| 1. |
- Adlercreutz, Dietlind, et al.
(författare)
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An enzymatic method for the synthesis of mixed-acid phosphatidylcholine
- 2004
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Ingår i: Journal of the American Oil Chemists Society. - : Wiley. - 0003-021X. ; 81:6, s. 553-557
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Tidskriftsartikel (refereegranskat)abstract
- The enzymatic synthesis of PC with decanoic acid in the sn-1 and hexanoic acid in the sn-2 position is described. The procedure comprises the following enzymatic steps: (i) treatment of egg yolk with phospholipase A(2) (PLA(2)) to hydrolyze egg yolk PC to 1-acyl lysophosphaticlylcholine (LPC) (ii) esterification of I-acyl LPC with hexanoic acid catalyzed by PLA(2) to yield PC with hexanoic acid in the sn-2 position; (iii) removal of the FA in the sn-1 position by lipase-catalyzed ethanolysis to yield 2-hexanoyl LPC; and finally (iv) introduction of decanoic acid in this position by lipase-catalyzed esterification of 2-hexanoyl LPC to yield 1-decanoyl-2-hexanoyl-PC. Two egg yolks with a weight of 16 g were required to obtain 160 mg of the desired product. The chemical purity of the PC product and the positional purity of the FA were around 99%. The method is applicable for the synthesis of other mixed-acid PC species as well.
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| 2. |
- Adlercreutz, Dietlind, et al.
(författare)
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Synthesis of phosphatidylcholine with defined fatty acid in the sn-1 position by lipase-catalyzed esterification and transesterification reaction.
- 2002
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 78:4, s. 403-411
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Tidskriftsartikel (refereegranskat)abstract
- The incorporation of caproic acid in the sn-1 position of phosphatidylcholine (PC) catalyzed by lipase from Rhizopus oryzae was investigated in a water activity-controlled organic medium. The reaction was carried out either as esterification or transesterification. A comparison between these two reaction modes was made with regard to product yield, product purity, reaction time, and byproduct formation as a consequence of acyl migration. The yield in the esterification and transesterification reaction was the same under identical conditions. The highest yield (78%) was obtained at a water activity (a(w)) of 0.11 and a caproic acid concentration of 0.8 M. The reaction time was shorter in the esterification reaction than in the transesterification reaction. The difference in reaction time was especially pronounced at low water activities and high fatty acid concentrations. The loss in yield due to acyl migration and consequent enzymatic side reactions was around 16% under a wide range of conditions. The incorporation of a fatty acid in the sn-1 position of PC proved to be thermodynamically much more favorable than the incorporation of a fatty acid in the sn-2 position.
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| 3. |
- Adlercreutz, Patrick, et al.
(författare)
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Enzymatic conversions of polar lipids. Principles, problems and solutions
- 2001
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Ingår i: Journal of Molecular Catalysis - B Enzymatic. - 1381-1177. ; 11:4-6, s. 173-178
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Forskningsöversikt (refereegranskat)abstract
- This text provides a brief overview of the principles of enzymatic lipid conversion and some recent advances in the enzymatic conversion of glycerophospholipids and galactolipids. Lipases and phospholipases are used to exchange fatty acids or the polar group in the lipids. The reactions can be carried out either as hydrolysis-esterification sequences or as one-step transferase reactions. The scope and limitations of the different methods are discussed.
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| 4. |
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| 5. |
- Barros, Raúl J., et al.
(författare)
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Effect of mass-transfer limitations on the selectivity of immobilized α-chymotrypsin biocatalysts prepared for use in organic medium
- 2000
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Ingår i: Biotechnology and Bioengineering. - 0006-3592. ; 67:3, s. 319-326
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Tidskriftsartikel (refereegranskat)abstract
- The selectivity of preparations of α-chymotrypsin immobilized on Celite or polyamide and carrying out syntheses of di- and tripeptides in acetonitrile medium were studied. The study concerns the effect of mass- transfer limitations on three different kinds of selectivity: acyl donor, stereo- and nucleophile selectivities, defined respectively as the ratio of initial rates with different acyl donors; the enantioselectivity factor (E); and the ratio of initial rates of peptide synthesis and hydrolysis of the acyl donor. Strong mass-transfer limitations caused by increased enzyme loading had a very strong effect on acyl donor selectivity, with reductions of up to 79%, and on stereoselectivity, with reductions of up to 77% in relation to optimum values, both on Celite. Nucleophile selectivity was not affected as strongly by mass-transfer limitations. Using a small molecule (AlaNH2) as nucleophile, the onset of these limitations caused only minor reductions in selectivity, while when using a larger nucleophilic species (AlaPheNH2) it was reduced by up to 60% when increasing enzyme loading on Celite from 2 to 100 mg/g. The different way these kinds of selectivity are affected by the onset of mass-transfer limitations can be explained by a combination of different aspects: the kinetic behavior of the enzyme toward nucleophile and acyl donor concentrations, the relative concentrations of reagents used in the reaction media, and their relative diffusion coefficients. In short, higher concentrations of nucleophile than acyl donor are generally used, and the nucleophile most often used in the experiments hereby described (AlaNH2) diffuses faster than the acyl donors employed. These factors combined are expected to give rise to concentration gradients inside porous biocatalyst particles higher for acyl donor than for nucleophile under conditions of mass-transfer limitations. This explains why acyl donor selectivity and stereoselectivity are much more influenced by mass transfer limitations than nucleophile selectivity.
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| 6. |
- Barros, Raúl J., et al.
(författare)
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Enhancement of immobilized protease catalyzed dipeptide synthesis by the presence of insoluble protonated nucleophile
- 1999
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Ingår i: Enzyme and Microbial Technology. - 0141-0229. ; 24:8-9, s. 480-488
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Tidskriftsartikel (refereegranskat)abstract
- α-Chymotrypsin immobilized on celite catalyzing a kinetically controlled dipeptide synthesis reaction in acetonitrile medium showed an odd behavior in response to additions of triethylamine to the reaction mixture. This base is used to deprotonate the nucleophilic reagent, l-alaninamide hydrochloride, in order to increase its nucleophilicity and solubility. However, the enzyme performance is apparently enhanced by additions of triethylamine below one equivalent (in the range 15-20 mm) while the used concentration of nucleophilic reagent is 30 mm. Under these conditions, the initial rate is up to 2.5 times higher and the nucleophile specificity is approximately 30% better than when one equivalent is added. The activating effect on initial rates of dipeptide synthesis was not observed when polyamide was used as support. Unlike polyamide, celite is a material with quite low porosity. Improvement of nucleophile specificity was observed using both supports. It is shown that this activation arises due to the presence of a separate dense liquid phase of insoluble l-alaninamide hydrochloride that intimately contacts with the enzyme preparation, and does not depend on the addition of triethylamine itself. Additions of l-alaninamide hydrochloride improved initial rates of synthesis more than 2.5-fold, and nucleophile specificity more than threefold. The initial rate activation was also observed when using non-porous glass beads to immobilize the enzyme at a loading of 5 mg enzyme g-1 glass but not at 1 mg enzyme g-1 glass when no mass transfer limitations in the immobilized enzyme layer are expected to occur. The results suggest that the presence of the separate phase helps to relieve mass transfer limitations on the system caused by overloading at the supports. One possible mechanism for the initial rate activation might be that the enzyme is partially desorbed from the support particles into the separate phase of nucleophile, and the better nucleophile specificity observed is due to increased local concentrations of the nucleophile within this phase. Copyright (C) 1999 Elsevier Science Inc. All rights reserved.
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| 7. |
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| 8. |
- Barros, Raúl J., et al.
(författare)
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Mass transfer studies on immobilized α-chymotrypsin biocatalysts prepared by deposition for use in organic medium
- 1998
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Ingår i: Biotechnology and Bioengineering. - 0006-3592. ; 59:3, s. 364-373
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Tidskriftsartikel (refereegranskat)abstract
- Mass transfer limitations were studied in enzyme preparations of α- chymotrypsin made by deposition on different porous support materials such as controlled pore glasses, Celite, and polyamides of different particle sizes. It is the onset of mass transfer limitations that determines the position of the activity optimum with respect to enzyme loading on each support. The evidence of various experiments indicates that internal diffusional limitations are the important mechanism for the observed mass transfer limitations. External diffusion was not found to play an important role under the conditions used, and it was also found that when immobilizing multilayers of enzyme the buried enzyme molecules are active to a large extent. An extreme situation is observed on Celite at very high loadings. Under these conditions, this support is expected to have its pores completely filled with packed enzyme molecules, and then it is the diffusion within the enzyme layer that determines the observed rate. As the enzyme loading increases, the area of contact between the deposited enzyme layers and the liquid solution inside the pores diminishes, causing a decrease on the observed rate of an intrinsically fast reaction which apparently is incongruous with the presence of more enzyme in the system. This work shows that mass transfer limitations can be an important factor when working with immobilized enzymes in organic media, and its study should be carried out in order to avoid undesired reduced enzyme activities and specificities.
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| 9. |
- Barros, Raúl J., et al.
(författare)
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Modeling the performance of immobilized α-chymotrypsin catalyzed peptide synthesis in acetonitrile medium
- 2001
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Ingår i: Journal of Molecular Catalysis - B Enzymatic. - 1381-1177. ; 11:4-6, s. 841-850
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Tidskriftsartikel (refereegranskat)abstract
- A model was developed which describes simultaneous reaction and internal diffusion for kinetically controlled, immobilized α-chymotrypsin-catalyzed, oligopeptide synthesis in acetonitrile medium. The model combines the equations that describe the intrinsic kinetics of four different reactions and the physical characteristics of three different support materials, as determined experimentally, to predict the apparent initial activity and nucleophile selectivity of the immobilized biocatalyst. The model is able to predict reasonably well the experimentally observed initial rate and nucleophile selectivity vs. enzyme loading profiles. The reduction in observed initial rate with enzyme loading when fast reactions are carried out with α-chymotrypsin immobilized on celite, and the larger influence of mass transfer limitations on the initial reaction rates than on nucleophile selectivities are correctly predicted by the numerical calculations. The model is general in terms of its application to other systems - enzymes, reactions, support materials and/or kinetic schemes - as long as the intrinsic kinetics and the characteristics of the enzyme and support material are known.
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| 10. |
- Barros, Raúl J., et al.
(författare)
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Physical characterization of porous materials and correlation with the activity of immobilized enzyme in organic medium
- 1998
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Ingår i: Biocatalysis and Biotransformation. - : Informa UK Limited. - 1024-2422 .- 1029-2446. ; 16:1, s. 67-85
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Tidskriftsartikel (refereegranskat)abstract
- A series of commonly used porous supports was characterized by determination of particle size distribution, porosity, surface area (total and distributions with pore diameters) and skeletal density. The performance of immobilized α-chymotrypsin catalyzed dipeptide synthesis in an acetonitrile medium was correlated with these physical properties.At high enzyme loading, when internal mass transfer limitations are expected to occur, the activity can be correlated with the support characteristic parameter. This is a combination of physical properties such as particle size, porosity, and volumetric porosity, which influence the substrate diffusion rate. At low enzyme loading the important parameter is the accessible surface area, which will determine how the enzyme is distributed in the pores of the support. When assessing the effect of the support material on enzymatic activity, the geometric considerations studied here should always be contemplated before making any assumptions about direct effects of support material on enzymatic catalytic properties.
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| 11. |
- Björup, Peter, et al.
(författare)
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Effects of acetonitrile-water mixtures on α-chymotrypsin catalyzed dipeptide synthesis
- 1996
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Ingår i: Biocatalysis and Biotransformation. - : Informa UK Limited. - 1024-2422 .- 1029-2446. ; 13:3, s. 189-200
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Tidskriftsartikel (refereegranskat)abstract
- α-Chymotrpysin (EC 3.4 21.1) was immobilized by deposition on celite and subsequent cross-linking with glutaraldehyde. The effects of different mixtures of aqueous buffer and acetonitrile on the immobilized preparation were evaluated using a dipeptide synthesis as model reaction. The initial reaction rate at 6-95% of water increased with increasing water content. The maximum yield of peptide had two maxima; the first one at 6% of water (92%) and the second one at 80% of water (39%). The presence of two maxima was due to severe enzyme inactivation at intermediate water contents (50-60%). The immobilisation procedure slowed the inactivation of α-chymotrypsin. Cross-linked enzyme was inactivated to a lesser extent than both free enzyme and enzyme that had been deposited on celite. The increased resistance to inactivation was, however, not sufficient to make peptide synthesis attractive at intermediate water contents (50-60%). In order to obtain good peptide yields, low water contents (below 10%) should be used.
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| 12. |
- Brocca, Stefania, et al.
(författare)
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Mutants provide evidence of the importance of glycosydic chains in the activation of lipase 1 from Candida rugosa
- 2000
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 9:5, s. 985-990
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Tidskriftsartikel (refereegranskat)abstract
- Sequence analysis of Candida rugosa lipase 1 (LIP1) predicts the presence of three N-linked glycosylation sites at asparagine 291, 314, 351. To investigate the relevance of sugar chains in the activation and stabilization of LIP1, we directed site mutagenesis to replace the above mentioned asparagine with glutamine residues. Comparison of the activity of mutants with that of the wild-type (wt) lipase indicates that both 314 and 351 Asn to Gln substitutions influence, although at a different extent, the enzyme activity both in hydrolysis and esterification reactions, but they do not alter the enzyme water activity profiles in organic solvents or temperature stability. Introduction of Gln to replace Asn35 is likely to disrupt a stabilizing interaction between the sugar chain and residues of the inner side of the lid in the enzyme active conformation. The effect of deglycosylation at position 314 is more difficult to explain and might suggest a more general role of the sugar moiety for the structural stability of lipase 1. Conversely, Asn291Gln substitution does not affect' the lipolytic or the esterase activity of the mutant that behaves essentially as the wt enzyme. This observation supports the hypothesis that changes in activity of Asn314Gln and Asn351Gln mutants are specifically due to deglycosylation.
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| 13. |
- Costes, David, et al.
(författare)
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Cross-linked crystals of hydroxynitrile lyase as catalyst for the synthesis of optically active cyanohydrins
- 2001
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Ingår i: Journal of Molecular Catalysis - B Enzymatic. - 1381-1177. ; 11:4-6, s. 607-612
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Tidskriftsartikel (refereegranskat)abstract
- Purified hydroxynitrile lyase (HNL) from Manihot esculenta was crystallized by the sitting-drop vapour-diffusion method. The bipyramidal crystals formed (10-20 μm) were cross-linked with different amounts of glutaraldehyde and used as biocatalyst for the synthesis of optically active cyanohydrins. The cross-linked crystals were more stable than Celite-immobilized enzymes when incubated in organic solvents, especially in polar solvents. After six consecutive batch reactions in dibutylether, the remaining activity of the cross-linked crystals was more than 70 times higher than for the immobilized enzymes. Nevertheless, the specific activity of the cross-linked crystals (per milligram protein) was reduced compared to the activity of immobilized enzymes. The product enantiopurity was independent of the type of enzyme preparation used.
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| 14. |
- Costes, David, et al.
(författare)
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Hydroxynitrile lyase-catalyzed synthesis of cyanohydrins in organic solvents Parameters influencing activity and enantiospecificity
- 1999
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Ingår i: Enzyme and Microbial Technology. - 0141-0229. ; 25:3-5, s. 384-391
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Tidskriftsartikel (refereegranskat)abstract
- (S)-Hydroxynitrile lyases from Hevea brasiliensis, Manihot esculenta, and Sorghum bicolor and (R)-hydroxynitrile lyase from Prunus amygdalus have been used as biocatalyst for the enantiospecific addition of hydrogen cyanide to aldehydes in organic solvents. The effects of the reaction parameters on the enzymatic reaction rate and product enantiomeric excess (e.e.) are presented. The reaction rate increased with the solvent hydrophobicity but highly hydrophobic solvents were not adapted to high hydrogen cyanide concentrations and provoked loss of activity and product e.e. In the synthesis of 3-phenylpropionaldehyde cyanohydrin catalyzed by (S)-hydroxynitrile lyases from H. brasiliensis, an e.e. value of 88 ± 1% was obtained under optimized reaction conditions. Lower enantiomeric excess values were obtained under conditions where the enzyme was inactivated: high hydrogen cyanide concentration, high solvent log P, low enzyme loading. At lower temperature (down to -5°C) the e.e. was increased for all four enzymes used. Enzymes from different sources used under identical optimized reaction conditions were found to yield cyanohydrins with very different enantiopurities. This intrinsic enantiospecificity is not an effect of spontaneous reactions independent of the enzyme. Copyright (C) 1999 Elsevier Science Inc.
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| 15. |
- Costes, David, et al.
(författare)
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Stability and stabilization of hydroxynitrile lyase in organic solvents
- 2001
-
Ingår i: Biocatalysis and Biotransformation. - : Informa UK Limited. - 1024-2422 .- 1029-2446. ; 19:2, s. 119-130
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Tidskriftsartikel (refereegranskat)abstract
- The stability of hydroxynitrile lyase from Hevea brasiliensis has been studied in organic solvents. In dry solvents, the enzyme had half-lives in the range 1400-2500 hours. The enzyme half-life was one order of magnitude lower if the medium was water saturated. The substrates, aldehyde and hydrogen cyanide, were found to promote enzyme deactivation. The deactivation increased with substrate concentration, but was reduced in hydrophilic solvent. At high substrate concentration (2 M) in tert-butyl methyl ether, the enzyme half-life was 1.7 h when incubated with hydrogen cyanide while it was 1.0 h with 3-phenylpropionaldehyde. The addition of polyethylenimine, 125 mg per g of enzyme preparation, increased the enzyme half-life to 110 h when incubated with hydrogen cyanide and to 3.2 h with 3-phenylpropanaldehyde in tert-butyl methyl ether. Albumin and poly(ethylene glycol) gave similar stabilization effect.
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| 16. |
- Egger, Dietlind, et al.
(författare)
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Characterization and optimization of phospholipase A2 catalyzed synthesis of phosphatidylcholine
- 1997
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Ingår i: BBA - Protein Structure and Molecular Enzymology. - 0167-4838. ; 1343:1, s. 76-84
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Tidskriftsartikel (refereegranskat)abstract
- The phospholipase A2 (PLA2) catalyzed synthesis and hydrolysis of phosphatidylcholine (PC) was studied in a water activity controlled organic medium. The aim of the study was to find the conditions most favorable for the synthetic reaction. To do this, the impact of various parameters such as water activity, substrate concentration and temperature on enzyme activity and equilibrium yield was determined. The PC to lysophosphatidylcholine (LPC) ratio at equilibrium increases with decreasing water activity and increasing fatty acid concentration, as can be expected from the law of mass action of an esterification reaction. The enzyme activity on the other hand decreases under conditions that favor the esterification. The best yield in the synthetic reaction is 60% at a water activity of 0.11 and an oleic acid concentration of 1.8 M. That is to our knowledge the highest yield ever reported in this reaction. Both the hydrolysis and synthesis reaction follow Michaelis-Menten kinetics, the apparent K(m) values are the same for PC and LPC, namely 4.9 mM. V(max) is 82.5 and 10.4 nmol h-1 mg-1 for the hydrolysis and synthesis reaction, respectively. Studies on PLA2 at water activity controlled conditions resulted in a more complete understanding of the enzymatic reaction and allowed to find the conditions most favorable for the synthetic reaction.
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| 17. |
- Hansson, Therese, et al.
(författare)
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Influence of water activity on the competition between β-glycosidase-catalysed transglycosylation and hydrolysis in aqueous hexanol
- 2001
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Ingår i: Enzyme and Microbial Technology. - 0141-0229. ; 29:8-9, s. 527-534
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Tidskriftsartikel (refereegranskat)abstract
- Five different β-glycosidases (Pyrococcus furiosus β-glucosidase, Sulfolobus solfataricus β-galactosidase, Caldocellum saccharolyticum β-glucosidase, almond β-glucosidase and Escherichia coli β-galactosidase) were evaluated as transglycosylation catalysts in hexanol containing various amounts of water. All enzymes catalysed both hydrolysis and transglycosylation of the glycosidic substrates (pentyl- and p-nitrophenyl-β-glucoside and p-nitrophenyl-β-galactoside). From the concentration ratio (alcohol/water) it was expected that the transglycosylation/hydrolysis ratio would decrease with increasing water activity in the hexanol. However, for all enzymes tested the selectivity for the alcohol increased with increasing water activity. This counteracted the effect of higher water concentration and in most cases the transglycosylation/hydrolysis ratio increased with increasing water activity. On the other hand, in hexanol/water two-phase systems, hydrolysis was by far the dominating reaction even though the total activity increased for all enzymes. The selectivity values were used to predict the maximal reaction yields in the kinetically controlled reactions. However, deviations were found in cases when the reactions became thermodynamically controlled: at high water contents secondary hydrolysis reduced the transglycosylation yields while higher transglycosylation yields than predicted were obtained at low water activity in some cases using enzymes poorly selective for the alcohol.
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| 18. |
- Jönsson, Åsa, et al.
(författare)
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Low reaction temperature increases the selectivity in an enzymatic reaction due to substrate solvation effects
- 1997
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Ingår i: Biotechnology Letters. - 0141-5492. ; 19:1, s. 85-88
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Tidskriftsartikel (refereegranskat)abstract
- Immobilized α-chymotrypsin was used as catalyst for studying temperature effects on acyl transfer reactions (acyl-donor: Bz-TyrOEt) in a water-immiscible organic solvent. The solubility of the two nucleophiles, Phe-NH2 and water, decreased with decreasing temperature. The relative decrease for the amide was larger (2.4-fold) than for water. Therefore the thermodynamic activity (estimated by the relative saturation) increased more for this substrate and hence the selectivity in the reaction was increased.
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| 19. |
- Jönsson, Åsa, et al.
(författare)
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Temperature effects on protease catalyzed acyl transfer reactions in organic media
- 1996
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Ingår i: Journal of Molecular Catalysis B: Enzymatic. - : Elsevier BV. - 1381-1177. ; 2:1, s. 43-51
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Tidskriftsartikel (refereegranskat)abstract
- The influence of reaction temperature on synthesis activity, product yield and nucleophile specificity for α-chymotrypsin and subtilisin Carlsberg were studied. The enzymes were immobilized on Celite and used in acetonitrile with a water content of 10%. Acyl-transfer reactions with Ac-PheOEt as acyl donor and 11 different amino acid amides and 3 dipeptides as nucleophiles were studied. The decrease in temperature from 25 to -1°C had a positive effect on the peptide yield in all reactions studied. The most efficient nucleophiles for the two enzymes α-chymotrypsin and subtilisin Carlsberg is arginine amide and glycine amide, respectively. When decreasing the reaction temperature the yield for α-chymotrypsin increased from 86 to 94% with arginine amide as nucleophile and for subtilisin the yield increased from 75 to 84% for glycine amide. The nucleophile specificity was determined as the p value, which describes the competition between nucleophile and water for the acyl enzyme. α-Chymotrypsin showed preference for both small and positively charged amino acid residues and subtilisin preferred small uncharged nucleophiles. The temperature did not affect the specificity order but all nucleophiles became more effective in the competition with water at low temperature. In addition, the results indicate that the temperature effect is more pronounced for the smaller nucleophiles.
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| 20. |
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| 21. |
- Jönsson, Åsa, et al.
(författare)
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The influence of water activity on the enantioselectivity in the enzyme- catalyzed reduction of 2-pentanone
- 1998
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Ingår i: Journal of Molecular Catalysis - B Enzymatic. - 1381-1177. ; 5:1-4, s. 273-276
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Tidskriftsartikel (refereegranskat)abstract
- The stereoselective reduction of 2-pentanone by alcohol dehydrogenase from Thermoanaerobium brockii was studied at controlled water activity and at different reaction temperatures. The reaction rate increased when water activity was increased from 0.32 to 0.96 and when raising the temperature from 5°C to 40°C. The enantioselectivity, E, reached a plateau value at high water activities. The enantioselectivity increased with decreasing reaction temperature.
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| 22. |
- Jönsson, Åsa, et al.
(författare)
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Thermodynamic and kinetic aspects on water vs. organic solvent as reaction media in the enzyme-catalysed reduction of ketones
- 1999
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Ingår i: BBA - Protein Structure and Molecular Enzymology. - 0167-4838. ; 1430:2, s. 313-322
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Tidskriftsartikel (refereegranskat)abstract
- The stereoselective reduction of ketones catalysed by alcohol dehydrogenase from Thermoanaerobium brockii was studied in different reaction media, hexane at controlled water activities, hexane with 2.5% water (biphasic) and pure water. The reactions were studied in the temperature range from -1 to 50°C. Increasing the water activity from 0.53 to 0.97 increased the reaction rate 16-fold. The rate was further enhanced in hexane when exceeding the water solubility and in pure water the rates were even higher. This was general for all ketones studied. At controlled water activity the entropy of activation (ΔS(≠)) was the dominating factor. Large negative ΔS(≠) values caused low reaction rates at low a(w). When increasing the carbon chain length of the substrate, for reactions in hexane, the decrease of reaction rate was mainly due to a decrease in ΔS(≠). In the comparison between hexane and pure water, ΔG(≠) values were higher in hexane due to higher ΔH(≠) values. The enantioselectivity (E value) increased from 2.6 at water activity 0.53 to 4.6 at water activity 0.97. Changing media from hexane (2.5%, v/v water) to pure water was not affecting the enantioselectivity or the specificity for different ketones. Copyright (C) 1999.
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| 23. |
- Kaur, Jasmedh, et al.
(författare)
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Water transfer kinetics in a water activity control system designed for biocatalysis in organic media
- 1997
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Ingår i: Enzyme and Microbial Technology. - 0141-0229. ; 21:7, s. 496-501
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Tidskriftsartikel (refereegranskat)abstract
- The performance of a water activity-controlling system in organic solvents using saturated salt solutions circulating in silicone tubing submerged in the solvent was studied. The mass transfer could be regarded as a flux through a cylindrical geometry. Integration over the barrier gave diffusion coefficients of water (D(aw)) which were similar for the different tubing sizes used. The driving force for the transfer was shown to be the difference in water activity and not the water concentration across the membrane. Hydrophilic solvents (ethyl acetate) gave higher transfer rates than more hydrophobic ones (diisopropyl ether). The D(aw) obtained in different solvents was influenced by the swelling behaviour of the tubing and the solubility of water. The water transfer was studied in a water 'producing' system consisting of 2.5 m silicone tubing submerged into a 250-ml tank reactor with a constant influx of water-saturated solvent. Different steady-state levels were obtained at different flow rates and the corresponding D(aw) values were calculated. The data obtained can be used to predict the required amount of tubing necessary to achieve a desired water transfer in a new application.
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| 24. |
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| 25. |
- Persson, Mattias, et al.
(författare)
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Effects of solvent, water activity and temperature on lipase and hydroxynitrile lyase enantioselectivity
- 2002
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Ingår i: Enzyme and Microbial Technology. - 0141-0229. ; 30:7, s. 916-923
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Tidskriftsartikel (refereegranskat)abstract
- The influence of the reaction conditions on the enantioselectivity of reactions catalysed by lipases or hydroxynitrile leases (HNLs) in organic solvents was investigated. The lipases catalysed kinetic resolution of chiral secondary alcohol, or chiral carboxylic acids and the HNLs catalysed asymmetric addition of hydrogen cyanide to aldehydes. The temperature effects on enantioselectivity, were studied in detail. From measurements of the enantiomeric ratio (C) at different temperatures the activation parameters DeltaDeltaH(#) and DeltaDeltaS(#) were determined. In the lipase-catalysed reactions the enthalpic and entropic effects on E always counteracted, while in a few of the HNL-catalysed reactions, DeltaDeltaH(#) and DeltaDeltaS(#) had opposite sign, and therefore the effects cooperated to give high E values (-RTInE = DeltaDeltaG(#) = DeltaDeltaH(#) - TDeltaDeltaS(#)). In all the HNL-catalysed reactions and most of the lipase-catalysed ones, the enantioselectivity increased with decreasing reaction temperature. However, in one of the lipase-catalysed reactions, the enantioselectivity decreased with decreasing temperature. The theoretical background of these observations wars discussed. In the HNL-catalysed reactions, the enantioselectivity increased with increasing water content up to water saturation, while in the lipase-catalysed reactions the opposite trend was found in one case and in the others no significant effect was observed. Solvent mixtures of diisopropylether and hexane were used to obtain solvents with different log P values. The log P value of the solvent did not influence the enantioselectivity in the HNL-catalysed reactions. while the enantioselectivity increased with increasing log P value in two of the lipase-catalysed reactions. The reaction temperature was shown to be a very useful way to influence enzyme selectivity and the effects obtained could be rationalised. The influence of the reaction medium (solvent and water activity) is much more difficult to rationalise and predict. (C) 2002 Elsevier Science Inc. All rights reserved.
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| 26. |
- Persson, Mattias, et al.
(författare)
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Factors governing the activity of lyophilised and immobilised lipase preparations in organic solvents
- 2002
-
Ingår i: ChemBioChem. - 1439-4227. ; 3:6, s. 566-571
-
Tidskriftsartikel (refereegranskat)abstract
- Active site titration and activity measurements were performed in hexane on lyophilised lipase preparations containing different amounts of phosphate buffer and lipase immobilised on porous polypropylene. Lyophilisation of Thermomyces lanuginosus lipase with large quantities of phosphate salts (200 mm) increased the specific activity fourfold, and the number of rapidly titratable active sites increased to 50% from the 73% observed when smaller amounts of phosphate buffer were used (20 mm) during lyophilisation. The phosphate buffer worked as an immobilisation matrix for the lipase, and the increase in specific activity was at least portly due to decreased mass transfer limitations. When lipase was immobilised on porous polypropylene, the specific activity was 770 times higher than that of the best freeze-dried preparation. At optimal enzyme loading, 93% of the enzyme molecules were titrated at a high rate; this indicates that this adsorption on a hydrophobic surface was a very efficient means of reducing moss transfer limitations and of immobilising the enzyme in its active conformation for use in organic solvents. The variation in specific activity with water activity was found to correlate very well with the variation in titratable active sites when lipases from Burkholderia cepacia and Thermomyces lanuginosus were immobilised on porous polypropylene. The catalytic activity per competent active site was thus constant over the whole range of water activities.
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| 27. |
- Persson, Mattias, et al.
(författare)
-
Immobilisation of lipases by adsorption and deposition : High protein loading gives lower water activity optimum
- 2000
-
Ingår i: Biotechnology Letters. - 0141-5492. ; 22:19, s. 1571-1575
-
Tidskriftsartikel (refereegranskat)abstract
- Two different immobilisation techniques for lipases were investigated: adsorption on to Accurel EP-100 and deposition on to Celite. The specific activities were in the same order of magnitude, 2.9 (μmol min -1 mg protein) when Celite was used as support and 2.3 (μmol min -1 mg -1 protein) when Accurel EP-100 was used as support, even if the amount of lipase loaded differed by 2 orders of magnitude. Immobilisation on Accurel EP-100 was the preferred technique since 40-100 times more protein can be loaded/per g carrier, thus yielding a more active catalyst. The water activity profiles in lipase catalysed esterification were influenced by the amount of protein adsorbed to Accurel EP-100. Higher protein loading (40 mg g -1) resulted in a bell-shaped water activity profile with highest specific activity (6.1 μmol min -1 mg -1 protein) at a(w) = 0.11, while an enzyme preparation with low protein loading (4 mg g -1) showed highest specific activity at a(w) = 0.75.
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| 28. |
- Persson, Mattias, et al.
(författare)
-
Preparation of lipases for use in organic solvents
- 2002
-
Ingår i: Enzyme and Microbial Technology. - 0141-0229. ; 31:6, s. 833-841
-
Tidskriftsartikel (refereegranskat)abstract
- The efficiency of different preparations of lipases was evaluated in organic solvents. Lipases from Humicola lanuginosa, Candida rugosa, Rhizomucor miehei and Pseudomonas cepacia were adsorbed onto the surfactant sorbitan monostearate (Span 60) and the specific activities were compared in hexane to crude powder (used straight from the bottle) and lipase freeze-dried from buffer solution. Lipases adsorbed on the surfactant were "activated" 1.9- to 150-fold compared to the crude lipase. The solubility of the lipase-surfactant preparation in the reaction media was extremely low and the preparation contained aggregates of micrometer size. In further comparisons lipase from H. lanuginosa was freeze-dried in the presence of KCl, crown ethers, immobilised by entrapment into a sol-gel and immobilised on porous polypropylene support (Accurel EP-100). Addition of potassium chloride before freeze-drying of the lipase increased the activity up to 46-fold compared to crude powder. The additive probably worked as an immobilisation matrix for the lipase. When 18-crown-6 was added to the lipase before freeze-drying a 40-fold increase in activity was achieved. In this case a low amount of additive (0.4 mg crown ether/g protein) was needed for activation indicating that specific interactions were involved in the activation. In order to obtain maximal activity, immobilisation on Accurel EP-100 and entrapment into a sol-gel were the best methods to use. The activities were 400- and 320-fold better than that of crude powder. The high activities obtained were due to an improved dispersion of the catalyst in the organic media. The protein loading that could be used when the lipase was adsorbed onto Accurel EP-100 was much higher than what could be used when the lipase was entrapped into a sol-gel. This makes the adsorption technique the best for practical applications. (C) 2002 Elsevier Science Inc. All rights reserved.
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| 29. |
- Svensson, Ingemar, et al.
(författare)
-
Effects of water activity on reaction rates and equilibrium positions in enzymatic esterifications
- 1994
-
Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 44:5, s. 549-556
-
Tidskriftsartikel (refereegranskat)abstract
- A technique of continuous water activity control was used to examine the effects of water activity on enzyme catalysis in organic media. Esterification catalyzed by Rhizopus arrhizus lipase was preferably carried out at a water activity of 0.33, which resulted in both maximal initial reaction rate and a high yield. When Pseudomonas lipase was used as catalyst it was beneficial to start the reaction at high water activity (giving the optimal reaction rate with this enzyme) and then shift to a lower water activity toward the end of the reaction to obtain a high yield. The apparent equilibrium constant of the reaction was influenced by the water activity of the organic solvent. © 1994 John Wiley & Sons, Inc.
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| 30. |
- Triantafyllou, Angeliki Öste, et al.
(författare)
-
Effects of sorbitol addition on the action of free and immobilized hydrolytic enzymes in organic media
- 1995
-
Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 45:5, s. 406-414
-
Tidskriftsartikel (refereegranskat)abstract
- The effect of the addition of sorbitol on the activity and stability of enzymes was examined by monitoring transesterification reactions performed in organic media at various water activities (aw = 0.08 to 0.97). Lipases from Chromobacterium viscosum and Candida rugosa immobilized on celite, and chymotrypsin, free or immobilized on celite, were used. When the sorbitol‐containing enzymes were employed, higher reaction rates and less hydrolysis were observed. Immobilization of chymotrypsin resulted in high activity and operational stability, while the nonimmobilized enzyme was stable only in the presence of sorbitol. The activity of all preparations diminished after washing them with pyridine to remove sorbitol. Furthermore, severe stability problems occurred in the preparations lacking sorbitol. Sorbitol treatment, even after removal of the sorbitol itself, improved the activity of nonimmobilized chymotrypsin relative to the washed control. On the other hand, washing to remove sorbitol had a negative effect on the activity of both coimmobilized lipase and coimmobilized chymotrypsin. Addition of a substrate analogue, N‐acetyl‐L‐phenylalanine, to chymotrypsin yielded a preparation that exhibited higher activity than both the control and its sorbitol‐containing counterpart. Differential scanning calorimetry measurements revealed that the chymotrypsin–sorbitol complex was stable against thermal denaturation, undergoing transition at a high temperature (89°C). The transition temperatures of the substrate‐containing chymotrypsin and of the control were identical (72°C). © 1995 John Wiley & Sons, Inc.
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| 31. |
- Triantafyllou, Angeliki Öste, et al.
(författare)
-
How do additives affect enzyme activity and stability in nonaqueous media?
- 1997
-
Ingår i: Biotechnology and Bioengineering. - 0006-3592. ; 54:1, s. 67-76
-
Tidskriftsartikel (refereegranskat)abstract
- The catalytic activities of lyophilized powders of α-chymotrypsin and Candida antarctica lipase were found to increase 4- to 8-fold with increasing amounts of either buffer salts or potassium chloride in the enzyme preparation. Increasing amounts of sorbitol in the chymotrypsin preparation produced a modest increase in activity. The additives are basically thought to serve as immobilization matrices, the sorbitol being inferior because of its poor mechanical properties. Besides their role as supports, the buffer species were indispensable for the transesterification activity of chymotrypsin because they prevented perturbations of the pH during the course of the reaction. Hence, increasing amounts of buffer species yielded a 100- fold increase in transesterification activity. Effects of pH changes were not as predominant in the peptide synthesis and the lipase-catalyzed reactions. Immobilization of the protease on celite resulted in a remarkable improvement of transesterification activity as compared to the suspended protease, even in the absence of buffer species. Immobilization of the lipase caused a small improvement of activity. The activity of the immobilized enzymes was further enhanced 3-4 times by including increasing amounts of buffer salts in the preparation. The inclusion of increasing amounts of sodium phosphate or sorbitol to chymotrypsin rendered the catalyst more labile against thermal inactivation. The denaturation temperature decreased with 7°C at the highest content of sodium phosphate, as compared to the temperature obtained for the denaturation of the pure protein. The apparent enthalpy of denaturation increased with increasing contents of the additives. The enhancement of hydration level and flexibility of the macromolecule upon addition of the compounds partly provides the explanation for the observed results.
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| 32. |
- Triantafyllou, Angeliki Öuste, et al.
(författare)
-
Polyacrylamides as immobilization supports for use of hydrolytic enzymes in organic media
- 1997
-
Ingår i: Biocatalysis and Biotransformation. - : Informa UK Limited. - 1024-2422 .- 1029-2446. ; 15:3, s. 185-203
-
Tidskriftsartikel (refereegranskat)abstract
- Polyacrylamide beads and celite were used for immobilization of bovine α-chymotrypsin and lipase B from Candida antarctica. Polyacrylamides with a high degree of cross-linking afforded high catalytic rates when used as immobilization matrices. Derivatization of the polymer with carboxyl and tertiary amino groups prior to immobilization caused no significant changes in α-chymotrypsin behavior. The water absorption isotherms for the different supports revealed that the higher the degree of cross-linking the higher the water content of the support is. Leveling off of the enzyme activity above a certain enzyme loading of the supports indicated mass transfer limitations. Diffusional limitations were less pronounced for the polymers with a high degree of cross-linking. This phenomenon may be attributed to their larger surface area. Diffusional limitations were obvious when celite and the polyacrylamides with a low degree of cross-linking were employed. Acetone precipitation of the enzyme was more favorable for enzyme activity than vacuum drying, particularly when the polyacrylamide was employed as the support. The acetone treatment resulted in a morphologically different surface structure of the polymer, as compared to the structure observed after vacuum drying.
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| 33. |
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| 34. |
- Wehtje, Ernst, et al.
(författare)
-
A new method to evaluate the thermal stability of lyophilized enzymes
- 1996
-
Ingår i: Biotechnology Techniques. - 0951-208X. ; 10:12, s. 947-952
-
Tidskriftsartikel (refereegranskat)abstract
- The thermal inactivation of lyophilized chymotrypsin was studied at controlled water activities. At 60 °C the enzyme showed good stability except at aw 0.97, whereas at 75 °C considerable inactivation occured at most water activities. Increasing the amount of buffer on the preparation decreased the stability significantly. The optimal temperature of enzymatic activity was increased 14 °C, when the water activity was decreased from 1 to 0.5.
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| 35. |
- Wehtje, Ernst, et al.
(författare)
-
Activity and operational stability of immobilized mandelonitrile lyase in methanol/water mixtures
- 1988
-
Ingår i: Applied Microbiology and Biotechnology. - 0175-7598. ; 29:5, s. 419-425
-
Tidskriftsartikel (refereegranskat)abstract
- The enzyme mandelonitrile lyase was covalently immobilized on solid support materials using different methods. Immobilization on porous silica using coupling with glutaraldehyde afforded preparations with high enzyme loading (up to 9% (w/w)). The immobilized enzyme was used in a packed bed reactor for the continuous production of d-mandelonitrile from benzaldehyde and cyanide. The influence of the flow rate, pH, substrate concentrations and enzyme loading on the reaction yield and the enantiomeric purity of the product was investigated. In order to suppress the competing spontaneous reaction, the enzymatic reaction must be rapid. A flow rate of 9.5 ml/min (0.1 M benzaldehyde and 0.3 M HCN) through a 3 ml reactor afforded a 86% yield of mandelonitrile with 92% enantiomeric excess. No leakage of enzyme occurred under continuous operation. One column was used continuously for 200 h without any decrease in yield or enantiomeric purity of the product. High concentrations of benzoic acid were shown to decrease the operational stability of the system.
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| 36. |
- Wehtje, Ernst, et al.
(författare)
-
Continuous control of water activity during biocatalysis in organic media
- 1993
-
Ingår i: Biotechnology Techniques. - 0951-208X. ; 7:12, s. 873-878
-
Tidskriftsartikel (refereegranskat)abstract
- This paper describes a newly developed technique to adjust and control the water activity in enzymatic reactions in organic media. A saturated salt solution of known water activity is circulated inside a silicone tube, submerged into the reaction medium. The circulating solution can both absorb and release water. Water activity control during lipase catalyzed esterification was demonstrated with diisopropyl ether as solvent.
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| 37. |
- Wehtje, Ernst, et al.
(författare)
-
Control of water activity in organic medium
- 1996
-
Ingår i: Annals of the New York Academy of Sciences. - : Wiley. - 0077-8923 .- 1749-6632. ; 799, s. 351-357
-
Tidskriftsartikel (refereegranskat)
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| 38. |
- Wehtje, Ernst, et al.
(författare)
-
Enantioselectivity of lipases : Effects of water activity
- 1997
-
Ingår i: Journal of Molecular Catalysis - B Enzymatic. - 1381-1177. ; 3:5, s. 221-230
-
Tidskriftsartikel (refereegranskat)abstract
- The enantioselectivity (E) of lipases in esterifications of secondary alcohols with decanoic acid was studied in organic media. The enantioselectivity of 2-octanol differed greatly among the lipases used. Candida antarctica lipase was extremely selective (E= 9 000) while Candida rugosa lipase was much less selective (E= 1.7). Other enzymes (Lipozyme and lipases from Pseudomonas and Rhizopus arrhizus) had intermediate selectivities. In all cases the enantioselectivity for an enzyme was unaffected by changes in water activity. Different methods of determining the enantioselectivity was used: reactions using single enantiomers as well as racemic mixtures. The effect of water activity on enantioselectivity and the enanatioselectivity values themselves were similar irrespective of the method used. The enantioselectivity of other alcohols were also found to be unaffected by the water activity. The enantioselectivity of Pseudomonas lipase was influenced by the organic solvent. The E decreased with increasing hydrophobicity, from 62 in acetonitrile to 40 in toluene and 33 in hexane. In none of these cases was the enantioselectivity affected by the water activity. However, for Lipozyme and Candida rugosa lipase in toluene a trend of increased E with increasing water activity was observed. In summary it can be stated that the water activity does not generally affect the enantioselectivity of the five lipases tested.
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| 39. |
- Wehtje, Ernst, et al.
(författare)
-
Formation of C—C bonds by mandelonitrile lyase in organic solvents
- 1990
-
Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 36:1, s. 39-46
-
Tidskriftsartikel (refereegranskat)abstract
- Mandelonitrile lyase (EC 4.1.2.10) catalyzes the formation of D‐mandelonitrile from HCN and benzaldehyde. Mandelonitrile lyase was immobilized by adsorption to support materials, for example, Celite. The enzyme preparations were used in diisopropyl ether for production of D‐mandelonitrile. In order to obtain optically pure D‐mandelonitrile it was necessary to use reaction conditions which favor the enzymatic reaction and suppress the competing spontaneous reaction, which yields a racemic mixture of D, L‐mandelonitrile. The effects of substrate concentrations, water content, and support materials on both the spontaneous and enzymatic reactions were studied. The enzymatic reaction was carried out under conditions where the importance of the spontaneous reaction was negligible and high enantiomeric purity of D‐mandelonitrile was achieved (at least 98% enantiomeric excess). The operational stability of the enzyme preparations was studied in batch as well as in continuous systems. It was vital to control the water content in the system to maintain an active preparation. In a packed bed reactor the enzyme preparations were shown to be active and stable. The reactors were run for 50 h with only a small decrease in product yield.
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| 40. |
- Wehtje, Ernst, et al.
(författare)
-
Improved activity retention of enzymes deposited on solid supports
- 1993
-
Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 41:2, s. 171-178
-
Tidskriftsartikel (refereegranskat)abstract
- Enzymes deposited on solid support usually show good stability when operated in organic solvents. Decreased stability of the enzyme preparations was noticed when low enzyme loadings were used (e.g., with Celite as support; less than 1 mg enzyme/g). It was possible to avoid the activity loss by the addition of an additive which protects the enzyme during the immobilization. Proteins (such as albumin, gelatin, and casein) and poly(ethylene glycol) were effective additives whereas amino acids, monomeric carbohydrates, and polysaccharides had no effect. The amount of additive needed for stabilization was shown to depend on the structure of the support, more additive being required for a support with high porosity. The stabilizing effect was investigated in a series of glyceryl‐controlled‐pore glass (CPG) with varying specific surface areas (9.5–180 m2/g). The minimum addition of albumin, giving full stabilization, on the different supports correlated to a monolayer coverage of the surface, approximately 2–3 mg protein/m2. The effect of the additive was less pronounced when increasing amounts of enzyme were immobilized (5–40 mg enzyme/g Celite). The effect of the additives was studied using mandelonitrile lyase, but α‐chymotrypsin and lipase P were also shown to be stabilized. © 1993 John Wiley & Sons, Inc.
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| 41. |
- Wehtje, Ernst, et al.
(författare)
-
Integrated enzymatic reactions and analysis
- 1992
-
Ingår i: Biotechnology Techniques. - 0951-208X. ; 6:2, s. 165-168
-
Tidskriftsartikel (refereegranskat)abstract
- Enzymatic reactions were performed in a modified auto-injector unit of a Shimadzu HPLC system. The reactions were analyzed by automated injections directly into the HPLC separation system. Two reactions were studied, and the enzymes mandelonitrile lyase and α-chymotrypsin were immobilized by adsorption onto a solid support, e.g., Celite and Chromosorb. The reactions were performed in various organic solvents e.g., diisopropyl ether, heptane/ethyl acetate mixtures and acetonitrile.
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| 42. |
- Wehtje, Ernst, et al.
(författare)
-
Lipases have similar water activity profiles in different reactions
- 1997
-
Ingår i: Biotechnology Letters. - 0141-5492. ; 19:6, s. 537-540
-
Tidskriftsartikel (refereegranskat)abstract
- The shape of the profiles of enzyme activity versus water activity for four different lipases were independent of the reaction used to determine the activity. The profile for each lipase (Rhizopus arrhizus, Pseudomonas sp., Candida rugosa and Lipozyme) in esterification, hydrolysis and transesterifications profiles were the same. In transesterification the yield was unaffected by the water activity but the hydrolysis yield increased with increasing a(w).
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| 43. |
- Wehtje, Ernst, et al.
(författare)
-
Reaction kinetics of immobilized α chymotrypsin in organic media 2. Effects of substrate partition
- 1993
-
Ingår i: Biocatalysis and Biotransformation. - : Informa UK Limited. - 1024-2422. ; 7:3, s. 163-176
-
Tidskriftsartikel (refereegranskat)abstract
- The reaction kinetics of αchymotrypsin (EC 3.4.21.1.) catalyzed esterification of N-protected phenylalanine with ethanol were studied. The enzyme was deposited on Chromosorb and reactions were performed mainly in water-saturated mixtures of ethyl acetate and heptane, but also other media were used, such as mixtures of ethyl acetate and acetonitrile. The hydrophobicity of the substrate was varied by using different N-protecting groups (acetyl, Cbz and Fmoc). The apparent Km obtained for the three substrates were 1.1, 7.8 and 17 mM, respectively. The apparent Vmax decreased as the hydrophobicity of the substrates increased. The reaction medium greatly affected the apparent kinetic parameters, Km and Vmax. Nonpolar media (increasing proportion of heptane in mixtures of ethyl acetate and heptane) increased the apparent Kmax and decreased the apparent Km. The effects on the apparent Km values could be correlated with the partitioning of the substrates between the reaction medium and an aqueous phase. In mixtures of acetonitrile and ethyl acetate both the apparent Km and the apparent Vmax decreased as the proportion of acetonitrile increased. The apparent Km and Vmax were also dependent on the water content in the reaction media as well as the buffer concentration and buffer pH.
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| 44. |
- Wehtje, Ernst, et al.
(författare)
-
Reaction kinetics of immobilized αchymotrypsin in organic media 1. Influence at solvent polarity
- 1993
-
Ingår i: Biocatalysis and Biotransformation. - : Informa UK Limited. - 1024-2422. ; 7:3, s. 149-161
-
Tidskriftsartikel (refereegranskat)abstract
- Esterification of N-acetyl phenylalanine with ethanol catalyzed by immobilized αchymotrypsin (EC 3.4.21.1) was studied in various reaction media. The effect of reaction medium polarity on enzymatic activity as well as equilibrium yield was measured. The reaction rate increased with increasing amounts of added water, reaching an optimum corresponding to water saturation of the reaction medium. Further additions of water resulted in decreased activity. Bell-shaped activity profiles were obtained for all reaction media tested. Reaction media consisting of pure solvents and of mixtures of solvents were used. The enzymatic activity and the equilibrium yield increased with decreased polarity of the medium. Maximum activity was found in a reaction medium consisting of 80% diisopropyl ether and 20% heptane. The enzymatic activity obtained at optimal water additions in the different solvents and solvents mixtures could be correlated to the solubility of water and the log P of the medium. The equilibrium yield of the reaction was much more closely correlated to the solubility of water than the log P. Much lower enzymatic activity was obtained when solvent mixtures producing water-miscible media were created, than in mixtures producing water-immiscible media, such as mixtures of acetonitrile and diisopropyl ether. The equilibrium yield could be increased by decreasing the water content in the reaction medium, which reduced the water activity.
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| 45. |
- Wehtje, Ernst, et al.
(författare)
-
Stabilization of Adsorbed Enzymes Used as Biocatalysts in Organic Solvents
- 1992. - C
-
Ingår i: Progress in Biotechnology. - 0921-0423. ; 8:C, s. 377-382
-
Bokkapitel (refereegranskat)abstract
- Enzymes immobilized by deposition onto a solid support at low enzyme loadings were shown to be stabilized by additives, such as proteins and PEG. The additive protects the enzyme from deactivation during immobilization. The amount of additive required to obtain full stabilization was dependent on the type of support material used and corresponded approximately to a monolayer coverage of the additive on the surface of the support. The stabilizing effect of the additive was less pronounced at high enzyme loadings.
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| 46. |
- Wehtje, Ernst, et al.
(författare)
-
Water activity and substrate concentration effects on lipase activity.
- 1997
-
Ingår i: Biotechnology and Bioengineering. - 1097-0290. ; 55:5, s. 798-806
-
Tidskriftsartikel (refereegranskat)abstract
- Catalytic activity of lipases (from Rhizopus arrhizus, Canadida rugosa, and Pseudomonas sp. was studied in organic media, mainly diisopropyl ether. The effect of water activity (a(w)) on V(max) showed that the enzyme activity in general increased with increasing amounts of water for the three enzymes. This was shown both for esterification and hydrolysis reactions catalyzed by R. arrhizus lipase. In the esterification reaction the K(m) for the acid substrate showed a slight increase with increasing water activities. On the other hand, the K(m) for the alcohol substrate increased 10-20-fold with increasing water activity. The relative changes in K(m) were shown to be independent of the enzyme studied and solvent used. The effect was attributed to the increasing competition of water as a nucleophile for the acyl-enzyme at higher water activities. In a hydrolysis reaction the K(m) for the ester was also shown to increase as the water activity increased. The effect of water in this case was due to the fact that increased concentration of one substrate (water), and thereby increased saturation of the enzyme, will increase the apparent K(m) of the substrate (ester) to be determined. This explained why the hydrolysis rate decreased with increasing water activity at a fixed, low ester concentration. The apparent V(max) for R. arrhizus lipase was similar in four of six different solvents that were tested; exceptions were toulene and trichloroethylene, which showed lower values. The apparent K(m) for the alcohol in the solvents correlated with the hydrophobicity of the solvent, hydrophobic solvents giving lower apparent K(m). (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 798-806, 1997.
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| 47. |
- Wehtje, Ernst, et al.
(författare)
-
Water activity control in enzymatic esterification processes
- 1997
-
Ingår i: Enzyme and Microbial Technology. - 0141-0229. ; 21:7, s. 502-510
-
Tidskriftsartikel (refereegranskat)abstract
- Lipase-catalyzed (Lipozyme IM 20 and Novozyme SP 435) esterification reactions in 5-methyl-2-hexanone were studied in order to investigate the practical application of a water activity control system consisting of a semipermeable silicone tubing and saturated salt solutions. The system used successfully in different reactor configurations [butch, continuous stirred-tank reactor (CSTR), and tubular] was described. The dimensions of the tubing could be calculated given the diffusion coefficient of water (D(aw)) through the tubing, the amount of enzyme, and the maximum accepted increase in water activity. Tubular reactors with continuous water activity control were constructed. The enzyme preparation was packed in a silicone tubing which was submerged into a saturated salt solution. A reactor Lipozyme was successfully used for two months without any decrease in product yield (72% conversion at a(w) = 0.33). By connecting a second tubular reactor (containing immobilized lipase and molecular sieves), the yield increased to 95%. A two-stage tubular reactor was used to catalyze esterification in pure substrates (equimolar amounts). The yield (after the second reactor) reached equilibrium (95% ester) at a water activity of 0.33.
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| 48. |
- Yang, Hong, et al.
(författare)
-
The enantiomeric purity of alcohols formed by enzymatic reduction of ketones can be improved by optimisation of the temperature and by using a high co-substrate concentration
- 1997
-
Ingår i: Biochimica et Biophysica Acta - General Subjects. - 0304-4165. ; 1336:1, s. 51-58
-
Tidskriftsartikel (refereegranskat)abstract
- The stereoselective reduction of ketones by alcohol dehydrogenase from Thermoanaerobium brockii was studied in organic reaction media, 2-Propanol was used as co-substrate to regenerate the coenzyme NADPH. The enantiomeric excess of the alcohol formed from the ketone decreased during the course of the reaction (from 53 to 0% e.e. in the formation of (R)-2-butanol). This was interpreted as being due to the reversibility of all the reactions involved. By using a large excess of 2-propanol this effect was suppressed. In the reduction of 2-butanone to (R)-2-butanol, the enantiomeric excess increased with increasing temperature, but in the reduction of a-pentanone to (S)-2-pentanol the enantiomeric excess decreased with increasing temperature, The data were evaluated in terms of free energy of activation of the reaction pathways leading to the different possible products.
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| 49. |
- Öste-Triantafyllou, Angeliki, et al.
(författare)
-
Calorimetric studies on solid α-chymotrypsin preparations in air and in organic solvents
- 1996
-
Ingår i: BBA - Protein Structure and Molecular Enzymology. - : Elsevier BV. - 0167-4838. ; 1295:1, s. 110-118
-
Tidskriftsartikel (refereegranskat)abstract
- Differential scanning calorimetry was the method to investigate the thermostability of chymotrypsin. The transition temperature decreased by approx. 30°C when the dry enzyme became highly hydrated. High degree of hydration corresponded to extensive conformational changes during protein denaturation, reflected by large enthalpy values. Sorbitol, lyophilized together with the enzyme, caused the destabilization of the complex within the whole range of water activities. When the enzyme was equilibrated through the apolar solvent, isooctane, stabilization of chymotrypsin was observed at high water activities, compared to equilibration in air. The presence of isooctane resulted in a remarkable stabilization of the chymotrypsin-sorbitol complex. A sorbitol concentration of 5 mmol/g of protein was prerequisite to induce stabilization when equilibrated through isooctane at high water activities. The transition enthalpy increased with increasing amounts of sorbitol. Different hydration isotherms were obtained for the air-equilibrated and solvent-equilibrated enzyme preparations. Increasing amounts of buffer salts within the chymotrypsin preparation caused the enhancement of both the temperature and the enthalpy of the transition at a water activity 0.97. Variations on the hydration of the preparations both offered the explanation to the thermal stability results and supported the evidence obtained from enzyme activity studies. Generally, the catalyst whose hydration was suppressed due to its environment exhibited low enzymatic activity.
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