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.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    In Memoriam Wim Beeftink (1926-2014)
    Schaminee, J.H.J. - \ 2014
    Stratiotes 46 (2014). - ISSN 0928-2297 - p. 86 - 87.
    Kinetic and structural analysis of two transferase domains inPasteurella multocida hyaluronan synthase
    Kooy, F.K. ; Beeftink, H.H. ; Eppink, M.H.M. ; Tramper, J. ; Eggink, G. ; Boeriu, C.G. - \ 2014
    Journal of Molecular Catalysis. B, Enzymatic 102 (2014). - ISSN 1381-1177 - p. 138 - 145.
    blood-group-b - enzymological characterization - conformational-changes - n-acetylglucosamine - crystal-structure - group-a - glycosyltransferase - polypeptide - mechanism - substrate
    Pasteurella multocida hyaluronan synthase (PmHAS) encompasses two transferase domains that elongatea growing hyaluronan (HA) oligosaccharide chain by addition of either GlcNAc or GlcUA residues froma corresponding UDP-sugar. Initial velocity studies of single-step elongations were conducted for bothdomains by independently varying the concentrations of the HA oligosaccharide and the UDP-sugar.Two-substrate models were discriminated by their goodness-of-fit parameters and by dead-end inhi-bition studies. A mechanistic shift from a steady-state ordered bi-bi to rapid equilibrium ordered bi-bimechanism was observed at the NAc-site between the HA6and HA8elongation. This shift was invokedby a minor reduction in turnover number kcat. Both NAc- and UA-transferase domains follow a sequentialkinetic mechanism, most likely an ordered one in which the UDP-sugar donor binds first, followed bythe HA oligosaccharide. After transfer of the sugar moiety, both products are released, first the elongatedHA oligosaccharide and then the UDP sugar. This mechanism was visualized with a structural model ofPmHAS that presented two flexible loops, one in each transferase domain; these loops form a bridgeabove the active site.
    Segregation of Granular Particles by Mass, Radius, and Density in a Horizontal Rotating Drum
    Arntz, M.M.H.D. ; Beeftink, H.H. ; Otter, W.K. den; Briels, W.J. ; Boom, R.M. - \ 2014
    AIChE Journal 60 (2014)1. - ISSN 0001-1541 - p. 50 - 59.
    size segregation - particulate systems - flow - simulation - cylinders - mixtures - dynamics - solids - behavior - motion
    The impact of particle properties on segregation and mixing of bidisperse granular beds in a rotating horizontal drum have been studied by discrete element method (DEM) simulations. Bidispersities in radius, density, and mass have pronounced influences on the stationary mixing pattern, although they hardly affect the granules’ flow regime. At 50% fill level, all beds mix well for a Froude number of 0.56, corresponding to a flow regime intermediate to cascading and cataracting, while segregation occurs both at lower (rolling and cascading regime) and higher (cataracting/centrifuging regime) Froude numbers. These observations are explained qualitatively by noticing that the angular drum velocity dictates the flow regime, which in turn determines the effectiveness and direction of four competing (de)mixing mechanisms: random collisions, buoyancy, percolation, and inertia. A further dozen particle properties have been varied, including the friction coefficients and elastic modulus, but these proved inconsequential to the steady-state degree of mixing.
    Structural and functional evidence for two separate oligosaccharide binding sites of Pasteurellamultocida hyaluronan synthase
    Kooy, F.K. ; Beeftink, H.H. ; Eppink, M.H.M. ; Tramper, J. ; Eggink, G. ; Boeriu, C.G. - \ 2013
    Advances in Enzyme Research 1 (2013)4. - ISSN 2328-4846 - p. 97 - 111.
    Pasteurella multocida hyaluronan synthase (PmHAS) is a bi-functional glycosyltransferase, containing a ß1,3-glucuronyltransferase and ß1,4-N-acetylglucosaminetransferase domain. PmHAS catalyzes the elongation of hyaluronan (HA) through the sequential addition of single monosaccharides to the non-reducing end of the hyaluronan chain. Research is focused on the relation between the length of the HA oligo- saccharide and the single-step elongation ki- netics from HA4 up to HA9. It was found that the turnover number kcat increased with length to maximum values of 11 and 14 s-1 for NAc- and UA-transfer, respectively. Interestingly, the spe- cificity constant kcat/KM increased with polymer length from HA5 to HA7 to a value of 44 mM-1·s-1, indicating an oligosaccharide binding site with increasing specificity towards a heptasaccha- ride at the UA domain. The value of kcat/KM re- mained moderately constant around 8 mM-1·s-1 for HA4, HA6, and HA8, indicating a binding site with significantly lower binding specificity at the NAc domain than at the UA domain. These find- ings are further corroborated by a structural homology model of PmHAS, revealing two dis- tinct sites for binding of oligosaccharides of different sizes, one in each transferase domain. Structural alignment studies between PmHAS and glycosyltransferases of the GT-A fold showed significant similarity in the binding of the UDP-sugars and the orientation of the ac- ceptor substrate. These similarities in substrate orientation in the active site and in essential amino acid residues involved in substrate bind- ing were utilized to localize the two HA oligo- saccharide binding sites.
    Process design for enzymatic peptide synthesis in near-anhydrous organic media
    Vossenberg, P. ; Beeftink, H.H. ; Stuart, M.A.C. ; Tramper, J. - \ 2013
    Biocatalysis and Biotransformation 31 (2013)5. - ISSN 1024-2422 - p. 255 - 268.
    catalyzed dipeptide synthesis - water activity control - industrial protease alcalase - immobilized lipases - precursor dipeptide - support material - 2-phase systems - solvents - biocatalysis - esterification
    This work is a case study on a process design for enzymatic peptide synthesis, which is based on and inspired by previously established data about the Alcalase-catalyzed coupling of an amino acid amide and a chemically synthesized activated N-protected amino acid carbamoylmethyl ester in near-anhydrous tetrahydrofuran. The choices with regard to Alcalase formulation, the type of reactor, method of controlling the water content, and whether or not to recycle the enzyme, are discussed. In addition, an estimate is given for the reactor size, volumes of solvent, amount of substrate, enzyme and molecular sieves, needed in order to meet a specific demand for peptides. We believe that this case study gives a good indication of the various choices that have to be made when designing a process for enzymatic peptide synthesis and the implications of these choices.
    Chain length distribution and kinetic characteristics of an enzymatically produced polymer
    Mulders, K.J.M. ; Beeftink, H.H. - \ 2013
    e-Polymers 13 (2013)1. - ISSN 1618-7229 - p. 261 - 272.
    monte-carlo-simulation - multiple attack mechanism - sequential reactions - actin-filaments - enzyme - model - transglycosylation - fragmentation - competition - reactors
    Non-processive enzymatic polymerization leads to a distribution of polymer chain lengths. A polymerization model was developed to investigate the relation between the extent of this distribution on one hand, and the polymerization start conditions and reaction kinetics on the other hand. The model describes changes in concentration of chains of length n as the result of two elongation reactions: elongation by monomer addition to length n-1 and elongation by monomer addition to length n. Polymerization reactions were assumed to be zero order in monomer concentration and to obey Michaelis-Menten kinetics with respect to polymer concentrations. In addition, the amount of enzyme available for each individual reaction (n n+1) is assumed to be Non-processive enzymatic polymerization leads to a distribution of polymer chain lengths. A polymerization model was developed to investigate the relation between the extent of this distribution on one hand, and the polymerization start conditions and reaction kinetics on the other hand. The model describes changes in concentration of chains of length n as the result of two elongation reactions: elongation by monomer addition to length n-1 and elongation by monomer addition to length n. Polymerization reactions were assumed to be zero order in monomer concentration and to obey Michaelis-Menten kinetics with respect to polymer concentrations. In addition, the amount of enzyme available for each individual reaction (n n+1) is assumed to be proportional to the concentration of polymer substrate of length n. The development of the shape of the chain length distribution was found to be independent of the value of the overall reaction rate constant; only the rate at which these shapes developed was influenced by the 1st-order rate constant. The value of the Michaelis parameter did affect the form of the chain length distribution curve since it affects the reaction order. An increase in reaction order was found to promote widening of the chain length distribution. Differences in kinetic parameters between the subsequent polymerization reactions, if any, were also found to have a large effect on the development of the chain length distribution. An increase in rate constants with chain length entailed a wider distribution; a more narrow distribution would require a decrease in rate constants with chain length.proportional to the concentration of polymer substrate of length n. The development of the shape of the chain length distribution was found to be independent of the value of the overall reaction rate constant; only the rate at which these shapes developed was influenced by the 1st-order rate constant. The value of the Michaelis parameter did affect the form of the chain length distribution curve since it affects the reaction order. An increase in reaction order was found to promote widening of the chain length distribution. Differences in kinetic parameters between the subsequent polymerization reactions, if any, were also found to have a large effect on the development of the chain length distribution. An increase in rate constants with chain length entailed a wider distribution; a more narrow distribution would require a decrease in rate constants with chain length.
    Effect of enzyme dehydration on alcalase-catalyzed dipeptide synthesis in near-anhydrous organic media.
    Vossenberg, P. ; Beeftink, H.H. ; Cohen Stuart, M.A. ; Tramper, J. - \ 2013
    Biotechnology Progress 29 (2013)4. - ISSN 8756-7938 - p. 870 - 875.
    peptide-synthesis - fatty-acid - esterification - solvents - kinetics - water - deactivation - formulations - hydrolases
    The effect of enzyme dehydration by molecular sieves on the coupling of phenylalanine amide and the carbamoylmethyl ester of N-protected phenylalanine in near-anhydrous tetrahydrofuran was investigated. This coupling was catalyzed by Alcalase covalently immobilized onto macroporous acrylic beads (Cov); these immobilized enzymes were hydrated prior to use. The dehydration kinetics of Cov by molecular sieve powder were determined by incubating Cov with different amounts of molecular sieve powder for different periods of time (0-80 h). Subsequently, the remaining coupling activity of Cov was measured. Dehydration-induced inactivation of Cov by molecular sieve powder was found to occur in three phases: (1) an initial, rapid, major dehydration-induced inactivation that takes place during the first activity measurement, (2) a phase of first-order inactivation, and (3) a plateau phase in activity. These dehydration kinetics were incorporated into a previously found reaction kinetics model. The resulting model was then used to fit progress curve data of the coupling in the presence of different amounts of molecular sieve powder. Upon establishment of parameter values, the model was used to predict independent data sets and found to work well.
    Dipeptide synthesis in near-anhydrous organic media: Long-term stability and reusability of immobilized Alcalase
    Vossenberg, P. ; Beeftink, H.H. ; Nuijens, T. ; Quaedflieg, P.J.L.M. ; Cohen Stuart, M.A. ; Tramper, J. - \ 2013
    Journal of Molecular Catalysis. B, Enzymatic 93 (2013). - ISSN 1381-1177 - p. 23 - 27.
    high initial activity - enzymatic esterification - subtilisin carlsberg - peptide-synthesis - shear - solvents - enzymes - deactivation - kinetics - water
    The long-term stability and re-use of Alcalase covalently immobilized onto macroporous acrylic beads (Cov) in tetrahydrofuran (THF) were investigated. Cov can be used to synthesize dipeptides under near-anhydrous conditions in THF. Cov was incubated with and without molecular sieves (beads or powder) in THF, in order to investigate whether its stability is affected by the presence of molecular sieves. After different incubation periods, the enzyme activity was determined in an aqueous environment. In addition, Cov was repeatedly recycled to examine its reusability. Without molecular sieve beads, Cov hardly inactivated in THF. With molecular sieve beads, Cov lost activity over time. Incubated Cov samples were rotated on a blood rotator, entailing mechanical forces between Cov and the molecular sieve beads. Mechanical damage of Cov by the molecular sieve beads was found to be the main reason for the instability of Cov. During reuse, intermediate rehydration of Cov also caused a small but significant activity loss.
    The influence of end walls on the segregation pattern in a horizontal rotating drum
    Arntz, M.M.H.D. ; Otter, W.K. den; Beeftink, H.H. ; Boom, R.M. ; Briels, W.J. - \ 2013
    Granular Matter 15 (2013)1. - ISSN 1434-5021 - p. 25 - 38.
    reversible axial segregation - granular-materials - mixtures - simulation - cylinders - dynamics - motion
    The influence of end walls on segregation of bidisperse granular beds in a short rotating horizontal drum is studied by a discrete element method. Whereas non-closed periodically continued drums segregate radially, all simulations of drums with end walls resulted in axial segregation with two bands at low friction between the particles and the end-wall, and three bands at high friction. Various simulations show irregular transitions between two approximately equally stable states, with rapid oscillations preceding the conversions. The formation of two axial bands decreases the energy dissipation by the bed, whereas neither radial segregation nor axial segregation into three bands reduced the power absorption at constant angular velocity. Roughening up the end-walls also increased the rate of axial segregation.
    Kinetics of Alcalase-catalyzed dipeptide synthesis in near-anhydrous organic media
    Vossenberg, P. ; Beeftink, H.H. ; Cohen Stuart, M.A. ; Tramper, J. - \ 2013
    Journal of Molecular Catalysis. B, Enzymatic 87 (2013). - ISSN 1381-1177 - p. 113 - 120.
    peptide-synthesis - enzyme-kinetics - proteases - solvents
    The coupling kinetics of phenylalanine amide and the carbamoylmethyl ester of N-protected phenylalanine in near-anhydrous tetrahydrofuran were investigated. This coupling was catalyzed by Alcalase covalently immobilized onto macroporous acrylic beads; these immobilized enzymes were hydrated prior to use. Near-anhydrous conditions (i.e. extremely low water activity) were maintained by a carefully chosen amount of molecular sieve powder. Kinetic characteristics were determined from reaction time courses up to full conversion at various initial concentrations of substrate and product. These progress curve data were fitted with different kinetic models to determine which of these models best approximates the kinetic properties of the immobilized Alcalase with respect to the coupling under study. An appropriate model of the coupling reaction is necessary in order to design a reactor and predict its performance adequately leading to the practical implementation of biocatalyic peptide synthesis. The kinetics of the coupling were found to be complex and to obey a two-substrate kinetic model with two competitive product inhibition terms. To reduce the effect of the product inhibition on immobilized Alcalase, a reactor should be designed in which at least glycolamide is selectively removed as this was found to be the strongest inhibitor.
    Chemo-enzymatic peptide synthesis : bioprocess engineering aspects
    Vossenberg, P. - \ 2012
    Wageningen University. Promotor(en): Hans Tramper; Martien Cohen Stuart, co-promotor(en): Rik Beeftink. - [S.l. : s.n. - ISBN 9789461733900 - 185
    bioproceskunde - eiwitsynthese - enzymactiviteit - bioprocess engineering - protein synthesis - enzyme activity

    Peptides, in particular oligopeptides, play an important role in the fields of health care, nutrition and cosmetics. Chemical synthesis is currently the most mature technique for the synthesis of peptides that range in length from 5 to 80 amino acids. Chemical synthesis is, however, expected to be more and more combined with enzyme-catalyzed synthesis, resulting in chemo-enzymatic approaches towards peptide synthesis. The racemization that hampers chemical synthesis can be prevented by forming the peptide bond enzymatically.

    In the work in this thesis the bioprocess engineering aspects of a specific chemo-enzymatic peptide synthesis route are studied. In this route, an N-protected, C‑terminally activated amino acid is coupled with a C-protected amino acid nucleophile. The coupling step is catalyzed by Alcalase. The initial idea was to also enzymatically catalyze the activation of the amino acid, which is used in the coupling step, and to carry out the coupling and activation steps in one pot. In the work of Chapter 2 lipase B from Candida antarctica (CalB) and Alcalase were used as a model system for such a one-pot chemo-enzymatic peptide synthesis, in order to investigate the (in)compatibility between the two enzymes. The rate of activity loss of native and immobilized CalB in the absence and presence of native and immobilized Alcalase was calculated from the rate of triacetin hydrolysis. It was shown that native Alcalase degrades native CalB under aqueous conditions. Immobilization of both or either CalB or Alcalase onto macroporous beads, however, effectively prevented hydrolysis of CalB by Alcalase.

    Due to the current impracticality of the enzyme-catalyzed activation step, the rest of the work in the thesis focuses on the Alcalase-catalyzed coupling step. The non-aqueous coupling in tetrahydrofuran (THF) of phenylalanine amide (Phe-NH2) and the carbamoylmethyl ester of phenylalanine (of which the amino group was benzyloxycarbonyl-protected, Z-Phe-OCam) was used as a model coupling reaction.

    In protease-catalyzed peptide synthesis the availability of water is essential, as a compromise must be made between on the one hand the overall enzymatic activity and, on the other hand, the rate of product synthesis. Water is essential for enzyme activity, but at the same time causes hydrolytic side reactions. In the work of Chapter 3 the model coupling reaction was catalyzed by cross-linked enzyme aggregates of Alcalase optimized for use in organic media (Alcalase CLEA-OM) at a range of water activity (aw) values, including the coupling in the presence of molecular sieves (i.e. at very low aw values). The rate of peptide synthesis could not be increased by increasing awvalueswithout significantly increasing the rate of hydrolysis, i.e. without significantly decreasing the synthesis / hydrolysis (S/H) ratio. Hydrolysis (in the present system, only the activated substrate, not the dipeptide product, may be hydrolyzed) was found to dominate above aw ≈0.2.To prevent hydrolysis, the presence of molecular sieves was found to be necessary. Nevertheless, the use of molecular sieves over longer periods of time should be carefully considered as they may dehydrate and thereby inactivate the enzyme in time.

    In the work of Chapter 4, besides CLEA-OM, also other Alcalase formulations were used to catalyze the model coupling reaction. The reaction was done in the presence of molecular sieves (i.e. under near-dry conditions). Hydration prior to drying (with anhydrous tert-butanol and anhydrous THF)of the Alcalase formulations resulted in a significant increase in rate of the subsequent dipeptide synthesis. Without such initial hydration, the enzymes seem to lack the water needed to maintain their catalytically active conformation. Repeated use in the presence of molecular sieves, without intermediate rehydration, led to inactivation of the enzyme. For three enzyme formulations this inactivation could be counteracted by intermediate rehydration. Inactivation of another enzyme formulation, Alcalase immobilized onto dicalite, was only partially reversible by hydration. Alcalase immobilized onto dicalite was found to be initially the most active in dipeptide synthesis. Nevertheless, due to its small particle size and its lack of operational stability, this formulation may not be the best choice for the synthesis of dipeptides in neat organic media on a large scale. The most promising enzyme formulation for this is Alcalase covalently immobilized onto macroporous acrylic beads (in this thesis abbreviated as Cov) due to its reasonable activity, its seemingly good operational stability, and its practical size and uniform spherical shape.

    If, for economic reasons, Cov should be reused repeatedly for dipeptide synthesis in organic media, its operational stability is important and thus its activity should not decrease significantly. The long-term stability and reuse of hydrated Cov in THF was investigated in the work of Chapter 5. Cov was incubated with and without molecular sieves (beads or powder) in anhydrous THF. After different incubation periods in THF, the enzyme activity was determined in an aqueous environment. In addition, Cov was repeatedly recycled in order to examine its reusability. The effect of reuse on the aqueous activity of Cov and on the Cov-catalyzed model coupling reaction in near-anhydrous THF was studied. Without molecular sieve beads, Cov hardly inactivated in THF. Nevertheless, when Cov was incubated with molecular sieve beads in THF in rotating reaction vials, Cov lost activity over time. Mechanical damage of Cov by the molecular sieve beads was found to be the main reason for the instability of Cov. In order to reuse Cov for the model coupling reaction in the presence of molecular sieves, it needs to be rehydrated in between the batches. Nevertheless, each intermediate rehydration step also caused a small but significant enzyme activity loss.

    In the work of Chapter 6, the coupling kinetics of the model coupling reaction, catalyzed by hydrated Cov, were investigated. Near-anhydrous conditions were maintained by a carefully chosenamount of molecular sieve powder (in contrast to molecular sieve beads, molecular sieve powder does not lead to mechanical damage of Cov). Kinetic characteristics were determined from reaction time courses up to full conversion at various initial concentrations of substrate and product. These progress curve data were fitted with different kinetic models to determine which of these models best approximates the kinetic properties of the immobilized Alcalase with respect to the coupling under study. It was found that the kinetics of the coupling can be described well with a two-substrate kinetic model with two inhibitory products. To reduce the effect of the product inhibition on Cov, a reactor should be designed in which at least glycolamide is selectively removed, as it was found to be the strongest inhibitor.

    In Chapter 4 it was shown that molecular sieves dehydrate and thereby reversibly inactivate the enzyme. In the work of Chapter 7 the effect of enzyme dehydration by molecular sieves on the Cov-catalyzed model coupling reaction was studied in detail. The dehydration kinetics of Cov by different amounts of molecular sieve powder were determined by incubating Cov with molecular sieve powder for different periods of time. Subsequently, the remaining coupling activity of Cov was measured. Dehydration-induced inactivation of Cov by molecular sieve powder seemed to occur in three phases: (1) an initial, rapid,major dehydration-induced inactivation that takes place during the first activity measurement (1 h), (2) a phase of first-order inactivation (20 h), and (3) a relatively low plateau phase in activity. These dehydration kinetics were incorporated into the reaction kinetics model described in Chapter 6. The resulting model was then used to fit progress curve data of the model coupling reaction in the presence of different amounts of molecular sieve powder. Using the estimated parameter values, the model was used to predict independent data sets and found to work well.

    The work of Chapter 8 is a case study about a process design for enzymatic peptide synthesis, which is based on the findings of the previous chapters. The choices with regard to Alcalase formulation, type of reactor, way to control the water content, and whether or not to recycle the enzyme, are discussed. In addition, an estimate is given for the reactor size, volumes of solvent, amounts of substrate, enzyme and molecular sieves, needed in order to produce a specific demand for peptides. We believe that this case study gives a good impression of the various choices that have to be made when designing a process for enzymatic peptide synthesis and the implications of these choices.

    Selecting optimal conditions for Alcalase CLEA-OM for synthesis of dipeptides in organic media
    Vossenberg, P. ; Beeftink, H.H. ; Nuijens, T. ; Cohen Stuart, M.A. ; Tramper, J. - \ 2012
    Journal of Molecular Catalysis. B, Enzymatic 75 (2012)3. - ISSN 1381-1177 - p. 43 - 49.
    industrial protease alcalase - controlled peptide-synthesis - controlled water activity - enzymatic-synthesis - support material - solvents - transesterification - acetonitrile - tripeptide - catalysis
    In protease-catalyzed peptide synthesis, the availability of water is essential, as a compromise must be made between on the one hand the overall enzymatic activity and, on the other hand, the rate of product synthesis. Water is essential for enzyme activity, but at the same time causes hydrolytic side reactions. We studied the coupling of the carbamoylmethyl ester of N-protected phenylalanine and phenylalanine amide in tetrahydrofuran catalyzed by Alcalase CLEA-OM at a range of water activity (aw) values, including the coupling in the presence of molecular sieves (i.e. at very low aw values). The hydrolytic side reaction (in the present system only the hydrolysis of substrate occurs) was found to dominate above an aw value of about 0.2. To prevent hydrolysis, the presence of molecular sieves was found to be necessary.
    Performance of Alcalase formulations in near dry organic media: Effect of enzyme hydration on dipeptide synthesis
    Vossenberg, P. ; Beeftink, H.H. ; Nuijens, T. ; Quaedflieg, P.J.L.M. ; Cohen Stuart, M.A. ; Tramper, J. - \ 2012
    Journal of Molecular Catalysis. B, Enzymatic 78 (2012). - ISSN 1381-1177 - p. 24 - 31.
    industrial protease alcalase - catalyzed peptide-synthesis - controlled water activity - subtilisin carlsberg - precursor dipeptide - support material - solvents - immobilization - transesterification - tripeptide
    The use of different Alcalase formulations for protease-catalyzed dipeptide synthesis was investigated by studying the coupling of the carbamoylmethyl ester of N-protected phenylalanine with phenylalanine amide in tetrahydrofuran in the presence of molecular sieves (i.e. under near dry conditions). Hydration prior to drying (with anhydrous tert-butanol and anhydrous tetrahydrofuran) of the Alcalase formulations resulted in a significant increase in rate of the subsequent dipeptide synthesis. Repeated use, in the presence of molecular sieves, without intermediate rehydration led to inactivation of the enzyme. For three enzyme formulations this inactivation could be counteracted by intermediate rehydration. Inactivation of another enzyme formulation was only partially reversible by hydration. Alcalase immobilized onto dicalite with glutaraldehyde was found to be the most active in dipeptide synthesis, i.e. the formulation that initially produces the largest amount of product per gram of total formulation per unit of time. Due to its small particle size and its lack of operational stability, this formulation may nevertheless not be the best choice for the synthesis of dipeptides in neat organic media on a large scale. The most promising enzyme formulation for this is Alcalase covalently immobilized onto macroporous acrylic beads due to its reasonable activity, its seemingly good operational stability, and its size and uniform shape
    Immobilization to prevent enzyme incompatibility with proteases
    Vossenberg, P. ; Beeftink, H.H. ; Cohen Stuart, M.A. ; Tramper, J. - \ 2011
    Biocatalysis and Biotransformation 29 (2011)6. - ISSN 1024-2422 - p. 288 - 298.
    candida-antarctica - peptide-synthesis - organic-synthesis - crystal-structure - water activity - lipase-b - deactivation - improvement - autolysis - proteins
    Enzyme incompatibility is a problem in multi-enzyme processes that involve a non-specific protease, such as Alcalase. An example is the one-pot enzymatic synthesis of peptides catalyzed by a lipase and a protease. The incompatibility between lipase B from Candida antarctica (CalB) and Alcalase was studied. To what extent immobilization of both or either CalB or Alcalase onto macroporous beads helps to prevent hydrolysis of CalB by Alcalase was evaluated. The rate of activity loss of native and immobilized CalB in the absence and presence of native and immobilized Alcalase was calculated from the rate of triacetin hydrolysis. Immobilization of both or either CalB or Alcalase onto macroporous beads was found to be effective in largely preventing hydrolysis of CalB by Alcalase
    Valorisation of Jatropha curcas: Solubilisation of proteins and sugars from the NaOH extracted de-oiled press cake
    Kootstra, A.M.J. ; Beeftink, H.H. ; Sanders, J.P.M. - \ 2011
    Industrial Crops and Products 34 (2011)1. - ISSN 0926-6690 - p. 972 - 978.
    antinutritional factors - acid pretreatment - products - biomass - crop
    In this study, we investigated the possibilities for increasing the valorisation of de-oiled Jatropha press cake (DO-JPC). The studied raw material is the by-product of the alkaline protein extraction of the DO-JPC: NaOH Extracted DO-JPC (NEDO-JPC). Protein solubilisation of NEDO-JPC was performed under neutral and acidic conditions (pH 2, 100 mM maleic acid), at elevated temperature (100, 120, and 140 °C), and at 5% (w/w) dry solids loading. After the treatment, the amount of solubilised protein was determined, as well as the solubilisation of polymeric sugars and formation of sugar degradation products furfural and 5-hydroxymethylfurfural (HMF). Although a clear influence is shown for temperature, no difference in protein solubilisation was found between treatments at pH 7 and pH 2. A maximum of 25% (w/w) of the available protein was solubilised, at 140 °C. The lignocellulose fraction of NEDO-JPC proved relatively recalcitrant to acid hydrolysis, suggesting a more intense treatment to be necessary to sufficiently increase accessibility for cellulolytic enzymes in a lignocellulosic bioethanol process. At €8.00 per tonne DO-JPC, it is concluded that the possibilities for valorisation of the protein fraction of NEDO-JPC at neutral and acid pH are limited, leaving the lignocellulose fraction as a source of valorisation to be investigated
    Energy Budget for the Cultured, Zooxanthellate Octocoral Sinularia flexibilis
    Khalesi, M.K. ; Beeftink, H.H. ; Wijffels, R.H. - \ 2011
    Marine Biotechnology 13 (2011). - ISSN 1436-2228 - p. 1092 - 1098.
    coral stylophora-pistillata - photosynthetically fixed carbon - porites-porites scleractinia - soft corals - hermatypic corals - light-absorption - symbiotic coral - bleached corals - quantum yield - reef corals
    The zooxanthellate octocoral Sinularia flexibilis is a producer of potential pharmaceutically important metabolites such as antimicrobial and cytotoxic substances. Controlled rearing of the coral, as an alternative for commercial exploitation of these compounds, requires the study of species-specific growth requirements. In this study, phototrophic vs. heterotrophic daily energy demands of S. flexibilis was investigated through light and Artemia feeding trials in the laboratory. Rate of photosynthetic oxygen by zooxanthellae in light (˜200 µmol quanta m-2¿s-1) was measured for the coral colonies with and without feeding on Artemia nauplii. Respiratory oxygen was measured in the dark, again with and without Artemia nauplii. Photosynthesis–irradiance curve at light intensities of 0, 50, 100, 200, and 400 µmol quanta m-2¿s-1 showed an increase in photosynthetic oxygen production up to a light intensity between 100 and 200 µmol quanta m-2¿s-1. The photosynthesis to respiration ratio (P/R¿>¿1) confirmed phototrophy of S. flexibilis. Both fed and non-fed colonies in the light showed high carbon contribution by zooxanthellae to animal (host) respiration values of 111–127%. Carbon energy equivalents allocated to the coral growth averaged 6–12% of total photosynthesis energy (mg C¿g - ¹ buoyant weight day - ¹) and about 0.02% of the total daily radiant energy. “Light utilization efficiency (e)” estimated an average e value of 75% 12 h - ¹ for coral practical energetics. This study shows that besides a fundamental role of phototrophy vs. heterotrophy in daily energy budget of S. flexibilis, an efficient fraction of irradiance is converted to useable energy
    Repeated segregation and energy dissipation in an axially segregated granular bed
    Arntz, M.M.H.D. ; Otter, W.K. den; Beeftink, H.H. ; Boom, R.M. ; Briels, W.J. - \ 2010
    Europhysics Letters 92 (2010)5. - ISSN 0295-5075 - p. 54004 - 54004.
    long drum mixer - horizontal rotating cylinder - simulations - dynamics - mixtures - patterns - model - core
    Discrete element simulations were used to study the segregation behaviour in a bed of bidisperse granules in a rotating drum. In the final state the large particles ended up in the upper part of the bed near the vertical walls. In order to arrive at this state, the system went through two cycles of structural changes, on top of which fast oscillations were observed between an axially segregated and a somewhat more mixed state. These oscillations were sustained by different angles of repose near the vertical walls and in the middle of the bed. Concomitantly with the structural changes, the system's energy dissipation went through two cycles after which it settled in the state requiring the least work of all traversed states, suggesting that the granular bed strives for minimal dissipation.
    Processing lignocellulosic by-product streams using organic acids
    Kootstra, A.M.J. - \ 2010
    Wageningen University. Promotor(en): Johan Sanders, co-promotor(en): Rik Beeftink; Elinor Scott. - S.l. : s.n. - ISBN 9789085858348 - 173
    lignocellulosehoudend afval - stro - bioethanol - biobased economy - bioraffinage - reststromen - lignocellulosic wastes - straw - bioethanol - biobased economy - biorefinery - residual streams
    Het doel van dit proefschrift is om de voorbehandeling van lignocelluloseachtige grondstoffen met organische zuren te bestuderen. Meer specifiek gesteld, richt dit onderzoek zich op de invloed van maleïnezuur en fumaarzuur op de suikerafbraak tijdens de voorbehandeling, en op de prestatie van deze zuren in de voorbehandeling zelf, vergeleken met zwavelzuur. De optimalisatie van de voorbehandeling met maleïnezuur houdt niet alleen in het maximaliseren van de suikeropbrengst en de gerelateerde baten, maar houdt ook rekening met nadelige kanten van de voorbehandeling die hogere kosten met zich meebrengen. Als laatste richt dit onderzoek zich op mogelijkheden om de toepassing van maleïnezuur uit te breiden naar andere, eiwithoudende lignocelluloseachtige grondstoffen. Omdat tarwe de meest verbouwde graansoort is in de Europese Unie, is tarwestro gekozen als grondstof voor het werk over voorbehandeling van lignocellulose in dit proefschrift. Voor het werk over eiwithoudende lignocelluloseachtige grondstoffen is gekozen voor het bijproduct van biodieselproductie uit oliezaden van Jatropha curcas; de ontoliede perskoek.
    Modeling of particle segregation in a rotating drum
    Arntz, M.M.H.D. - \ 2010
    Wageningen University. Promotor(en): Remko Boom; W.J. Briels, co-promotor(en): W.K. den Otter; Rik Beeftink. - [S.l. : S.n. - ISBN 9789085858027
    vermenging - korrels - vaten - rotatie - simulatiemodellen - modelleren - mixing - granules - drums - rotation - simulation models - modeling
    Mixing of granular solids is a processing step in a wide range of industries. The fundamental phenomena in granule mixing are still poorly understood, making it difficult to a priori predict the effectiveness of mixing processes.
    While mixing of granules is easy when the particles are homogeneous in size, shape and density and other properties, in practice they are not. With such a mixture, homogenizing is far more complex, since the heterogeneous particles tend to segregate, and special care has to be taken in the design of the mixing process to avoid this.
    In view of the practical need for better understanding and control of solids mixing, the work in this thesis has two closely coupled objectives. The first objective is to obtain a better understanding of segregation mechanisms. This insight should enable the enhancement of mixing and at the same time suppress segregation, or vice versa, namely the deliberate and controlled segregation of a mixture. The second objective is to provide guidelines for mixing operations that can be derived from insights extracted from the data on mixing behaviour at different rotational velocities and fill levels. From this perspective, we here report an extensive numerical study of mixing and segregation in a bed of bidisperse granules in a rotating horizontal drum, which is the simplest relevant geometry in industrial practice.
    Two types of segregation can occur: fast radial segregation during which smaller or denser particles accumulate along the axis of rotation; and slow axial segregation with fully segregated bands of small and large particles perpendicular to the rotating axis, with in general particle bands of large particles adjacent to the end walls. This thesis reports on both radial and axial segregation phenomena in a horizontally rotating drum.
    While visual observation of the particle bed was used as a qualitative observation technique to determine the degree of mixing/segregation, in parallel a more quantitative method was developed as well, which was based on calculating the entropy over the systems. By subdividing the system with a lattice, calculating the entropy of mixing in each cell of the lattice, and summarizing them over the system, a measure for the degree of overall segregation was obtained. By using different grids (a 3D mesh, a 2D set of slices perpendicular to the axis, or 2D bars parallel to the axis), different types of segregation could be distinguished.
    The radial segregation dynamics were investigated in semi-2D (very short) drums, which inhibits axial segregation. Diagrams were prepared that visualise the mixing behaviour as function of the Froude number (rotational speed) for systems with different bidisperse systems. It was found that while almost all systems showed radial segregation at low Fr (rolling regime), and most showed inverted radial segregation at high Fr (cataracting or centrifuging regime), at Fr ≈ 0.56 all systems became radially mixed. This could be understood by assuming a percolation mechanism. In the moving layer on top of the load, smaller particles percolate in between the moving larger particles, down to the centre of the load, as long as the motion is not too fast. The same phenomenon is inverted at high speeds. In between, the flowing layer is expanded in such a way that many large voids are present, which makes the percolation mechanism less selective on the particle size. The little segregation that occurs is negligible, since the two phenomena described above work in different directions. Surprisingly this transitional Fr number is the same for all investigated systems.
    Since axial segregation is always preceded by radial segregation, it is logical to also study axial segregation. This was done by studying longer drums, which allows axial segregation to develop along the axis. Axial segregation was found for most systems; its occurrence is mostly dictated by differences in size.
    It was found that for drums that have intermediate length, surprising dynamic behaviour results. The axial segregation developed with low and high frequency oscillations. While the low frequency oscillations could be understood as the development and migration of segregated areas in the system, the higher frequency oscillations, with a period of 10 to 20 revolutions, were not identified before. This oscillatory behaviour is probably coupled to the use of intermediately sized drums, as this behaviour has not been seen with very long drums. We ascribe the oscillations to the influence of the (vertical) end walls, which expose the adjacent particles to different forces than those particles inside the drum load. These differences induce an axial flow in the system. The particles adjacent to the vertical walls tend to be lifted higher than the particles far away from the vertical walls. This creates a concave profile of the load surface throughout the drum, inducing the particles (in the rolling regime) to follow a path away from the vertical walls towards the centre of the drum. Once past the centre, the particles flow back to the vertical walls in response to the locally convex bed profile.
    Even in this particular flow profile the percolation mechanism is of importance: smaller particles percolate through the flowing layer and end up deeper inside the bed, while the larger particles accumulate on top of the flowing layer and are conveyed back to the vertical walls. Due to the percolation of the small particles the final end configuration must clearly be a banding configuration of large-small-large particle bands. Prolonged rotation of the bed increases the concave form of the flowing layer. This induces fast oscillations and a sudden mixing of a part of the large particle band with the small particle band, giving fast mixing and leading to a configuration, in which a small-particle band is formed below the large-particles bands. Subsequently segregation into three bands (large-small-large) slowly occurs again, after which the asymmetry in the angel of repose further increases. The configuration, in which larger particles accumulate on top of the bed adjacent to the end walls, coincides with a minimum in energy dissipation, which is not present when the systems segregates radially or axially into three pure bands.
    The effect found implies that the end walls are important in the dynamics of axial segregation. This effect is studied further by varying the end wall properties. The above mentioned fast and slow oscillations vanish in systems that have smoother end walls, while also the rate of segregation decreases; nevertheless the same axially segregated three band (large-small-large) state of mixing resulted finally. Reducing the friction further to completely smooth end walls however changed the final configuration into a two-banded system. Systems with no end wall at all, simulated through periodic end walls, only gave radial segregation over the (considerable) simulated time span. We expect here that as long as there is still a driving force for axial segregation, the absence of the induction of axial flow by the end walls make the transition very slow or impossible. The formation of two axial bands lowers the energy dissipation by the bed, whereas neither radial segregation nor axial segregation into three bands reduced the power absorption at constant angular velocity.
    While the oscillatory behaviour is relevant in its own right, their study also allows shedding some light on the fundamental mechanisms underlying the segregation mechanisms, and especially the transition from radial to axial segregation. The fact that this is dependent on not only the properties of the granular materials, but also on the geometry and design of the drum, implies that these findings have relevance to the design and operation of processes.
    Optimization of the dilute maleic acid pretreatment of wheat straw
    Kootstra, A.M.J. ; Beeftink, H.H. ; Scott, E.L. ; Sanders, J.P.M. - \ 2009
    Biotechnology for Biofuels 2 (2009). - ISSN 1754-6834 - p. 31 - 31.
    cellulose hydrolysis - enzymatic-hydrolysis - ethanologenic yeast - high-temperature - organic-acids - corn stover - pig diets - d-xylose - biomass - degradation
    Background - In this study, the dilute maleic acid pretreatment of wheat straw is optimized, using pretreatment time, temperature and maleic acid concentration as design variables. A central composite design was applied to the experimental set up. The response factors used in this study are: (1) glucose benefits from improved enzymatic digestibility of wheat straw solids; (2) xylose benefits from the solubilization of xylan to the liquid phase during the pretreatment; (3) maleic acid replenishment costs; (4) neutralization costs of pretreated material; (5) costs due to furfural production; and (6) heating costs of the input materials. For each response factor, experimental data were fitted mathematically. After data translation to €/Mg dry straw, determining the relative contribution of each response factor, an economic optimization was calculated within the limits of the design variables. Results - When costs are disregarded, an almost complete glucan conversion to glucose can be reached (90% from solids, 7%-10% in liquid), after enzymatic hydrolysis. During the pretreatment, up to 90% of all xylan is converted to monomeric xylose. Taking cost factors into account, the optimal process conditions are: 50 min at 170°C, with 46 mM maleic acid, resulting in a yield of 65 €/Mg (megagram = metric ton) dry straw, consisting of 68 €/Mg glucose benefits (from solids: 85% of all glucan), 17 €/Mg xylose benefits (from liquid: 80% of all xylan), 17 €/Mg maleic acid costs, 2.0 €/Mg heating costs and 0.68 €/Mg NaOH costs. In all but the most severe of the studied conditions, furfural formation was so limited that associated costs are considered negligible. Conclusions - After the dilute maleic acid pretreatment and subsequent enzymatic hydrolysis, almost complete conversion of wheat straw glucan and xylan is possible. Taking maleic acid replenishment, heating, neutralization and furfural formation into account, the optimum in the dilute maleic acid pretreatment of wheat straw in this study is 65 €/Mg dry feedstock. This is reached when process conditions are: 50 min at 170°C, with a maleic acid concentration of 46 mM. Maleic acid replenishment is the most important of the studied cost factors
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