Previous studies on inhibition of the Escherichia coli glycerol kinase enzyme have suggested that subunit-subunit or domain bridging interactions form part of the network in communicating ligand binding to inhibition. In this study, five amino acids were identified to be in close proximity to an Arg369 residue which is a domain bridging residue. Three of the amino acid residues (Q37, Y39 and Q104) are in domain I of the enzyme subunit, while the other two (M308 and Q314) are in domain II of the enzyme subunit. To evaluate the importance of each domain bridging residue in IIAGlc inhibition, alanine substitutions were made of the residues, and the kinetic properties characterized with respect to IIAGlc inhibition. Kinetic parameters obtained for each variant glycerol kinase enzyme was compared to values obtained for the Wild Type enzyme to assess the importance of the amino acid residue in IIAGlc inhibition. The effects of the substitutions on FBP inhibition as well as catalysis of the enzyme were also analyzed by obtaining kinetic parameters for each of the variant enzymes. The results from this study indicate that the domain I bridging interactions with Arg369 are important in IIAGlc regulation of the E. coli glycerol kinase enzyme. The domain II bridging interactions appear to be unimportant in regulating IIAGlc inhibition. Two of the domain I bridging residues studied were also found to be important in FBP inhibition. These results indicate that some the domain bridging residues seen to be involved in IIAGlc regulation also appear to be involved in FBP regulation. In catalysis, with the exception of Q314, the rest of the domain I and II bridging residues appear to be important for substrate binding and/or catalysis.

Plants use several strategies to defend themselves against microbial pathogens. These include basal resistance, which is induced in response to pathogen encoded effector proteins, and resistance (R) protein-mediated resistance that is activated upon direct or indirect recognition of pathogen encoded avirulence protein(s). The activation of Rmediated signaling is often associated with generation of a signal, which, upon its translocation to the distal uninfected parts, confers broad-spectrum immunity against related or unrelated pathogens. This phenomenon known as systemic acquired resistance (SAR) is one of the well-established forms of induced defense response. However, the molecular mechanism underlying SAR remains largely unknown. Induction of plant defense is often associated with a fitness cost, likely because it involves reprogramming of the energy-providing metabolic pathways. Glycerol metabolism is one such pathway that feeds into primary metabolism, including lipid biosynthesis. In this study, I evaluated the role of glycerol-3-phosphate (G3P) in host-pathogen interaction. Inoculation with the hemibiotrophic fungal pathogen Colletotrichum higginsianum led to increased accumulation of G3P in wild-type plants. Mutants impaired in biosynthesis of G3P showed enhanced susceptibility, suggesting a correlation between G3P levels and basal defense. Conversely, increased biosynthesis of G3P correlated with enhanced resistance. The Arabidopsis genome encodes one copy of glycerol kinase (GK), which catalyzes phosphorylation of glycerol to G3P, and five copies of G3P dehydrogenase (G3Pdh), which catalyze reduction of dihydroxyacetone phosphate to G3P. Analysis of plants mutated in various G3Pdh's showed that plastidal lipid biosynthesis was only dependent on the GLY1 isoform but the pathogen induced G3P pool required the function of GLY1 and two other G3Pdh isoforms. Interestingly, compromised G3P biosynthesis in GK and G3Pdh mutants also compromised SAR, which was restored when G3P was provided exogenously. Detailed biochemical analysis showed that G3P was transported to distal tissues and that this process was dependent on a lipid transfer protein, DIR1. Together, these results show that G3P plays an important role in both basal- and induced-defense responses.

Les besoins actuels en produits de fertilisation et notamment pour le traitement des carences chez le végétal sont en constante augmentation. Cependant, de plus en plus soucieuse de l’environnement, les industries productrices de ces compléments alimentaires souhaitent proposées des solutions de traitements plus durables tant au niveau de leur production que de leur devenir une fois épandue dans la parcelle. Par ailleurs, il a été établi que les cellules végétales peuvent être considérées comme de vraies usines à molécules. Les hydroxyacides en C3 fabriqués par la machinerie cellulaire présentent par exemple de nombreuses propriétés de chélation et de transport. La présence de fonction hydroxyles et carboxyliques leur confèrent en effet la capacité de pouvoir enserrer des métaux et de permettre leur déplacement entre tous les compartiments des cellules, mais aussi entre les différents organes de la plante. Par ailleurs, la présence de ces mêmes molécules au sein des réactions biologiques (respiration, photosynthèse) font d’elles des métabolites assimilables et donc potentiellement dégradables. Les travaux de cette thèse ont donc eu pour objectif de mettre en place une catalyse chimio-enzymatique afin de produire les molécules les plus semblables aux hydroxyacides en C3 végétaux. Dans cette démarche de biomimétisme, nous avons mis au point un protocole de fabrication d’hydroxyacides à partir de glycérol et de carbonate de glycérol dans des conditions similaires au milieu vivant : dans l’eau, à des températures faibles et à pression atmosphérique. Nous avons ainsi réussi à synthétiser des hydroxyacides tel que l’acide glycérique, le 2-oxo1,3-dioxolan-4-carboxylique et l’hémiacétal du carbonate de glycérol. Les travaux de thèse ont également permis de mettre au point une méthode analytique complète pour la visualisation et la quantification de la majorité des hydroxyacides en C3 élaborés selon ce protocole opératoire. Cette méthode regroupe des techniques de caractérisations globales telles que l’Infra-rouge, l’HPLC, l’HPIC, et des analyses plus fines comme la LCMS de haute résolution et la RMN 1D et 2D. Nous avons par ailleurs optimisé la synthèse des hydroxyacides en C3 à partir du glycérol et du carbonate de glycérol grâce à la mise en place d’un plan d’expérience. Les molécules fabriquées présenteraient des propriétés de complexants susceptibles d’être utilisés en agronomie dans le but de remplacer les chélatants actuels issues de réactions plus polluantes de la pétrochimie et générant des substances nocives pour la santé des sols agricoles une fois appliqués. / The current need for fertilization products, and particulary for the treatment of plant deficiencies, is constantly increasing. However, more and more environmentally concerned, industries producing these food complements aim at finding more sustainable treatment solutions both in preparing these substances and in controlling their degradation once on crops. Moreover, it is established that plant cells can be considered as true molecular factories. For example, the C3 hydroxy acids produced by the cellular machinery have many chelating and transport properties. The presence of hydroxyl and carboxylic functions give them the ability to enclose the metals and to allow their displacement through all the compartments of the cells and also through the different organs of the plant. Moreover, as they are involved in biological reactions (respiration, photosynthesis), these molecules can potentially be used in cellular metabolism or degraded. The work presented herein has been designed with the aim of producing the most similar hydroxy C 3 –acids’s plant molecules by establishing a chemo-enzymatic catalysis. Inspired by biological and chemical natural processes, we have developed a protocol for manufacturing hydroxy acids from glycerol and glycerol carbonate under natural conditions ie in water, at low temperature and at atmospheric pressure. We have also produced hydroxy acids such as glyceric acid, 2-oxo1,3-dioxolan-4-carboxylic acid and glycerol carbonate hemiacetal. The thesis also allowed us to develop a complete analytical method for the observation and the quantification of the majority of C3 hydroxy acids, produced according to our operating protocol. This method combines global characterization techniques such as HPLC, HPIC and more complex analyses such as HR-LCMS and 1D and 2D NMR. We have also optimized the synthesis of C3 hydroxyacids from glycerol and glycerol carbonate through the implementation of an experimental design. Our bioinspired molecules are potential complexing agents that could be used in agronomy, in order to replace the current chelating molecules obtained from petrochemistry, known for having harmful impact on agricultural soils.

Aspectos fundamentais das reações de eletrooxidação do glicerol e álcoois similares em ouro foram estudados por meio da comparação com a eletrooxidação de álcoois similares: 1-propanol, 2-propanol, propano-1,2-diol e propano-1,3-diol, além de sorbitol e glicose. Testes eletroquímicos foram feitos em soluções ácida, neutra e alcalina. Também foram realizados testes em meio alcalino com os possíveis subprodutos de oxidação do glicerol: dihidroxiacetona, gliceraldeído, glicerato, hidroxipiruvato, mesoxalato, tartronato, oxalato e formato. Por fim, os produtos de reação foram analisados por FTIR in situ e HPLC. Conforme esperado, o ouro foi praticamente inativo para todos os álcoois nos meios neutro e ácido, e muito ativo no meio alcalino. Entretanto, os dados de RDE mostraram que a formação de alcóxido não é a responsável pela atividade, como era proposto até então. Comparando a eletrooxidação do glicerol com álcoois similares, foi possível notar que a presença de hidroxilas vizinhas na molécula é a propriedade mais importante para render altas densidades de corrente (para a eletrooxidação de álcoois em geral) sobre ouro. Ela facilita a quebra das ligações C-C favorecendo a formação de produtos mais oxidados, conforme observado por FTIR e HPLC. As medidas de FTIR in situ também mostraram que pode haver neutralização e até acidificação nas proximidades do eletrodo em meio alcalino (se a concentração de base não for suficientemente alta), devido à formação de subprodutos ácidos, o que pode levar a uma mudança no mecanismo da reação. Medidas de RDE indicam que é possível controlar a seletividade dos produtos de eletrooxidação de glicerol e outros álcoois sobre ouro pela convecção artificial, entretanto, o controle pelo potencial ou concentração são limitados, conforme constatado pela análise de produtos. Os testes realizados com os subprodutos e análise de produtos permitiram refinar a rota reacional de eletrooxidação do glicerol. / Fundamental aspects of glycerol and similar alcohols electrooxidation reactions on gold were studied by comparing with similar alcohols: 1-propanol, 2-propanol, propane-1,2-diol and propane-1,3-diol, and also sorbitol and glucose. Electrochemical measurements were performed in acidic, neutral and alkaline solutions. Moreover, CV tests were performed in alkaline environment with the main possible glycerol oxidation byproducts: dihydroxyacetone, gyceraldehyde, glycerate, hydroxypyruvate, mesoxalate, tartronate, oxalate and formate. Finally, the reaction products were analyzed by in situ FTIR and HPLC. As expected, gold was practically inactive for all alcohols in acidic and neutral conditions, but highly active in alkaline medium. However, RDE data showed that the alkoxide formation is not the responsible for the high activity, as proposed until now. By comparing the glycerol electrooxidation with the other alcohols, it was possible note that the vicinal hydroxyl groups is the key property to yield the high current densities (for alcohols electrooxidation in a general way) seen on gold. It favors the C-C bonding break, that leads to more oxidized products, as seen by FTIR and HPLC data. In addition, FTIR measurements showed that neutralization and even acidification can occur near to the electrode in alkaline medium (if the base concentration is not high enough), due to the formation of acidic byproducts that can also lead to different reaction mechanism. RDE experiments indicated that it is possible to control the selectivity of glycerol and other alcohols electrooxidation products on gold by artificial convection, but control by the potential or concentration are limited, as shown by products analysis. The CV tests with byproducts and products analysis allowed to refine the glycerol electrooxidation reaction pathways.

Il existe une forte demande de l'industrie pour des technologies permettant de réduire la teneur en alcool des vins. Bien que des levures à faible rendement alcool aient été développées par ingénierie génétique, les approches non-OGM sont aujourd'hui largement privilégiées. Nous avons mis en œuvre et comparé différentes stratégies d'évolution adaptative, à partir d'une souche œnologique commerciale, afin de réorienter le flux carboné vers la formation de glycérol aux dépens de l'éthanol. Après 200 générations en conditions de stress salin, nous avons obtenu des souches évoluées présentant une augmentation de la production de glycérol de 50 à 70 %, capables de diminuer de 0,45 à 0,80 % (v/v) la teneur en alcool de vins naturels ou synthétiques. Cette réorientation s'accompagne d'une accumulation de succinate et de 2,3-butanediol et d'une réduction de la vitesse fermentaire. Les mutants ont une survie accrue en conditions de stress salin et carence en glucose. Afin d'identifier les mécanismes sous-jacents, nous avons réalisé des analyses du transcriptome, du métabolome (exo- et endo-) et du génome des souches évoluées. Nous avons montré que le phénotype des mutants n'est pas dû à une dérégulation ou à des mutations des gènes de la voie de synthèse du glycérol mais à de larges modifications du métabolisme carboné, énergétique et redox. Le génome des souches évoluées présente des pertes d'hétérozygotie qui pourraient contribuer au phénotype observé. L'étude génétique d'une souche évoluée montre une origine multigénique des traits métaboliques. Une analyse de cartographie de QTL en utilisant une approche « bulk sequencing » a été initiée pour identifier les mutations impliquées dans le caractère fort glycérol/faible éthanol. Ces travaux ont ainsi permis de développer et caractériser des souches œnologiques faibles productrices d'alcool et de fournir un cadre pour l'identification des bases moléculaires impliquées.Mots-clefs : S. cerevisiae, fermentation œnologique, évolution adaptative, éthanol, glycérol, transcriptome, métabolome, génome. / There is a strong demand from the industry for technologies to reduce the alcohol content of wine. Although low-alcohol yield yeasts have been developed by genetic engineering, GMO-free approaches are now widely preferred. We have implemented and compared different strategies for adaptive evolution to redirect the carbon flux towards glycerol formation at the expense of ethanol. After 200 generations salt stress conditions, we obtained evolved strains with glycerol production increased by 50 to 70%, able to decrease from 0.45 to 0.80% (v/v) the alcohol content of natural or synthetic wines. This shift is accompanied by an accumulation of succinate and 2,3-butanediol and a reduced fermentation rate. Mutants also exhibit a better survival under salt stress and glucose restriction conditions. To identify the underlying mechanisms, we analysed the transcriptome, metabolome (endo- and exo-) and genome of the evolved strains. We showed that the evolved phenotype is not due to deregulation or mutations of genes of involved in the glycerol synthesis pathway but to major changes in carbon, energy and redox metabolism. The genome of the evolved strains revealed loss of heterozygosity which could contribute to the observed phenotype. The genetic study of an evolved strain shows that the metabolic traits are under multigenic control. A QTL mapping analysis using a "bulk sequencing" approach was initiated to identify mutations involved in the high glycerol/low ethanol trait. This work has enabled the development and characterization of low alcohol wine yeast strains and has provided a framework for the identification of the underlying molecular bases.Key-words: S. cerevisiae, wine fermentation, adaptative evolution, ethanol, glycerol, transcriptome, metabolome, genome.

L'augmentation de la production mondiale de glycérine, en raison de la croissance dans l'utilisation du biodiesel, et la tendance à remplacer les plastifiants dérivés de l'anhydride phtalique dans l'industrie du PVC, ont conduit à la recherche de la plastification du PVC avec des esters de glycérol. Cette étude a utilisé une méthodologie pour la conception du produit, avec une approche du troisième paradigme du génie chimique. Plusieurs esters de glycérol ont été produits avec neuf acides carboxyliques, et l'efficacité et la performance comme plastifiant du PVC ont été évaluées.L'évaluation expérimentale comprend la détermination de la capacité à former des films et six tests de performance utilisés dans l'industrie. La modélisation moléculaire utilise les interactions binaires entre une molécule de plastifiant et une molécule modèle de PVC. Avec les résultats expérimentaux de la performance des films avec le PVC, et en appliquant des méthodes d'évaluation multicritère pour la sélection d'alternatives, trois modèles ont été développés pour la sélection des plastifiants pour des applications générales et une utilisation médicale. Enfin, nous avons étudié expérimentalement la production en laboratoire de deux esters, qui ont montré une performance appropriée selon l'évaluation multicritère. Deux modèles on été construits pour simuler la production, et en utilisant une optimisation multi-objectif des modèles et un outil d'analyse multi-critères, nous avons établi les conditions dans lesquelles une étude de changement d'échelle devrait être réalisée pour une production industrielle. / Phtalates are the most important plasticizers used in the polymer industry. However, their fossil origin and results of recent test have showed a potential negative effect on human health, have encouraged polymer industry to demand free phthalate plasticizers. At the same time, biodiesel industry produces a glut of glycerol, leading the scientific community to seek new applications for this substance. A methodology for the evaluation of new free-phthalate plasticizers for PVC applied to a new type of plasticizers, esters of glycerol, is proposed. This thesis presents an overview of the performance of eight esters derived from glycerol as new plasticizers for poly(vinyl chloride) - PVC and an outline of its compatibility with the polymer based in a method to predict the plasticization effect of glycerol esters, constructed with molecular modeling.Having several alternatives of new PVC-plasticizers, three multicriteria methods were used to construct models to select the best alternatives: multiattribute utility theory - MAUT, Measuring Attractiveness by a Categorical Based Evaluation Technique - Macbeth and Rough sets. With these methods the preferences of a group of experts was modeled.In order to establish the conditions in which this new type of plasticizers must be produced at industrial scale, a model of the process is presented. A parametric identification was done. Also, a multicriteria optimization, in Pareto’s sense, was performed using an evolutionary algorithm. Besides, a decision aid tool based on two different functions to minimize costs and maximize productivity, respectively, was conducted with the technique Macbeth.

No description available.

Des nouvelles voies de synthèse sont proposées pour la préparation des hydroxyuréthanes et polyhydroxyuréthanes glycériliques, sans isocyanate, à partir de composés biosourcés fournisseurs de groupements cyclocarbonates hautement réactifs vis-à-vis des amines, diamines, polyamines. Les fournisseurs de groupement cyclocarbonate sont le carbonate de glycérol et le nouveau synthoon bifonctionnel l’acrylate de carbonate de glycérol. Des hydroxyuréthanes ou polyhydroxyuréthanes glycéryliques polyoxygénés et polyazotés ont été ainsi élaborés via ces nouveaux intermédiaires cyclocarbonates glycériliques en substitution des carbonates cycliques d’origine fossile comme les carbonates d’alkylènes. La stratégie d’élaboration des polyhydroxyuréthanes glycériliques à haut poids moléculaire s’appuie sur la réactivité séquencée de l’acrylate de carbonate de glycérol : réaction d’Aza-Michaël sur le groupement éthylénique de l’acrylate et réaction d’aminolyse par ouverture de cycles carbonates à 5- chaînons. Selon la nature des polyamines, on prépare des polymères de poids moléculaire supérieur à 40000 g/mol, d’aspects gel, mousse ou résine selon que les pontages linéaires ou interchaînes prédominent. Des réseaux chimiques intramoléculaires sont constitués par l’établissement des liaisons hydrogène entre les sites O-H ou N-H et l’oxygène du groupement carbamoyle du squelette glycérilique avec comme conséquence la réduction de la susceptibilité du groupe carbamoyle à l’hydrolyse et l’amélioration des propriétés de résistance chimique et mécanique des polyhydroxyuréthanes glycériliques. / New synthesis routes have been developed for the preparation of glycerilic hydroxyurethanes and polyhydroxyurethanes without the use of isocyanates. They were prepared from biosourced cyclocarbonates, highly reactive towards amines, diamines and polyamines. The providers of cyclocarbonate function are glycerol carbonate and glycerol acrylate carbonate which is a bifunctional reactive. Glycerilic hydroxyurethanes and polyhydroxyurethanes with high oxygenated and nitrogenated potential were then designed via these new glycerilic cyclocarbonates’ intermediates, substitutes of fossil cyclic carbonates such as ethylene or propylene carbonates. The strategy development of high molecular weights glycerilic polyhydroxyurethanes is based on the sequenced reactivity of the glycerol acrylate carbonate: Aza-Michaël reaction on the ethylenic function of the acrylate and aminolysis reaction of the 5-membered cyclic carbonate function. According to the nature of the amine, polyhydroxyurethanes up to 40,000 g/mol were obtained. They can be obtained as foams, gels, resins, depending on the predominance of linear or cross-linked bridges. Intramolecular chemical networks were elaborated by hydrogen bonding between O-H or N-H sites with the oxygen atom of the carbamoyl function of the glycerilic skeleton. That is the reason why carbamoyle functions are less susceptible to hydrolysis and why the chemical and mechanical resistance properties of the glycerilic polyhydroxyurethanes are increased.

Energy demand is projected to increase by 56% before 2040 and this will lead to the fast depletion of fossil fuels. Currently, biomass is the only sustainable source of organic carbon and liquid fuels. One major method of converting biomass involves the utilization of heterogeneous catalysts. However, there is still a lack of understanding in the reaction mechanisms and surface interactions between biomass-derived oxygenates and catalysts. Specifically, three important reactions are investigated: i) dehydration of glycerol, ii) hydrolysis of cellulose and cellobiose, and iii) hydrodeoxygenation of bio-oil. Some important concepts are gathered and provide insight into the most attractive conversion strategies. These concepts include the role of Lewis and Brønsted acid sites, synergistic effect between defect sites and functional groups, the advantage of weak acid sites, steric effect imposed by aromatic substituents, and the evolution of surface species in catalyst deactivation. These studies show that a deep understanding of surface chemistry can help to elucidate elementary reaction steps, and there is great potential in using heterogeneous catalysts for the conversion of biomass into targeted fuels and chemicals.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Having caused the deaths of more than 10 million individuals since 2000 with most of them occurring in Africa, malaria remains a serious disease that requires undivided attention. To this end a detailed kinetic model of Plasmodium falciparum glycolysis was constructed, validated and used to determine potential drug targets for the development of novel, effective antimalarial therapies. The kinetic model described the behaviour of the glycolytic enzymes with a set of ordinary differential equations that was solved to obtain the steady state fluxes and concentrations of internal metabolites. The model included a glycerol branch represented in a single fitted equation. This present study set out to detect, characterise, and incorporate into the model the enzymes that constitute the glycerol branch of P. falciparum glycolysis. The kinetic parameters of glycerol 3-phosphate dehydrogenase (G3PDH), the first enzyme in the branch and catalyst of the dihydroxyacetone phosphosate (DHAP) reducing reaction, was determined and added to the detailed kinetic model. The model was subsequently validated by comparing its prediction of steady state fluxes with experimentally measured fluxes. Once it was evident that the predictions of the unfitted model agreed with experimentally measured fluxes, metabolic control analysis was performed on this branched system to ascertain the distribution of control over the steady state flux through the glycerol branch. The control G3PDH exercised over its own flux was less than expected due to the enzyme’s sensitivity to changes in NADH and thus the redox balance of the cell. Attempts were made to detect the enzymes responsible for the conversion of glycerol 3-phosphate (G3P) to glycerol. Very low levels of glycerol kinase activity was observed. Although G3P-dependent release of inorganic phosphate was detected results were inconclusive as to whether a non-specific phosphatase also mediated the conversion. Overall, the expansion of the model to include G3PDH did not affect the steady state metabolite concentrations and flux adversely. / AFRIKAANSE OPSOMMING: Vanaf die jaar 2000 het malaria die dood van meer as 10 miljoen mense veroorsaak. Die meeste sterftes het in Afrika voorgekom —’n aanduiding van hoe ernstige siekte dit is en een wat onverdeelde aandag moet geniet. Om hierdie rede is ’n gedetaileerde kinetiese model van glikoliese in Plasmodium falciparum gebou, gevalideer en gebruik om potensiële dwelm teikens te identifiseer vir die ontwikkeling van nuwe, meer effektiewe anti-malaria terapieë. Die kinetiese model beskryf die gedrag van die glikolitiese ensieme in terme van gewone differensiële vergelykings wat opgelos is om die bestendige toestand fluksies en interne metaboliet konsentrasies te bepaal. Die model sluit ’n gliserol-tak in wat deur ’n enkele aangepaste vergelyking verteenwoordig word. Hierdie studie het voorgeneem om die ensieme van die gliserol-tak van P. falciparum glikoliese te identifiseer, karakteriseer en in die model te inkorporeer. Ons het die kinetiese parameters van die eerste ensiem in die gliserol-tak, gliserol 3-fosfaat dehidrogenase (G3PDH), die katalis van die dihidroksiasetoon fosfaat(DHAP) reduserende reaksie, bepaal. Die kinetiese parameters is by die gedetaileerde model gevoeg. Validering het plaasgevind deur die model se voorspellings met eksperimenteel bepaalde waardes te vergelyk. Toe dit duidelik geword het dat die voorspellings van die model met die eksperimenteel bepaalde fluks ooreenstem, is metaboliese kontrole analiese op die vertakte sisteem uitgevoer. Dit is gedoen om vas te stel hoe die bestendige toestand fluks deur die gliserol-tak beheer word. G3PDH het nie volle beheer oor sy eie fluks nie, in teenstelling met ons vergewagtinge. Daar is gepoog om vas te stel watter ensieme verantwoordelik is vir die produksie van gliserol vanuit gliserol 3-fosfaat (G3P). ’n Lae gliserolkinase aktiwiteit is waargeneem. Alhoewel G3P afhanklike vrystelling van anorganise fosfaat waargeneem is, is dit nie duidelik vanuit die resultate of die proses deur ’n nie-spesifieke fosfatase uitgevoer word nie. Die uitbreiding van die model om ’n G3PDH vergelyking in te sluit het nie die bestendige toestand metaboliet konsentrasies en fluks negatief geaffekteer nie.