rheology - viscoelasticity - cell walls - enzymes - enzymology - fermentation - food industry - food technology - tomatoes
<p>The effect of some physical processes and enzymatic hydrolysis on the physicochemical properties of tomato suspensions was studied.<p>Concentration degree has a large effect on the apparent viscosity and the storage modulus of suspensions after being diluted to a standardized water insoluble solids level. Besides decrease in average particle size, microscopic fracture of the cellulosic microfibrillar network during concentration are thought to be responsible for this phenomenon. By wet sieving it was shown that the bulk of the particles has a size between 45-180 μm. The tomato cell wall seems to be highly deformable. The 90- 180 μm wet sieve fraction had highest apparent viscosity and yield stress as well before as after homogenization.<p>Homogenization of tomato suspensions as well as of strawberry sauce led to an increase in the apparent viscosity and storage modulus, whereas that of apple sauce led to a decrease in the apparent viscosity. The difference in behaviour upon homogenization is due to a difference in fracture behaviour of the plant cells, which is probably a result of the cell wall structure, especially the microfibrillar cellulose structure.<p>Incubation of tomato suspensions with highly purified well specified polysaccharide degrading enzymes resulted in a decrease in rheological parameters. By homogenization the apparent viscosity increased to higher values compared to that of the non enzyme treated tomato suspension. The enzyme preparations gave rise to more serum separation. Hydrolysis of diluted hot break paste by pectin esterase from oranges or fungi resulted in a much higher yield stress, which is probably due to the formation of a calcium pectinate network. The apparent viscosity became only slightly higher. Serum viscosity increased initially, after which it decreased to about 50% of the original value.<p>The main physical problem of tomato suspensions is the formation of a serum layer on top of it. Several mechanisms are responsible: uniaxial compression of the weak particle network due the gravitational force and drainage of serum as a result of unevenness in the surface. The physical behaviour of tomato suspensions can be better understood by considering the tomato cell wall as a concentrated, composite gel consisting of cellulose microfibrils embedded in a "jelly" matrix of pectic and hemicellulosic substances.
Reactiewegen van (bio)chemische omzettingen beter doorgronden.
Voragen, A.G.J. - \ 1995
Voedingsmiddelentechnologie 28 (1995)7. - ISSN 0042-7934 - p. 66 - 67.
chemische samenstelling - chemie - enzymen - enzymologie - fermentatie - voedsel - voedselsamenstelling - voedingsmiddelen - voedingswaarde - eigenschappen - kwaliteit - chemical composition - chemistry - enzymes - enzymology - fermentation - food - food composition - foods - nutritive value - properties - quality
The genus Lolium : taxonomy and genetic resources
Loos, B.P. - \ 1994
Agricultural University. Promotor(en): Jos van der Maesen; Ronald van den Berg. - S.l. : Loos - ISBN 9789073771116 - 101
grassen - poaceae - lolium - genenbanken - genetische bronnen - germplasm - hulpbronnenbehoud - genetische bronnen van plantensoorten - taxonomie - plantkunde - iso-enyzmen - enzymologie - plantenanatomie - plantenmorfologie - grasses - gene banks - genetic resources - resource conservation - plant genetic resources - taxonomy - botany - isoenzymes - enzymology - plant anatomy - plant morphology
<p>Several aspects of variation within the genus <em>Lolium,</em> and more in detail within <em>Lolium perenne</em> (perennial ryegrass) have been highlighted. As the results are extensively discussed in each chapter, the general discussion is focused on two aspects of the research.<p><u>Speciation</u><br/>It is clear that the genus <em>Lolium is</em> a very variable genus. The variation within the species reduces the clarity of separation of the species. Stebbins (1956) found the differences between <em>Lolium</em> and <em>Festuca</em> not sufficient to justify two separate genera. He also states that the family of <em>Poaceae is</em> a phylogenetically derived family, and therefore of comparatively recent origin. Differences between species and between genera are still developing. In older families, intermediate forms or species have become extinct, therefore genera and species delimitations are clearer in these families, e.g. the <em>Papaveraceae</em> (Stebbins, 1956). Producing additional information besides morphological data, such as cytogenetic studies and biochemical studies, is of little use for these families as genera and species delimitations are easily made with simple morphological characters. In families of more recent origin, such as the <em>Poaceae,</em> as many characteristics as possible should be used to establish the relationships between the species. In Chapters 2, 3 and 4 several types of characters have been analyzed for the genus <em>Lolium. No</em> phylogenetic analysis was performed with these data, only phenetic analysis due to the nature of the characters used. Results indicate that all species, as mentioned in the general introduction, can be recognized, although species delimitations are not unambiguous. Only for <em>L. persicum</em> and <em>L. temulentum</em> the results indicate that these species could possibly be two varieties of one species. All species show diagnostic characters for one ore more of the different type of characters. Although the overlap of species is often unsatisfactory, joining of species would be even more artificial. As speciation is a continuous process it cannot be predicted at which point in time species are going to be sufficiently delimited.<p>Man has had large influence on the speciation within <em>Lolium.</em> This is illustrated by the three weedy species within the genus. <em>L. remotum is</em> known as a weed in flax (Hjelmqvist, 1950), <em>L. temulentum</em> and <em>L. persicum</em> are known as weeds in cereals (e.g. Dore, 1950). All three are mimicry weeds, the morphology of the seeds andlor the habit of the plant is similar to the crop in which it grows. Until a few decades back, these three species had a significant impact as weeds, but due to enhanced seed cleaning techniques the distribution area of these species has largely decreased (Hubbard, 1954).<p>Other examples of the influence of man on the genus <em>Lolium,</em> are the species <em>L. perenne</em> and <em>L. multiflorum.</em> Their distribution area has largely increased due to sowing by man. Scholz (1975) stated that man has had an enormous influence on the development of both species. According to Scholz (1975), this influence started no more than a few thousand years back, with the cutting and burning of forest for replacement by grassland for cattle, and the discovery of hay making. This has made it almost impossible to determine in which parts of the world both species are indigenous. Not only the distribution area of both species is influenced by man but also the phenotype. Selection changes the phenotype in favour of character states desired by man, such as increased yield. Tyler (1979) observed that after a period in which the standard of management is relaxed, natural phenotypes reoccur. This is confirmed with the results from Chapter 5: Dutch perennial ryegrass populations, collected after a period of more relaxed management, have a distinct phenotype compared to cultivars. Tyler (1979) also indicated that the differences between wild and cultivated forms are extremely blurred for <em>L. perenne.</em> This statement is confirmed by the results from Chapter 6: for allozymic variation, cultivars show absolutely no reduction in variation compared to natural populations. Ellenberg (1963) calls the type of plant as <em>L. perenne</em> semi-domesticated, as the crop is not harvested each year but only kept at an acceptable production level using reseeding. During each phase of their lifecycle, populations are exposed to selection forces. Leading to the situation that in grasslands cultivars are often mixed with plants that have been exposed to enviromnental selection for a number of years. This makes the distinction between natural and cultivated grassland extremely vague.<p>Chapter 5 illustrates, as management is the factor that optimizes the amount of genetic variation found within a location, the enormous influence man has had and still has on the amount of variation in phenotypes of <em>L. perenne.</em> Reduction of the influence of man would probably lead to the existence few differing perennial ryegrass phenotypes, and could in some areas even mean extinction of perennial ryegrass. In the Netherlands, foreland and salt marshes are the only original habitats for grassland (Bink et al., 1984). Although <em>L. perenne is</em> a species with much competitive ability, it would suffer from a large reduction of distribution area in case management of grasslands was totally abandoned. Because mainly under man-made conditions, e.g. fertilizing, treading, intensive grazing, drainage, <em>L. perenne</em> expresses this competitive nature.<p>For <em>L. rigidum</em> the influence of man is less strong. <em>L. rigidum is</em> used in some parts of the world (e.g. Australia) as a cultivated fodder crop, but in Europe this is not current. In Europe the fate of <em>L. rigidum</em> depends on the perspectives of <em>L. rigidum</em> as a fodder crop in dry areas or as a crossing parent in breeding programmes. Next to its presence in cultivation <em>L. rigidum is</em> well capable to maintain itself under less cultivated circumstances, this in contrast to, especially, <em>L. perenne.</em> Hartley (1956) states that <em>L. rigidum</em> originates from the Mediterranean region and that <em>L. perenne</em> and <em>L. multiflorum</em> originate from the Eurasian region. The ancestral species of the genus <em>Lolium is</em> supposed to have originated in the Mediterranean region (Malik, 1967). This would indicate that <em>L. rigidum</em> could be the wild form for both cultivated species. The relation between wild and cultivated is often confirmed by the reduction of genetic variation within the cultivated forms. Brown (1978) mentions two examples, based on allozyme variation, for which this assumption is valid. <em>Lycopersicon pimpinellifolium</em> has 61 % unique allelic forms compared to those in <em>L. esculentum.</em> Both species share 37% of the allelic variants and 2% is unique for <em>L. esculentum. Oryza perennis</em> has 47% unique peroxidase alleles, and 22% unique esterase alleles, compared to 0. <em>sativa.</em> Both species share 53 % and 78 % of the alleles respectively. The results from Chapter 3 do not indicate a reduction in allelic variation within <em>L. perenne</em> and <em>L. multiflorum,</em> compared to <em>L. rigidum.</em> This indicates that, if <em>L. perenne</em> and <em>L. multiflorum</em> indeed did arise from <em>L. rigidum,</em> this speciation is of recent origin. Phylogenetic relations between species cannot be determined on basis of these data.<p><em>L. loliaceum is</em> not known as a weed nor as a crop plant, it mainly grows under poor and maritime conditions. The influence of man on populations of this species is not large, therefore it is not likely that this species becomes extinct nor that its distribution area will suddenly increase. Phenotypic developments are expected to be gradual and slow.<br/>For <em>L. canariense</em> the same holds true as for <em>L. loliaceum,</em> it is not a crop nor a weed and grows under poor conditions, making it a stable and localized species.<p>The screening of the <em>Lolium</em> species for allozymic variation, added little to the species determination within the genus <em>Lolium.</em> The pattern of allozyme diversity could hardly be linked with taxonomic classification (Chapter 3); mainly because all allelic variants were common in each population screened. As pointed out in the discussion of Chapter 3, this maybe caused by the small number of enzyme systems screened. A question that can be asked is whether increase of the number of allozymes could lead to better results for genotypic screening. In Chapters 3 and 6 the calculated diversity statistics for the cross-breeding <em>Lolium</em> species were above the average for other wind- pollinated cross-breeding species (Hamrick & Godt, 1990). These statistics indicated that a larger proportion than average of the loci screened were polymorphic, and also that the average heterozygosity of the loci was far above the mean. Extension of the number of loci screened would therefore most likely result in finding monomorphic loci or less variable polymorphic loci and would not enhance the results. In literature, analyses of <em>L. perenne</em> populations for several other allozymes are reported. These allozymes are Glutamate- oxaloacetate-transaminase (GOT, Hayward & McAdam, 1977; Arcioni et al., 1988; Charmet et al., 1993), Isocitrate dehydrogenase (IDH: Lallemand et al., 1991; Charmet et al., 1993), Peroxidase (PRX: Charmet et al., 1993) and Superoxide dismutase (SOD: Charmet et al., 1993). All authors report results that confirm the expectation that higher number of allozymes screened do not improve the elucidation of speciation. Again, the within- population variation is too large compared to the betweenpopulation variation.<p>Another option would be to make use of molecular markers, e.g. restriction fragment length polymorphism (RFLP). Few reports are known for <em>Lolium</em> species, using these techniques. Darbyshire & Warwick (1992) report on the results for one <em>L. perenne</em> population, which was compared with several other grass populations classified in 26 <em>Festuca</em> species and the genera <em>Vulpia, Poa</em> and <em>Puccinella.</em> Eleven restriction endonucleases and twelve restriction fragments from chloroplast DNA of <em>Petunia hybrida</em> Vilm. were used in this analysis. In total 341 <em></em> bands were observed of which 108 (31.7%) <em></em> were polymorphic. Of these 108 bands, 34 <em></em> were detected in the <em>L. perenne</em> population. Only one plant was analyzed from each population. Chloroplast DNA variation in other <em>Lolium</em> species (Lehväslaiho et al., 1987 <em>;</em> Soreng et al., 1990) is <em></em> only reported for one <em>L. multiflorum</em> population, using five restrictionenzymes and direct end labelling. Again only one plant has been analyzed and compared with a large set of populations and genera mainly from the family <em>Poaceae. L. multiflorum</em> differs in 11 bands on a total of 144 shared bands with <em>Festuca pratensis.</em> Only one report is known (Wu et al., 1992) <em></em> on the between-population variation within <em>L. perenne</em> for RFLP's. Five cultivars of perennial ryegrass were screened, using 2 <em></em> restriction enzymes and 37 <em></em> probes from <em>Festuca pratensis.</em> Twenty-four (65 %) <em></em> of these probes hybridized, resulting in on average 69% <em></em> polymorphism between the five cultivars . On average 3.2 different banding patterns were observed for each restriction enzyme-probe combination. Again only one plant was analyzed for each population.<p>No reports on the between-species and the within-population variation are known for any of the <em>Lolium</em> species.<p>The results from Chapter 3 and Chapter 6 <em></em> indicate that substantial variation is found within populations of the cross-breeding <em>Lolium</em> species, which makes results based on only one plant per population unreliable (Wu et al, 1992). <em></em> It remains necessary to analyse a minimum number of plants for the crossbreeding <em>Lolium</em> species, unless an acceptable bulk sample can be taken. This would be desirable as otherwise the cost and time needed to analyse a population using molecular markers could be limiting. The danger of using a bulk sample would be that no differences between populations and even between species can be observed (as would be the case for a bulk sample when screening for allozyme variation). Screening of five enzyme systems resulted in a maximum of 10 bands observed <em>(</em> 13 <em>,</em> if the heterozygous bands were also counted), in case a plant was heterozygous (maximum variation) for each enzyme. This is a much better result as reported by Darbyshire & Warwick (1992), 34 <em></em> bands out of 132 <em></em> restriction enzyme-probe combinations. It is equal to the theoretical maximum number of bands reported by Wu et al. (1992), 96 <em></em> bands in case of heterozygosity at all 48 restriction enzyme-probe combinations. The preliminary conclusion would therefore be that RFLP analysis will not greatly enhance the distinction of crossbreeding <em>Lolium</em> species and populations.<p>For the inbreeding <em>Lolium</em> species the analysis of few plants is sufficient. The observed variation would probably increase compared to the observed allozyme diversity (Chapter <em>3:</em> fixation for four of the five enzymes), as the number of possible markers would greatly increase using RFLP's. The use of molecular markers for the screening of inbreeding <em>Lolium</em> populations would therefore be a valuable extension of the knowledge on these species.<p><u>Genetic resources: <em>in situ</em> conservation</u><br/>In the general introduction three research questions were mentioned, concerning the genetic resources of <em>L. perenne.</em> What are the answers to these questions after analyzing the results from four years of research? Firstly, the Dutch populations do form, morphologically, a distinguishable group of forms within the genetic variation for perennial ryegrass. This conclusion is based on the observation of morphological characters only, as the heritability of these characters is better determined than for agronomically important traits as winter hardiness, spring growth etc. The date of ear emergence is one of the most important characters, both agronomically and for the recognition of breeders rights. Results indicate that genetic variation for this character is substantial in the Netherlands: in natural surroundings, populations varying from very early till very late heading could be collected. It is expected that if the Dutch populations show this amount of genetic variation for morphological characters, results can be analogous for agronomical characters. The morphological variation found within the Dutch populations is not comparable with the variation found in the cultivars used in these trials. Although date of ear emergence indicates that some Dutch populations are heading as late as the cultivars, morphologically they are distinct. The Dutch populations are a.o. more prostrate growing and shorter at ear emergence, this could indicate that this phenotype is natural for <em>L. perenne</em> in the Netherlands. The fact that Dutch populations are morphologically clearly distinct from the cultivars and that there was also substantial variation observed between Dutch populations, indicates that <em>in situ</em> conservation is a realistic option for <em>L. perenne.</em> Weibull (1989) gives several advantages and disadvantages for the <em>in situ</em> conservation of forages. The advantages are: continued co-evolution of populations and the possibility to study the ecology of the species. It is also possible to make successive collections, and it avoids space and time consuming activities for storage en regeneration for genebanks. A combination of the genetic resources conservation objective with other objectives like nature conservation could be an option. This possibility is clearly illustrated with the present results, as all Dutch populations were collected in areas managed by nature conservation organisations.<p>Disadvantages are that it is difficult to determine how many sites, and which sites should be preserved to optimize genetic variation. Natural populations are vulnerable to external factors, such as human influences and extreme weather conditions. Also the costs of the maintenance of conservation sites maybe high, and access of breeders can be a problem in case of a combination with nature conservation objectives.<p>For the allozyme variation no differences between the Dutch populations and the cultivars were found. Allelic variants were very common in all populations, the cultivars showed much larger differences in allelic frequencies than the Dutch populations. <em>In situ</em> conservation would be very successful in retaining genetic diversity at the allozyme level. The data were not useful for selection of accessions for genebanks. Phillips et al. (1993) reported for <em>Avena sterilis</em> L. (inbreeder), the wild progenitor of <em>A. sativa</em> L., the possibility to separate populations in six different groups based on 23 loci. Selection of genebank accessions can be facilitated using these six groups, combined with morphological data.<p>Francisco-Ortega et al. (1992) observed for <em>Chamaecytisus proliferus</em> (L. fil.) Link a totally different pattern. Morphologically this species can be separated into seven subspecies, which are morphologically distinct and ecologically each occupy a distinct niche. Allozyme diversity shows no differentiation between these seven subspecies.<p>Just like in the genus Lolium, almost all allelic variants are common and widespread, and the within-population variation is very large. Also in this case allozyme data were considered of no use for the selection of genebank accessions.<p>Generally, the usefulness of screening for allozyme variation varies substantially. Compatibility behaviour and age of the genus/species are the major factors, explaining the value of this kind of data.
Characterization of an endochitinase able to rescue the carrot somatic embryo variant ts11
Jong, A.J. de - \ 1994
Agricultural University. Promotor(en): A. van Kammen; Sacco de Vries. - S.l. : De Jong - ISBN 9789054852483 - 137
somatische embryogenese - weefselkweek - embryokweek - planten - embryologie - groei - plantenontwikkeling - enzymen - enzymologie - fermentatie - somatic embryogenesis - tissue culture - embryo culture - plants - embryology - growth - plant development - enzymes - enzymology - fermentation
<p>Cultured carrot cells secrete proteins, many of which are glycosylated, into the culture medium. A correlation has been found between somatic embryogenesis and the presence or absence of some of these secreted proteins, and evidence has been obtained that one or more secreted glycoproteins are actually essential for somatic embryo formation. The starting point of the experiments described in this thesis was the temperature-sensitive carrot cell line ts11, originally identified on the basis of the temperature- sensitive arrest in the transition of globular to heart stage somatic embryos. The arrest in ts11 embryo development at the nonpermissive temperature could be lifted by addition of medium proteins, secreted by wildtype cells, to the culture medium. The major goal of the study presented in this thesis was to identify the secreted proteins, that were able to rescue the arrested ts11 embryos.<p>In chapter 1 a brief introduction in zygotic and somatic embryogenesis is presented, followed by an overview of what is currently known about the first essential steps of the development of the zygotic embryo and of the formation of embryogenic cells and somatic embryos in vitro. Based on these studies, it is discussed whether analogous cellular mechanisms control early zygotic embryogenesis and the formation of embryogenic cells in tissue culture.<p>In chapter 2 the experiments are described that demonstrate that is 11 embryos can be rescued by a single secreted protein of 32 kD. The amino acid sequences of two tryptic peptides of this protein shared homology with several plant endochitinases. Biochemical analysis showed that the 32-kD protein is an acidic endochitinase.<p>In chapter 3 the results of a search for putative products of endochitinase activity effective in ts l 1 rescue, are presented. A molecule produced by <em>Rhizobium,</em> the N-acetylglucosamine-containing lipo-oligosaccharide, NodR1v-V(Ac, C18:4), appeared to be effective in stimulating the formation of ts11 embryos with a similar efficiency as the 32-kD endochitinase.<p>In chapter 4 evidence is presented that a decreased amount of an otherwise fully functional endochitinase is closely correlated with the window of sensitivity of ts1 1 cells to addition of the 32-kD endochitinase. Morphological observations suggest that the original ts l 1 mutation is quite pleiotropic and does not only affect embryogenesis in this line.<p>In chapter 5 experiments are described to identify a 32-kD endochitinase cDNA. The deduced amino acid sequence of the isolated cDNAs was found to be nearly identical to the amino acid sequences of the 32-kD endochitinase-derived peptides. The EP3 cDNA sequences suggested that the 32-kD endochitinse is a class IV chitinase.<p>Finally, in chapter 6 the significance of chitinases and lipo-oligosaccharides for plant development in general is discussed.
Effect microbieel fytase in het voer op de opfokresultaten van gespeende biggen
Peet-Schwering, C. van der - \ 1993
Praktijkonderzoek varkenshouderij 7 (1993)2. - ISSN 1382-0346 - p. 10 - 12.
spenen - toevoegingen - voedersupplementen - enzymen - enzymologie - fermentatie - productiviteit - rentabiliteit - dierhouderij - weaning - additives - feed supplements - enzymes - enzymology - fermentation - productivity - profitability - animal husbandry
De vervanging van voederfosfaat door fytase leidt niet tot een verbetering van de opfokresultaten van gespeende biggen. Als het calciumgehalte verlaagd wordt, leidt fytase echter ook niet tot een verslechtering van de resultaten
Enzyme recovery using reversed micelles
Dekker, M. - \ 1990
Agricultural University. Promotor(en): K. van 't Riet; B.H. Bijsterbosch. - S.l. : Dekker - 109
fermentatie - voedselbiotechnologie - enzymen - enzymologie - immobilisatie - technieken - biotechnologie - chemische industrie - biochemie - biologie - scheiding - fractionering - zuiveren - fermentation - food biotechnology - enzymes - enzymology - immobilization - techniques - biotechnology - chemical industry - biochemistry - biology - separation - fractionation - purification
<p>The objective of this study was to develop a liquid-liquid extraction process for the recovery of extracellular enzymes. The potentials of reaching this goal by using reversed micelles in an organic solvent have been investigated.<p>Reversed micelles are aggregates of surfactant molecules containing an inner core of water molecules, dispersed in a continuous organic solvent medium. The considerable biotechnological potential of these systems is derived principally from the ability of the water droplets to dissolve enzymes without loss of activity. Enzymes can be transported from a bulk aqueous phase to a reversed micellar phase and visa versa.<p>The distribution coefficient of an enzyme between a reversed micellar and an aqueous phase depends on the interactions which are possible between the enzyme and the reversed micelle. When ionic surfactants are used, electrostatic interactions have been shown to be the most important ones. The distribution can therefore be controlled by adjusting pH and ionic strength. The optimum pH for transfer depends on the size and titration behaviour of the enzyme. The extraction to a reversed micellar phase therefore shows enzyme selectivity.<p>Using the possibility to vary the distribution coefficient a continuous forward and back extraction process has been developed (Chapter 2). In two mixer/settler units the enzyme α-amylase is concentrated using a recirculating reversed micellar phase of the cationic surfactant trioctylmethylammonium chloride and the cosurfactant octanol in isooctane.<p>During the forward extraction some inactivation of the enzyme occurs by a complexation between the enzyme and the surfactant in the aqueous phase (Chapter 3). The extraction process has been modelled in terms of mass transfer and inactivation of the enzyme in all phases. As predicted by the model the extraction efficiency can be optimized by reducing the concentration of enzyme in the first aqueous phase through increasing the distribution coefficient (by the addition of a nonionic surfactant to the reversed micellar phase) and by increasing the mass transfer rate during the forward extraction. The observed enzyme recovery values correlate quite well with the values predicted by the model.<p>An important parameter of the extractions is the mass transfer rate of the enzyme to and from the reversed micellar phase. During forward extraction the rate of mass transfer is controlled by diffusion in the aqueous phase. The back extraction rate, however, is governed by the interfacial process of coalescence of the reversed micelles with the bulk interface. This process is strongly dependent on the pH, probably due to interactions of the surfactant with charged groups on the enzyme (Chapter 4).<p>An alternative process for the recovery of the enzyme from the reversed micellar phase uses the temperature effect on the phase behaviour of the system (Chapter 5). By increasing the temperature some aqueous phase is expelled from the organic phase, enabling the enzyme to be recovered in this phase. This phenomenon was applied successfully in an extraction process with two centrifugal extractors.<p>The applicability of the process to fermentation broths has to be subjected to further investigation, but some established general features are discussed (Chapter 6). In conclusion it can be stated that reversed micellar extraction of enzymes is a selective process with an enormous potential to purify and concentrate proteins in one operation.<p>The study described in this thesis was performed in a partnership between the Departments of Food Science (Food and Bioprocess Engineering Group), Biochemistry and Physical and Colloid Chemistry. The project was financed by the Netherlands Technology Foundation (STW).
Die Verbesserung der Impraegnierbarkeit von Fichtenholz mittels chemischer und enzymatischer Vorbehandlung
Militz, H. - \ 1990
Agricultural University. Promotor(en): W. Pilnik; A.G.J. Voragen. - S.l. : Militz - 220
bosbouw - bomen - enzymen - hout - houtchemie - houteigenschappen - vloeistoffen (liquids) - gassen - permeabiliteit - organische verbindingen - houtverduurzaming - enzymologie - fermentatie - toedieningswijzen - picea abies - forestry - trees - enzymes - wood - wood chemistry - wood properties - liquids - gases - permeability - organic compounds - wood preservation - enzymology - fermentation - application methods
<p>Finely ground spruce ( <em>Picea abies</em> (L.) Karst. ) was incubated with different enzyme preparations. The enzyme concentration, incubation time, temperature and buffer concentration were varied. The nature and quantity of uronic acids and neutral sugars released from the cell walls were determined. The most effective enzyme preparations were shown to be those with a broad cellulolytic and hemicellulolytic spectrum of activity. Specific pectinases were found not to be particularly effective. Sapwood and heartwood were broken down in equal degrees. Enzymatic preliminary treatment of intact spruce improved the permeability of the timber. The extent of the improvement was dependent on the enzyme preparation applied, the enzyme concentration, the incubation time, the incubation temperature and the origin of the timber. Enzymatic preliminary treatment was more effective in sapwood than in heartwood. Scanning electron microscopy made it possible to demonstrate the decomposition of the torus and the margo of the bordered pits as well as the decomposition of the pits between tracheids and parenchyma. The compression strength and the modulus of elasticity were not reduced. Preliminary treatment of intact spruce with alkali, acids and chelating agents did not result in improved permeability.
Nieuwe technieken voor het meten van de verteerbaarheid van grondstoffen en mengvoeders voor varkens : in vitro methoden, de in vitro automaat = New techniques to measure the digestibility of feedstuffs and diets for pigs : in vitro methods, the in vitro automate
Meer, J.M. van der; Schraa, R. - \ 1989
Lelystad : IVVO (Rapport / IVVO no. 206) - 34
verteerbaarheid - enzymologie - experimenten - voer - in vitro - varkens - technieken - digestibility - enzymology - experiments - feeds - pigs - techniques
Voor de waardering van varkensvoeders, grondstoffen en mengvoeders is een in-vitro-methode ontwikkeld, die de in vivo schijnbare organische stofverteerbaarheid voorspelt. De methode is gebaseerd op een aantal opeenvolgende incubaties bij 40 graden Celsius met "handels" verteringsenzymen. Voor deze methode is tevens een automaat ontwikkeld, die de handelingen van de analist overneemt
Enzymatic degradation of plant cell walls and the use of in vitro procedure for evaluation
Bediye, S. ; Wever, G. ; Meer, J.M. van der - \ 1987
Lelystad : IVVO (Intern rapport / Instituut voor Veevoedingsonderzoek 217) - 19
biologische behandeling - enzymen - enzymologie - voer - fermentatie - biological treatment - enzymes - enzymology - feeds - fermentation
CAF : een door calcium geactiveerd protease in spierweefsel : een literatuuroverzicht en enige ervaringen met de bepaling van het enzym = CAF : a calcium activated protease of muscle tissue : a review and some experience with the enzyme assay
Garssen, G.J. ; Boxtel, H.L. van - \ 1986
Zeist : IVO "Schoonoord (I.V.O.-rapport B-284) - 31
dierlijke producten - enzymen - enzymologie - fermentatie - vlees - vleeswaren - spieren - malsheid - textuur - animal products - enzymes - enzymology - fermentation - meat - meat products - muscles - tenderness - texture
Enzymatische sterolbepaling
Essers, M.L. ; Frijns, L.M.H. ; Muuse, B.G. - \ 1983
Wageningen : RIKILT (Verslag / RIKILT 83.49) - 11
enzymologie - cholesterol - sterolen - digitonine - boter - enzymology - sterols - digitonin - butter
Momenteel wordt het sterolgehalte gravimetrisch bepaald met behulp van Digitonine (NEN 6350). Internationaal (IUPAC) wil men deze methode vervangen door een enzymatische methode omdat deze vooral geschikt is voor kleine hoeveelheden monster. In dit onderzoek werd nagegaan of deze methode in onze praktijk geschikt is. De enzymatische bepalingen werden uitgevoerd met behulp van de cholesterol enzymkit van Boehringer. Vergeleken werden de sterolgehalten verkregen met de digitonine en met de enzymatische methode in een aantal produkten (botervetten, margarines en halvarines).
Properties of hydrogenase from Megasphaera elsdenii
Dijk, C. van - \ 1980
Landbouwhogeschool Wageningen. Promotor(en): C. Veeger, co-promotor(en): S.G. Mayhew. - Wageningen : Van Dijk - 111
enzymen - enzymologie - fermentatie - isolatie - peptostreptococcus - synthese - enzymes - enzymology - fermentation - isolation - synthesis
This thesis is concerned with the purification and properties of hydrogenase from the obligate anaerobic rumen bacterium <em>Megasphaera elsdenii.</em> In chapter 1 the motives underlying this thesis, the physiological role of hydrogenase in some heterotrophs, including <em>Megasphaera elsdenii,</em> as well as a comparison of physico-chemical and kinetic properties of purified hydrogenase preparations from microorganisms, showing great variations in physiology and taxonomy, are given. The physiological role and the physico-chemical and kinetic properties of the hydrogenases from <em>Megasphaera elsdenii</em> and <em>Clostridium pasteurianum</em> show great similarities.<p/>In chapter 2 the procedure for the anaerobic purification of hydrogenase from <em>Megasphaera elsdenii</em> is given. Two activity bands are separated on DEAE-cellulose chromatography, of which one (fraction I; see Table 1 of this chapter) represents a different hydrogenase or a complex of the enzyme in fraction II. Kinetics with the purified enzyme of the hydrogen production activity at pH 8, with the electron donors flavodoxin hydroquinone, reduced ferredoxin and methyl viologen semiquinone show non-linear double reciprocal plots of the activity versus the electron donor concentration. Two kinetic models were developed, with an identical general rate equation for the hydrogen production activity, which describe a random mechanism for the reaction of the oxidized enzyme with a proton and a reduced electron donor. These models also indicate that the hydrogenase accepts the two electrons necessary for hydrogen production in two separate, independent steps. Hydrogen oxidation with methyl and benzyl viologen shows, in contrast to hydrogen production, Michaelis Menten kinetics. In chapter 3 effectors of the hydrogenase activity, such as salts (which have hydrophylic properties) Me <sub><font size="-1">2</font></sub> SO and ethylene glycol (which have hydrophobic properties), and oxidants (oxygen, ferricyanide, Cl <sub><font size="-1">2</font></sub> Ind, (bi)sulphite) are described. The data show that the more chaotropic the anion of the salt the greater the increase in activity, per mole salt used. Both Me <sub><font size="-1">2</font></sub> SO and ethylene glycol inhibit the hydrogen evolution activity, however, Me <sub><font size="-1">2</font></sub> SO stimulates the hydrogen oxidation activity, while ethylene glycol does not affect this activity. Careful oxidation of the reduced enzyme with (bi)sulphite or Cl <sub><font size="-1">2</font></sub> Ind irreversibly increases the activity of the enzyme by about 60%; in contrast, oxygen and ferricyanide inactivate the enzyme. This irriversibly oxidized enzyme, which shows identical kinetic properties to the reduced enzyme, is more resistant to the effects of oxygen, Me <sub><font size="-1">2</font></sub> SO and storage, than the reduced enzyme. Difficulties encountered in ascribing redox potentials to the observed EPR spectra of hydrogenase at several redox states, such as the effects of temperature on the Nernst equation, on the apparent pH and on the midpoint potentials of the redox species, are described.<p/>In chapter 4 the theoretical aspects are described to determine the hydrogen production activity spectrophotometrically in a series coupled redox reactions as function of the pH and redox potential, as well as the limitations of this method. The hydrogen production activity is determined from the decrease in concentration of the reduced electron donor, or increase in the concentration of the oxidized electron donor. The changes in concentration of the reduced or oxidized electron donor do not equal the amount of hydrogen produced but only represent a measure of the actual amount of hydrogen produced.<p/>In chapter 5 the method as described in chapter 4 is used to study the effects of pH and redox potential on the hydrogen production activity. The hydrogen production activity is strongly pH- and redox potential-dependent; the activity patterns observed as function of pH and redox potential are independent of the nature of the electron donor used and thus represent a property of the enzyme. If at a given pH the activities are considered to express the degree of reduction of the enzyme the dependence of the activity on the redox potential represents a n=2 type redox titration curve with an 'apparent midpoint potential, which corresponds with the potential of the hydrogen electrode at that pH. To explain the effects of redox potential, proton and electron donor concentration on the hydrogenase activity Model I of chapter 2 was slightly adapted without changing its general rate equation for the hydrogen production activity. This model also explains the 'loss' of electrons of the reduced enzyme in EPR spectroscopy as well as the formation of HD together with DD during H <sub><font size="-1">2</font></sub> -D <sub><font size="-1">2</font></sub> O exchange experiments.
A study on the NAD(P)+ transhydrogenase from Azotobacter vinelandii
Krul, J. - \ 1975
Landbouwhogeschool Wageningen. Promotor(en): C. Veeger. - Wageningen : [s.n.] - 101
azotobacter - pseudomonadaceae - enzymen - enzymologie - fermentatie - oxidoreductasen - enzymes - enzymology - fermentation - oxidoreductases
<em>Azotobacter vinelandii</em> transhydrogenase shows a pronounced polymerizing depolymerizing character (Chapter 3 and 5). Several factors seem to influence this phenomenon. With purified enzyme, obtained by a new purification method (Chapter 3), several parameters influencing the association-dissociation behaviour were investigated (Chapter 5).<p/>It was found that dissociation of transhydrogenase can be obtained at a minimum concentration of 1 μM of the substrate NADP <sup><font size="-1">+</font></SUP>. A reversal of this effect is obtained with NADPH depending on the initial concentration of NADP <sup><font size="-1">+</font></SUP>used. No clear effect of 2'-AMP on the structure is found in contrast to the results obtained with the <em>Pseudomonas</em> enzyme (Louie et al., 1972). Increase of the pH to about pH 9.5 results in a dissociation of the polymerized structures present in purified transhydrogenase as described by Middleditch at al. (1972). A very marked associating effect of divalent metal ions is found (Chapter 3 and 5). Large aggregates are formed, still catalytically active, that can be seen with the electron microscope and even under the phase contrast microscope (Chapter 5).<p/>The aggregates thus formed are not soluble, and addition of EDTA does not result in a clear reversal of the effects induced by the metal. Besides an associating effect divalent metal ions also cause some dissociation of the filamentous structures into rozettes, rings and cylinders with a perpendicular striping (Chapter 5).<p/>The effect on the highly polymerized structures of thiol reducing agents was investigated. A clear dissociating effect of β-mercaptoethanol was obtained while reduced lipoamide and dithiothreitol are much less effective (Chapter 5). Addition of ammonium sulphate to purified transhydrogenase results initially in dissociation followed by the formation of microcristalline structures (Chapter 5). As the effects of the reagents on the morphological structure are totally different, it must be concluded that these result from a combination of different types of binding (interaction). The dissociating effect of NADP <sup><font size="-1">+</font></SUP>at low concentration points to a strong effect of this nucleotide m the total structure of the enzyme as a very local interaction (at the catalytical side only) hardly can result in such a drastic change in appearance.<p/>As divalent metal ions cause a pronounced association this leads to the conclusion that probably charge neutralization of negative groups within the protein causes this phenomenon. On the other hand decrease of positive charges (upon increasing the pH) results in dissociation of the enzyme.<p/>The role of thiol reducing agents also remains obscure as only β-mercaptoethanol has a pronounced effect whereas a more powerfull reducing agent as dithiothreitol has much less effect. This probably must be attributed either to a difference in steric hindrance or the differences in hydrophobicity of these agents. The significance of the different association-dissociation steps for the catalytic mechanism is not clear at this time. In the catalytic reaction it is found that divalent metal ions have a pronounced effect on the kinetic patterns (Chapter 3).<p/>It is discussed that these effects must be due to modification of the enzyme structure rather than of the substrate structure.<p/>A remarkable inhibition by anions is also found in the reduction of NADP <sup><font size="-1">+</font></SUP>or S-NAD <sup><font size="-1">+</font></SUP>by NADH. From the kinetic inhibition picture obtained with phosphate and sulphate a pingpong mechanism might be favoured but the inhibition by nitrate points to the existance of a rapid ternary complex mechanism (Chapter 3).<p/>Spectral studies of Van den Broek et al. (1971) showed a pronounced binding of NADP <sup><font size="-1">+</font></SUP>to the oxidized enzyme and an absence of 2'-AMP binding. It is however found (Chapter 6) that the spectral effects of NADP <sup><font size="-1">+</font></SUP>and 2'-AMP strongly depend on the presence of phosphate. The fluorescence emission quenching by NADP <sup><font size="-1">+</font></SUP>is much stronger in the presence of phosphate than in the absence and concomitantly the dissociation constant becomes lower in the presence of phosphate than in the absence. From the effect on the fluorescence emission it can be derived that there are two binding sites for NADP <sup><font size="-1">+</font></SUP>. Also a more pronounced shift of the emission peak towards shorter wavelengths is observed in the presence of phosphate. No effects of 2'-AMP on the emission characteristics are found unless phosphate is present, while the quenching effect induced by phosphate. is reversed by 2'-AMP.<p/>By studying the absorption difference spectrum also binding of phosphate can be observed; addition of 2'-AMP reverses the spectral effects initially induced by phosphate (Chapter 6). The chemical reduction of the enzyme was reported to require two moles of reductant per mole of enzyme bound flavin (Van den Broek et al., 1971). Reexamination of these experiments show however that 1 mole of reductant suffices to get complete reduction of the enzyme bound flavin. (Chapter 6).<p/>An interesting effect of the ionic strength on the equilibrium constant of the transhydrogenase catalyzed reaction was found (Chapter 4). The shift of this constant in relation to the ionic strength and hence the chemical nature of the products and substrates involved is discussed. It is concluded that the folded form of the pyridine nucleotides is responsible for the slope as obtained in the log K vs.VI plot. The conformation of the pyridine nucleotides was studied with N.M.R. (Chapter 4), in the absence and presence of neutral salt.<p/>No definite conclusions can be drawn from the data obtained but both electrostatics interactions and folding do attribute to the proton shifts obtained.
Broeibestrijding met chemische middelen in voordroogkuil, in vers - en voorgedroogd gras
Schukking, S. ; Hengeveld, A.G. - \ 1972
Wageningen : [s.n.] (Mededelingen / Instituut voor bewaring en verwerking van landbouwprodukten no. 401, 415)
enzymen - enzymologie - fermentatie - hooi - kuilvoerbereiding - voedergrassen - enzymes - enzymology - fermentation - hay - silage making - fodder grasses
Occurrence and properties of bacterial pectate lyases
Rombouts, F.M. - \ 1972
Landbouwhogeschool Wageningen. Promotor(en): W. Pilnik. - Wageningen : Pudoc - ISBN 9789022004128 - 132
biochemie - plantkunde - enzymen - enzymologie - fermentatie - fruitgewassen - lyasen - microbiële fysiologie - micro-organismen - fysiologie - groenten - biochemistry - botany - enzymes - enzymology - fermentation - fruit crops - lyases - microbial physiology - microorganisms - physiology - vegetables
<p/>Some 100 pectolytic bacteria belonging to different genera and species, were obtained by isolation from vegetables and by screening of culture collections. The crude enzyme preparations of 19 of these strains were typed by mutual comparison. Differences in the composition of five commercial fungal 'pectinase' preparations were also studied. Purified endo pectate lyase of <em>Arthrobacter</em> which was studied in detail, appeared to attack pectate far 'less randomly', than endo pectate lyases of Bacillus polymyxa or <em>Pseudomonas.</em> The best substrates for pectate lyases were not pectates but 21 to 44% esterified pectins. A new method for the determination of the number average degree of polymerization of pectic substances was introduced. The literature on pectolytic enzymes was reviewed.
Methode voor het bepalen van de broeigevoeligheid in monsters voordroogkuil
Schukking, S. ; Hengeveld, A.G. - \ 1971
Wageningen : [s.n.] (Mededelingen / Instituut voor bewaring en verwerking van landbouwprodukten no. 379) - 14
rundvee - drogen - enzymen - enzymologie - fermentatie - kuilvoer - kuilvoerbereiding - voedergrassen - cattle - drying - enzymes - enzymology - fermentation - silage - silage making - fodder grasses
Verslag van de cursus "Enzymatische bepalingen in de levensmiddelenchemie", georganiseerd op 5 en 6 maart door de Firma Boehringer
Hessel - de Heer, J.C.M. - \ 1970
Zeist : I.V.O. (Rapport / Instituut voor Veeteeltkundig Onderzoek "Schoonoord" no. C 142) - 7
chemische samenstelling - enzymologie - voedselindustrie - voedseltechnologie - voedingsmiddelen - technieken - chemical composition - enzymology - food industry - food technology - foods - techniques
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