Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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Profile hidden Markov models trained on aligned KEGG Orthology sequences for enzyme annotation
Rodenburg, Y.A. ; Ridder, D. de; Govers, F. ; Seidl, M.F. - \ 2019
Wageningen University & Research
annotation - bioinformatics - enzymes - hidden Markov models - HMM - homology - KEGG - Kyoto Encyclopedia of Genes and Genomes - proteins
Profile hidden Markov models trained on aligned KEGG Orthology sequences for enzyme annotation. These HMMs were used to reconstruct metabolic networks for the manuscript: The genome of Peronospora belbahrii reveals high heterozygosity, a low number of canonical effectors and CT-rich promoters
Smullen van beschimmeld stro
Hendriks, Wouter ; Cone, John - \ 2018
biobased economy - animal nutrition - biomass - fibres - enzymes - digestibility - straw
Delicious mouldy straw : animal nutrition
Hendriks, Wouter ; Cone, John - \ 2018
biobased economy - animal nutrition - biomass - fibres - enzymes - digestibility - straw
The impact of xylanase, phytase and their combination on performance, nutrient utilization and nitrogen/energy balance in nile tilapia Oreochromis niloticus
Maas, R.M. ; Verreth, J.A.J. ; Verdegem, M.C.J. ; Dersjant-Li, Yueming ; Schrama, J.W. - \ 2017
- 4 p.
Nile tilapia - enzymes - Energy balance
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 - 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

Abstract

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 - plant breeding - genetic engineering - 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

K.A. Bednarska

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.
Heliotropium europaeum Poisoning in Cattle and Analysis of its Pyrrolizidine Alkaloid Profile
Shimshoni, J.A. ; Mulder, P.P.J. ; Bouznach, A. ; Edery, N. ; Pasval, I. ; Barel, S. ; Khaliq, M.A.E. ; Perl, S. - \ 2015
Journal of Agricultural and Food Chemistry 63 (2015)5. - ISSN 0021-8561 - p. 1664 - 1672.
metabolic-activation - senecio-jacobaea - toxicosis - livestock - enzymes - calves - milk
Pyrrolizidine alkaloids (PAs) are carcinogenic and genotoxic phytochemicals found exclusively in angiosperms. The ingestion of PA-containing plants often results in acute and chronic toxicities in man and livestock, targeting mainly the liver. During February 2014, a herd of 15-18-month-old mixed-breed beef cattle (n = 73) from the Galilee region in Israel was accidently fed hay contaminated with 12% Heliotropium europaeum (average total PA intake was 33 mg PA/kg body weight/d). After 42 d of feed ingestion, sudden death occurred over a time period of 63 d with a mortality rate of 33%. Necropsy and histopathological examination revealed fibrotic livers and moderate ascites, as well as various degrees of hyperplasia and fibrosis of bile duct epithelial cells. Elevated ¿-glutamyl-transferase and alkaline phosphatase levels were indicative of severe liver damage. Comprehensive PA profile determination of the contaminated hay and of native H. europaeum by LC-MS/MS revealed the presence of 30 PAs and PA-N-oxides, including several newly reported PAs and PA-N-oxides of the rinderine and heliosupine class. Heliotrine- and lasiocarpine-type PAs constituted 80% and 18% of the total PAs, respectively, with the N-oxides being the most abundant form (92%). The PA profile of the contaminated hay showed very strong resemblance to that of H. europaeum
Isomalto/Malto-Polysaccharide, A Novel Soluble Dietary Fiber Made Via Enzymatic Conversion of Starch
Leemhuis, H. ; Dobruchowska, J.M. ; Ebbelaar, M. ; Faber, F. ; Buwalda, P.L. ; Maarel, M.J.E.J. ; Kamerling, J.P. ; Dijkhuizen, L. - \ 2014
Journal of Agricultural and Food Chemistry 62 (2014)49. - ISSN 0021-8561 - p. 12034 - 12044.
including resistant starch - lactobacillus-reuteri 121 - structural-characterization - amylose content - chain-length - oligosaccharides - enzymes - 4,6-alpha-glucanotransferase - dextransucrase - dextranase
Dietary fibers are at the forefront of nutritional research because they positively contribute to human health. Much of our processed foods contain, however, only small quantities of dietary fiber, because their addition often negatively affects the taste, texture, and mouth feel. There is thus an urge for novel types of dietary fibers that do not cause unwanted sensory effects when applied as ingredient, while still positively contributing to the health of consumers. Here, we report the generation and characterization of a novel type of soluble dietary fiber with prebiotic properties, derived from starch via enzymatic modification, yielding isomalto/malto-polysaccharides (IMMPs), which consist of linear (a1 ¿ 6)-glucan chains attached to the nonreducing ends of starch fragments. The applied Lactobacillus reuteri 121 GTFB 4,6-a-glucanotransferase enzyme synthesizes these molecules by transferring the nonreducing glucose moiety of an (a1 ¿ 4)-glucan chain to the nonreducing end of another (a1 ¿ 4)-a-glucan chain, forming an (a1 ¿ 6)-glycosidic linkage. Once elongated in this way, the molecule becomes a better acceptor substrate and is then further elongated with (a1 ¿ 6)-linked glucose residues in a linear way. Comparison of 30 starches, maltodextrins, and a-glucans of various botanical sources, demonstrated that substrates with long and linear (a1 ¿ 4)-glucan chains deliver products with the highest percentage of (a1 ¿ 6) linkages, up to 92%. In vitro experiments, serving as model of the digestive power of the gastrointestinal tract, revealed that the IMMPs, or more precisely the IMMP fraction rich in (a1 ¿ 6) linkages, will largely pass the small intestine undigested and therefore end up in the large intestine. IMMPs are a novel type of dietary fiber that may have health promoting activity.
Galacto-oligosaccharide production with immobilized ß-galactosidase in a packed-bed reactor vs. free ß-galactosidase in a batch reactor
Warmerdam, A. ; Benjamins, E. ; Leeuw de, T.F. ; Broekhuis, T.A. ; Boom, R.M. ; Janssen, A.E.M. - \ 2014
Food and Bioproducts Processing 92 (2014)4. - ISSN 0960-3085 - p. 383 - 392.
bacillus-circulans - lactose hydrolysis - eupergit-c - covalent immobilization - enzymatic-synthesis - enzymes - supports - temperature - milk - art
We report here that the usage of immobilized enzyme in a continuous packed bed reactor (PBR) can be a good alternative for GOS production instead of the traditional use of free enzyme in a batch reactor. The carbohydrate composition of the product of the PBR with immobilized enzyme was comparable to that of the batch reactor with free enzyme. The stability of the immobilized enzyme at a lactose concentration of 38% (w/v) and at 50¿C was very high: the half-life time of the immobilized enzyme was approximately 90 days. The enzymatic productivity of GOS production using immobilized enzyme in a PBR can be more than six times higher than that of GOS production with free enzyme in a batch reactor. Besides, when aiming for an equal volumetric productivity to the batch process in designing a PBR, the volume of the PBR can be much smaller than that of the batch reactor, depending on the enzyme dosage and the run time of a single batch
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.

Two-step enzymatic fingerprinting of sugar beet pectin
Remoroza, C.A. ; Broxterman, S.E. ; Gruppen, H. ; Schols, H.A. - \ 2014
Carbohydrate Polymers 108 (2014). - ISSN 0144-8617 - p. 338 - 347.
galacturonic acid distribution - aspergillus-niger - esterified oligogalacturonides - endopolygalacturonase - quantification - degradation - oligomers - enzymes - lyase
A two-step enzymatic fingerprinting method was introduced to analyze a highly methylesterified and acetylated sugar beet pectin having a degree of methylesterification (DM) of 62 and acetylation of 30. A cocktail of pectolytic enzymes, including endo-polygalacturonase II (endo-PGII) and pectin lyase (PL), was used for the first digestion. The endo-PGII and PL resistant pectin fragments were isolated and subjected to a second digestion using fungal pectin methylesterase and endo-PGII. After the two sequential digestions, 78% of the total GalA residues present in the parental pectin were recovered as mono- and oligomers, which were used to quantitatively describe the parental SBP. For this reason, the descriptive parameters degree of blockiness (DBabs), degree of hydrolysis by PG (DHPG) and degree of hydrolysis by PL (DHPL) were established for both digestions. The first digestion revealed the presence of short blocks of nonesterified GalA residues and blocks of partly methylesterified and acetylated GalA residues in the parental SBP, in addition to blocks of highly methylesterified and acetylated GalA residues. The second digestion revealed the presence of blocks of methylesterified, partly methylesterified and/or acetylated GalA residues in a sequence not to be degradable by neither endo-PGII nor by PL. The acetyl groups were present in an blockwise manner. Application of the method to two differently prepared DM 50 SBPs showed that the two pectins differ in the ratio of blocks of nonesterified and blocks of partly methylesterified and acetylated GalA residues.
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.

Natural products – learning chemistry from plants
Staniek, A. ; Bouwmeester, H.J. ; Fraser, P.D. ; Kayser, O. ; Martens, S. ; Tissier, A. ; Krol, A.R. van der; Wessjohann, L. ; Warzecha, H. - \ 2014
Biotechnology Journal 9 (2014)3. - ISSN 1860-6768 - p. 326 - 336.
escherichia-coli - benzylisoquinoline alkaloids - saccharomyces-cerevisiae - vanillin production - synthetic biology - organic-synthesis - biosynthesis - biocatalysis - artemisinin - enzymes
Plant natural products (PNPs) are unique in that they represent a vast array of different structural features, ranging from relatively simple molecules to very complex ones. Given the fact that many plant secondary metabolites exhibit profound biological activity, they are frequently used as fragrances and flavors, medicines, as well as industrial chemicals. As the intricate structures of PNPs often cannot be mimicked by chemical synthesis, the original plant providers constitute the sole source for their industrial, large-scale production. However, sufficient supply is not guaranteed for all molecules of interest, making the development of alternative production systems a priority. Modern techniques, such as genome mining and thorough biochemical analysis, have helped us gain preliminary understanding of the enzymatic formation of the valuable ingredients in planta. Herein, we review recent advances in the application of biocatalytical processes, facilitating generation of complex PNPs through utilization of plant-derived specific enzymes and combinatorial biochemistry. We further evaluate the options of employing heterologous organisms harboring PNP biosynthetic pathways for the production of secondary metabolites of interest.
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