Functional analyses of plant-specific histone deacetylases : Their role in root development, stress responses and symbiotic interactions
Li, Huchen - \ 2017
Wageningen University. Promotor(en): T. Bisseling, co-promotor(en): O. Kulikova. - Wageningen : Wageningen University - ISBN 9789463436816 - 188
plants - histones - enzymes - roots - development - symbiosis - gene expression - molecular biology - root nodules - mycorrhizas - planten - histonen - enzymen - wortels - ontwikkeling - symbiose - genexpressie - moleculaire biologie - wortelknolletjes - mycorrhizae
Plants have a sessile lifestyle. To ensure survival, they develop a potential to respond to environmental cues to set up an adaptive growth and development. This adaptation involves transcriptional reprogramming of the genome through chromatin-based mechanisms relying on the dynamic interplay of transcription factors (TFs), post-translational modification of histones, the deposition of histone variants, DNA methylation, and nucleosome remodeling. This thesis is focused on a role of one group of histone post-translational modifiers, plant-specific histone deacetylases (HDTs), in plant development under control condition and variable stresses/symbiotic interactions.
It is well known that HDTs are involved in plant responses to environmental stresses. However, whether they play a role in regulating plant growth and development is elusive. In this thesis it is shown that Arabidopsis thaliana AtHDT1/2 regulate the cell fate switch from division to expansion in the Arabidopsis root. Knock-down of AtHDT1/2 (hdt1,2i) causes that this switch occurs earlier and results in less cells in the root meristem. This process slows down root growth. One target of AtHDT1/2, AtGA2ox2, is identified here. Its overexpression displays the same root phenotype as hdt1/2i , and its knock-out partially rescues hdt1,2i root meristem phenotype. AtGA2ox2 inactivates gibberellin (GA4) whose application increases root meristem cell number in WT, but not in hdt1,2i. Based on these data, we conclude that AtHDT1/2 repress the transcription of AtGA2ox2, and likely fine-tunes GA homeostasis to regulate the switch from cell division to expansion in root tips.
HDTs respond to salt stress in Arabidopsis seedlings. Halotropism is a novel reported tropism allowing roots to avoid a saline environment. Whether the AtHDT1/2-AtGA2ox2 module is operational in halotropism is studied here. We show that hdt1,2i mutants respond more severe in halotropism. AtHDT1/2, as well as AtGA2ox2 display asymmetric localization patterns in halotropism with AtHDT1/2 reduced and AtGA2ox2 induced at high salt side of root tips. Our data indicate that their asymmetric patterns likely results in less GA at high salt side of root tips and this is required for halotropism establishment. In line with this, both constitutive expression of AtHDT2 and exogenous GA application reduce halotropic response. A reduction of GA in root tips causes an earlier switch from cell division to expansion. We discuss that this earlier switch enables roots rapidly to bend away from saline environment.
It has been shown that HDTs play a role under biotic stress in rice and tobacco leaves. We demonstrate that they are also involved in response to biotic stress in Arabidopsis leaves. Arabidopsis hdt2 mutants are more susceptible to virulent Pseudomonas syringae pv. tomato PstDC3000, whereas AtHDT2 overexpression mutants are more resistant. In addition, we detected a translocation of AtHDT2 from nucleolus to nucleoplasm after the perception of flagellin22 in Arabidopsis leaf cells. This translocation is not observed under abiotic stress. A mechanism controlling this translocation is identified. AtMPK3 is activated under biotic stress, it interacts with and phosphorylates AtHDT2. This leads to the accumulation of AtHDT2 in nucleoplasm where it contributes to the repression of defense genes.
During the interaction with symbiotic microorganisms, plants could develop a symbiotic organ/structure. For example, legumes of which Medicago truncatula is a model, can form root nodules or arbuscules by interacting with rhizobia or arbuscular mycorrhiza.
We show that nodule-specific knock-down of MtHDT1/2/3 (MtHDTs RNAi) blocks nodule primordia development and affects the function of nodule meristem. This is consistent with their roles in controlling cell division during root development and suggests that the function of nodule and root meristems is closely related. However, MtHDT2 gains a new sub-nuclear localization pattern in nodule meristem by using a not yet known mechanism, different from that in root meristem. This suggests that these two meristems have different transcriptional landscapes. In the nodule infection zone MtHDTs are also expressed and in MtHDTs RNAi the intracellular release of rhizobia is markedly reduced. Expression of MtHMGR1 and its paralogs, encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductases are down-regulated in MtHDTs RNAi. It has been shown MtHMGR1 interacts with MtDMI2, a component of Nod factor signalling pathway, to control rhizobial infection. Knock-down of MtHMGR1/MtDMI2, as well as inhibiting MtHMGRs enzymatic activity blocks nodule primordia development and rhizobial infection in nodule primordia/mature nodules. This phenotype partially resembles MtHDTs RNAi phenotype. We discuss that MtHDTs regulate expression of MtHMGRs and in this way affect Nod factor signalling and control nodule development.
Similar to nodule symbiosis, during arbuscular mycorrhizal symbiosis cells in the cortex are also intracellularly infected. We show that MtHDT2 is also induced in these arbuscule containing cells. Knock-down of MtHDT2 (MtHDT2i) significantly reduces the intracellular infection of the hyphae on the mycorrhized root segments, indicating that MtHDT2 control mycorrhizal intracellular infection. We discuss whether MtHDTs can regulate mycorrhizal/rhizobial infection in a similar way.
The data obtained in this thesis and the published information related to these subjects are discussed at the end. HDTs are key players in plant responses to environmental cues, whereas they respond to abiotic factors and biotic factors differently. They are also key regulators of plant growth and development that is clearly demonstrated in this thesis on examples of root and nodule development. I also propose a role of AtHDT1/2 in response to salt signal to fine-tune the switch from cell division to expansion in root tips during halotropism.
Prof. Richard Kranenburg: Bacteriën als fabriekjes van de bio-economie
Kranenburg, R. van - \ 2017
Wageningen University & Research
industriële microbiologie - bacteriën - biobased economy - microbiële afbraak - enzymen - genetische verandering - onderzoek - industrial microbiology - bacteria - biobased economy - microbial degradation - enzymes - genetic change - research
Video over micro-organismen in de biobased economy
Trimming proline dehydrogenase : protein and cofactor minimization
Huijbers, Mieke M.E. - \ 2017
Wageningen University. Promotor(en): Willem van Berkel. - Wageningen : Wageningen University - ISBN 9789463430517 - 181
proline - thermus thermophilus - enzymes - amino acids - binding proteins - catalysts - proline - thermus thermophilus - enzymen - aminozuren - bindende eiwitten - katalysatoren
Proline is one of the proteinogenic amino acids and one of the most abundant amino acids in the cell. Next to serving as one of the non-essential amino acids, proline also has a central role in metabolism. In Chapter 1, the different functions of this imino acid are described, as well as the proline metabolic enzymes. The focus is on the enzyme proline dehydrogenase (ProDH), which catalyzes the flavin-dependent conversion of L-proline to Δ1-pyrroline-5-carboxylate (P5C). Malfunctioning of this enzyme has severe implications for human health and has been associated with tumorigenesis and schizophrenia.
This thesis deals with the engineering and biochemical characterization of Thermus thermophilus ProDH (TtProDH) in order to gain more insight into the structure-function relationship of this thermo-resistant flavoenzyme. TtProDH is a membrane-associated protein and recombinant soluble forms of the enzyme have only been obtained in limited amounts. Chapter 2 describes the heterologous production of TtProDH in Escherichia coli. Using maltose-binding protein (MBP) as solubility tag, high yields of active holoenzyme are obtained. The MBP-tag can be efficiently removed from the fusion protein with trypsin, yielding native TtProDH. This enzyme is thermotolerant as well as solvent tolerant; however, both fused and clipped TtProDH are prone to aggregation. In Chapter 3, we show that the hydrophobic N-terminal helix of TtProDH is responsible for this non-native self-association. Phe10 and Leu12, located at the protein surface, were replaced by glutamates, generating the F10E/L12E (EE) variant of MBP-TtProDH. This more polar variant exclusively forms tetramers and exhibits excellent catalytic features. Specific removal of the MBP-tag of the EE variant is less easy than for WT, as trypsinolysis of the fusion enzyme leads to degradation of TtProDH. Since the MBP tag does not influence the spectral and catalytic properties of the enzyme, further experiments were performed with MBP-tagged variants of TtProDH.
ProDH has a distorted (βα)8 TIM-barrel fold which is conserved throughout the PutA/ProDH family. In contrast, the N-terminal sequence of ProDH is poorly conserved. TtProDH contains, next to the distorted TIM-barrel, three N-terminal helices, αA, αB and αC, of which the function is not well understood. In Chapter 4, we describe the characterization of helical arm-truncated variants, lacking respectively one (ΔA), two (ΔAB), or three (ΔABC) N-terminal helices. All three variants show flavin properties that are highly similar to EE, indicating no changes in the microenvironment of the flavin isoalloxazine ring. ΔA and ΔAB are highly active tetramers, whereas removal of the complete N-terminal arm (ΔABC) results in poorly active dimers. Furthermore, EE, ΔA and ΔAB rapidly react with the suicide inhibitor N-propargylglycine, while ΔABC is not capable of forming a flavin adduct with N-propargylglycine. This indicates that helix αC has a crucial role in both the oligomerization and activity of TtProDH. Closer examination revealed an ionic interaction as well as a hydrophobic patch between helices αC and α8, the latter helix being crucial for substrate recognition. To investigate the functional role of helix αC in further detail, additional enzyme variants were created that disrupt the interactions between both helices. While disrupting the ionic interaction had minor effects, disrupting the hydrophobic patch leads to dimer formation, loss of activity and decreased reactivity with N-propargylglycine. This supports that helix αC is crucial for TtProDH catalysis and tetramerization through positioning of helix α8.
The quaternary structure of TtProDH was investigated in more detail in Chapter 5. Two ionic interactions at the dimeric interface were selectively disrupted by changing Asp205 and Glu207 of TtProDH variants EE, ΔA, ΔAB and ΔABC into lysines. These KK-variants form monomers (except for EE KK, which forms dimers) and have improved catalytic properties at moderate temperatures compared to their non-KK counterparts. However, their melting temperatures are decreased by more than 20 °C. This indicates that a trade-off is made between thermostability and catalytic activity.
In Chapter 6, we studied the cofactor binding of TtProDH. Flavoenzymes contain either FAD or FMN as cofactor. FAD often binds to a Rossmann fold, while FMN prefers a TIM-barrel or flavodoxin-like fold. Proline dehydrogenase is denoted as an exception: it possesses a TIM barrel-like fold while binding FAD. To study the cofactor binding of TtProDH, we produced MBP-TtProDH EE in its apoform using a riboflavin auxotrophic E. coli strain. Reconstitution of the enzyme with either FAD or FMN revealed that MBP-TtProDH has no preference for FAD as cofactor. Kinetic parameters of both holo-FAD and holo-FMN are similar, as are the dissociation constants for FAD and FMN release. We show that the holo form of MBP-TtProDH, as produced in E. coli TOP10 cells, contains about three times more FMN than FAD. In addition, we obtained the crystal structure TtProDH ΔABC, which shows no electron density for an AMP moiety of the cofactor. This indicates the presence of mainly FMN in the enzyme. The capability of TtProDH to display equal properties with both cofactors is unique for flavoenzymes, and classification of TtProDH as an FAD-containing enzyme should be reconsidered.
In Chapter 7, we discuss the novel findings described in this thesis and put them in a broader perspective. We have created a minimalist ProDH that is an excellent catalyst, but is deprived of all structural features that are unnecessary for in vitro functioning. Our results expand the knowledge on the structure-function relationship of ProDHs, and give insight into enzyme functionality from an industrial perspective. We also discuss how this knowledge might be used in future studies for a better understanding of the properties of eukaryotic ProDHs, with a special interest in the human enzyme.
In vitro fermentation and immunomodulating characteristics of dietary fibres
Rösch, C. - \ 2016
Wageningen University. Promotor(en): Harry Gruppen; Henk Schols. - Wageningen : Wageningen University - ISBN 9789462577954 - 130
dietary fibres - degradation - enzymes - immunomodulatory properties - cytokines - glycosides - fermentation - voedingsvezels - degradatie - enzymen - immunomodulerende eigenschappen - cytokinen - glycosiden - fermentatie
Dietary fibres are a diverse group of substances, indigestible by human digestive enzymes, but (partially) fermentable in the human large intestine by the resident microbiota. Many health beneficial effects of fibres such as lowering blood cholesterol levels or increasing stool bulk have been reported. For some fibres, immunomodulating properties have been shown. Other studies investigate the degradation fate of fibres by the bacteria. In this PhD thesis BMDCs from TLR2/4 knock out mice were validated to be unresponsive to naturally present contaminants like LPS and proved to be a good tool to analyse the immune response of dietary fibres. A variety of 44 fibres, was tested on these immune cells and all fibres were found to modulated the immune system differently. Also, different immunomodulating properties of an oat and barley β-glucan having rather similar chemical structures, were found. The insoluble fraction of the β-glucans induced highest amounts of cytokines. As a consequence, sample preparation such as drying, dispersing and heating were shown to affect the immunomodulatory properties. The in vitro fermentation characteristics of barley β-glucan and sugar beet pectin and the immunomodulatory properties of their degradation products on BMDCs were compared and shown to be substrate and degradation product specific. This study showed, that glycosidic degradation products of both fibres induced higher amounts of cytokines than their intact polysaccharide. An in vitro batch fermentation of soluble, indigestible maltodextrins by human faecal inocula was monitored and the activity of carbohydrate degrading enzymes, produced by the microbiota, was analysed. Results revealed that the maltodextrin was only slowly and incompletely fermented, despite the high potential of microbial enzymes present to degrade typical starch linkages.
Overall, this thesis showed that dietary fibres interact and influence the immune system dependent on their individual chemical fine structure. Additionally, an evaluation of the health impact of dietary fibres can only be complete when also glycosidic fermentation products are considered.
Alkaline pretreatments of lignin-rich by-products and their implications for enzymatic degradation
Murciano-Martinez, P. - \ 2016
Wageningen University. Promotor(en): Harry Gruppen, co-promotor(en): Mirjam Kabel. - Wageningen : Wageningen University - ISBN 9789462576629 - 156
degradation - enzymes - pretreatment - byproducts - lignin - food chemistry - delignification - sugarcane bagasse - degradatie - enzymen - voorbehandeling - bijproducten - lignine - voedselchemie - delignificatie - suikerrietbagasse
The increasing interest in plant biomass based biofuels and chemicals arouses mainly from the increased awareness of a possible finiteness of fuels. The current main challenge to produce such biofuels and biochemicals is economic efficiency, but also knowledge concerning type and effectiveness of both thermally assisted chemical and enzymatic treatments, needed to generate fermentable sugars, is lacking. The subject of this thesis is to gain understanding of the effect of both sulphuric acid and NaOH catalysed pretreatments of sugar cane bagasse and oil palm empty fruit bunches on subsequent enzymatic saccharification, with a focus on the NaOH catalysed pretreatments. The fate of the main polymers present, lignin, cellulose and xylan, was studied and also single-activity xylan degrading enzymes, all from Rasamsonia emersonii, were studied for their mode-of-action.
DNA : the recipe book for all the processes in the plant : all cells have the same generic information
Heuvelink, E. ; Kierkels, T. - \ 2015
In Greenhouses : the international magazine for greenhouse growers 4 (2015)4. - ISSN 2215-0633 - p. 12 - 13.
dna - plantenveredeling - genetische modificatie - transfer rna - messenger rna - ribosomen - eiwitten - aminozuren - enzymen - mutaties - dna - plant breeding - genetic engineering - transfer rna - messenger rna - ribosomes - proteins - amino acids - enzymes - mutations
It’s sometimes called a blueprint: DNA, the carrier of genetic information. But the term recipe book covers it better. It explains how the plant can respond to changing conditions. Plant breeders take advantage of natural variations in DNA. Genetic modification can make their job easier.
Kinetic modelling of enzymatic starch hydrolysis
Bednarska, K.A. - \ 2015
Wageningen University. Promotor(en): Tiny van Boekel; Remko Boom, co-promotor(en): Anja Janssen. - Wageningen : Wageningen University - ISBN 9789462573086 - 159
hydrolyse - enzymen - zetmeel - stochastische modellen - verwerking - hydrolysis - enzymes - starch - stochastic models - processing
Kinetic modelling of enzymatic starch hydrolysis – a summary
The dissertation entitled ‘Kinetic modelling of enzymatic starch hydrolysis’ describes the enzymatic hydrolysis and kinetic modelling of liquefaction and saccharification of wheat starch. After the background information about the enzymes, the substrate and the basics of the model in the first chapter, we describe a model predicting the outcome of wheat starch liquefaction by α-amylase from Bacillus licheniformis at 50°C in chapter 2. We demonstrate the ability of the model to predict starch hydrolysis products larger than the oligosaccharides considered in the existing models. The model in its extended version follows all the products of wheat starch hydrolysis separately, and despite the quantitative differences, the qualitative predictions are satisfactory. We also show that the difference between the experimental and computed data might stem from the inaccuracy of the subsite map.
In the following chapters the model is used to find a better description of the hydrolysis data at two temperatures (50°C and 80°C), by varying the energy values of the subsite map and evaluating the inhibition. We hypothesize that a subsite map that is based on the cleavage patterns of linear, short molecules does not account for the complexity of hydrolysis of amylopectin. The branched structure of amylopectin molecules influences the composition of the hydrolysis products by restricting the access to some of the bonds. The presence of branches creates steric obstacles for the enzyme. The used α-amylase has difficulties hydrolysing and accommodating α-(1,6)-glycosidic bonds, which imposes on the hydrolysis of the α-(1,4)-glycosidic bonds located in its proximity. On this basis, we analyse the subsite maps in detail and suggest which of the subsites are crucial when making predictions about the product composition of starch hydrolysates. On top of that we propose new subsite maps that allow a quantitative description of the experimental data.
After the model was shown to work at different experimental conditions, we also test it at increased the dry matter content during wheat starch hydrolysis. We follow both the liquefaction by BLA and the saccharification process by glucoamylase from Aspergillus niger at low moisture content. The liquefaction model, is used to predict all of the products of wheat starch hydrolysis at higher dry matter contents (30-60 w/w%). The liquefaction model also creates the substrate matrices representing maltodextrins to be used in the saccharification model. The saccharification of liquefacts to glucose is followed with a new mechanistic model, also using the assumptions of the subsite theory. The saccharification model predicts all of the reaction products using the subsite maps of glucoamylase available in literature.
The findings described in the thesis are summarized and put in context in the general discussion. We demonstrate how the parameters of the liquefaction model at low moisture contents were chosen. The outcomes of the model are also compared with the experimental data at 30-60 w/w%. Next, we test our liquefaction model with starch hydrolysis data at 5 and 60 w/w% taken from literature, to verify both the approach we used and the validity of the parameters we obtained in previous chapters. The method used to improve the subsite maps is also tested on another enzyme, Bacillus amyloliquefaciens α-amylase. After discussing the factors that influence saccharification at high dry matter contents, we conclude the chapter with describing the potential of stochastic modelling and its practical use.
Enzymcocktails uit Wageningen : Nog effectiever, nog goedkoper
Joppen, L. ; Visser, J. ; Voragen, A.G.J. - \ 2015
Agro&Chemie Performis B.V.
enzymen - biomassaconversie - plantenvezels - bedrijfsvoering - onderzoek - enzymbereidingen - biobased economy - enzymes - biomass conversion - plant fibres - management - research - enzyme preparations
In een bedrijvencomplex in Wageningen werken ruim 40 wetenschappers aan de ontwikkeling van enzymen en enzymcocktails voor de afbraak van plantenvezels. Dyadic, met een Amerikaanse eigenaar, maar met diepe roots in Nederland, ontwikkelt enzymen en enzymcocktails voor verschillende toepassingen, waaronder ook de productie van bio-ethanol op basis van tweedegeneratie biomassa.
Enzyme-assisted separation and hydrolysis of gluten : options for intensification
Hardt, N.A. - \ 2014
Wageningen University. Promotor(en): Remko Boom, co-promotor(en): Atze Jan van der Goot. - Wageningen : Wageningen University - ISBN 9789462571228 - 165
gluten - graaneiwitten - scheiding - enzymen - hydrolyse - voedseltechniek - watergehalte - watergebruik - gluten - cereal proteins - separation - enzymes - hydrolysis - food engineering - water content - water use
The food industry is one of the largest water consumers in industry. Using large amounts of water, however, is undesirable from an environmental point of view because freshwater is a scarce good in many regions of the world and undesirable from an economic point of view because high water loadings require high amounts of energy for dehydration and signify high amounts of wastewater. This thesis uses wheat, one of the major crops in human nutrition, to study the influence of low water concentrations on two relevant processes in wheat processing:
The separation of starch and gluten. Separation is often performed using 10–15 L water per kg dry matter. Instead, starch and gluten can be separated by inducing shear using 0.5 L water per kg dry matter. In this thesis we make use of xylanases to hydrolyze arabinoxylan present in wheat, thereby releasing the water associated with arabinoxylan. In doing so, shear-induced starch–gluten separation is performed at even more concentrated conditions. The influence of arabinoxylan hydrolysis in wheat dough at low water contents is studied in chapters 2 and 3.The hydrolysis of gluten. Hydrolysis is currently performed using approximately 4 L water per kg dry mater. In this thesis we perform gluten hydrolysis at solid concentrations of up to 70%, thereby investigating the changes in the hydrolysis reaction and the functionality of the resulting hydrolysates. Wheat gluten hydrolysis at low water contents is studied in chapters 4, 5 and 6.
This thesis consists of seven chapters. Chapter 1 gives a general introduction of the thesis. In chapter 2, wheat dough rheology at low water contents below 40% and the influence of xylanases is studied. A reduction in water content from 43.5–44.8% (representing optimal Farinograph water absorption) to 34% (the lowest water content where a dough forms) results in a non-linear increase in the dough consistency, elastic modulus G’, and a decrease in the maximum creep compliance Jc,max of 1–2 orders of magnitude. Addition of xylanases has the same effect on the dough consistency, G’ and Jc,max as an increase in water content of 2–5% (on a water basis). Tan δ is hardly and Jel not influenced by xylanase addition showing that the influence of xylanases on the mechanism of hydration is negligible.
In chapter 3, shear-induced starch–gluten separation with the help of xylanases is studied at water contents from 43.5% to 34%. Addition of xylanases at the standard water content of 43.5% results in a slurry without any separation. As a result, lower water contents are used. At water contents below 40%, the local formation of gluten clusters is observed with and without xylanases addition. However, opposed to shear-induced separation at 43.5% water without xylanase, the gluten patches do not migrate to the center of the cone because of the densely packed dough and an inhomogeneity in the shear field. Nevertheless, gluten clusters can be concentrated up to 60% (N×5.7) protein. Similar to chapter 2, xylanase addition allows water savings of 3–5% (on a water basis).
Chapter 4 introduces enzymatic wheat gluten hydrolysis at high solid concentrations and describes the influence of high-solid hydrolysis on the resulting functional properties of the gluten hydrolysates. Wheat gluten can be hydrolyzed at solid concentrations of up to 60% (w/w). The water solubility of the dried hydrolysates is independent of the solid concentration during hydrolysis, just like the foam stabilizing properties at degrees of hydrolysis (DH%) below 8% At DH% above 8%, high solid concentrations even increase the foam stabilizing properties of the resulting hydrolysates, which is related to the presence of more peptides with a molecular mass >25 kDa. Furthermore, an increase in solid concentration results in an increase of the volumetric productivity.
Despite the advantages of high-solid gluten hydrolysis, we also observe lower hydrolysis rates in high-solid gluten hydrolysis compared to low-solid gluten hydrolysis at constant enzyme-to-substrate ratios. The factors causing this hydrolysis rate limitation are investigated in chapter 5. It is shown that enzyme inhibition, the water activity, and mass transfer limitations do not impede the hydrolysis up to 50% solids. However, the hydrolysis rate limitation can be explained by a second-order enzyme auto-inactivation rate along with the higher enzyme concentrations used. At solid concentrations above 50%, the hydrolysis rate further decreases due to mass transfer limitations. Furthermore, the addition of enzyme after 24 h at high solid concentrations hardly increases the DH%, suggesting that the maximum attainable DH% decreases at high solid concentrations. This DH% limitation is explained by a reduced enzyme activity due to a decline in water activity.
Based on the findings in chapters 4 and 5, a direct hydrolysis of gluten present in wheat flour at high solid concentrations is investigated in chapter 6, thereby omitting the starch–gluten separation. At a constant protein concentration, the protease activity is higher for wheat flour hydrolysis (at 40% total solids) than for vital wheat gluten hydrolysis (at 7.2% total solids) in the initial 6 h of hydrolysis, despite the high starch content in wheat flour and consequently lower water content. This is related to the starch granules in wheat flour, preventing the aggregation of (native) gluten. At wheat flour concentrations above 50% and for longer reaction times the positive effect of starch disappears. This is explained by mass transfer limitations and reduced water activities in the wheat flour slurry or dough, respectively.
Chapter 7 summarizes and generalizes the main findings of this thesis and compares the current status in starch–gluten separation and gluten hydrolysis with the concentrated separation and hydrolysis processes developed in this study. Water and energy savings of at least 50% are possible when separating and hydrolyzing at concentrated conditions. In the end, future prospects in high-solid wheat gluten hydrolysis are briefly discussed.
Elucidation of strigolactone biosynthesis in the host plant rice and the signal perception in the parasitic plant Striga hermonthica
Zhang, Y. - \ 2014
Wageningen University. Promotor(en): Harro Bouwmeester, co-promotor(en): Carolien Ruyter-Spira. - Wageningen : Wageningen University - ISBN 9789462570191 - 208
striga hermonthica - parasitaire planten - biosynthese - signaaltransductie - oryza sativa - rijst - enzymen - plantenfysiologie - striga hermonthica - parasitic plants - biosynthesis - signal transduction - oryza sativa - rice - enzymes - plant physiology
Strigolactones (SLs) are a newly identified class of plant hormones regulating plant architecture, including shoot and root branching. Plants also secrete blends of SLs into the rhizosphere, where they stimulate colonisation of the host roots by arbuscular mycorrhizal (AM) fungi, beneficial organisms for the host. But SLs also induce the seed germination of root parasitic plants, such as Striga, which can have a big negative impact on crop yield. A better insight in how the different SLs are synthesized by the host and how the parasitic plant Striga perceives them could help to develop crops with proper AM colonisation and Striga resistance at the same time. In this thesis, two cytochrome P450 enzymes responsible for the last step in SL formation and SL structural diversification in rice were identified. In addition, the F-Box protein MAX2 of Striga (ShMAX2), a SL signalling component, was characterised, representing the first example from a root parasitic plant species, which is paving the way for furthering our understanding of how SLs are perceived by these parasites. The knowledge gained in this thesis brings us a significant step closer to the possibility to improve crop breeding strategies for parasitic weed resistance.
Introducing enzyme selectivity as a quantitative parameter to describe the effects of substrate concentration on protein hydrolysis
Butré, C.I. - \ 2014
Wageningen University. Promotor(en): Harry Gruppen, co-promotor(en): Peter Wierenga; Stefano Sforza. - Wageningen : Wageningen University - ISBN 9789462570238 - 199
eiwitten - eiwittechnologie - eiwitafbraak - hydrolyse - enzymen - concentratie - proteins - protein engineering - protein degradation - hydrolysis - enzymes - concentration
To understand the differences in peptide composition that result from variations in the conditions of enzymatic hydrolysis of proteins (e.g. substrate concentration) the mechanism of hydrolysis needs to be understood in detail. Therefore, methods and tools were developed to characterize and quantify the peptides formed during enzymatic protein hydrolysis. The information obtained was used to introduce a novel quantitative parameter: the selectivity of the enzyme towards the individual cleavage sites in the substrate, within the given specificity of the enzyme applied. The selectivity describes the rate of hydrolysis of a cleavage site compared to the rate of hydrolysis of all cleavage sites in the parental protein. Large differences in the selectivity of the enzyme towards the cleavage sites after the same type of amino acid residues in a protein were found. For β-lactoglobulin hydrolyzed by Bacillus licheniformis protease the selectivity was found to vary between 0.003 % and 17 % or even 0 for some cleavage sites. The effects of increasing substrate concentration and pH on the hydrolysis were studied. An increase in substrate concentration results in lower kinetics of hydrolysis, related to the available amount of water. This also resulted in significant changes in the enzyme selectivity towards the cleavage sites for which the enzyme has a high selectivity. Changing the pH of hydrolysis resulted in large changes in the kinetics of hydrolysis as well as in the enzyme selectivity. Due to the detailed analysis of the peptide composition, certain a-specific peptides were identified. It was shown that these originate from spontaneous cleavage of formed peptides. The changes in the mechanism of hydrolysis were compared to simulation data. The simulation data were obtained from a stochastic model based on random selection of the substrate and the cleavage site, given the specificity of the enzyme. A quite good agreement was obtained between simulated and experimental data. The parameters and methods developed in this study to describe the mechanism of hydrolysis can potentially be used for more complex systems.
Water holding capacity and enzymatic modification of pressed potato fibres
Ramasamy, U. - \ 2014
Wageningen University. Promotor(en): Harry Gruppen, co-promotor(en): Mirjam Kabel. - Wageningen : Wageningen University - ISBN 9789461739643 - 156
aardappelpulp - aardappelen - vezels - celwandstoffen - polysacchariden - waterbergend vermogen - hydrolyse - enzymen - potato pulp - potatoes - fibres - cell wall components - polysaccharides - water holding capacity - hydrolysis - enzymes
Cell wall polysaccharides (CWPs) contribute to the water holding capacity (WHC) of fibre rich feeds, such as pressed potato fibres (PPF). However, the role of CWPs on the WHC of PPF was unidentified so far.
PPF was characterized to be abundant in arabinogalactan (AG) linked rhamnogalacturonan-I (RG-I), homogalacturonan (HG) and cellulose, next to which xyloglucan (XG) contributed the most of the hemicellulosic CWPs. The CWP network in potatoes was loosened upon starch extraction of potatoes and solubilized HG-RG-I-AG.
Analyses of the WHCs upon enzyme treatments indicated that the WHC of PPF was mainly caused by a network of insoluble, non-cellulosic CWPs such as pectic CWPs (HG-RG-I-AG) and XG. Findings in this thesis showed that AGs were better degraded than xyloglucans (XGs). Since XGs were found to be equally important in contributing to the WHC as AGs, the substantial removal of AGs, as well as XGs, should be advantageous to lower the WHC.
Other than lowering the WHC, the use of a pectinase-rich preparation improved the recovery of starch from potatoes by the degradation of mainly pectic CWPs, in particular pectic AG side chains and HG. The degradation of arabinan was observed to be inhibited by components in potato juice (PJ).
Text mining for metabolic reaction extraction from scientific literature
Risse, J.E. - \ 2014
Wageningen University. Promotor(en): Ton Bisseling; Jack Leunissen, co-promotor(en): P.E. van der Vet. - Wageningen : Wageningen University - ISBN 9789461739001 - 138
metabolomica - gegevensanalyse - databanken - text mining - publicaties - wetenschappelijk onderzoek - moleculaire biologie - thesauri - enzymen - metabolieten - metabolomics - data analysis - databases - text mining - publications - scientific research - molecular biology - thesauri - enzymes - metabolites
Science relies on data in all its different forms. In molecular biology and bioinformatics in particular large scale data generation has taken centre stage in the form of high-throughput experiments. In line with this exponential increase of experimental data has been the near exponential growth of scientific publications. Yet where classical data mining techniques are still capable of coping with this deluge in structured data (Chapter 2), access of information found in scientific literature is still limited to search engines allowing searches on the level keywords, titles and abstracts. However, large amounts of knowledge about biological entities and their relations are held within the body of articles. When extracted, this data can be used as evidence for existing knowledge or hypothesis generation making scientific literature a valuable scientific resource. To unlock the information inside the articles requires a dedicated set of techniques and approaches tailored to the unstructured nature of free text. Analogous to the field of data mining for the analysis of structured data, the field of text mining has emerged for unstructured text and a number of applications has been developed in that field.
This thesis is about text mining in the field of metabolomics. The work focusses on strategies for accessing large collections of scientific text and on the text mining steps required to extract metabolic reactions and their constituents, enzymes and metabolites, from scientific text. Metabolic reactions are important for our understanding of metabolic processes within cells and that information provides an important link between genotype phenotype. Furthermore information about metabolic reactions stored in databases is far from complete making it an excellent target for our text mining application.
In order to access the scientific publications for further analysis they can be used as flat text or loaded into database systems. In Chapter 2we assessed and discussed the capabilities and performance of XML-type database systems to store and access very large collections of XML-type documents in the form of the Medline corpus, a collection of more than 20 million of scientific abstracts. XML data formats are common in the field of bioinformatics and are also at the core of most web services. With the increasing amount of data stored in XML comes the need for storing and accessing the data. The database systems were evaluated on a number of aspects broadly ranging from technical requirements to ease-of-use and performance. The performance of the different XML-type database systems was measured Medline abstract collections of increasing size and with a number of different queries. One of the queries assessed the capabilities of each database system to search the full-text of each abstract, which would allow access to the information within the text without further text analysis. The results show that all database systems cope well with the small and medium dataset, but that the full dataset remains a challenge. Also the query possibilities varied greatly across all studied databases. This led us to conclude that the performances and possibilities of the different database types vary greatly, also depending on the type of research question. There is no single system that outperforms the others; instead different circumstances can lead to a different optimal solution. Some of these scenarios are presented in the chapter.
Among the conclusions of Chapter 2is that conventional data mining techniques do not work for the natural language part of a publication beyond simple retrieval queries based on pattern matching. The natural language used in written text is too unstructured for that purpose and requires dedicated text mining approaches, the main research topic of this thesis. Two major tasks of text mining are named entity recognition, the identification of relevant entities in the text, and relation extraction, the identification of relations between those named entities. For both text mining tasks many different techniques and approaches have been developed. For the named entity recognition of enzymes and metabolites we used a dictionary-based approach (Chapter 3) and for metabolic reaction extraction a full grammar approach (Chapter 4).
In Chapter 3we describe the creation of two thesauri, one for enzymes and one for metabolites with the specific goal of allowing named entity identification, the mapping of identified synonyms to a common identifier, for metabolic reaction extraction. In the case of the enzyme thesaurus these identifiers are Enzyme Nomenclature numbers (EC number), in the case of the metabolite thesaurus KEGG metabolite identifiers. These thesauri are applied to the identification of enzymes and metabolites in the text mining approach of Chapter 4. Both were created from existing data sources by a series of automated steps followed by manual curation. Compared to a previously published chemical thesaurus, created entirely with automated steps, our much smaller metabolite thesaurus performed on the same level for F-measure with a slightly higher precision. The enzyme thesaurus produced results equal to our metabolite thesaurus. The compactness of our thesauri permits the manual curation step important in guaranteeing accuracy of the thesaurus contents, whereas creation from existing resources by automated means limits the effort required for creation. We concluded that our thesauri are compact and of high quality, and that this compactness does not greatly impact recall.
In Chapter 4we studied the applicability and performance of a full parsing approach using the two thesauri described in Chapter 3 for the extraction of metabolic reactions from scientific full-text articles. For this we developed a text mining pipeline built around a modified dependency parser from the AGFL grammar lab using a pattern-based approach to extract metabolic reactions from the parsing output. Results of a comparison to a modified rule-based approach by Czarnecki et al.using three previously described metabolic pathways from the EcoCyc database show a slightly lower recall compared to the rule-based approach, but higher precision. We concluded that despite its current recall our full parsing approach to metabolic reaction extraction has high precision and potential to be used to (re-)construct metabolic pathways in an automated setting. Future improvements to the grammar and relation extraction rules should allow reactions to be extracted with even higher specificity.
To identify potential improvements to the recall, the effect of a number of text pre-processing steps on the performance was tested in a number of experiments. The one experiment that had the most effect on performance was the conversion of schematic chemical formulas to syntactic complete sentences allowing them to be analysed by the parser. In addition to the improvements to the text mining approach described in Chapter 4I make suggestions in Chapter 5 for potential improvements and extensions to our full parsing approach for metabolic reaction extraction. Core focus here is the increase of recall by optimising each of the steps required for the final goal of extracting metabolic reactions from the text. Some of the discussed improvements are to increase the coverage of the used thesauri, possibly with specialist thesauri depending on the analysed literature. Another potential target is the grammar, where there is still room to increase parsing success by taking into account the characteristics of biomedical language. On a different level are suggestions to include some form of anaphora resolution and across sentence boundary search to increase the amount of information extracted from literature.
In the second part of Chapter 5I make suggestions as to how to maximise the information gained from the text mining results. One of the first steps should be integration with other biomedical databases to allow integration with existing knowledge about metabolic reactions and other biological entities. Another aspect is some form of ranking or weighting of the results to be able to distinguish between high quality results useful for automated analyses and lower quality results still useful for manual approaches. Furthermore I provide a perspective on the necessity of computational literature analysis in the form of text mining. The main reasoning here is that human annotators cannot keep up with the amount of publications so that some form of automated analysis is unavoidable. Lastly I discuss the role of text mining in bioinformatics and with that also the accessibility of both text mining results and the literature resources necessary to create them. An important requirement for the future of text mining is that the barriers around high-throughput access to literature for text mining applications have to be removed. With regards to accessing text mining results, there is a long way to go for many applications, including ours, before they can be used directly by biologists. A major factor is that these applications rarely feature a suitable user interface and easy to use setup.
To conclude, I see the main role of a text mining system like ours mainly in gathering evidence for existing knowledge and giving insights into the nuances of the research landscape of a given topic. When using the results of our reaction extraction system for the identification of ‘new’ reactions it is important to go back to the actual evidence presented for extra validations and to cross-validate the predictions with other resources or experiments. Ideally text mining will be used for generation of hypotheses, in which the researcher uses text mining findings to get ideas on, in our case, new connections between metabolites and enzymes; subsequently the researcher needs to go back to the original texts for further study. In this role text mining is an essential tool on the workbench of the molecular biologist.
Enzymatic modification and characterization of xanthan
Kool, M.M. - \ 2014
Wageningen University. Promotor(en): Harry Gruppen, co-promotor(en): Henk Schols. - Wageningen : Wageningen University - ISBN 9789461738653 - 136
xanthan - modificatie - enzymen - karakterisering - xanthan - modification - enzymes - characterization
In this thesis an enzymatic approach for the modification and characterization of xanthans was introduced. Complete backbone degradation of xanthan by cellulases was obtained independent on the molar composition of a xanthan sample. It was shown that only xanthan segments that occurred in a disordered xanthan conformation were susceptible to enzymatic backbone degradation. HILIC-ELSD-MS analysis revealed the presence of six different xanthan repeating units (RUs). All RUs consisted of the same pentasaccharide structure, with different acetyl and pyruvate substitution patterns. Interestingly the presence of an acetyl group at the O-6 position of the outer mannose unit was shown. Analysis of 5 xanthan samples showed that 5–19% of all acetyl groups present are positioned on the outer mannose. Furthermore, the relative abundance of the RUs present in xanthan samples can vary, even when their molar compositions are the same.
Analysis of the transitional behavior of xanthan based on the enzymatic release of the six types of RUs showed that the acetyl groups on the outer mannose, and not on the inner mannose, as was previously reported, are responsible for the stabilization of xanthans conformation. It was proposed that acetylation of the outer mannose also determines the functional properties of a xanthan solution. Furthermore, it was postulated that 1) The RUs that are either acetylated on the outer mannose units or solely acetylated on the inner mannose units are block wise distributed over the xanthan molecule. 2) Pyruvylated RUs and unsubstituted RUs are randomly distributed.
Screening for xanthan modifying enzymes resulted in the discovery of the first two acetyl esterases being active towards xanthan. AXE3, a xylan acetyl esterase produced by Myceliophthora thermophila C1, showed to be specific for the removal of the acetyl groups at the inner mannose unit and was only active towards the disordered xanthan conformation. YesY, a pectin acetyl esterase produced by Bacillus subtilis strain 168, specifically removed the acetyl groups at the outer mannose units and its activity is not influenced by xanthans conformation.
Nieuwe verwaarding algenbiomassa : literatuurstudie en praktische screening van enkele ontsluitingsmethoden
Kootstra, A.M.J. ; Schipperus, R. ; Berg, W. van den; Grobben-Gaastra, S.A. ; Weide, R.Y. van der - \ 2013
Lelystad : PPO AGV (PPO rapport 555) - 26
algen - bioraffinage - biomassaconversie - celwanden - chemische behandeling - enzymen - biobased economy - algae - biorefinery - biomass conversion - cell walls - chemical treatment - enzymes
Om de eiwitten, oliën, koolhydraten, carotenoïden en andere nuttige stoffen aanwezig in algencellen los van elkaar te kunnen benutten, is het nodig om de celwanden van de algen open te breken, ofwel om de algen te ontsluiten. Uiteraard is het van belang om dit te doen op een manier die de te winnen componenten zo min mogelijk beschadigt. Dit rapport bestaat uit twee delen. Het eerste is een literatuurstudie waarin een aantal algensoorten wordt omschreven, met waar mogelijk nadruk op de celwandstructuur. Verder wordt een aantal methoden beschreven om de celwanden te openen of te verzwakken. In het tweede deel van het rapport worden een aantal uitgevoerde proeven beschreven waarbij algen zijn behandeld met enzymen, schoonmaakazijn, of ethanol.
Metabolic shifts in microorganisms: the case of Lactococcus lactis
Goel, A. - \ 2013
Wageningen University. Promotor(en): Willem de Vos; B. Teusink, co-promotor(en): D. Molenaar. - S.l. : s.n. - ISBN 9789461737342 - 184
lactococcus lactis - metabolisme - enzymactiviteit - enzymen - systeembiologie - metabolische studies - lactococcus lactis - metabolism - enzyme activity - enzymes - systems biology - metabolic studies
A commonly observed organismal response to changing growth rate is a metabolic shift from one mode of metabolism to another. This phenomenon is potentially interesting from a fundamental and industrial perspective because it can influence cellular choices and can limit the capacity of industrial microorganisms to channel nutrients to desired products. The mechanistic cause of the metabolic shift may vary between species, but the presence of such shifts from bacteria to man suggests functional relevance, which may be understood through an evolutionary perspective. One of the many existing hypotheses (reviewed in Chapter 2) states that protein investment costs affect the metabolic strategy employed, and that the implemented strategy is the result of a cost-benefit analysis. To test this experimentally, we performed a global multi-level analysis using the model lactic acid bacterium Lactococcus lactis subsp. cremoris MG1363, which shows a distinct, anaerobic version of the bacterial Crabtree/Warburg effect: at low growth rates it produces “mixed-acids” (acetate, formate and ethanol) and at high growth rates it produces predominantly lactate from glucose.
We first standardized growth conditions and established an in vivo–like enzyme assay medium mimicking the intracellular environment for enzyme activity measurements of growing cells of L. lactis (Chapter 3). With standardized experimental procedures we characterized at multiple cellular levels, glucose-limited chemostat cultures of L. lactis at various growth rates. More than a threefold change in growth rate was accompanied by metabolic rerouting with, surprisingly, hardly any change in transcription, protein ratios, and enzyme activities (Chapter 4). Even ribosomal proteins, constituting a major investment of cellular machinery, scarcely changed. Thus, contrary to the original hypothesis, L. lactis displays a strategy where its central metabolism appears always prepared for high growth rate and it primarily employs the regulation of enzyme activity rather than alteration of gene expression. Only at the highest growth rate and during batch growth – conditions associated with glucose excess – we observed down-regulated stress protein levels and up-regulated glycolytic protein levels. We conclude from this that for glucose, transcription and protein expression largely follow a binary feast / famine logic in L. lactis.
To delve deeper into the mechanism of regulation of the shift in L. lactis, we tested a mixed-acid fermentative lactose-utilizing L. lactis MG1363 derivative and showed that there is a strong positive correlation between glycolytic flux and the extent of homolactic fermentation: a correlation caused by metabolic regulation (Chapter 5). We subsequently provided new evidence for a causal relationship between the concentration of the glycolytic intermediate, fructose-1,6-bisphosphate (FBP) and the metabolic shift. We showed that 2,5-anhydromannitol, which converts to a non-metabolizable FBP analogue in vivo, almost doubles the flux towards lactate when taken up by the cells. In vitro the activating effect of the analogue on lactate dehydrogenase is similar to native FBP, whereas it had no effect on the enzyme phosphotransacetylase (part of the mixed-acid pathway). The activation concentration of the analogue, however, is much lower than normal intracellular FBP concentrations. This may imply that the activation of lactate dehydrogenase in vivo requires a much higher concentration of FBP, but this remains to be resolved. We subsequently put the regulatory relationships of glycolytic flux, FBP, the redox potential and allosteric effectors on enzymes of the glycolytic and downstream pathways together in a mathematical model to test and investigate whether these interactions can explain the metabolic shift (Chapter 6). Although the model was not able to consistently fit combined data from the chemostats at various dilution rates, and in vivo–NMR data of glucose pulsed non-growing cells, we found for the best fitted model that the parameters most influencing the metabolic shift were those involved in regulation by FBP and inorganic phosphate.
In conclusion, L. lactis seems to be always prepared for high growth rate as it carries a high overcapacity of enzymes, a property retained even after evolving for 800 generations under constant environmental conditions. Moreover, its growth rate-related metabolic shift does not appear to be an outcome of growth-rate optimization with protein cost as a major driver. At the mechanistic level, the choice of the strategy is regulated via alterations in metabolite levels, with FBP (and probably phosphate) exerting a central role.
Fate of rapeseed meal polysaccharides during digestion in pigs and poultry : effect of processing and enzyme addition
Pustjens, A.M. - \ 2013
Wageningen University. Promotor(en): Harry Gruppen, co-promotor(en): Mirjam Kabel; Walter Gerrits. - S.l. : s.n. - ISBN 9789461736604 - 184
raapzaad - raapzaadmeel - polysacchariden - spijsvertering - varkens - pluimvee - voedermiddelbewerking - enzymen - rapeseed - rapeseed oilmeal - polysaccharides - digestion - pigs - poultry - feed processing - enzymes
In this thesis, the fate of non-starch polysaccharides (NSP) from rapeseed meal (RSM) during fermentation in vitro and in vivo was studied. The aim was to understand and improve the fermentation of NSP from RSM in poultry and pigs, by processing and enzyme addition. First, the NSP-structures in RSM were characterized as being branched arabinan, arabinogalactan type II, homogalacturonan, glucurono-xylan, XXGG- and XXXG-type xyloglucan, and cellulose. Second, RSM was processed using shear, heat, and acid prior to in vitro incubation, in the presence or absence of pectolytic enzymes. Acid-treatment combined with pectolytic enzymes was the best option to improve NSP-solubilization in vitro. Unprocessed and acid-extruded RSM with or without addition of enzymes were fed to broilers. In broilers, 22% of the NSP in unprocessed RSM could be fermented, which only significantly improved to 38% by addition of commercial pectolytic enzymes. In broilers’ excreta, XXXG-type xyloglucan, (glucurono-)xylan, arabinan, and cellulose remained unfermented. Unprocessed and acid-extruded RSM was also fed to growing pigs and NSP-fermentation was followed along the digestive tract. In pigs, at the terminal ileum 22% of the NSP was cumulatively fermented and total tract around 70% was fermented. Acid-extrusion improved total tract NSP-fermentability in pigs numerically by 4% points. Water-soluble carbohydrates were nearly completely fermented. In the feces some rhamnogalacturonan, (branched) arabinan, linear xylan, XXXG-type xyloglucan, galactomannan, and cellulose remained. Surprisingly, during alkaline extraction of the broilers’ excreta and pigs’ feces, around 40% (w/w) of the insoluble carbohydrates was released as glucosyl- and/or uronyl-rich carbohydrates, probably originally present via ester-linkages or hydrogen-bonding within the cellulose-lignin network. These linkages are expected to hinder complete NSP-fermentation.
Targeted discovery and functional characterisation of complex-xylan degrading enzymes
Gool, M.P. van - \ 2012
Wageningen University. Promotor(en): Harry Gruppen, co-promotor(en): Henk Schols. - S.l. : s.n. - ISBN 9789461733528 - 224
enzymen - xylaan - xylanolytische micro-organismen - myceliophthora thermophila - bodemschimmels - enzymes - xylan - xylanolytic microorganisms - myceliophthora thermophila - soil fungi
This thesis describes the development of a screening method to discover efficient hemicellulase producers in a wide range of fungi. The method is based on the potential of soil fungi to degrade soluble and insoluble xylan-rich substrates, by assigning various individual enzyme activities. Released mono- and oligosaccharides were monitored by high performance anion exchange chromatography and matrix assisted laser desorption/ionisation time-of-flight mass spectrometry. In addition, the released amounts of acetic acid were measured. Fungal strains, grown on wheat straw, with an efficient production of hemicellulolytic enzymes were Aspergillus lentulus, Penicllium pinophilum, and Trichoderma harzianum.
Next, two glycoside hydrolase (GH) family 10 endo-xylanases and two GH11 endo-xylanases of Myceliophthora thermophila C1 (C1) were subjected to a detailed characterisation. The degradation products from xylan-rich substrates, varying in type and degree of substituents, displayed that both intra- and interfamily differences occurred for these endo-xylanases. The different degradation profiles were correlated with a difference in the predicted secondary protein structure of the GH10 xylanases and with a minor difference in the primary protein structure of the GH11 xylanases. An additional cellulose binding domain attached to the endo-xylanases did not enhance the degradation of insoluble substrates.
In addition, synergy studies were performed on acetyl (xylan) esterases with glucuronidases. A concerted action of acetyl (xylan) esterases from different carbohydrate esterase families as well as synergy with glucuronidases of different GH families was observed.
Characterising the cellulose synthase complexes of cell walls
Mansoori Zangir, N. - \ 2012
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Luisa Trindade. - S.l. : s.n. - ISBN 9789461732958 - 162
planten - celwanden - cellulose - biosynthese - enzymen - enzymactiviteit - eiwitten - katalyse - genetische kartering - genomica - plants - cell walls - cellulose - biosynthesis - enzymes - enzyme activity - proteins - catalysis - genetic mapping - genomics
One of the characteristics of the plant kingdom is the presence of a structural cell wall. Cellulose is a major component in both the primary and secondary cell walls of plants. In higher plants cellulose is synthesized by so called rosette protein complexes with cellulose synthases (CESAs) as the catalytic subunits of the complex. The objective of the research presented in this thesis was to generate more in-depth knowledge in cellulose biosynthesis and to this aim better characterize and understand the cellulose synthase complex and its components by notably investigating the similarities and differences between the CESAs in the primary and secondary cellulose complex and identifying the various interacting proteins forming the complex in the plant cell wall. KORRIGAN and specific isoforms of sucrose synthase were shown to be co-localized and physically interact with the CESAs in the Cellulose Synthase Complex at the plasma membrane supporting their participation in cellulose biosynthesis in Arabidopsis.
Enzyme-catalyzed modification of poly(ethersulfone) membranes
Nady, N. - \ 2012
Wageningen University. Promotor(en): Remko Boom; Han Zuilhof, co-promotor(en): Karin Schroen; Maurice Franssen. - S.l. : s.n. - ISBN 9789461731456 - 172
membranen - oppervlakteverandering - laccase - enzymen - kunststoffen - membranes - surface modification - laccase - enzymes - plastics
The robustness of a membrane is determined by the properties of the base polymer and the functionality of its surface. One of the most popular polymers used for membrane preparation is polyethersulfone (PES), which has excellent thermo-physical properties, but the surface properties are in need of improvement to reduce membrane fouling by adsorption of e.g. protein and live cells, which cause sever flux decline during filtration. Therefore, it is not strange that a wide range of modification methods has been published to reduce surface hydrophobicity of PES membranes. However, the methods that are currently suggested are all rather offer random control over the resulting surface structure and may be environmentally adverse
This study presents enzyme-initiated grafting of PES membranes as the first successful example of an environmentally friendly modification of PES membranes. Various phenolic acids, such as 4-hydroxybenzoic acid and gallic acid (3,4,5-trihydroxybenzoic acid), were coupled to the membrane in aqueous medium at room temperature using laccase from Trametes versicolor as catalyst. This enzyme is able to oxidize phenolic compounds to their corresponding free radicals that are subsequently grafted onto PES membranes, introducing polar groups (OH, COOH) on the membrane surface by formation of a covalent C-O linkage as was proven by spin density calculations and IRRAS.
We succeed in altering the surface properties of PES membranes using laccase-catalyzed modification method. It was found that the surface structure or shape that can be tuned through both the modification conditions and the modifier structure, has a significant role in prevention of adsorption rather than surface hydrophilicity as is often assumed. Membrane flux is hardly influenced (10% reduction), and foulant (e.g., bovine serum albumin, dextrin, tannin, and pathogenic bacterium Listeriamonocytogenes) repellence is greatly increased.
In conclusion, the enzyme-catalyzed modification method shows a remarkable flexibility, and allows careful tuning of the membrane properties in such a way that membrane fouling can be suppressed. Besides, the modification method does not influence the bulk properties of the membrane adversely, the modification layer is resistant to a wide range of pH, and the costs of this modification on industrial scale are reasonable, which makes this modification method an interesting eco-friendly alternative to currently used methods.