Galacto-oligosaccharide production with immobilized ß-galactosidase in a packed-bed reactor vs. free ß-galactosidase in a batch reactor
Warmerdam, A. ; Benjamins, E. ; Leeuw de, T.F. ; Broekhuis, T.A. ; Boom, R.M. ; Janssen, A.E.M. - \ 2014
Food and Bioproducts Processing 92 (2014)4. - ISSN 0960-3085 - p. 383 - 392.
bacillus-circulans - lactose hydrolysis - eupergit-c - covalent immobilization - enzymatic-synthesis - enzymes - supports - temperature - milk - art
We report here that the usage of immobilized enzyme in a continuous packed bed reactor (PBR) can be a good alternative for GOS production instead of the traditional use of free enzyme in a batch reactor. The carbohydrate composition of the product of the PBR with immobilized enzyme was comparable to that of the batch reactor with free enzyme. The stability of the immobilized enzyme at a lactose concentration of 38% (w/v) and at 50¿C was very high: the half-life time of the immobilized enzyme was approximately 90 days. The enzymatic productivity of GOS production using immobilized enzyme in a PBR can be more than six times higher than that of GOS production with free enzyme in a batch reactor. Besides, when aiming for an equal volumetric productivity to the batch process in designing a PBR, the volume of the PBR can be much smaller than that of the batch reactor, depending on the enzyme dosage and the run time of a single batch
Kinetic Characterization of Galacto-Oligosaccharide (GOS) Synthesis by Three Commercially Important b-Galactosidases
Warmerdam, A. ; Zisopoulos, F.K. ; Boom, R.M. ; Janssen, A.E.M. - \ 2014
Biotechnology Progress 30 (2014)1. - ISSN 8756-7938 - p. 38 - 47.
bacillus-circulans - aspergillus-oryzae - lactose hydrolysis - kluyveromyces-lactis - enzymatic-synthesis - escherichia-coli - skim milk - purification - onpg
Many b-galactosidases show large differences in galacto-oligosaccharide (GOS) production and lactose hydrolysis. In this study, a kinetic model is developed in which the effect of lactose, glucose, galactose, and oligosaccharides on the oNPG converting activity of various b-galactosidases is quantified. The use of oNPG as a competing substrate to lactose yields more information than can be obtained by examining only the conversion of lactose itself. The reaction rate with lactose or oligosaccharides as substrate relative to that with water as acceptor is much higher for the b-galactosidase of Bacillus circulans than the bgalactosidases of Aspergillus oryzae and Kluyveromyces lactis. In addition, the bgalactosidase of B.circulans has a high reaction rate with galactose as acceptor, in contrast to those of A. oryzae and K. lactis. The latter two are strongly inhibited by galactose. These differences explain why b-galactosidase of B. circulans gives higher yields in GOS production than other b-galactosidases. Many of the reaction rate constants for the b-galactosidase isoforms of B. circulans increase with increasing molecular weight of the isoform. This indicates that the largest isoform b-gal-A is most active in GOS production. However, its hydrolysis rate is also much higher than that of the other isoforms, which results in a faster hydrolysis of oligosaccharides as well.
Molecular assembly, interfacial rheology and foaming properties of oligofructose fatty acid esters
Kempen, S.E.H.J. van; Schols, H.A. ; Linden, E. van der; Sagis, L.M.C. - \ 2014
Food & Function 5 (2014)1. - ISSN 2042-6496 - p. 111 - 122.
lipase-catalyzed synthesis - surface-active properties - solid-phase synthesis - air-water-interface - enzymatic-synthesis - beta-lactoglobulin - chain-length - sugar esters - dilatational rheology - candida-antarctica
Two major types of food-grade surfactants used to stabilize foams are proteins and low molecular weight (LMW) surfactants. Proteins lower the surface tension of interfaces and tend to unfold and stabilize the interface by the formation of a visco-elastic network, which leads to high surface moduli. In contrast, LMW surfactants lower the surface tension more than proteins, but do not form interfaces with a high modulus. Instead, they stabilize the interface through the Gibbs-Marangoni mechanism that relies on rapid diffusion of surfactants, when surface tension gradients develop as a result of deformations of the interface. A molecule than can lower the surface tension considerably, like a LMW surfactant, but also provide the interface with a high modulus, like a protein, would be an excellent foam stabilizer. In this article we will discuss molecules with those properties: oligofructose fatty acid esters, both in pure and mixed systems. First, we will address the synthesis and structural characterization of the esters. Next, we will address self-assembly and rheological properties of air/water interfaces stabilized by the esters. Subsequently, this paper will deal with mixed systems of mono-esters with either di-esters and lauric acid, or proteins. Then, the foaming functionality of the esters is discussed.
Effects of Carbohydrates on the oNPG Converting Activity of ß-Galactosidases
Warmerdam, A. ; Wang, J. ; Boom, R.M. ; Janssen, A.E.M. - \ 2013
Journal of Agricultural and Food Chemistry 61 (2013)26. - ISSN 0021-8561 - p. 6458 - 6464.
bacillus-circulans - physiological consequences - enzymatic-synthesis - aspergillus-oryzae - lactose hydrolysis - oligosaccharides - confinement
The effects of high concentrations of carbohydrates on the o-nitrophenyl ß-D-galactopyranoside (oNPG) converting activity of ß-galactosidase from Bacillus circulans are studied to get a better understanding of the enzyme behavior in concentrated and complicated systems in which enzymatic synthesis of galacto-oligosaccharides is usually performed. The components that were tested were glucose, galactose, lactose, sucrose, trehalose, raffinose, Vivinal GOS, dextran-6000, dextran-70 000, and sarcosine. Small carbohydrates act as acceptors in the reaction. This speeds up the limiting step, which is binding of the galactose residue with the acceptor and release of the product. Simultaneously, both inert and reacting additives seem to cause some molecular crowding, which results in a higher enzyme affinity for the substrate. The effect of molecular crowding on the enzyme activity is small compared to the effect of carbohydrates acting in the reactions as acceptors. The effects of reactants on ß-galactosidases from B. circulans, A. oryzae, and K. lactis are compared. KEYWORDS: galacto-oligosaccharides, GOS, ß-galactosidase, Bacillus circulans, enzyme activity, carbohydrates, crowding, galactosyl transfer, transgalactosylation, Aspergillus oryzae, Kluyveromyces lactis
The effect of diesters and lauric acid on rheological properties of air/water interfaces stabilized by oligofructose lauric acid monoesters
Kempen, S.E.H.J. van; Schols, H.A. ; Linden, E. van der; Sagis, L.M.C. - \ 2013
Journal of Agricultural and Food Chemistry 61 (2013)32. - ISSN 0021-8561 - p. 7829 - 7837.
enzymatic-synthesis - esters - esterification - oligosaccharides - viscoelasticity - lipase - shear
In this study, the rheological properties of interfaces stabilized by oligofructose fatty acid esters were elucidated. First, the properties of interfaces stabilized by monoesters (ME), diesters (DE), lauric acid (LA), oligofructose (OF), and mixtures of ME with DE, LA, or OF were studied. Second, the properties of interfaces stabilized by the crude product (CP) containing ME, DE, LA, and OF were studied. The dependency of the dilatational modulus on frequency and deformation amplitude indicated the possible formation of a soft glass phase for ME, and a viscous interface for DE. When ME and DE were mixed at a ratio of 0.8/0.2, the experimental results suggest that the interfacial structure consists of islands of a glass phase formed by ME, dispersed in a 2D viscous phase of DE. CP stabilized interfaces, where the ratio ME/DE was higher, lead to a different rheological response. The ratio ME/DE plays an important role in the surface properties of the CP. This may have significant consequences for applications in macroscopic systems such as foams.
Computer-aided solvent screening for biocatalysis
Abildskov, J. ; Leeuwen, M.B. van; Boeriu, C.G. ; Broek, L.A.M. van den - \ 2013
Journal of Molecular Catalysis. B, Enzymatic 85-86 (2013). - ISSN 1381-1177 - p. 200 - 213.
2-phase partitioning bioreactor - vapor-liquid-equilibria - polycyclic aromatic-hydrocarbons - quantum-mechanics calculations - molecular-dynamics simulation - fluctuation solution theory - unifac group-contribution - organic-solvents - enzymatic-synthesis - therm
A computer-aidedsolventscreening methodology is described and tested for biocatalytic systems composed of enzyme, essential water and substrates/products dissolved in a solvent medium, without cells. The methodology is computationally simple, using group contribution methods for calculating constrained properties related to chemical reaction equilibrium, substrate and product solubility, water solubility, boiling points, toxicity and others. Two examples are provided, covering the screening of solvents for lipase-catalyzed transesterification of octanol and inulin with vinyl laurate. Esterification of acrylic acid with octanol is also addressed. Solvents are screened and candidates identified, confirming existing experimental results. Although the examples involve lipases, the method is quite general, so there seems to be no preclusion against application to other biocatalysts
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.
Biocatalytic synthesis of new copolymers from 3-hydroxybutyric acid and a carbohydrate lactone
Kakasi-Zsurka, S. ; Todea, A. ; But, A. ; Paul, C. ; Boeriu, C.G. ; Davidescu, C. ; Nagy, L. ; Kuki, A. ; Keki, S. ; Peter, F. - \ 2011
Journal of Molecular Catalysis. B, Enzymatic 71 (2011)1-2. - ISSN 1381-1177 - p. 22 - 28.
ring-opening polymerization - lipase-catalyzed synthesis - enzymatic-synthesis - ionic liquids - polyesters
Lipase-catalyzed reaction of 3-hydroxybutyric acid with d-glucono-d-lactone at 5:1 molar ratio and 80 °C yielded a mixture of moderate molecular weight linear and cyclic oligomers. The most efficient biocatalyst, Candida antarctica B lipase (Novozyme 435), allowed the synthesis of new oligomeric compounds with ring-opened gluconolactone units included in the oligomeric chain, without previous derivatization of the sugar, or activation of the acid monomer. The reaction medium nature had an important influence on the product composition. Although the main copolymer amount was synthesized in tert-butanol/dimethylsulfoxide medium, the highest polymerization degrees, up to 9 for the copolymer, and 10 for the 3-hydroxybutyric acid homopolymer co-product, were achieved in solventless conditions.
Regio- and stereoselective glucosylation of diols by sucrose phosphorylase using sucrose or glucose 1-phosphate as glucosyl donor
Renirie, R. ; Pukin, A. ; Lagen, B. van; Franssen, M.C.R. - \ 2010
Journal of Molecular Catalysis. B, Enzymatic 67 (2010)3-4. - ISSN 1381-1177 - p. 219 - 224.
enzymatic-synthesis - catalyzed synthesis - bifidobacterium-adolescentis - water activity - glycosides
Previously it has been shown that glycerol can be regioselectively glucosylated by sucrose phosphorylase from Leuconostoc mesenteroides to form 2-O-alpha-D-glucopyranosyl-glycerol (Coedl et al., Angew. Chem. Int. Ed. 47 (2008) 10086-10089). A series of compounds related to glycerol were investigated by us to determine the scope of the alpha-glucosylation reaction of sucrose phosphorylase. Both sucrose and glucose 1-phosphate (GIP) were applied as glucosyl donor. Mono-alcohols were not accepted as substrates but several 1,2-diols were readily glucosylated, proving that the vicinal diol unit is crucial for activity. The smallest substrate that was accepted for glucosylation appeared to be ethylene glycol, which was converted to the monoglucoside for 69%. Using high acceptor and donor concentrations :up to 2.5 M), sucrose or GIP hydrolysis (with H2O being the 'acceptor') can be minimised. In the study cited above, a preference for glucosylation of glycerol on the 2-position has been observed. For 1,2-propanediol however, the regiochemistry appeared to be dependent on the configuration of the substrate. The (R)enantiomer was preferentialy glucosylated on its 1-position (ratio 2.5:1), whereas the 2-glucoside is the major product for (S)-1,2-propanediol (1:4.1). d.e.(p)s of 71-83% were observed with a preference for the (S)-enantiomer of the glucosides of 1,2-propanediol and 1,2-butanediol and the (R)-enantiomer of the glucoside of 3-methoxy-1,2-propanediol. This is the first example of stereoselective glucosylation of a non-natural substrate by sucrose phosphorylase. 3-Amino-1,2-propanediol, 3-chloro-1,2-propanediol, 1-thioglycerol and glyceraldehyde were not accepted as substrates. Generally, the glucoside yield is higher when sucrose is used as a donor rather than GIP. due to the fact that the released phosphate is a stronger inhibitor of the enzyme (in case of Cl P) than the released fructose (in case of sucrose). Essentially the same results are obtained with sucrose phosphorylase from Blfidobacterium adolescentis.
Rational engineering of Lactobacillus acidophilus NCFM maltose phosphorylase into either trehalose or kojibiose dual specificity phosphorylase
Nakai, H. ; Petersen, B.O. ; Westphal, Y. ; Dilokpimol, A. ; Hachem, M.A. ; Duus, J.O. ; Schols, H.A. ; Svensson, B. - \ 2010
Protein Engineering, Design & Selection 23 (2010)10. - ISSN 1741-0126 - p. 781 - 787.
thermoanaerobacter-brockii - enzymatic-synthesis - crystal-structure - enzymes - cloning - brevis - gene - oligosaccharides - glucoamylase - purification
Lactobacillus acidophilus NCFM maltose phosphorylase (LaMP) of the (a/a)6-barrel glycoside hydrolase family 65 (GH65) catalyses both phosphorolysis of maltose and formation of maltose by reverse phosphorolysis with ß-glucose 1-phosphate and glucose as donor and acceptor, respectively. LaMP has about 35 and 26% amino acid sequence identity with GH65 trehalose phosphorylase (TP) and kojibiose phosphorylase (KP) from Thermoanaerobacter brockii ATCC35047. The structure of L. brevis MP and multiple sequence alignment identified (a/a)6-barrel loop 3 that forms the rim of the active site pocket as a target for specificity engineering since it contains distinct sequences for different GH65 disaccharide phosphorylases. Substitution of LaMP His413–Glu421, His413–Ile418 and His413–Glu415 from loop 3, that include His413 and Glu415 presumably recognising the a-anomeric O-1 group of the glucose moiety at subsite +1, by corresponding segments from Ser426–Ala431 in TP and Thr419–Phe427 in KP, thus conferred LaMP with phosphorolytic activity towards trehalose and kojibiose, respectively. Two different loop 3 LaMP variants catalysed the formation of trehalose and kojibiose in yields superior of maltose by reverse phosphorolysis with (a1, a1)- and a-(1,2)-regioselectivity, respectively, as analysed by nuclear magnetic resonance. The loop 3 in GH65 disaccharide phosphorylase is thus a key determinant for specificity both in phosphorolysis and in regiospecific reverse phosphorolysis.
Selective Synthesis of Unsaturated N-Acylethanolamines by Lipase-Catalyzed N-Acylation of Ethanolamine with Unsaturated Fatty Acids
Plastina, P. ; Vincken, J.P. ; Gruppen, H. ; Witkamp, R.F. ; Gabriele, B. - \ 2009
Letters in Organic Chemistry 6 (2009)6. - ISSN 1570-1786 - p. 444 - 447.
enzymatic-synthesis - amide surfactants - carboxylic-acids - ionic liquids - solvent - anandamide - endocannabinoids - amidation - pressure - amines
The selective synthesis of unsaturated N-acylethanolamines 1b-6b by lipase-catalyzed direct condensation between unsaturated fatty acids 1a-6a and ethanolamine is reported. Reactions were carried out in hexane at 40 °C, in the presence of Candida antarctica Lipase B as the catalyst, to give the corresponding amides 1b-6b with yields ranging from 80 to 88%.
Effects of cultivation conditions on folate production by lactic acid bacteria
Sybesma, W. ; Starrenburg, M. ; Tijsseling, L. ; Hoefnagel, M.H.N. ; Hugenholtz, J. - \ 2003
Applied and Environmental Microbiology 69 (2003)8. - ISSN 0099-2240 - p. 4542 - 4548.
gtp cyclohydrolase-i - neural-tube defects - folic-acid - streptococcus-lactis - lactococcus-lactis - enzymatic-synthesis - purification - homocysteine - biosynthesis - strain
A variety of lactic acid bacteria were screened for their ability to produce folate intracellularly and/or extracellularly. Lactococcus lactis, Streptococcus thermophilus, and Leuconostoc spp. all produced folate, while most Lactobacillus spp., with the exception of Lactobacillus plantarum, were not able to produce folate. Folate production was further investigated in L. lactis as a model organism for metabolic engineering and in S. thermophilus for direct translation to (dairy) applications. For both these two lactic acid bacteria, an inverse relationship was observed between growth rate and folate production. When cultures were grown at inhibitory concentrations of antibiotics or salt or when the bacteria were subjected to low growth rates in chemostat cultures, folate levels in the cultures were increased relative to cell mass and (lactic) acid production. S. thermophilus excreted more folate than L. lactis, presumably as a result of differences in the number of glutamyl residues of the folate produced. In S. thermophilus 5,10-methenyl and 5-formyl tetrahydrofolate were detected as the major folate derivatives, both containing three glutamyl residues, while in L. lactis 5,10-methenyl and 10-formyl tetrahydrofolate were found, both with either four, five, or six glutamyl residues. Excretion of folate was stimulated at lower pH in S. thermophilus, but pH had no effect on folate excretion by L. lactis. Finally, several environmental parameters that influence folate production in these lactic acid bacteria were observed; high external pH increased folate production and the addition of p-aminobenzoic acid stimulated folate production, while high tyrosine concentrations led to decreased folate biosynthesis
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.