| 1. |
- Adlercreutz, Dietlind, et al.
(författare)
-
An enzymatic method for the synthesis of mixed-acid phosphatidylcholine
- 2004
-
Ingår i: Journal of the American Oil Chemists Society. - : Wiley. - 0003-021X. ; 81:6, s. 553-557
-
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.
|
|
| 2. |
- Adlercreutz, Dietlind, et al.
(författare)
-
Synthesis of phosphatidylcholine with defined fatty acid in the sn-1 position by lipase-catalyzed esterification and transesterification reaction.
- 2002
-
Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 78:4, s. 403-411
-
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.
|
|
| 3. |
- Adlercreutz, Patrick, et al.
(författare)
-
Enzymatic conversions of polar lipids. Principles, problems and solutions
- 2001
-
Ingår i: Journal of Molecular Catalysis - B Enzymatic. - 1381-1177. ; 11:4-6, s. 173-178
-
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.
|
|
| 4. |
|
|
| 5. |
- Barros, Raúl J., et al.
(författare)
-
Effect of mass-transfer limitations on the selectivity of immobilized α-chymotrypsin biocatalysts prepared for use in organic medium
- 2000
-
Ingår i: Biotechnology and Bioengineering. - 0006-3592. ; 67:3, s. 319-326
-
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.
|
|
| 6. |
- Barros, Raúl J., et al.
(författare)
-
Enhancement of immobilized protease catalyzed dipeptide synthesis by the presence of insoluble protonated nucleophile
- 1999
-
Ingår i: Enzyme and Microbial Technology. - 0141-0229. ; 24:8-9, s. 480-488
-
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.
|
|
| 7. |
|
|
| 8. |
- Barros, Raúl J., et al.
(författare)
-
Mass transfer studies on immobilized α-chymotrypsin biocatalysts prepared by deposition for use in organic medium
- 1998
-
Ingår i: Biotechnology and Bioengineering. - 0006-3592. ; 59:3, s. 364-373
-
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.
|
|
| 9. |
- Barros, Raúl J., et al.
(författare)
-
Modeling the performance of immobilized α-chymotrypsin catalyzed peptide synthesis in acetonitrile medium
- 2001
-
Ingår i: Journal of Molecular Catalysis - B Enzymatic. - 1381-1177. ; 11:4-6, s. 841-850
-
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.
|
|
| 10. |
- Barros, Raúl J., et al.
(författare)
-
Physical characterization of porous materials and correlation with the activity of immobilized enzyme in organic medium
- 1998
-
Ingår i: Biocatalysis and Biotransformation. - : Informa UK Limited. - 1024-2422 .- 1029-2446. ; 16:1, s. 67-85
-
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.
|
|
