167 research outputs found
Coenzyme recognition in para-hydroxybenzoate hydroxylase
No full textBiochemistry is the science that studies the chemistry of life. This 'biological' chemistry includes growth, differentiation, movement, conductivity, immunity, transport and storage. During these processes proteins play an important role. The building blocks of proteins are amino acids, of which twenty are known. With these building blocks at hand it is possible to construct numerous proteins with many specific functions. A protein is not an elongated chain of amino acid residues but a compact very well defined three-dimensional structure. Two basic substructures are known in a protein, a cylindricalα-helix and an elongatedβ-strand. A number of theseα-helices and/orβ-strands connected by loop regions form a protein domain and a protein is built up of one or more domains. Furthermore, proteins can contain certain motifs (folds), structural conserved patterns. A large group of proteins with similar function and/or structure are called a protein family.A special group of proteins, called enzymes or biocatalysts, are able to increase the rate of a chemical reaction by lowering the activation energy of that reaction. Enzymes are highly specific, because they influence the reactivity of the substrate in such a way that the substrate is quickly and efficiently converted into a product. Moreover, flexible/dynamic movements in enzymes may play an important role during catalysis, because enzymes are not always rigid bodies. To control the reaction, enzymes often need cofactors. Some examples are the already mentioned dinucleotides NAD(P)H and FAD, that play a role in electron transfer (redox) reactions. Generally speaking, these cofactors bind very specific to a protein. A well-known binding motif for NAD(P)H and FAD in different enzyme families is the Rossmann fold (Chapter 1), discovered by Michael Rossmann in 1974.The NAD(P)H cofactor binds to the enzyme, electron transfer takes place and finally, the oxidized cofactor is released. In some proteins, the mode of NADPH binding is unknown. One example is p -hydroxybenzoate hydroxylase (PHBH), a flavoprotein monooxygenase that belongs to the family of FAD-dependent aromatic hydroxylases.FAD-dependent Aromatic HydroxylasesFAD-dependent aromatic hydroxylases play a role in the biodegradation of aromatic compounds. In nature, these compounds occur in plant polymers (lignin) as well as in proteins, steroïds and terpenes. During this century, the natural pool of aromatic compounds has been extended with products of industrial origin. Many of these synthetic compounds (pesticides, herbicides, fungicides and detergents) place a heavy burden on the environment and accumulate in soil and sludge. Microbial FAD-dependent aromatic hydroxylases catalyze the conversion of natural and synthetic aromatic substrates into products that can be further degraded to carbon dioxide and water. Recently, it was found that these enzymes are also involved in the biosynthesis of steroïds, plant hormones and antibiotics. PHBH is the archetype (prototype) of the family of FAD-dependent aromatic hydroxylases. In Wageningen, research on PHBH and related enzymes is embedded in the Wageningen Graduate School of Environmental Chemistry & Toxicology.p -Hydroxybenzoate Hydroxylasep -Hydroxybenzoate hydroxylase is isolated from the soil bacterium Pseudomonas fluorescens . This microbe can grow on 4-hydroxybenzoate (POHB) and other aromatic compounds as sole carbon source. PHBH catalyzes the conversion of POHB into 3,4-dihydroxybenzoate (DOHB) in the presence of NADPH and molecular oxygen. DOHB is a common intermediate in the aerobic degradation of plant material. After ring cleavage of DOHB and further degradation, the final products acetyl coenzyme A and succinate are fed into the citric acid cycle to provide energy for the cell.p -Hydroxybenzoate hydroxylase has been subject to detailed kinetic and structural studies. The three-dimensional structure of PHBH is built up of three domains (Chapter 1). The first domain is the FAD-binding domain with the specific Rossmann fold for binding the ADP part of FAD. The second domain is the substrate-binding domain and the third domain (interface domain) is important for the interaction with another PHBH subunit, because PHBH exists as a dimer.The structure of the enzyme-substrate complex is known in atomic detail. Recently, it was found that the flavin ring is able to move between an "open" and "closed" conformation. This flavin mobility is important for substrate binding and product release. However, unknown is the NADPH-binding site and where the reaction between NADPH and FAD takes place. Related questions are:-Which amino acids play a role in cofactor binding?-Is there a particular sequence motif for cofactor binding?Which amino acids are responsible for the coenzyme specificity and involved in binding of the 2'-phosphate moiety of NADPH?Another very important question concerns the effector role of the substrate. Upon binding of the aromatic substrate the flow of electrons from NADPH to FAD is 105 times enhanced. However, the molecular principles of this control are poorly understood. In this thesis we have tried to shed more light on the coenzyme recognition by PHBH.Flavin ring mobilityIn Chapter 2 the FAD in PHBH is substituted by a modified FAD, normally present in alcohol oxidase from methylotrophic yeasts. The crystal structure of p -hydroxybenzoate hydroxylase with this flavin analog not only represents the first crystal structure of an enzyme reconstituted with a modified flavin, but also provides direct evidence for the presence of an arabityl sugar chain in the modified form of FAD. The reconstituted enzyme-substrate complex shows that the flavin ring attains the "open" conformation. In the native enzyme-substrate complex the flavin ring is located in the "closed" conformation. The rate of flavin reduction by NADPH is much more rapid as compared to the native enzyme-substrate complex, suggesting that the mobility of the flavin ring is essential for the efficient reduction of the enzyme/substrate complex.Amino acids involved in NADPH bindingTo investigate the mode of NADPH binding, several amino acid residues were replaced by site-directed mutagenesis. The amino acids were selected on the basis of earlier results from chemical modification, crystallographic and modeling studies. Chapters 3, 4, 6 and 8 describe the properties of single mutants. It is concluded that Arg33, Gln34, Tyr38, Arg42, Arg44, His162 and Arg269 are involved in NADPH binding.Structural motif for NADPH bindingPHBH contains two conserved sequence motifs, both involved in FAD binding. Chapter 5 describes a new unique sequence motif for the family of FAD-dependent aromatic hydroxylases, putatively involved in both FAD and NAD(P)H binding. From the recently determined crystal structure of phenol hydroxylase it is deduced that this sequence motif is also structurally conserved. Chapter 6 and 7 show that only His162 of this novel motif is directly important for the binding of NADPH.Coenzyme specificityChapter 8 describes the cloning, purification and characterization of PHBH from Pseudomonas species CBS3. This is the first PHBH enzyme with known sequence that is active with NADH. Based on sequence analysis and homology modelling it is proposed that the helix H2 region is important for the binding of the 2'-phosphate moiety of NADPH. In Chapter 9 , the coenzyme specificity of PHBH from Pseudomonas fluorescens was addressed in further detail. Multiple replacements in helix H2 showed that Arg33 and Tyr38 are crucially involved in determining the coenzyme specificity. For the first time, a PHBH enzyme was constructed, which is more efficient with NADH.Effector specificitySubstrate binding is essential for a rapid reduction of FAD. This allows the subsequent attack of oxygen and the formation of the flavinhydroperoxide hydroxylating species. The question arises whether the stimulating effect of substrate binding on flavin reduction is caused by a large conformational change or merely due to subtle rearrangements in the active site. Chapter 10 describes the crystal structure of the substrate-free enzyme. This study shows that no large conformational changes take place upon substrate (analog) binding. The stimulating role of POHB is probably caused by several subtle effects. Stabilisation of the phenolate form of the substrate results in distribution of the electronic charges in the active site. These charge distributions influence the dynamic equilibrium between the "open" and "closed" conformation of FAD in such a way that the nicotinamide ring of NADPH and the isoalloxazine ring of FAD become optimally oriented for efficient reduction.</p
Eutectic solvents as a novel extraction system for microalgae biorefinery
No full textThe common lipid extraction from microalgae involves sequential of energy-intensive processes and the use of harmful organic solvents. Eutectic solvents (ES), a novel class of designer solvents, hold a great potential as alternative solvents. Not only they are easy to prepare and have tailorable properties, but they are also able to permeabilize the cell wall of microalgae. However, the lack of vapor pressure makes the solvent regeneration difficult. Therefore, in this thesis, a semi-hydrophobic eutectic solvent was developed to extract lipids from microalgae, with a viable solvent regeneration step. Moreover, a preliminary biorefinery process was also explored.In Chapter 2, we screened several semi-hydrophobic ES. The combination of polar imidazole and nonpolar hexanoic acid showed tuneable hydrophobicity depending on the composition. At low imidazole presence, the mixture dissolved lipids and the lipid solubility decreased at higher imidazole content. This principle was then applied to separate the ES from the dissolved lipids. With this approach, about 75% of lipids could be recovered with a high purity (> 85%).Since retrieving imidazole from the altered ES was difficult, we explored other auxiliaries which can also shift the ES hydrophobicity (Chapter 3). Polar antisolvents, such as water, methanol, and ethanol, were observed to reduce the lipid solubility in the ES. The reduction of lipid solubility increases with the antisolvent polarity and amount. Furthermore, since the antisolvents were (moderately) volatile, a large amount of antisolvents could be loaded to obtain higher purity (up to 100%) and recovery (> 90%) when compared to the previous approach. With this approach, more than 90% of ES could be regenerated through evaporating the antisolvents. Methanol was selected as the best antisolvent as it offered the ease of regeneration without sacrificing the lipid recovery.The novel ES was then applied to intact Nannochloropsis oceanica for lipid extraction (Chapter 4). The yield of lipids extracted with the ES was comparable to the standard Bligh & Dyer method using chloroform/methanol. The extraction yield was found to benefit from the low imidazole content (0 – 15 mol%; hydrophobic state), a high temperature, longer incubation time, and high solvent-to-biomass ratio. Interestingly, the moisture from the wet biomass enhanced the lipid extraction, which was found to decrease with freeze-dried biomass. In addition, supplementation of water could reverse the adverse effect of drying. This result implied that the disruption and drying of biomass was not necessary to ensure the high lipid yield.We also developed further the ES-based process towards microalgae biorefinery (Chapter 5). First, the separation of algae lipid from the ES was performed by addition of methanol at low temperature (-20 °C). With this approach, 60% of the lipids could be recovered, which was significantly lower than the finding in Chapter 3. Furthermore, the ES were reusable after three iterative cycles without significant drop of extraction efficiency. Besides that, the process was found to be scalable with slightly lower efficiency and recovery. Furthermore, denatured proteins and carbohydrates could be obtained from the defatted biomass through an aqueous extraction.Several key findings and challenges from this thesis are summarized in Chapter 6. Besides that, some ideas for tackling the current drawbacks of the proposed process are also discussed. Additionally, we include recommendations for further research to gain understanding of molecular interactions between the ES components and target molecules. Ultimately, task-specific ES for biorefining could be tailored based on the obtained knowledge
Biorefinery : recovery of valuable biomolecules
No full textInaugural speech Wageningen University, 23 April 201
Biorefinery : recovery of valuable biomolecules
No full textInaugural speech Wageningen University, 23 April 201
Surface plasmon resonance imaging as a screening platform in biopharmaceutical development
No full textDevelopment of biopharmaceutical products requires extensive research on a multitude of steps that are involved in the delivery of a final product. In this chapter, a general introduction on biopharmaceuticals is provided, including two examples of biopharmaceutical products (erythropoietin and monoclonal antibodies) that have been studied throughout this research. The following paragraphs will highlight a typical production process for biopharmaceuticals consisting of both upstream and downstream processing. In addition, product characterization using a variety of analytical tools is required to determine the product quality, based on several critical quality attributes of biopharmaceuticals that need to be assessed. An overview of the most widely applied analytical tools, including the product characteristics that can be determined, is provided. This is followed by a more detailed description of the principles of Surface Plasmon Resonance (SPR) and SPR imaging (SPRi), which was the main analytical technology that has been applied in this research. The use and potential applications of SPRi during various steps in biopharmaceutical development have been explored and will be further discussed throughout this work.</p
Biorefinery of functional biomolecules from algae
No full textAlgae have been regarded as a promising source of several biomolecules with industrial interest such as proteins, carbohydrates, lipids and pigments. To obtain such biomolecules, it is necessary to implement several extraction and fractionation steps which often result in poor yields, low purities and high costs. To overcome these issues, novel processes are needed, in which the concepts of minimum processing, integration of unit operations, in situ extraction and recyclability are applied. In this PhD thesis several fractionation strategies are presented in which such concepts are implemented. The main goal is to efficiently refine algal biomass into fractions containing functional biomolecules, in particular proteins. The fractionation strategies investigated here are based on mechanical disintegration, using bead milling, and chemical dissolution by means of ionic liquids as green solvents. Furthermore, it is demonstrated how minimum processing can lead, not only to low energy consumptions, but to fractions which display technical functionality comparable to animal derived protein isolates. Algae fraction are therefore envisioned as novel animal-free protein ingredients which can be used as foaming, emulsifying and gelling agents in food products.</p
Harvesting and cell disruption of microalgae
No full textMicroalgae are a potential feedstock for various products. At the moment, they are already used as feedstock for high-valuable products (e.g. aquaculture and pigments). Microalgae pre-dominantly consist out of proteins, lipids and carbohydrates. This makes algae an interesting feedstock for various bulk-commodities. To successfully produce bulk-commodities, a multi-product biorefinery should be adopted that aims on production of both bulk- and high value co-products. In the downstream process, however, harvesting- and cell disruption are technological hurdles for cost effective multi-product biorefinery. Flocculation is considered as a low-cost harvesting process. Flocculating microalgae at high salinities used to be not feasible We demonstrated that marine microalgae can successfully be flocculated and harvested by using cationic polymers. In the second part of this thesis we studied Pulsed Electric Field (PEF) as potential cheap and non-disruptive technology to open microalgae. PEF-treatment evokes openings/’holes’ in micro-organisms. PEF in combination with a pre-treatment to weaken the cell wall resulted in release of proteins from microalgae at low energy consumption. Recent advances in technology development learned that harvesting of micro-algae is no longer a bottleneck. Future research and development should focus on cell disruption and mild extraction technologies. Costs for the biorefinery will decrease by process simplification. For that unit operations for cell disruption and extraction need to be integrated. This project was part of a large public private partnership program AlgaePARC biorefinery (www.AlgaePARC.com). Objective of this program is to develop a more sustainable and economically feasible microalgae production process. For that all biomass components (e.g. proteins, lipids, carbohydrates) should be used at minimal energy requirements and minimal costs while keeping the functionality of the different biomass components. Biorefining of microalgae is very important for the selective separation and use of the different functional biomass components.</p
The seroepidemiology of a neglected zoonotic and livestock pathogen in free-ranging bovids : Leptospirosis in African buffaloes (syncerus caffer)
No full textFunding: This research was funded by Wellcome Trust, grant number 216634/Z/19/Z to M.H.M and grant number 222941/Z/21/Z to W.G. Sample collection and W.G, T.K., and M.M. were funded by the South African government through the South African Medical Research Council and the National Research Foundation South African Research Chair Initiative [grant #86949]. The APC was funded by the Wellcome Trust. Author Contributions: Conceptualization, M.H.M., W.G. and M.M.; methodology, M.H.M., W.G., A.P. and M.M.; formal analysis, M.H.M. and W.G.; writing—original draft preparation, M.H.M.; writing—review and editing, W.G., A.P., T.J.K. and M.M.; visualization, M.H.M.; supervision, M.M.; project administration, M.H.M. and M.M.; funding acquisition, M.H.M. and M.M. All authors have read and agreed to the published version of the manuscript.Peer reviewe
Digital twin in high throughput chromatographic process development for monoclonal antibodies
No full textThe monoclonal antibody (mAb) industry is becoming increasingly digitalized. Digital twins are becoming increasingly important to test or validate processes before manufacturing. High-Throughput Process Development (HTPD) has been progressively used as a tool for process development and innovation. The combination of High-Throughput Screening with fast computational methods allows to study processes in-silico in a fast and efficient manner. This paper presents a hybrid approach for HTPD where equal importance is given to experimental, computational and decision-making stages. Equilibrium adsorption isotherms of 13 protein A and 16 Cation-Exchange resins were determined with pure mAb. The influence of other components in the clarified cell culture supernatant (harvest) has been under-investigated. This work contributes with a methodology for the study of equilibrium adsorption of mAb in harvest to different protein A resins and compares the adsorption behavior with the pure sample experiments. Column chromatography was modelled using a Lumped Kinetic Model, with an overall mass transfer coefficient parameter (kov). The screening results showed that the harvest solution had virtually no influence on the adsorption behavior of mAb to the different protein A resins tested. kov was found to have a linear correlation with the sample feed concentration, which is in line with mass transfer theory. The hybrid approach for HTPD presented highlights the roles of the computational, experimental, and decision-making stages in process development, and how it can be implemented to develop a chromatographic process. The proposed white-box digital twin helps to accelerate chromatographic process development.BT/Bioprocess EngineeringBT/Design and Engineering Educatio
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