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Engineering Geobacillus thermodenitrificans to introduce cellulolytic activity; expression of native and heterologous cellulase genes
Daas, Martinus J.A. ; Nijsse, Bart ; Weijer, Antonius H.P. van de; Groenendaal, Bart W.A.J. ; Janssen, Fons ; Oost, John van der; Kranenburg, Richard van - \ 2018
BMC Biotechnology 18 (2018)1. - ISSN 1472-6750
CBP - Cellulase - Geobacillus - Metagenome - β-Xylosidase
Background: Consolidated bioprocessing (CBP) is a cost-effective approach for the conversion of lignocellulosic biomass to biofuels and biochemicals. The enzymatic conversion of cellulose to glucose requires the synergistic action of three types of enzymes: exoglucanases, endoglucanases and β-glucosidases. The thermophilic, hemicellulolytic Geobacillus thermodenitrificans T12 was shown to harbor desired features for CBP, although it lacks the desired endo and exoglucanases required for the conversion of cellulose. Here, we report the expression of both endoglucanase and exoglucanase encoding genes by G. thermodenitrificans T12, in an initial attempt to express cellulolytic enzymes that complement the enzymatic machinery of this strain. Results: A metagenome screen was performed on 73 G. thermodenitrificans strains using HMM profiles of all known CAZy families that contain endo and/or exoglucanases. Two putative endoglucanases, GE39 and GE40, belonging to glucoside hydrolase family 5 (GH5) were isolated and expressed in both E. coli and G. thermodenitrificans T12. Structure modeling of GE39 revealed a folding similar to a GH5 exo-1,3-β-glucanase from S. cerevisiae. However, we determined GE39 to be a β-xylosidase having pronounced activity towards p-nitrophenyl-β-d-xylopyranoside. Structure modelling of GE40 revealed its protein architecture to be similar to a GH5 endoglucanase from B. halodurans, and its endoglucanase activity was confirmed by enzymatic activity against 2-hydroxyethylcellulose, carboxymethylcellulose and barley β-glucan. Additionally, we introduced expression constructs into T12 containing Geobacillus sp. 70PC53 endoglucanase gene celA and both endoglucanase genes (M1 and M2) from Geobacillus sp. WSUCF1. Finally, we introduced expression constructs into T12 containing the C. thermocellum exoglucanases celK and celS genes and the endoglucanase celC gene. Conclusions: We identified a novel G. thermodenitrificans β-xylosidase (GE39) and a novel endoglucanase (GE40) using a metagenome screen based on multiple HMM profiles. We successfully expressed both genes in E. coli and functionally expressed the GE40 endoglucanase in G. thermodenitrificans T12. Additionally, the heterologous production of active CelK, a C. thermocellum derived exoglucanase, and CelA, a Geobacillus derived endoglucanase, was demonstrated with strain T12. The native hemicellulolytic activity and the heterologous cellulolytic activity described in this research provide a good basis for the further development of G. thermodenitrificans T12 as a host for consolidated bioprocessing.
Complete Genome Sequence of Geobacillus thermodenitrificans T12, A Potential Host for Biotechnological Applications
Daas, Tijn ; Vriesendorp, Bastienne ; Weijer, Tom van de; Oost, John van der; Kranenburg, Richard van - \ 2018
Current Microbiology 75 (2018)1. - ISSN 0343-8651 - p. 49 - 56.
In attempt to obtain a thermophilic host for the conversion of lignocellulose derived substrates into lactic acid, Geobacillus thermodenitrificans T12 was isolated from a compost heap. It was selected from over 500 isolates as a genetically tractable hemicellulolytic lactic acid producer, requiring little nutrients. The strain is able to ferment glucose and xylose simultaneously and can produce lactic acid from xylan, making it a potential host for biotechnological applications. The genome of strain T12 consists of a 3.64 Mb chromosome and two plasmids of 59 and 56 kb. It has a total of 3.676 genes with an average genomic GC content of 48.7%. The T12 genome encodes a denitrification pathway, allowing for anaerobic respiration. The identity and localization of the responsible genes are similar to those of the denitrification pathways found in strain NG80-2. The hemicellulose utilization (HUS) locus was identified based on sequence homology against G. stearothermophilus T-6. It appeared that T12 has all the genes that are present in strain T-6 except for the arabinan degradation cluster. Instead, the HUS locus of strain T12 contains genes for both an inositol and a pectate degradation pathway. Strain T12 has complete pathways for the synthesis of purine and pyrimidine, all 20 amino acids and several vitamins except D-biotin. The host-defense systems present comprise a Type II and a Type III restriction-modification system, as well as a CRISPR-Cas Type II system. It is concluded that G. thermodenitrificans T12 is a potentially interesting candidate for industrial applications.
Lignocellulolytic capacities of Geobacillus thermodenitrificans: towards consolidated bioprocessing
Daas, Martinus J.A. - \ 2017
University. Promotor(en): John van der Oost; Richard van Kranenburg. - Wageningen : Wageningen University - ISBN 9789463431644 - 180
lactic acid - thermophiles - geobacillus - processing - bioenergy - melkzuur - thermofielen - verwerking - bio-energie
The growing demand for consumables and energy, combined with increasing consciousness over environmental issues like global warming, faces us with the challenge to find alternatives for fossil resources. Alternative production methods for energy, like windmills, solar panels and hydroelectricity plants, are far developed and have become economically competitive to fossil resourcebased production processes. However, the production of many (bulk) chemicals and products is still dominated by the petroleum industry. One such chemical is lactic acid, a fermentation product of many bacteria and a compound that is gaining interest as a building block for poly lactic acid (PLA). PLA is a polymer used to produce bioplastics, and thereby provides an alternative to petroleumbased plastic production. As described in Chapter 1, economically feasible production of lactic acid is envisioned through consolidated bioprocessing (CBP). In a CBP process, pretreated lignocellulosic biomass is hydrolyzed to fermentable sugars and those sugars are subsequently fermented to desired product in one reaction vessel. The organism of choice for this hydrolyzation and fermentation is preferentially a thermophile, capable of enzyme production and lactic acid fermentation. Species from the genus Geobacillus have many of the desired characteristics, and in Chapter 2 we have enriched and isolated facultative anaerobic (hemi)cellulolytic Geobacillus strains from compost samples. By selecting for growth on both cellulose and xylan, 94 strains were isolated. Subsequent screening for lactic acid production was carried out from C6 and C5 sugar fermentations and a selection of the best lactic acid producers was made. The denitrifying Geobacillus thermodenitrificans T12 was selected for further research and was rendered genetically accessible with a transformation efficiency of 1.7×105 CFU/µg of plasmid DNA. In fermentations on a mixture of glucose and xylose, a total of 20.3 g of lactic acid was produced with a yield of 0.94 g product/g sugar consumed. In addition, we demonstrated that strain T12 is capable of direct conversion of beechwood xylan to mainly lactic acid in minimal media. Chapter 3 describes the genome sequencing and several features of G. thermodenitrificans T12. The genome of strain T12 consists of a 3.64 Mb chromosome and two plasmids of 59 kb and 56 kb. It has a total of 3.676 genes with an average genomic GC content of 48.7%. The T12 genome encodes a denitrification pathway, allowing for anaerobic respiration. The identity and localization of the responsible genes is similar to those of the denitrification pathways found in strain NG80-2. The host-defence systems present comprise a Type II and a Type III restriction-modification system, as well as a CRISPR-Cas Type II system that could potentially be exploited as a genome editing tool for thermophiles. Furthermore, the hemicellulose utilisation (HUS) locus of strain T12 appeared to have orthologues for all the genes that are present in strain T-6 except for the arabinan degradation cluster. Instead, the HUS locus of strain T12 contains genes for both an inositol and a pectate degradation pathway. The HUS-locus associated gene, GtxynA1, encodes an extracellular endoxylanase of strain T12, and belongs to the family 10 glycoside hydrolases (GH10). In Chapter 4, we describe the cloning, expression and characterization of GtXynA1. The recombinant endoxylanase was purified to homogeneity and showed activity between 40°C and 80°C, with an optimum activity at 60°C, while being active between pH 3.0 to 9.0 with an optimum at pH 6.0. Its thermal stability was high and GtXynA1 showed 85% residual activity after 1 h of incubation at 60°C. Highest activity was demonstrated towards wheat arabinoxylan (WAX), beechwood xylan (BeWX) and birchwood xylan (BiWX). GtXynA1 can degrade WAX and BeWX producing mainly xylobiose and xylotriose. To determine its mode of action, we compared the hydrolysis products generated by GtXynA1 with those from the well-characterized GH10 endoxylanase produced from Aspergillus awamori (AaXynA). The main difference in the mode of action between GtXynA1 and AaXynA on WAX is that GtXynA1 is less hindered by arabinosyl substituents and can therefore release shorter oligosaccharides. The extensive hydrolysis of branched xylans makes this enzyme particularly suited for the conversion of a broad range of lignocellulosic substrates.
The enzymatic conversion of cellulose to glucose requires the synergistic action of three types of enzymes: exoglucanases, endoglucanases and β-glucosidases. The thermophilic, hemicellulolytic Geobacillus thermodenitrificans T12 was shown to be a potential candidate for CBP but lacks the desired endo and exoglucanases needed for the conversion of cellulose. In Chapter 5 we report the heterologous expression of endoglucanases and exoglucanases by G. thermodenitrificans T12, in an attempt to complement the enzymatic machinery of this strain and its suitability for consolidated bioprocessing. A metagenome screen was performed on the metagenome of 73 G. thermodenitrificans strains using HMM profiles of all known CAZy families that contain endo and/or exoglucanases. Two putative endoglucanases, GE39 and GE40, belonging to glucoside hydrolase family 5 were isolated and expressed in both E. coli and G. thermodenitrificans T12. Structure modeling of GE39 revealed a folding similar to a GH5 exo-1,3-βglucanase from S. cerevisiae. However, we determined GE39 to be a β-xylosidase having most activity towards p-nitrophenyl-β-dxylopyranoside. Structure modelling of GE40 revealed a protein architecture similar to a GH5 endoglucanase from B. halodurans, and its endoglucanase activity was confirmed by enzymatic analysis against 2-HE-cellulose, CM-cellulose and barley β-glucan. In addition, we successfully expressed the earlier characterized Geobacillus sp. 70PC53 endoglucanase celA and the C. thermocellum exoglucanase celK in strain G. thermodenitrificans T12. The native hemicellulolytic activity and the heterologous cellulolytic activity described in this research provide a good basis for the further development of Geobacillus thermodenitrificans T12 as a host for consolidated bioprocessing. In Chapter 6, we provided more insight in the genetic variation of the hemicellulolytic utilization cluster of G. thermodenitrificans. This variation is far greater than described before and gives ample opportunities for the further development of Geobacillus spp. for hemicellulose degradation. The production of cellulases in Geobacillus species is demonstrated to be successful, and we have expanded on that knowledge with the expression of both endo and exoglucanases from C. thermocellum. However, in line with previous studies, direct cellulose fermentation by geobacilli is not yet achieved, most likely due to insufficient cellulase production and/or secretion. With a rapidly expanding genetic toolbox for thermophiles, now including a thermostable Cas9, we expect that the successful development of Geobacillus spp. for consolidated bioprocessing is just a matter of time.
Biochemical characterization of the xylan hydrolysis profile of the extracellular endo-xylanase from Geobacillus thermodenitrificans T12
Daas, Tijn ; Martínez, Patricia Murciano ; Weijer, Tom van de; Oost, John van der; Vos, Willem M. de; Kabel, Mirjam A. ; Kranenburg, Richard van - \ 2017
BMC Biotechnology 17 (2017)1. - ISSN 1472-6750
Biotechnology - Endo-xylanase - Geobacillus - Thermophile - Xylan
Background: Endo-xylanases are essential in degrading hemicellulose of various lignocellulosic substrates. Hemicellulose degradation by Geobacillus spp. is facilitated by the hemicellulose utilization (HUS) locus that is present in most strains belonging to this genus. As part of the HUS locus, the xynA gene encoding an extracellular endo-xylanase is one of the few secreted enzymes and considered to be the key enzyme to initiate hemicellulose degradation. Several Geobacillus endo-xylanases have been characterized for their optimum temperature, optimum pH and generation of degradation products. However, these analyses provide limited details on the mode of action of the enzymes towards various substrates resulting in a lack of understanding about their hydrolytic potential. Results: A HUS-locus associated gene (GtxynA1) from the thermophile Geobacillus thermodenitrificans T12 encodes an extracellular endo-xylanase that belongs to the family 10 glycoside hydrolases (GH10). The GtxynA1 gene was cloned and expressed in Escherichia coli. The resulting endo-xylanase (termed GtXynA1) was purified to homogeneity and showed activity between 40 °C and 80 °C, with an optimum activity at 60 °C, while being active between pH 3.0 to 9.0 with an optimum at pH 6.0. Its thermal stability was high and GtXynA1 showed 85% residual activity after 1 h of incubation at 60 °C. Highest activity was towards wheat arabinoxylan (WAX), beechwood xylan (BeWX) and birchwood xylan (BiWX). GtXynA1 is able to degrade WAX and BeWX producing mainly xylobiose and xylotriose. To determine its mode of action, we compared the hydrolysis products generated by GtXynA1 with those from the well-characterized GH10 endo-xylanase produced from Aspergillus awamori (AaXynA). The main difference in the mode of action between GtXynA1 and AaXynA on WAX is that GtXynA1 is less hindered by arabinosyl substituents and can therefore release shorter oligosaccharides. Conclusions: The G. thermodenitrificans T12 endo-xylanase, GtXynA1, shows temperature tolerance up to 80 °C and high activity to a variety of xylans. The mode of action of GtXynA1 reveals that arabinose substituents do not hamper substrate degradation by GtXynA1. The extensive hydrolysis of branched xylans makes this enzyme particularly suited for the conversion of a broad range of lignocellulosic substrates.
Thermostable cas9 nucleases
Oost, J. van der; Daas, M.J.A. ; Kengen, S.W.M. ; Vos, W.M. de - \ 2016
Octrooinummer: WO2016198361, verleend: 2016-12-15.
Thermostable Cas9 nucleases. The present invention relates to the field of genetic engineering and more particularly to nucleic acid editing and genome modification. The present invention provides an isolated Cas protein or polypeptide fragment thereof having an amino acid sequence of SEQ ID NO: 1 or a sequence of at least 77% identity therewith, wherein the Cas protein or polypeptide is capable of DNA cleavage at a temperature in the range 50°C and 100°C inclusive. The invention further provides isolated nucleic acid molecules encoding said Cas9 nucleases, expression vectors and host cells. The Cas9 nucleases disclosed herein provide novel tools for genetic engineering at elevated temperatures and are of particular value in the genetic manipulation of thermophilic organisms; particularly microorganisms.
Isolation of a genetically accessible thermophilic xylan degrading bacterium from compost
Daas, Tijn ; Weijer, Tom van de; Vos, Willem M. de; Oost, John van der; Kranenburg, Richard van - \ 2016
Biotechnology for Biofuels 9 (2016). - ISSN 1754-6834
CMC - Compost - Electroporation - Fermentation - Geobacillus - Lactic acid - Thermophile - Xylan
Background: Due to the finite nature of global oil resources we are now faced with the challenge of finding renewable resources to produce fuels and chemicals in the future. Lactic acid has great potential as a precursor for the production of bioplastics alternatives to conventional plastics. Efficient lactic acid fermentation from non-food lignocellulosic substrates requires pretreatment and saccharification to generate fermentable sugars. A fermentation process that requires little to no enzyme additions, i.e. consolidated bioprocessing would be preferred and requires lactic acid-producing organisms that have cellulolytic and/or hemicellulolytic activity. Results: To obtain candidate production strains we have enriched and isolated facultative anaerobic (hemi) cellulolytic bacterial strains from compost samples. By selecting for growth on both cellulose and xylan, 94 Geobacillus strains were isolated. Subsequent screening for lactic acid production was carried out from C6 and C5 sugar fermentations and a selection of the best lactic acid producers was made. The denitrifying Geobacillus thermodenitrificans T12 was selected for further research and was rendered genetically accessible. In fermentations on a mixture of glucose and xylose, a total of 20.3 g of lactic acid was produced with a yield of 0.94 g product/g sugar consumed. In addition, strain T12 is capable of direct conversion of beech wood xylan to mainly lactic acid in minimal media. Conclusions: We have demonstrated that G. thermodenitrificans T12 is genetically accessible and produces lactic acid as its main fermentation product on glucose, xylose and a mixture thereof. Strain T12 was additionally used for the direct conversion of xylan to lactic acid. The genetic accessibility of the T12 strain provides a solid basis for the development of this strain into a host for consolidated bioprocessing of biomass to lactic acid.
Isolation and Screening of Thermophilic Bacilli from Compost for Electrotransformation and Fermentation: Characterization of Bacillus smithii ET 138 as a New Biocatalyst
Bosma, E.F. ; Weijer, A.H.P. van de; Daas, M.J.A. ; Oost, J. van der; Vos, W.M. de; Kranenburg, R. van - \ 2015
Applied and Environmental Microbiology 81 (2015)5. - ISSN 0099-2240 - p. 1874 - 1883.
genetic tool development - lactic-acid - simultaneous saccharification - clostridium-thermocellum - industrial platform - ethanol - lignocellulose - coagulans - bacteria - licheniformis
Thermophilic bacteria are regarded as attractive production organisms for cost-efficient conversion of renewable resources to green chemicals, but their genetic accessibility is a major bottleneck in developing them into versatile platform organisms. In this study, we aimed to isolate thermophilic, facultatively anaerobic bacilli that are genetically accessible and have potential as platform organisms. From compost, we isolated 267 strains that produced acids from C5 and C6 sugars at temperatures of 55°C or 65°C. Subsequently, 44 strains that showed the highest production of acids were screened for genetic accessibility by electroporation. Two Geobacillus thermodenitrificans isolates and one Bacillus smithii isolate were found to be transformable with plasmid pNW33n. Of these, B. smithii ET 138 was the best-performing strain in laboratory-scale fermentations and was capable of producing organic acids from glucose as well as from xylose. It is an acidotolerant strain able to produce organic acids until a lower limit of approximately pH 4.5. As genetic accessibility of B. smithii had not been described previously, six other B. smithii strains from the DSMZ culture collection were tested for electroporation efficiencies, and we found the type strain DSM 4216T and strain DSM 460 to be transformable. The transformation protocol for B. smithii isolate ET 138 was optimized to obtain approximately 5 × 103 colonies per µg plasmid pNW33n. Genetic accessibility combined with robust acid production capacities on C5 and C6 sugars at a relatively broad pH range make B. smithii ET 138 an attractive biocatalyst for the production of lactic acid and potentially other green chemicals
|Validation study to evaluate the reproducibility of a candidate in vitro potency assay of Newcastle disease vaccines and to establish the suitability of a candidate biological reference preparation to evaluate the reproducibility
Claassen, I.J.T.M. ; Maas, H.A. ; Oei, H.L. ; Daas, A. ; Milne, C. - \ 2004
Pharmeuropa 1 (2004). - ISSN 1684-7075 - p. 1 - 15.
Het-Energiehuis: energiebesparing via internet passend bij leefstijlen van huishoudens: eindrapport
Bottema, S. ; Klijnsma, X. ; Schönbeck, H. ; Veenstra, A. ; Burg, S.W.K. van den; Slingerland, S. ; Spaargaren, G. ; Oei, R. ; Daas, A. - \ 2003
Wageningen : Environmental Policy (ENP) - ISBN 9067547115 - 55 p.
Toward the recognition of structure-function relationships in galactomannans.
Daas, P. ; Grolle, K. ; Vliet, T. van; Schols, H.A. ; Jongh, H.H.J. de - \ 2002
Journal of Agricultural and Food Chemistry 50 (2002). - ISSN 0021-8561 - p. 4282 - 4289.
In this paper the determination of the physical/rheological characteristics is described for a series of commercial galactomannans of which the structural details have been reported previously. Both solubility of the galactomannans and rheological properties of galactomannan solutions and galactomannan/xanthan mixtures were determined. Using a statistical analysis approach an attempt was undertaken to recognize correlations between structural and rheological data. The best correlation found was between the abundance of galactose substituents at a regular distance (type of galactomannan) and the storage modulus (G') of mixed galactomannan/xanthan gels, underscoring the hypothesis that branching hinders the formation of a network with xanthan gum. Also, the G' for the group of locust bean gums correlated with the degree of blockiness, that is, the size and occurrence of nonsubstituted regions on the mannose backbone. In addition, galactomannans displayed an apparent decrease in gelling ability with increasing average molecular weight. That G' also relates to the type of galactomannan can therefore partly be attributed to differences in average molecular weight for the various galactomannan types. However, within the series of locust bean gums tested, also an increase of G' with molecular weight was observed. This can be explained by the decreasing number of loose ends of the polymers and the concomitant increasing efficiency in network participation with increasing molecular weight.
|Revealing the chemical fine structure of galactomannans helps us to understand their functionality
Schols, H.A. ; Daas, P.J.H. ; Grolle, K. ; Vliet, T. van; Jongh, H.H.J. de - \ 2001
In: The eleventh gums and stabilisers for the food industry conference, Wrexham 2-6 July 2001 Wrexham : The North East Wales Institute - p. 48 - 48.
Study of methyl esther distribution in pectin with endo-polygalacturonase and high-performance size-exclusion chromatography
Daas, P.J.H. ; Voragen, A.G.J. ; Schols, H.A. - \ 2001
Biopolymers 58 (2001). - ISSN 0006-3525 - p. 195 - 203.
A method was developed that enables the study of the methyl ester distribution in the polymers of pectin on a molecular level. Endo-polygalacturonase was used to extensively degrade three 70␖ethyl esterified pectins. The molecular weight distribution of the non- and enzymatically degraded pectins was determined with high-performance size-exclusion chromatography. Next, the molecular weight distribution was converted into a degree of polymerization distribution of galacturonan fragments. Monte Carlo methods were employed for the reconstruction of the parental polymers from their enzymatic degradation products. The results for the random methyl esterified pectin revealed that the enzyme-degradable sites were indeed randomly distributed, which confirmed the correctness of the procedure developed. The two other pectins studied differed greatly in the amount of non-, low-, and high-esterified regions present in the reconstructed pectic molecules of a given molecular mass. That the approach developed is able to reveal such detailed information makes it unique. The information on the fragmental composition of pectic polymers obtained is an important addition to the study of the methyl ester distribution and the functional properties of pectin.
Nonesterified galacturonic acid sequence homology of pectins
Daas, P.J.H. ; Boxma, B. ; Hopman, A.M.C.P. ; Voragen, A.G.J. ; Schols, H.A. - \ 2001
Biopolymers 58 (2001). - ISSN 0006-3525 - p. 1 - 8.
The methyl ester distribution of pectins was studied with a recently developed enzymatic method. Endopolygalacturonase of Kluyveromyces fragilis was used to degrade pectin and the composition of the degradation products was determined with high-performance anion-exchange chromatography at pH 5. Three characteristics indicative for the distribution of nonesterified galacturonic acid residues were obtained: the percentage of nonesterified galacturonic acid residues liberated of the total number of nonesterified galacturonic acid in the undigested polymer, the proportion of nonesterified mono-, di-, and trigalacturonic acid released, and the ratio of the sum of the peak areas of methyl ester containing oligomers divided by the sum of the peak areas of the nonesterified oligomers detected. From these characteristics and the degree of methyl esterification, the mean sequence similarity of the methyl ester distributions was calculated. Computational techniques commonly employed in the determination of the sequence similarity of DNA and proteins were used to discriminate the various types of distributions found and to construct a distance tree. In general, three types of methyl ester distributions could be discerned in pectin: random, high, and blockwise esterified. This report is the first to describe a parametric approach for the comparison of the substituent distribution in polymers. The importance of this novel approach in the study of the methyl ester distribution and the functional properties of pectin is discussed
Embryonic origins of health: Long term effects of IVF in human and livestock
Boerjan, M.L. ; Daas, J.H.G. den; Dieleman, S.J. - \ 2000
Theriogenology 53 (2000). - ISSN 0093-691X - p. 537 - 547.
|Enzymes as tools for structural studies of pectins
Voragen, A.G.J. ; Daas, P.J.H. ; Schols, H.A. - \ 2000
In: Bioactive Carbohydrate Polymers, Proceedings of the Phytochemical Society of Europe, Vol. 44 : Bioactive Carbohydrate Polymers, Oslo, 1999 / Paulsen, B.S., Dordrecht : Kluwer Academic Publishers - ISBN 9780792361190 - p. 129 - 145.
Effects of different reproduction techniques: AI, MOET or IVP, on health and welfare of bovine offspring
Wagtendonk-de Leeuw, A.M. van; Mullaart, E. ; Roos, A.P.W. ; Merton, J.S. ; Daas, J.H.G. den; Kemp, B. ; Ruigh, L. de - \ 2000
Theriogenology 53 (2000)2. - ISSN 0093-691X - p. 575 - 598.
|HPLC of oligosaccharides : New developments in detection and peak identification
Schols, H. ; Kabel, M. ; Bakx, E. ; Daas, P. ; Alebeek, G.J.W.M. van; Voragen, F. - \ 2000
In: Association Andrew van Hook, Comptes Rendus, 7th Symposium International, Les séparations chromatographiques dans l'analyse et les process sucriers : 7th Symposium International, Les séparations chromatographiques dans l'analyse et les process sucriers, Reims, 2000 / M. Mathlouthi. - Reims : Association Andrew van Hook, 2000 - p. 39 - 45.
On the galactosyl distribution of commercial galactomannans
Daas, P.J.H. ; Schols, H.A. ; Jongh, H.H.J. de - \ 2000
Carbohydrate Research : an international journal 329 (2000). - ISSN 0008-6215 - p. 609 - 619.
|Determination of the distribution of non-esterified galacturonic acid in pectin with endo-polygalacturonase
Daas, P.J.H. ; Alebeek, G.J.W.M. van; Voragen, A.G.J. ; Schols, H.A. - \ 2000
In: Proceedings 'Gums and Stabilisers for the Food Industry 10' : Gums and Stabilisers for the Food Industry 10, Wrexham, Wales, 1999 / Williams, P.A., Philips, G.O., Cambridge, UK : Royal Society of Chemistry - ISBN 9780854048205 - p. 3 - 18.
Characterization of non-esterified galacturonic acid sequences in pectin with endopolygalacturonase
Daas, P.J.H. ; Voragen, A.G.J. ; Schols, H.A. - \ 2000
Carbohydrate Research : an international journal 326 (2000). - ISSN 0008-6215 - p. 120 - 129.
A method was developed that enabled the study of non-esterified galacturonic acid sequences (so-called blocks) in pectin. Endopolygalacturonase of Kluyveromyces fragilis was used to extensively degrade pectin, and the composition of the galacturonic acid molecules produced was determined with high-performance anion-exchange chromatography at pH 5. With this technique, the amount of non-esterified mono-, di-, and trigalacturonic acid released was determined. In addition, the relative amounts of methyl-esterified oligomers — up to 10 galacturonic acid residues — could be observed. By comparing the percentages of non-esterified mono-, di-, and trigalacturonic acids released, pectins with large enzyme-degradable blocks could be distinguished from pectins with small enzyme-degradable blocks. High percentages of mono- and digalacturonic acid were found for pectins containing small non-esterified blocks. The total area of all peaks corresponding to methyl-esterified oligomers was found to be indicative for the distribution of these blocks. The higher the ratio of the methyl- to non-esterified peak areas, the more closely associated blocks are present. Randomly esterified pectins, with degrees of methyl esterification of 50 and higher, contained smaller, more clustered blocks than commercial extracted pectins of comparable degrees of esterification. The approach developed enables a very detailed study of the methyl-ester distribution of pectin to be carried out and is a very important addition in the study of the functional behavior of this complex polymer