Analysis of the polymerization initiation and activity of Pasteurella multocida heparosan synthase PmHS2, an enzyme with glycosyltransferase and UDP-sugar hydrolase activity
Chavaroche, A.A.E. ; Broek, L.A.M. van den; Springer, J. ; Boeriu, C. ; Eggink, G. - \ 2011
Journal of Biological Chemistry 286 (2011)3. - ISSN 0021-9258 - p. 1777 - 1785.
hyaluronan synthase - chemoenzymatic synthesis - identification - biosynthesis - glycosidases - transferase - mechanism - polymers - distinct - sulfate
Heparosan synthase catalyzes the polymerization of heparosan [-4GlcUAß1-4GlcNAca1-]n by transferring alternatively the monosaccharide units from UDP-GlcUA and UDP-GlcNAc to an acceptor molecule. Details on the heparosan chain initiation by Pasteurella multocida heparosan synthase PmHS2 and its influence on the polymerization process have not been reported yet. By site directed mutagenesis of PmHS2, the single action transferases PmHS2-GlcUA+ and PmHS2-GlcNAc+ were obtained. When incubated together in the standard polymerization conditions, the PmHS2-GlcUA+/PmHS2-GlcNAc+ showed comparable polymerization properties as determined for PmHS2. We investigated the first step occurring in heparosan chain initiation by the use of the single action transferases and by studying the PmHS2 polymerization process in the presence of heparosan templates and various UDP-sugar concentrations. We observed that PmHS2 favored the initiation of the heparosan chains when incubated in the presence of an excess of UDP-GlcNAc. It resulted in a higher number of heparosan chains with a lower average molecular weight or in the synthesis of two distinct groups of heparosan chain length, in the absence or in the presence of heparosan templates, respectively. These data suggest that PmHS2 transfers GlcUA from UDP-GlcUA moiety to a UDP-GlcNAc acceptor molecule to initiate the heparosan polymerization; as a consequence not only the UDP-sugar concentration but also the amount of each UDP-sugar is influencing the PmHS2 polymerization process. In addition, it was shown that PmHS2 hydrolyzes the UDP-sugars; UDP-GlcUA being more degraded than UDP-GlcNAc. However, PmHS2 incubated in the presence of both UDP-sugars favors the synthesis of heparosan polymers over the hydrolysis of UDP-sugars
Glycosyl hydrolases from Bifidobacterium adolescentis DSM20083 : Their role in the metabolism and synthesis of oligosaccharides.
Broek, L.A.M. van den - \ 2005
Wageningen University. Promotor(en): Fons Voragen. - Wageningen : WUR - ISBN 9789085041436 - 188
bifidobacterium adolescentis - hydrolasen - glycosidasen - oligosacchariden - synthese - bifidobacterium adolescentis - hydrolases - glycosidases - oligosaccharides - synthesis
Nowadays, there is an increasing interest for food, which is health beneficial for humans. Prebiotics e.g. is used to stimulate the growth of bacteria in the gut that have a positive effect on human health. It is claimed that bifidobacteria in the colon improve health and well being of humans. The first aim of this study was to identify and characterize enzymes from Bifidobacterium adolescentis, which can degrade non-digestible oligosaccharides. To make an inventory what kind of enzymes are present it can be predicted what kind of oligosaccharides can stimulate the growth of bifidobacteria. The second aim was to synthesize new oligosaccharides by the characterized enzymes to produce new sugars, which can be used as preferentially prebiotics for Bifidobacterium adolescentis. Different enzymes were cloned and characterized and most of the enzymes were able to produce new oligosaccharides.
Oligosaccharide synthesis by the hyperthermostable b-glucosidase from Pyrococcus furiosus: kinetics and modelling
Bruins, M.E. ; Strubel, M. ; Lieshout, J.F.T. van; Janssen, A.E.M. ; Boom, R.M. - \ 2003
Enzyme and Microbial Technology 33 (2003)1. - ISSN 0141-0229 - p. 3 - 11.
escherichia-coli - enzymatic-synthesis - bacillus-circulans - hydrolysis - galactosidase - lactose - temperature - disaccharides - glycosidases - glucoamylase
Oligosaccharides can be synthesised from monosaccharides or disaccharides, using glycosidases as a catalyst. To investigate the potential of this synthesis with beta-glycosidase from Pyrococcus furiosus we determined kinetic parameters for substrate conversion and product formation from cellobiose, lactose, glucose and galactose. The obtained parameters for initial rate measurements of disaccharide conversion were also used for the interpretation of experiments in time. The model for cellobiose gave a good description of the experiments. The enzyme was found to be uncompetitively inhibited by cellobiose and competitively inhibited by glucose. Lactose conversion however, could not be modelled satisfactorily; apparently additional reactions take place. Monosaccharide condensation also yielded oligosaccharides, but much slower. The use of a hyperthermostable, enzyme was found to be positive. More substrate could be dissolved at higher temperatures, which benefited all reactions.
Oligosaccharide production with thermophilic enzymes
Bruins, M.E. - \ 2003
Wageningen University. Promotor(en): Remko Boom, co-promotor(en): Anja Janssen. - [S.I.] : S.n. - ISBN 9789058088406 - 119
oligosacchariden - productie - thermofiele micro-organismen - glycosidasen - voedselverwerking - oligosaccharides - production - thermophilic microorganisms - glycosidases - food processing
The goal of the research reported in this thesis was to develop a process concept for the tailor made production of oligosaccharides. These specific non-digestible oligosaccharides can be used as prebiotics. They promote the growth of beneficial bacteria in the gastrointestinal (GI) tract. Commercial prebiotic oligosaccharides are often not pure oligosaccharides, but mixtures. In this thesis focus is on the production of oligosaccharides of higher purity.
Our main interest was in a production process at elevated temperatures. This can have many advantages, amongst which is the possibility to increase the substrate concentration. We used a thermophilicβ-glycosidase from Pyrococcus furiosus . Enzymes from thermophilic microorganisms have unique characteristics such as high temperature-, chemical- and pH stability. Applications with thermophilic enzymes are summarised in chapter 2. The main advantages of performing processes at higher temperatures are the reduced risk of microbial contamination, lower viscosity, improved transfer rates and improved solubility of substrates. However, co-factors, substrates or products might be unstable or other side reactions may occur.
One route of oligosaccharide production is the synthesis from monosaccharides or disaccharides, using glycosidases as a catalyst. Monosaccharides can be condensated to disaccharides and disaccharides can be transglycosylated to trisaccharides. To investigate the potential of this synthesis withβ-glycosidase fromPyrococcus furiosus we determined kinetic parameters for substrate conversion and product formation from cellobiose, lactose, glucose and galactose. The obtained parameters for initial rate measurements of disaccharide conversion were also used for the interpretation of experiments in time. The model for cellobiose gave a good description of the experiments. The enzyme was found to be uncompetitively inhibited by cellobiose and competitively inhibited by glucose. Lactose conversion however, could not be modelled satisfactorily; apparently additional reactions take place. Monosaccharide condensation also yielded oligosaccharides, but much slower. The use of a hyperthermostable enzyme was found to be positive. More substrate could be dissolved at higher temperatures, which benefited all reactions. This research is described in chapter 3.
Besides the advantage of higher substrate solubility, temperature also influences enzyme kinetics. In chapter 4, the thermostable Pyrococcus furiosus-glycosidase was applied for oligosaccharide production from lactosein a kinetically controlled reaction. The experiments showed that higher temperatures are beneficial for the absolute as well as relative oligosaccharide yield.
However, at reaction temperatures of 80°C and higher, the inactivation rate of the enzyme in the presence of sugars was increased by a factor 2, compared to the inactivation rate in the absence of sugars. This increased enzyme inactivation was caused by the occurrence of Maillard reactions between the sugar and the enzyme. The browning of our reaction mixture due to Maillard reactions was modelled by a cascade of a 0 thand 1 storder reaction and related to enzyme inactivation. From these results we conclude that modification of only a small number of amino-groups already gives complete inactivation of the enzyme.
Reduction of Maillard reactions can be done by altering process conditions or through modification of the enzyme, either chemically or by altering the enzyme structure through genetic modifications. Chemical modification of the enzyme was studied. The enzyme was covalently immobilised on Eupergit. Unfortunately, the immobilisation did not reduce Maillard reactivity.
Further reaction optimisation required a down-stream processing method for oligosaccharide separation. This was also essential for the production of a pure oligosaccharide product. Two methods for oligosaccharide purification are described in chapter 5.
Oligosaccharides were produced in a condensation reaction using the -glycosidase from Pyrococcus furiosus. With a 60% (w/w) galactose solution as the substrate and oligosaccharide yield of 18% (w/w) was obtained. The feasibility of a Simulated Moving Bed (SMB) for downstream separation was investigated by modelling. The required parameters were determined experimentally with column experiments. The components could be separated with an SMB into a 91% pure product stream and a 99% pure galactose stream. This galactose stream can be recycled to the enzyme reactor. Also nanofiltration can be used for oligosaccharide purification. This system was also modelled and the results were compared to those that can be achieved with SMB.
It is also possible to produce transgalacto-oligosaccharides in a more conventional way, with lactose as a substrate. Production is much cheaper when compared to a galactose-based process. Separation of oligosaccharides from this reaction via SMB was also studied.
The size of all separation units is still considerably large and further optimisation is necessary to make a process for the production of specific high purity galacto-oligosaccharides cost-effective.
Various aspects of the process are discussed further in chapter 6. Emphasis is on the specific influence of temperature on the process and on further optimisation of the downstream processing of oligosaccharides.
Engineering of β-glycosidases from hyperthermophilic Archaea
Kaper, T. - \ 2001
Wageningen University. Promotor(en): W.M. de Vos; J. van der Oost. - S.l. : S.n. - ISBN 9789058085047 - 162
glycosidasen - thermofiele micro-organismen - eiwittechnologie - glycosidases - thermophilic microorganisms - protein engineering
Hyperthermophilic Archaea are microorganisms that grow optimally above 80°C. To be able to live at these temperature extremes their cell components display extreme resistance towards thermal degradation. This characteristic is an attractive feature for use of their enzymes in industrial processes. Examples of thermozymes with potential applications in food and pharmaceutical industry are b -Glycosidases, enzymes that specifically hydrolize b -linked glycosidic bonds, which are present in for instance cellulose or the milk sugar lactose. .
Using the b -glucosidase CelB from Pyrococcus furiosus as a model enzyme, molecular determinants of substrate recognition and catalysis in b -glycosidases have been studied by rational design and directed evolution approaches. A 3D model of CelB was established, and its active site was compared to that of a related enzyme with a distinct specificity, the 6-phospho-b -galactosidase LacG of Lactococcus lactis. The substrate specificity of CelB was adjusted by engineering a phosphate-binding site, which resulted in a significant improvement in the hydrolysis of 6-phospho-b-glycosides. In a second study, the active sites of b -glucosidase CelB was compared to that of a b -mannosidase BglB from Pyrococcus horikoshii , and the substrate affinities and activities of the two enzymes could be swapped by exchange of unique residues in their active sites.
The b -glucosidase CelB of P. furiosus was also compared to that of the related b -glycosidase LacS of the hyperthermophile Sulfolobus solfataricus. While the enzymes are very similar regarding catalytic mechanism and substrate specificity, they have not been stabilized to withstand high temperatures in the same way. While CelB is relatively sensitive to detergents, LacS is readily inactivated in the presence of salts. This strongly suggests that CelB is mainly stabilized by hydrophobic interactions, while ion-pair interactions contribute most to the stability of LacS.
In one of the first laboratory evolution studies on proteins from a hyperthermophile, CelB has been optimized for low-temperature catalysis. In several CelB mutants this was accomplished with retention of wild-type stability. Increased activity at low temperatures seemed to result from mutations that increase protein flexibility. In a second directed evolution study, the genes coding for CelB and LacS were shuffled to functional b -glycosidase hybrids. The hybrids of this DNA shuffling experiment were screened for thermostability and hydrolysis of lactose at 70 °C. Several thermostable high-performance mutants were isolated and characterized. The hybrids consisted of an N-terminal LacS stretch, followed by a CelB core. This resulted in a hybrid active site structure, which could explain the altered catalytic properties.
Finally, site-directed CelB mutants from the previous mentioned studies have been tested for their ability to catalyze oligosaccharide synthesis. Indeed, several variants showed increased yields in galacto-oligosaccharide synthesis with lactose as a substrate, compared to wild-type CelB.
The studies described in the thesis are illustrative for the differences in protein engineering by rational design versus by directed evolution. While rational design can give an initial change in activity or substrate specificity, directed evolution is more likely to be successful for fine-tuning of enzyme properties.
A molecular analysis of L-arabinan degradation in Aspergillus niger and Aspergillus nidulans
Flipphi, M.J.A. - \ 1995
Agricultural University. Promotor(en): A.J.J. van Ooyen; J. Visser. - S.l. : Flipphi - ISBN 9789054853923 - 165
aspergillus - celwanden - koolhydraten - cellulose - celmembranen - fermentatie - voedselbiotechnologie - glycosidasen - polysacchariden - genexpressie - pleiotropie - moleculaire genetica - aspergillus - cell walls - carbohydrates - cellulose - cell membranes - fermentation - food biotechnology - glycosidases - polysaccharides - gene expression - pleiotropy - molecular genetics
This thesis describes a molecular study of the genetics ofL-arabinan degradation in Aspergillus niger and Aspergillus nidulans. These saprophytic hyphal fungi produce an extracellular hydrolytic enzyme system to depolymerize the plant cell wall polysaccharideL-arabinan. Chapter 1 surveys the occurrence, properties and applications ofL-arabinanolytic enzymes (arabinases). The A.niger system, which constitutes an endolytic endo-1,5-α-L-arabinase (ABN A) and two distinct α-L-arabinofuranosidases (ABF A and ABF B), has been a frequent subject of investigation in the past and represents the best characterizedL-arabinanolytic system to date. These three enzymes are all glycosylated. Current knowledge on the induction of fungal arabinase expression is summarized in this Chapter. Furthermore, the structure of the polysaccharide substrate and its function in the plant cell wall matrix are introduced.
In Chapters 2 to 5, the cloning and characterization of the structural genes coding for the three glycosyl hydrolases from the A. nigerL-arabinan-degrading complex are described. A. niger abf A and abf B ar e the first eukaryotic ABF-encoding genes to be isolated and sequenced, abn A is the first ABN-encoding gene published. Chapter 2 reports on the isolation of the abf A gene encoding ABF A, the minor extracellular ABF. This gene could be cloned by utilizing ABF Aspecific cDNA as the probe. This cDNA was immunochemically identified from a cDNA library generated fromL-arabitol-induced myceliurn of an A. nigerD-xylulose kinase mutant. This mutant is unable to grow onL-arabitol and features enhanced expression of all three arabinases when transferred to medium containing this pentitol as sole carbon source. In Chapter 3 , the cloning of the ABN A-encoding gene (abn A) is described. This gene was isolated following the same strategy as with abf A, although a second cDNA library had to be generated first. The induction process was immunochemically monitored in order to establish the proper induction conditions for the new library. The abn A gene and the gene product were characterized by DNA sequence analyses of the cloned genomic DNA and the cDN A. The N-terminal amino acid sequences of ABN A and a CNBr-derived peptide were determined. Several transcription initiation sites and one polyadenylation site could be identified. The structural region codes for a protein of 321 amino acids and is interrupted by three introns. Extracellular ABN A consists of 302 amino acid residues with a deduced molecular weight of 32.5 kDa and a theoretical pl of 3.5. For the protein, an apparent pl of 3.0 and an apparent molecular weight of 43 kDa, determined upon SDS-PAGE, were previously reported. Chapter 4 documents the isolation and characterization of the abf B gene, coding for the major extracellular ABF. The determination of N-terminal amino acid sequences from ABF B and CNBr-generated peptides allowed the design of deoxyoligonucleotide mixtures which enabled the cloning of abf B. When utilized as primers in a polymerase chain reaction (PCR), ABF B-specific amplification products emerged, one of which was used to probe the gene. The abf B gene and the gene product were characterized by DNA sequence analyses of the cloned genomic DNA and of ABF B- specific cDNA isolated from the library described in Chapter 3. Several transcription initiation sites and one polyadenylation site could be identified. The structural region is a single open reading frame and codes for a protein of 499 amino acids. The mature enzyme consists of 481 amino acid residues with a deduced molecular weight of 50.7 kDa and a theoretical pl of 3.8. An apparent pl of 3.5 and an apparent molecular weight of 67 kDa, determined upon SDS-PAGE, were previously reported. The abf B gene product was suggested to be identical to the ABF purified and characterized by Kaji and Tagawa (Biochim Biophys Acta 207 : 456-464 (1970)). Considering the non-amino acid content of the latter protein, a molecular weight of 64 kDa could be deduced for ABF B. In Chapter 5 , the abf A gene and its gene product were characterized by DNA sequence analyses of the genomic DNA and of the cDNA for which the isolation was described in Chapter 2. The N-terminal amino acid sequences of ABF A and a CNBr-derived peptide were determined. One transcription initiation site and two polyadenylation sites could be identified. The structural region is interrupted by seven introns and codes for a protein of 628 amino acids. Mature ABF A consists of 603 amino acid residues with a deduced molecular weight of 65.4 kDa and a theoretical pl of 3.7. For this ABF, an apparent pi of 3.3 and an apparent molecular weight of 83 kDa, determined upon SDS-PAGE, were previously documented.
Although the three enzymes are all active against (1->5)-α-glycosidic bonds betweenL-arabinofuranosides, ABF A, ABF B and ABN A are genetically unrelated. ABF A was found to be N -glycosylated whereas ABF B and ABN A were not - these enzymes are only O -glycosylated. For each gene, arabinaseoverproducing strains were generated by introducing multiple gene copies in A.niger or in A.nidulans uridine auxotrophic strains through co-transformation. Transformants were isolated upon primary selection for uridine prototrophy. Subsequent overproduction of the genes introduced was demonstrated in these recombinant strains upon growth on sugar beet pulp, both immunochemically and by assaying enzyme activity. abf A was shown to be expressed in the heterologous host A.nidulans, despite the absence of an abf A gene equivalent in this organism. High-copy number A.niger abf B transformants featured impaired secretion of other extracellular proteins upon growth on sugar beet pulp. ABN A overproduction was found to be limited to approximately five times the wild-type level in A.niger abn A transformants, but not in A.nidulans transformants. Such a limitation was not observed in case of the ABFs.
In Chapters 5 and 6, the regulation ofL-arabinan degradation is addressed. The structural genes seem to be regulated mainly at the transcriptional level. Additional copies of either A13F-encoding gene in A.niger were shown to result in a reduction, but not in total silencing of the expression of the wild-type ABN Aencoding gene upon induction with either sugar beet pulp orL-arabitol ( Chapter 5 ). The reduction of the expression level of abn A correlated with the abf gene dosage. The repression effected by extra abf B gene copies was more stringent and more persistent than that elicited by additional abf A copies. Although observed with both inducers, these phenomena were more outspoken and more persistent on sugar beet pulp. Similar, but more moderate effects were observed towards the expression of the other abf gene in multiple copy abf A- and abf B-transformants. It was proposed that the abf genes titrate two distinct gene activators both involved in coordination of arabinase gene expression. However, the three genes were shown to respond differently upon a mycelial transfer toL-arabitol-containing medium, indicating that gene-specific factors are also involved. Four distinct sequence motifs were found in common in the promoter regions of the three genes. One of these elements is identical to the A.nidulans CREA-motif, which has been shown to mediate carbon catabolite repression on several A.nidulans enzyme systems. Arabinase expression in A.niger is known to be repressed in the presence ofD-glucose. Two other motifs are highly homologous to cAMP-responsive elements described in other organisms. For the fourth motif no functional analogues could be found, but the element was found to be present in several other fungal genes which are not involved inL-arabinan degradation at all. It is therefore likely that none of these common elements confer system-specific regulation.
The presumed involvement ofL-arabitol in the induction process of fungal arabinases was further emphasized by the induction characteristics of an A. nidulans mutant unable to grow on the end-product ofL-arabinan degradation,L-arabinose, nor onL-arabitol ( Chapter 6).L-Arabitol is an intermediate ofL-arabinose catabolism in Aspergilli. This mutant was shown to lack NAD +-dependentL-arabitol dehydrogenase activity resulting inL-arabitol accumulation, both intracellularly and in the culture medium, wheneverL-arabinose is present. Upon submerged growth on various carbon sources in the presence ofL-arabinose, the mutant featured enhanced expression of the enzymes involved in extracellularL-arabinan degradation, and of those of the intracellularL-arabinose catabolism. The co-substrates on which the mutant secreted large amounts of arabitol simultaneously exhibited high arabinase expression and featured reduced growth.L-Arabitol secretion and enzyme production were also observed on a mixed carbon source ofD-glucose andL-arabinose, resulting in normal growth. Hence, in the presence ofL- arabinose, the carbon catabolite repression conferred byD-glucose in the wild-type, is overruled in the mutant.
In Chapter 7 , ABN A is shown to have remote sequence similarity with four bacterial xylanolytic glycosyl hydrolases (three β-D-xylosidases and an endo-1,4-β-D-xylanase), three of which feature activity against para -nitrophenyl-α-L-arabinofuranoside. This synthetic compound is commonly utilized to assay potential ABF activity, whereas it is known to be an inhibitor of the fourth enzyme. The homology became evident only after multi pie-sequence alignments and hydrophobic cluster analysis. It was proposed that these enzymes share a binding site for a terminal non-reducing α-linkedL-arabinofuranosyl residue and that they all belong to glycosyl hydrolase family 43. Implications from these suggestions were discussed. The ABFs could not be assigned to an established glycosyl hydrolase family.
Based on theL-arabinolytic system of the brown-rot fungus Monilinia fructigena, the sequence similarity found amongst ABF A and bacterial pullulan-degrading enzymes, and ABF expression levels under carbon starvation conditions and onD-glucose as the carbon source, distinct functions inL-arabinan and plant cell-wall degradation were proposed for ABF A and ABF B. ABF A would be essentially cell-wall associated and act to degradeL-arabinan fragments generated by ABN A. ABF B activity would be important for the primary release of small amounts ofL-arabinose which initiate induction of various endolytic systems to degrade plant cell walls, and thus function in substrate sensing. In line with these considerations, the involvement of other, not yet identified glycosyl hydrolases inL-arabinan degradation by A.niger was suggested.
Induction and repression of arabinase gene expression are further discussed in Chapter 7 . The results of the studies in A.niger (Chapter 5) and A.nidulans (Chapter 6) were interpreted in a mutual context. The identity of the lowmolecular-weight compound directly responsible for induction of arabinase gene expression, was addressed. BothL-arabinose andL-arabitol are likely candidates to fulfil such a role. However, it was not possible to weigh the actual inductive capacities ofL-arabinose andL-arabitol due to their in vivo convertibility and the carbon catabolite repression elicited by the pentose. Competition for such a compound provides an alternative explanation for the phenomena observed in Chapter 5. The involvement of the transcriptional repressor CREA in arabinase gene expression is not limited to the direct repression of structural and regulatory genes of theL-arabinan-degrading system. It also plays a role in inducer exclusion and end-product repression, two processes shown to be eminently involved in the regulation ofL-arabinan degradation in wild-type A.nidulans. Fungal growth rate was suggested to be related to derepression of theL-arabinan-degrading system. The possible involvement of cAMP in arabinase gene expression, as suggested by the presence of potential cis -acting cAMP-responsive elements in the structural genes, was considered. Various ways by which cAMP might modulate arabinase synthesis were surveyed.
The calcium-available phosphorus ratio in broiler diets related to the calcium and phosphorus retention and bone development at variable dietary phytate P and microbial phytase contents
Klis, J.D. van der; Gerritsen, C.L.M. - \ 1994
Beekbergen : ID-DLO, Branch Beekbergen (Spelderholt uitgave no. 616) - 28
beendervorming - beenderen - vleeskuikens - calcificatie - calcium - voer - glycosidasen - mineraalmetabolisme - mineralen - fosfor - skeletontwikkeling - skelet - bone formation - bones - broilers - calcification - calcium - feeds - glycosidases - mineral metabolism - minerals - phosphorus - skeletal development - skeleton
|The efficacy of phytase in laying hen diets with different feedstuff compositions at 24 and 36 weeks of age
Klis, J.D. van der; Versteegh, H.A.J. - \ 1994
Beekbergen : ID-DLO, Branch Beekbergen (Spelderholt report no. 623) - 17
samenstelling - enzymen - voer - glycosidasen - hennen - vitaminen - composition - enzymes - feeds - glycosidases - hens - vitamins
2e ronde leghennen: geexpandeerd voer en fytase bieden voordelen
Reuvekamp, B.F.J. - \ 1994
Praktijkonderzoek voor de Pluimveehouderij 5 (1994)1. - ISSN 0924-9087 - p. 17 - 20.
dierhouderij - dierlijke meststoffen - derivaten - enzymen - voer - warmtebehandeling - hennen - fosfaten - fosforpentoxide - productiviteit - rentabiliteit - drijfmest - vitaminetoevoegingen - vitaminen - vleeskuikens - samenstelling - glycosidasen - voedermiddelbewerking - animal husbandry - animal manures - derivatives - enzymes - feeds - heat treatment - hens - phosphates - phosphorus pentoxide - productivity - profitability - slurries - vitamin supplements - vitamins - broilers - composition - glycosidases - feed processing
Tijdens de tweede ronde met leghennen zijn o.a. geëxpandeerd voer en fytase onderwerpen van onderzoek. Met geëxpandeerd voer kunnen gunstige technische resultaten behaald worden. Door de toepassing van fytase kan een lager fosfaatgehalte in de mest worden bereikt, waarschijnlijk zonder dat de technische resultaten veranderen.
De nutritionele waardering van Natuphos-fytase in pluimveevoeders
Doesum, J.H. van; Geerse, C. - \ 1994
Praktijkonderzoek voor de Pluimveehouderij 5 (1994)1. - ISSN 0924-9087 - p. 7 - 10.
samenstelling - enzymen - voer - kippen - glycosidasen - voedingswaarde - pluimvee - vitaminetoevoegingen - vitaminen - composition - enzymes - feeds - fowls - glycosidases - nutritive value - poultry - vitamin supplements - vitamins
Door fytase toe te voegen aan het voer, komt meer fosfor beschikbaar voor het dier. Bij vleeskuikens wordt door Gist-brocades geadviseerd dat 500 FTU overeenkomt met 1 g Monocalciumfosfaat (MCP-P). Voor leghennen is het advies dat 300 FTU overeenkomt met 1 g MCP-P.
Fytase in de opfokperiode van slachtkuikenmoederdieren
Haar, J.W. van der - \ 1992
Praktijkonderzoek voor de Pluimveehouderij 3 (1992)2. - ISSN 0924-9087 - p. 20 - 22.
diervoedering - dierlijke meststoffen - samenstelling - enzymen - glycosidasen - drijfmest - vitaminetoevoegingen - jonge dieren - vleeskuikenouderdieren - animal feeding - animal manures - composition - enzymes - glycosidases - slurries - vitamin supplements - young animals - broiler breeders
De milieuproblematiek vereist dat er minder mineralen in de mest terechtkomen. Met de toevoeging van fytase aan het voer kan een betere fosforbenutting worden verkregen, waardoor er minder fosfor in de mest komt.
Downstream processing of polysaccharide degrading enzymes by affinity chromatography
Somers, W.A.C. - \ 1992
Agricultural University. Promotor(en): K. van 't Riet; F.M. Rombouts. - S.l. : Somers - ISBN 9789054850540 - 163
fermentatie - voedselbiotechnologie - glycosidasen - chromatografie - enzymen - polysacchariden - affiniteitschromatografie - fermentation - food biotechnology - glycosidases - chromatography - enzymes - polysaccharides - affinity chromatography
The objective of this study was the development of affinity matrices to isolate and purify a number of polysaccharide degrading enzymes and the application of these adsorbents in the large- scale purification of the enzymes from fermentation broths. Affinity adsorbents were developed for endo-polygalacturonase and α-amylase.
The isolation of two of these enzymes was realized using the specific affinity of the enzymes for the corresponding substrates, viz. pectate and starch. Normally interaction between an enzyme and its substrate is accompanied by hydrolysis of the polymeric substrate, resulting in total biodegradation. By specific modification of the substrate it is possible to obtain adsorbents which are capable of binding the enzyme while being resistant against biodegradation.
Pectate is the natural substrate for endo-polygalacturonase. Alginate, a substrate analogue for pectate, is able to bind endo-polygalacturonase while it is not hydrolyzed by the enzyme. Rigid beads can be obtained by calcium complexation of the alginate. The pH and ionic strength of the incubation medium influence the strength of the interaction between endo-polygalacturonase and alginate beads. Adsorption end desorption can be controlled by these two parameters. In this way the enzyme can be isolated and purified from complex mixtures. The adsorbent can be regenerated at least a hundred times in a continuous process (Chapter 2).
The adsorption of the enzyme to the matrix was subject of further study. By determining relevant mass transport parameters such as adsorption equilibrium parameters, diffusion coefficients and rate parameters it appeared to be possible to describe the adsorption process in mathematical terms. The velocity of adsorption is determined by the diffusion velocity of the enzyme in the beads and not by the reaction kinetics of the complex formation. The velocity of the desorption process is also determined by the diffusion velocity of the enzyme out of the bead (Chapter 3).
The most important substrate for α-amylase is starch. Alpha-amylase is used on a large scale for the enzymic conversion of starch into limit dextrins and other oligosaccharides. By means of a chemical crosslinking procedure of starch an adsorbent is obtained which is capable of binding the enzyme while it is degraded only to a limited extent. The adsorption and desorption characteristics of the interaction between enzyme and adsorbent were studied. It appears that the enzyme has the highest affinity for the adsorbent at the pH where it has its maximum catalytic activity. The interaction is biospecific and this principle allows a very selective isolation of the enzyme. The interaction between enzyme and adsorbent is essentially insensitive to changes in ionic strength of the medium. Desorption can be accomplished by a shift of pH or a raise in temperature of the incubation medium (Chapter 4).
The adsorption characteristics were further evaluated. Continuous use of the adsorbent in an isolation process of α-amylase results in a slow biodegradation of the matrix. This effect is accompanied by an increase of capacity of the adsorbent for the enzyme. It appears that the adsorbent can be used repeatedly for the isolation of the enzyme, the biodegradation is made up for by an improved mass transfer of the enzyme into the matrix combined with the increased capacity. The rate of adsorption is determined by the diffusion rate of the enzyme into the porous gel (Chapter 5).
For the direct application of adsorbents in fermentation broths a number of techniques have been proposed. One of these is the use of a fluid bed column. This imposes a few demands on the density and the diameter of the adsorbent. Particles, suitable for use in fluid bed columns were developed by inclusion of crosslinked starch in alginate particles and by the preparation of an alginate/starch copolymer bead. The adsorption characteristics of these adsorbents are comparable with those of crosslinked starch (Chapter 6).
In conclusion it can be stated that affinity separations for endo-polygalacturonase and α-amylase prove to be a selective process with good potential for a one step purification of these enzymes from a fermentation broth. In addition the procedure of adsorbent preparation offers good opportunities to prepare affinity adsorbents for other hydrolases.
This study was performed in a partnership between the Department of Food Science (Food and Bioprocess Engineering Group and Food Chemistry and Food Microbiology Group) and the Department of Genetics. The project was financed by the Netherlands Technology Foundation (STW).
Affinity purification of polysaccharide degrading enzymes with crosslinked substrates
Rozie, H.J. - \ 1992
Agricultural University. Promotor(en): F.M. Rombouts. - S.l. : Rozie - ISBN 9789054850533 - 122
fermentatie - voedselbiotechnologie - glycosidasen - adsorptie - adsorberende middelen - fermentation - food biotechnology - glycosidases - adsorption - adsorbents
The aim of this work was to find economically favourable, affinity based, purification methods for several polysaccharide splitting bulk enzymes. The framework in which this study is done is described in Chapter 1.
Chapter 2 describes the adsorption of endo-polygalacturonase (endoPG) from a commercial enzyme preparation (Rapidase) to calcium alginate beads. Approximately 75% of the various polygalacturonase activities from Rapidase can be adsorbed at pH 4.4 by calcium alginate beads as well as by crosslinked sodium alginate powder. Equilibrium experiments were conducted to determine a parameter (k) that represents the degree of interaction between endoPG and the adsorbent. This parameter can be influenced by a change in pH and ionic strength of the adsorbate. At pH 3.8 the degree of interaction is 20 times larger than at pH 4.2. There is increased adsorption when the ionic strength is lowered, but a small amount of CaCl 2 is required to keep the calcium alginate beads stable.
Despite the resemblance in structure between L-guluronate blocks and polygalacturonate, a lower k value was found when the alginate, used for the preparation of the beads, contained a larger proportion of guluronic acid residues. There is no evidence that L-guluronic blocks in the alginate chain are responsible for the large affinity of endo-PG to this adsorbent. The influence of the pH and the ionic strength and the lack of endoPG inhibition by sodium alginate are indicative for ionic interactions between endoPG and the alginate chains.
Ionic interactions were of no importance in the interaction between ct-amylase and crosslinked starch as is described in the chapters 3 and 4. Crosslinked potato starch was prepared as an affinity adsorbent for bacterial α-amylase. To this end, reaction parameters for crosslinking in an ethanol/water solvent were investigated (Chapter 3). The degree of crosslinking, and consequently the suitability of crosslinked starch as an adsorbent for α-amylase, changed by altering these parameters. An increase in the degree of crosslinking of the adsorbent caused lower affinity for bacterial α-amylase which resulted in an unfavourable decrease in adsorption capacity and a favourable decrease of degradation of the adsorbent by the enzyme.
The adsorption and desorption characteristics of two bacterial α-amylases (B.subtilis, B.licheniformis) on crosslinked potato starch are described in Chapter 4. A capacity of about 185 mg (B.subtilis) and 71 mg (B.licheniformis) protein per g adsorbent can be realized. However, at 4 °C a smaller adsorption constant (K a ) was measured for the enzyme from B.subtilis (0.53 * 10 5L/mole) than for the B.licheniformis enzyme (3.8 * 10 5L/mole). The K a decreases with increasing temperature suggesting that association is caused by van der Waals forces. Comparison of the adsorption of the α-amylases to crosslinked starch with the activity of the enzymes on their natural substrate reveals that the velocity constant of the backward reaction of the enzyme-adsorbent complex increases strongly with increasing temperatures (B.subtilis α -amylase, k 2 (20 °C)/ k 2 (4 °C) ≈30). Desorption can be accomplished by a raise in temperature. Glycerol (20%) is added to the desorption buffer to stabilize the enzymes and protect the adsorbent against enzymic attack. The optimal desorption temperature for the B.subtilis enzyme is 60 °C. For the B.licheniformis enzyme this value is 70 °C or even higher. The adsorption velocity of α-amylases to freshly crosslinked starch is low due to the low accessibility of the adsorbent. This can easily be improved by enzymatic modification. Thus, bacterial α-amylases can be adsorbed and desorbed within short time spans (10 min) in sufficiently high amounts to make such an affinity purification process economically feasible.
In order to facilitate the purification of xylanases from Aspergillus niger, an affinity adsorbent has been developed from oat spelts xylan (Chapter 5). A suitable adsorbent was only obtained by crosslinking oat spelts xylan with epichlorohydrin in water but not in ethanol or ethanol water mixtures. After some initial degradation of the adsorbent (approx. 4%), no further biodegradation was measured with a reused adsorbent. Up to 60% of the xylanase activity from an Aspergillus niger enzyme mixture (50 mU/ml) was adsorbed at pH 4 The degree of adsorption to crosslinked xylan of four fractions of this preparation, previously separated by DEAE-Biogel A chromatography, varied between 40 and 90%.
Adsorption was strongly dependent on pH and ionic strength and desorption was easily accomplished by an increase in ionic strength. In addition to xylanases, polygalacturonases also adsorbed to the matrix probably due to the D-glucuronic acid moieties in xylan. No significant adsorption of β-D-xylosidase, α-L-arabinofuranosidase, β-D-galactosidase, β-(1,4)- galactanase, β-(1-3/6)-D-galactanase or cellulase activities was found.
The binding behaviour of four commercial fungal enzyme preparations on crosslinked xylan is presented in Chapter 6. The xylanase activity in Pectinol Al (Röhm GmbH, Darmstadt, Germany) was efficiently purified with crosslinked xylan. The specific endo-xylanase activity increased from 5.5 U/mg up to 160 U/mg. Two proteins were found with SDS-PAGE in purified Pectinol (29 and 51 kD) whereas a K m of 1.1 mg/ml was measured. Equilibrium adsorption studies revealed a rather low capacity for the Pectinol endo- xylanase (1.5 mg xylanase/g adsorbent). The calculated K a was 4 * 10 6L/mole.
Some endo-xylanases were also adsorbed by cation exchange material. However, from crosslinked xylan chromatography and additional FPLC studies it appeared that the adsorption properties of crosslinked xylan were not only due to the cation-binding properties of this adsorbent.
Chapter 7 is an evaluation of the foregoing chapters. The adsorption properties of three kinds of economically important polysaccharide splitting enzymes are studied in this work. A cheap substrate analog and crosslinked substrates were used as adsorbents. The magnitude of the capacities of calcium alginate and crosslinked starch towards endo-polygalacturonases and α-amylases, respectively, is such that commercial applications can be considered. Only laboratory applications are foreseen for crosslinked xylan as affinity adsorbent for specific endo-xylanases since the capacity of this adsorbent is rather low.
Characterization and mode of action of xylanases ␁and some accessory enzymes
Kormelink, F.J.M. - \ 1992
Agricultural University. Promotor(en): A.G.J. Voragen. - S.l. : Kormelink - ISBN 9789054850526 - 175
glycosidasen - glycosidases
Three endo-(l,4)-β-D-xylanases; (Endo I, Endo II, and Endo III), a (1,4)-β-xylosidase and an (1,4)-β-D-arabinoxylan arabinofuranohydrolase (AXH) were purified from a culture filtrate produced by Aspergillus awamori CMI 142717. In addition to these enzymes, an acetyl xylan esterase (AE) was purified from a culture filtrate produced by Aspergillus niger DS 16813.
The enzymes were characterized by determining specific activities, molecular weight, isoelectric point, kinetic parameters (K m ,V max ), optimum pH and optimum temperature.
Arabinoxylan oligosaccharides were derived from alkali-extracted wheat arabinoxylans by complete digestion with Endo I and III. The structures of unknown oligosaccharides were elucidated by 1H-n.m.r. spectroscopy. From these structures a model was proposed for the mode of action of Endo I and Endo III towards arabinoxylans. The same oligosaccharides were also used to specify the action of (1,4)-β-xylosidase, AXH and two α-L-arabinofuranosidases towards these arabinofuranosylated arabinoxylan oligosaccharides.
The interaction between the purified enzymes was studied by degradation of xylans from rice bran, oat spelt, wheat-flour, larchwood, and birchwood by single and combined actions of these enzymes on these substrates. The cooperativity was monitored by the amount of reducing sugars and by the types of products released.
|Effekt van tarwefytase op de Ca- en P-verteerbaarheid onder invloed van maalfijnheid, voorweken en pelleteren = Effect of wheat phytase on the digestibility of Ca and P as influenced by fineness of grinding, soaking and pelleting
Kemme, P.A. ; Jongbloed, A.W. - \ 1989
Lelystad : I.V.V.O. (Rapport / Instituut voor Veevoedingsonderzoek no. 202) - 31
calcium - droge stof - esterasen - voer - glycosidasen - mineralen - voedingswaarde - fosfor - varkens - voedermiddelbewerking - dry matter - esterases - feeds - glycosidases - minerals - nutritive value - phosphorus - pigs - feed processing