| 1. |
- Bassanini, Ivan, et al.
(författare)
-
Dicarboxylic esters : Useful tools for the biocatalyzed synthesis of hybrid compounds and polymers
- 2015
-
Ingår i: Beilstein Journal of Organic Chemistry. - : Beilstein Institut. - 2195-951X .- 1860-5397. ; 11, s. 1583-1595
-
Forskningsöversikt (refereegranskat)abstract
- Dicarboxylic acids and their derivatives (esters and anhydrides) have been used as acylating agents in lipase-catalyzed reactions in organic solvents. The synthetic outcomes have been dimeric or hybrid derivatives of bioactive natural compounds as well as functionalized polyesters.
|
|
| 2. |
- Berggren, Johanna, et al.
(författare)
-
Revascularization of Free Skin Grafts Overlying Modified Hughes Tarsoconjunctival Flaps Monitored Using Laser-Based Techniques
- 2019
-
Ingår i: Ophthalmic Plastic and Reconstructive Surgery. - 1537-2677. ; 35:4, s. 378-382
-
Tidskriftsartikel (refereegranskat)abstract
- Purpose: It has recently been shown that the flap pedicle does not supply blood to a tarsoconjunctival graft in the modified Hughes procedure in patients. This raises questions concerning the rate of revascularization of the free skin graft commonly used to reconstruct the anterior lamella. The aim of this study was, thus, to monitor the course of revascularization in free skin grafts overlying modified Hughes tarsoconjunctival flaps, using laser-based techniques.Methods: Free skin grafts from the upper eyelid or upper arm in 9 patients were used to cover a tarsoconjunctival flap according to the modified Hughes procedure. Blood perfusion was monitored using laser speckle contrast imaging, and vascular reactivity was studied with laser Doppler velocimetry after heating the tissue to 44°C. Measurements were made at the time of surgery (baseline) and at 1, 3, 8, and 16 weeks postoperatively.Results: The gradual increase in perfusion of the free skin grafts during the healing process indicates revascularization. A slight increase in perfusion was seen already after 1 week. Perfusion reached 50% of the baseline after 3 weeks, and complete restoration of perfusion was seen after 8 weeks. The vascular function monitored with heat-induced hyperemia increased in a similar fashion.Conclusions: Full-thickness skin grafts revascularize within 3 to 8 weeks, despite overlying a tarsoconjunctival flap, which has recently been reported to be avascular. This provides further evidence that it should be possible to repair large eyelid defects using free full-thickness eyelid grafts.
|
|
| 3. |
- Berggren, Johanna V, et al.
(författare)
-
Laser Speckle Contrast Imaging of a Rotational Full-Thickness Lower Eyelid Flap Shows Satisfactory Blood Perfusion
- 2021
-
Ingår i: Ophthalmic Plastic and Reconstructive Surgery. - 1537-2677. ; 37:4, s. 139-141
-
Tidskriftsartikel (refereegranskat)abstract
- Full-thickness eyelid flaps from the lower eyelid are frequently used to repair larger upper eyelid defects. Perfusion monitoring has recently been implemented in several reconstructive surgical procedures, however, perfusion monitoring of a rotational eyelid flap has not yet been described. The authors' employed laser speckle contrast imaging to monitor blood perfusion in a rotational flap from the lower eyelid, used to cover a large tumor defect in the upper eyelid. Perfusion in the flap decreased by only 50% during surgery and was almost completely restored 5 weeks later at flap division (91%). The excellent surgical outcome in the present case is deemed to be the result of satisfactory blood perfusion of the flap.
|
|
| 4. |
- Berggren, Johanna V, et al.
(författare)
-
Laser Speckle Contrast Imaging of the Blood Perfusion in Glabellar Flaps Used to Repair Medial Canthal Defects
- 2022
-
Ingår i: Ophthalmic Plastic and Reconstructive Surgery. - 1537-2677. ; 38:3, s. 274-279
-
Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The glabellar flap is a common technique for surgical repair after tumor excision in the medial canthal area. However, the outcome may be affected by partial flap necrosis. Little is known about the impact of surgery on blood perfusion and the postoperative course of reperfusion due to the absence of reliable and noninvasive perfusion monitoring techniques. The aim of this study was to use a modern imaging technique to assess blood perfusion in glabellar flaps.METHODS: Glabellar flaps were used to repair medial canthal defects following tumor excision in 7 patients. Blood perfusion was monitored using laser speckle contrast imaging: during surgery, immediately postoperatively (0 weeks), and at follow-up, 1, 3, and 6 weeks after surgery.RESULTS: Perfusion decreased gradually along the length of the flap, and reached a minimum 15 mm from the flap base. Perfusion in the proximal 20 mm of the flap was completely restored after 1 week, while the distal part of the flap was gradually reperfused over 6 weeks. Both the functional and esthetic surgical outcomes were excellent.CONCLUSIONS: The rapid reperfusion of the glabellar flap may be explained by its connection to the vascular network via the flap pedicle. In flaps longer than 20 mm, the distal part can be considered a free skin transplant, and a combination of a glabellar flap and a free skin graft could then be considered.
|
|
| 5. |
- Berglund, Per, et al.
(författare)
-
2-Methylalkanoic acids resolved by esterification catalysed by lipase from Candida rugosa : Alcohol chain length and enantioselectivity
- 1993
-
Ingår i: Tetrahedron Asymmetry. - 0957-4166. ; 4:8, s. 1869-1878
-
Tidskriftsartikel (refereegranskat)abstract
- Enantiomerically pure (R)-2-methyldecanoic acid and (S)-2-methyl-1-decanol were prepared in a multi gram scale by esterification reactions catalysed by lipase from Candida rugosa. The enantiomeric ratios (E-values) were determined as a function of the chain length of the alcohol used as the complementary substrate in cyclohexane. In the resolution of 2-methyldecanoic acid the highest value (E = 37 ± 5) was obtained, when either 2-hexanol, 1-heptanol or 1-octanol were used. In contrast, when resolving 2-methyloctanoic acid, the E-values increased continually with increasing chain length of the alcohol used. 1-Hexadecanol gave the highest value: E > 100. The E-values were determined from the enantiomeric excess (ee) of the product at a conversion below 0.4. After two consecutive esterification reactions enantiomerically pure (R)-2-methyldecanoic acid, >99.8% ee, and after subsequent reduction of the ester produced, (S)-2-methyl-1-decanol, 96.7% ee, were obtained.
|
|
| 6. |
|
|
| 7. |
|
|
| 8. |
- Berglund, Per, et al.
(författare)
-
Reversed enantiopreference of Candida rugosa lipase supports different modes of binding enantiomers of a chiral acyl donor
- 1998
-
Ingår i: Journal of Molecular Catalysis - B Enzymatic. - 1381-1177 .- 1873-3158. ; 5:1-4, s. 283-287
-
Tidskriftsartikel (refereegranskat)abstract
- Molecular modelling identifies two different productive modes of binding the enantiomers of a 2-methyldecanoic acid ester to the active site of Candida rugosa lipase (CRL). The fast reacting S-enantiomer occupies the previously identified acyl-binding tunnel of the enzyme, whereas the R- enantiomer leaves the tunnel empty. The modelling suggested that if both enantiomers were forced to bind to the active site leaving the tunnel empty, the enzyme would reverse its enantiopreference to become R-enantioselective. To test this hypothesis, we designed a structural analogue to 2- methyldecanoic acid, 2-methyl-6-(2-thienyl)hexanoic acid, which was expected to be too bulky to fit its acyl moiety into the acyl-binding tunnel. The CRL- catalysed hydrolysis of the ethyl ester of this substrate resulted in the preferential conversion of the R-enantiomer as predicted by molecular modelling. This represents the first kinetic evidence supporting the existence of two different modes of binding the enantiomers of a 2- methyldecanoic acid ester to the active site of CRL. We have shown that a rational 3D based approach in combination with substrate engineering can be used to predict and control the stereochemical outcome of a lipase catalysed reaction.
|
|
| 9. |
- Berglund, Per, et al.
(författare)
-
Switched enantiopreference of Humicola lipase for 2-phenoxyalkanoic acid ester homologs can be rationalized by different substrate binding modes
- 1999
-
Ingår i: Tetrahedron. - 0957-4166 .- 1362-511X. ; 10:21, s. 4191-4202
-
Tidskriftsartikel (refereegranskat)abstract
- Humicola lanuginosa lipase was used for enantioselective hydrolyses of a series of homologous 2-phenoxyalkanoic acid ethyl esters. The enantioselectivity (E-value) of the enzyme changed from an (R)-enantiomer preference for the smallest substrate, 2-phenoxypropanoic acid ester, to an (S)-enantiomer preference for the homologous esters with longer acyl moieties. The E-values span the range from E=13 (R) to E=56 (S). A molecular modeling study identified two different substrate-binding modes for each enantiomer. We found that the enantiomers favored different modes. This discovery provided a model that offered a rational explanation for the observed switch in enantioselectivity. (C) 1999 Elsevier Science Ltd. All rights reserved.
|
|
| 10. |
- Bernhardt, Peter, et al.
(författare)
-
Molecular basis of perhydrolase activity in serine hydrolases
- 2005
-
Ingår i: Angewandte Chemie International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 44:18, s. 2742-2746
-
Tidskriftsartikel (refereegranskat)abstract
- (Chemical Equation Presented) Changing substrates: A mutation that forms a cis-proline-peptide bond in a loop structure close to the active site of an aryl esterase from Pseudomonas fluorescens converts the enzyme into a perhydrolase (see picture). The switch in activity is explained by a new hydrogen bond formed between a backbone carbonyl oxygen atom and the peroxy deacylation intermediate.
|
|
| 11. |
- Bohman, Elin, et al.
(författare)
-
Novel Evidence Concerning Lacrimal Sac Movement Using Ultra-High-Frequency Ultrasound Examinations of Lacrimal Drainage Systems
- 2021
-
Ingår i: Ophthalmic Plastic and Reconstructive Surgery. - 1537-2677. ; 37:4, s. 334-340
-
Tidskriftsartikel (refereegranskat)abstract
- PURPOSE: Current hypothesis regarding the mechanism of active tear drainage is based on studies performed ex vivo or under nonphysiological conditions. Novel ultra-high-frequency ultrasound has the advantage of generating images with superior resolution, enabling measurements of low flow in small vessels, and the tracking of tissue motion in real time. The purpose of this study was to investigate the lacrimal drainage system and active drainage using this modality.METHODS: The upper lacrimal drainage system was investigated with 40-70 MHz ultrasound in 22 eyes in 13 patients. Irrigation confirmed a lacrimal obstruction in 10 eyes. Motion tracking was used to map movement of the lateral lacrimal sac wall and to measure flow when possible.RESULTS: The anatomy of the upper lacrimal drainage system was mapped in vivo, including the proximal canaliculi, which have not previously been imaged. The lacrimal sac lumen is slit shaped in its resting state but is distended when irrigated or if a nasolacrimal duct obstruction is present. Thus, the healthy lacrimal sac is not a cavity, and the medial retinaculum does not act against a stretched structure. Motion tracking visualized the "lacrimal pump," showing that the direction of motion of the lateral lacrimal sac wall is mainly in the sagittal plane during blinking.CONCLUSIONS: Ultra-high-frequency ultrasound allows detailed physiological monitoring of the upper lacrimal drainage system in vivo. Our findings suggest that current theories of active tear drainage need to be reappraised.
|
|
| 12. |
- Branneby, Cecilia, et al.
(författare)
-
Aldol Additions with Mutant Lipase : Analysis by Experiments and Theoretical Calculations
- 2004
-
Ingår i: Journal of Molecular Catalysis B. - : Elsevier BV. - 1381-1177 .- 1873-3158. ; 31:4-6, s. 123-128
-
Tidskriftsartikel (refereegranskat)abstract
- A Ser105Ala mutant of Candida antarctica lipase B has previously been shown to catalyze aldol additions. Quantum chemical calculations predicted a reaction rate similar to that of natural enzymes, whereas experiments showed a much lower reaction rate. Molecular dynamics simulations, presented here, show that the low reaction rate is a consequence of the low frequencies of near attack complexes in the enzyme. Equilibrium was also considered as a reason for the slow product formation, but could be excluded by performing a sequential reaction to push the reaction towards product formation. In this paper, further experimental results are also presented, highlighting the importance of the entire active site for catalysis.
|
|
| 13. |
- Branneby, Cecilia, et al.
(författare)
-
Carbon-Carbon Bonds by Hydrolytic Enzymes
- 2003
-
Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 125:4, s. 874-875
-
Tidskriftsartikel (refereegranskat)abstract
- Enzymes are efficient catalysts in synthetic chemistry, and their catalytic activity with unnatural substrates in organic reaction media is an area attracting much attention. Protein engineering has opened the possibility to change the reaction specificity of enzymes and allow for new reactions to take place in their active sites. We have used this strategy on the well-studied active-site scaffold offered by the serine hydrolase Candida antarctica lipase B (CALB, EC 3.1.1.3) to achieve catalytic activity for aldol reactions. The catalytic reaction was studied in detail by means of quantum chemical calculations in model systems. The predictions from the quantum chemical calculations were then challenged by experiments. Consequently, Ser105 in CALB was targeted by site-directed mutagenesis to create enzyme variants lacking the nucleophilic feature of the active site. The experiments clearly showed an increased reaction rate when the aldol reaction was catalyzed by the mutant enzymes as compared to the wild-type lipase. We expect that the new catalytic activity, harbored in the stable protein scaffold of the lipase, will allow aldol additions of substrates, which cannot be reached by traditional aldolases
|
|
| 14. |
- Branneby, Cecilia, 1974-
(författare)
-
Exploiting enzyme promiscuity for rational design
- 2005
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Enzymes are today well recognized in various industrial applications, being an important component in detergents, and catalysts in the production of agrochemicals, foods, pharmaceuticals, and fine chemicals. Their large use is mainly due to their high selectivity and environmental advantage, compared to traditional catalysts. Tools and techniques in molecular biology offer the possibility to screen the natural sources and engineer new enzyme activities which further increases their usefulness as catalysts, in a broader area. Although enzymes show high substrate and reaction selectivity many enzymes are today known to catalyze other reactions than their natural ones. This is called enzyme promiscuity. It has been suggested that enzyme promiscuity is Nature’s way to create diversity. Small changes in the protein sequence can give the enzyme new reaction specificity. In this thesis I will present how rational design, based on molecular modeling, can be used to explore enzyme promiscuity and to change the enzyme reaction specificity. The first part of this work describes how Candida antarctica lipase B (CALB), by a single point mutation, was mutated to give increased activity for aldol additions, Michael additions and epoxidations. The activities of these reactions were predicted by quantum chemical calculations, which suggested that a single-point mutant of CALB would catalyze these reactions. Hence, the active site of CALB, which consists of a catalytic triad (Ser, His, Asp) and an oxyanion hole, was targeted by site-directed mutagenesis and the nucleophilic serine was mutated for either glycine or alanine. Enzymes were expressed in Pichia pastoris and analyzed for activity of the different reactions. In the case of the aldol additions the best mutant showed a four-fold initial rate over the wild type enzyme, for hexanal. Also Michael additions and epoxidations were successfully catalyzed by this mutant. In the last part of this thesis, rational design of alanine racemase from Geobacillus stearothermophilus was performed in order to alter the enzyme specificity. Active protein was expressed in Escherichia coli and analyzed. The explored reaction was the conversion of alanine to pyruvate and 2-butanone to 2-butylamine. One of the mutants showed increased activity for transamination, compared to the wild type.
|
|
| 15. |
|
|
| 16. |
|
|
| 17. |
- Branneby, Cecilia, et al.
(författare)
-
Rational redesign of a lipase to an aldolase
- 2003
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 42:28, s. 8633-8633
-
Tidskriftsartikel (refereegranskat)
|
|
| 18. |
- Cammenberg, Maria, et al.
(författare)
-
Molecular basis for the enhanced lipase-catalyzed N-acylation of 1-phenylethanamine with methoxyacetate
- 2006
-
Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 7:11, s. 1745-1749
-
Tidskriftsartikel (refereegranskat)abstract
- One of the commercial methods for preparing enantiopure amines is lipase-catalyzed kinetic resolution, although lipases catalyze, aminolysis with only low activity. Interestingly, in 1997 Balkenhohl et al. used, ethyl methoxyacetate instead of ethyl butyrate as an acylation reagent for the aminolysis of 1-phenylethanamine and increased the reaction rate more than a 100-fold. This method has been applied to other aminolysis reactions, but the molecular basis for the enhanced rate is not understood. A moecular-modeling study of the transition-state analogue for the aminolysis showed that an interaction between the beta-oxygen atom in methoxyacetate and the amine nitrogen atom might be a key factor in the rate enhancement. Other acylation reagents, such as methyl 3-methoxypropionate and methyl 4-methoxybutyrate, were chosen to test the influence of this interaction because these molecules can be spatially arranged to have similar to that in the acylation with methyoxyacetate. The initial aminolysis rates were improved (11-fold and sixfold, respectively) compared to that with butyrate. In with 1-phenylethanol afforded the same rate with all acyl donors.
|
|
| 19. |
- Carlqvist, Peter, et al.
(författare)
-
Exploring the Active-Site of a Rationally Redesigned Lipase for Catalysis of Michael-Type Additions
- 2005
-
Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 6, s. 331-336
-
Tidskriftsartikel (refereegranskat)abstract
- Michael-type additions of various thiols and alpha,beta-unsaturated carbonyl compounds were performed in organic solvent catalyzed by wild-type and a rationally redesigned mutant of Candida antarctica lipase B. The mutant locks the nucleophilic serine 105 in the active-site; this results in a changed catalytic mechanism of the enzyme. The possibility of utilizing this mutant for Michael-type additions was initially explored by quantum-chemical calculations on the reaction between acrolein and methanethiol in a model system. The model system was constructed on the basis of docking and molecular-dynamics simulations and was designed to simulate the catalytic properties of the active site. The catalytic system was explored experimentally with a range of different substrates. The k(cat) values were found to be in the range of 10(-3) to 4 min(-1), similar to the values obtained with aldolase antibodies. The enzyme proficiency was 10(7). Furthermore, the Michael-type reactions followed saturation kinetics and were confirmed to take place in the enzyme active site.
|
|
| 20. |
- Carlqvist, P., et al.
(författare)
-
Rational design of a lipase to accommodate catalysis of Baeyer-Villiger oxidation with hydrogen peroxide
- 2003
-
Ingår i: Journal of Molecular Modeling. - : Springer Science and Business Media LLC. - 1610-2940 .- 0948-5023. ; 9:3, s. 164-171
-
Tidskriftsartikel (refereegranskat)abstract
- The mechanism and potential energy surface for the Baeyer-Villiger oxidation of acetone with hydrogen peroxide catalyzed by a Ser105-Ala mutant of Candida antarctica Lipase B has been determined using ab initio and density functional theories. Initial substrate binding has been studied using an automated docking procedure and molecular dynamics simulations. Substrates were found to bind to the active site of the mutant. The activation energy for the first step of the reaction, the nucleophilic attack of hydrogen peroxide on the carbonyl carbon of hydrogen peroxide, was calculated to be 4.4 kcal mol(-1) at the B3LYP/6-31+G* level. The second step, involving the migration of the alkyl group, was found to be the rate-determining step with a computed activation energy of 19.9 kcal mol(-1) relative the reactant complex. Both steps were found to be lowered considerably in the reaction catalyzed by the mutated lipase, compared to the uncatalyzed reaction. The first step was lowered by 36.0 kcal mol(-1) and the second step by 19.5 kcal mol(-1). The second step of the reaction, the rearrangement step, has a high barrier of 27.7 kcal mol(-1) relative to the Criegee intermediate. This could lead to an accumulation of the intermediate. It is not clear whether this result is an artifact of the computational procedure, or an indication that further mutations of the active site are required.
|
|
| 21. |
- Engström, Karin, et al.
(författare)
-
Kinetic resolution of diarylmethanols using a mutated variant of lipase CALB
- 2012
-
Ingår i: Tetrahedron. - : Elsevier BV. - 0040-4020 .- 1464-5416. ; 68:37, s. 7613-7618
-
Tidskriftsartikel (refereegranskat)abstract
- An enzymatic kinetic resolution of diarylmethanols via acylation has been developed. This was achieved by the use of a mutated variant of CALB that accepts larger substrates compared to the wild type. By the use of diarylmethanols with two differently sized aryl groups, enantioselective transformations were achieved. A larger size-difference led to a higher enantioselectivity. In addition, substrates with electronically different aryl groups, such as phenyl and pyridyl, also gave an enantioselective reaction. The highest E value was observed with a substrate where steric and electronic effects were combined.
|
|
| 22. |
|
|
| 23. |
|
|
| 24. |
- Eriksson, Magnus, et al.
(författare)
-
Enzymatic One-Pot Route to Telechelic Polypentadecalactone Epoxide : Synthesis, UV Curing, and Characterization
- 2009
-
Ingår i: Biomacromolecules. - : American Chemical Society (ACS). - 1525-7797 .- 1526-4602. ; 10:11, s. 3108-3113
-
Tidskriftsartikel (refereegranskat)abstract
- In an enzymatic one-pot procedure immobilized lipase B from Candida antarctica was used to synthesize semicrystalline diepoxy functional macromonomers based on glycidol, pentadecalactone, and adipic acid. By changing the stoichiometry of the building blocks. macromonomers of controlled molecular weight front 1400 to 2700 g mol(-1) could be afforded. The enzyme-catalyzed reaction went to completion (conversion >= 95%) within 24 h at 60 degrees C. After removal of the enzyme, the produced macromonomers were used for photopolymerization without any purification. The macromonomers readily copolymerized cationically with a cycloaliphatic diepoxide (Cyracure UVR-6110; CA-dE) to high conversion. The cross-linked copolymers formed a durable film with a degree of crystallinity depending on the macromonomer size and amount of CA-dE used, without CA-dE the macromonomers homopolymerized only to a low degree. Combined with CA-dE conversions of 85-90% were determined by FT-Raman spectroscopy. The films became more durable once reinforced with CA-dE, increasing the cross-link density and reducing the crystallinity of the PDL segments in the films.
|
|
| 25. |
- Eriksson, Magnus G., et al.
(författare)
-
One-pot enzymatic polycondensation to telechelic methacrylate-functional oligoesters used for film formation
- 2011
-
Ingår i: POLYM CHEM. - Cambridge : ROYAL SOC CHEMISTRY. - 1759-9954 .- 1759-9962. ; 2:3, s. 714-719
-
Tidskriftsartikel (refereegranskat)abstract
- Based on largely renewable monomers, an enzymatic one-pot polycondensation route towards functional oligomers with targeted molecular weights and end-groups was developed. This one-pot synthesis was performed by combining Candida antarctica lipase B (CALB), 2-hydroxyethyl methacrylate (HEMA), ethylene glycol, and divinyl adipate under reduced pressure (72 mbar) at 60 degrees C. The polymerization went to completion (>95% conversion for all monomers) within 24 h and the fraction of methacrylate end-groups was >90%. Three targeted dimethacrylate functional oligomers with molecular weights of 920, 1700 and 2500 g mol(-1) (degrees of polymerization 4, 8, and 13 respectively) were synthesized. The oligomer products were characterized by NMR, MALDI-TOF MS and SEC. The dimethacrylate functional oligomers were further UV homopolymerized or combined with a tetrathiol crosslinker to demonstrate the potential to produce novel networks with tunable thermal properties dependent on chain length of the telechelic building blocks. This research is the first to demonstrate methacrylate functionalization and condensation polymerization in a one step process, which expands the growing toolbox for polymer/material chemists towards an increased throughput in available macromonomers used in material design.
|
|
| 26. |
- Eriksson, Magnus, 1973-
(författare)
-
Lipase-Catalyzed Syntheses of Telechelic Polyesters
- 2010
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Telechelic polyesters have successfully been synthesized with lipase-catalyzed polymerization. The produced telechelics had a high degree of difunctionalization, high purity (requiring little or no workup) and controlled degree of polymerization. The syntheses were performed in one-pot one-step reaction systems. The use of protection/deprotection chemistry was not necessary, since the lipase selectivity was utilized in the syntheses. Two different types of lipase-catalyzed polymerizations were applied – ring-opening polymerization and polycondensation. In ring-opening polymerization telechelics were produced by a combination of initiation, α-functionalization, and linking through termination, w-functionalization. In polycondensation different types of end-cappers were used to synthesize telechelics. Several exampels of functional groups were used for end-functionalization - epoxide, methacrylate and tetraallyls. Enzyme kinetic schemes describing the different functionalization methods of polyesters are presented and discussed. Stoichiometry and different reaction conditions have been studied to understand the effects these functions have on the final structure of the synthesized telechelics. Polyesters are classified as biodegradable, and can also be synthesized from materials that can be extracted or fermented from renewable sources like plants. Lipase-catalysts have several beneficial attributes, like high selectivity, they are renewable and biodegradable, are non-toxic and metal-free and can operate under mild reaction conditions. The focus of this thesis has been on lipase-catalyzed syntheses and characterization of the produced telechelics, in addition some materials have been produced. Some uses of telechelics are surface modification, materials for block co-polymers, functional films and biomedical applications.
|
|
| 27. |
- Eriksson, Magnus, et al.
(författare)
-
One-Pot Enzymatic Route to Tetraallyl Ether Functional Oligoesters : Synthesis, UV Curing, and Characterization
- 2010
-
Ingår i: Journal of Polymer Science Part A. - : Wiley. - 0887-624X .- 1099-0518. ; 48:23, s. 5289-5297
-
Tidskriftsartikel (refereegranskat)abstract
- An enzymatic one-pot route in bulk was used to synthesize tetraallyl ether (tAE) functional oligomers based on divinyl adipate, 1,4-butanediol and trimethylolpropane diallyl ether. By using lipase B from Candida antarctica as catalyst and varying the stoichiometric ratio of monomers, it was possible to reach targeted molecular weights (from 1300 to 3300 g mol(-1)) of allyl-ether functional polyesters. The enzyme catalyzed reaction reached completion (>98% conversion based on all monomers) within 24 h at 60 degrees C, under reduced pressure (72 mbar) resulting in similar to 90% yield after filtration. The tAE-functional oligoesters were photopolymerized, without any purification other than removal of the enzyme by filtration, with thiol functional monomers (dithiol, tetrathiol) in a 1: 1 ratio thiol-ene reaction. The photo-initiator, 2,2-dimethoxy-2-phenylacetophenone, was used to improve the rate of reaction under UV light. High conversions (96-99% within detection limits) were found for all thiol-ene films as determined by FT-Raman spectroscopy. The tAE-functional oligoesters were characterized by NMR, MALDI, and SEC. The UV-cured homopolymerized films and the thiol-ene films properties were characterized utilizing DSC and DMTA.
|
|
| 28. |
|
|
| 29. |
|
|
| 30. |
- Fransson, Linda
(författare)
-
Enzyme substrate solvent interactions : a case study on serine hydrolases
- 2008
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Reaction rates and selectivities were measured for transacylation of fatty acid esters in solvents catalysed by Candida antarctica lipase B and by cutinase from Humicola insolens. With these enzymes classical water-based enzymology can be expanded to many different solvents allowing large variations in interaction energies between the enzymes, the substrates and the surrounding. Further ,hydrolysis reactions catalysed by Bacillus subtilis esterase 2 were investigated. Thermodynamics analyses revealed that the enzyme contribution to reaction rate acceleration compared to acid catalysis was purely entropic. On the other hand, studies of differences in activation entropy and enthalpy between enantiomers and between homologous esters showed that high substrate specificity was favoured by enthalpic stabilisation. Solvent was found to have a profound effect on enzyme catalysis, affecting both reaction rate and selectivity. Differences in substrate solubility will impact enzyme specificity since substrate binding is an equilibrium between enzyme-bound substrate and substrate in free solution. In addition, solven tmolecules were found to act as enzyme inhibitors, showing both competitive and non-competitive behaviour. In several homologous data series enthalpy-entropy compensation relationships were encountered. A possible extrathermodynamic relationship between enthalpy and entropy can easily be lost under co-varying errors propagated from the experiments. From the data in this thesis, one instance was found of a real enthalpy-entropy compensation that could be distinguished from statistical errors, while other examples could not be verified.
|
|
| 31. |
- Fransson, Linda, et al.
(författare)
-
Minor Enantiomer Recycling-Effect of Two Reinforcing Catalysts on Product Yield and Enantiomeric Excess
- 2010
-
Ingår i: ChemCatChem. - : Wiley. - 1867-3880 .- 1867-3899. ; 2:6, s. 683-693
-
Tidskriftsartikel (refereegranskat)abstract
- Kinetic modeling of a recycling procedure in which the minor product enantiomer from an enantioselective catalytic reaction is selectively retransformed to starting material by a second chiral catalyst demonstrates that the enantiomeric excess of the product is not affected by the relative amounts of the two catalysts, but that the yield increases when the amount of the catalyst for the product-forming reaction is increased. The yield, but not the enantiomeric excess, is also affected by the initial substrate concentration. The recycling process is compared to sequential processes in which either the second catalyst is added after completion of the first reaction or in which the two catalysts are added simultaneously. In the sequential processes, high enantioselectivity can be obtained at the expense of product yield, whereas under recycling conditions both high enantiomeric excess and high yield can be achieved. Experimental data from a recycling procedure providing qualitative support for results from kinetic modeling are presented.
|
|
| 32. |
- Fransson, Linda, 1971-
(författare)
-
Molecular modelling - understanding and prediction of enzyme selectivity.
- 2009
-
Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Molecular modelling strategies for evaluation of enzyme selectivity wereinvestigated with a focus on principles of how molecular interactionscould be evaluated to provide information about selectivity. Althoughmolecular modelling provides tools for evaluation of geometrical andenergy features of molecular systems, no general strategies for evaluationof enzyme selectivity exist. Geometrical analyses can be based uponinspection and reasoning about molecular interactions, which provide aneasily accessible way to gain information, but suffer from the risk of biasput in by the modeller. They can also be based on geometrical features ofmolecular interactions such as bond lengths and hydrogen-bond formation.Energy analyses are appealing for their modeller independenceand for the possibility to predict not only stereopreference, but also itsmagnitude.In this thesis, four examples of enantio- or regioselective serinehydrolase-catalysed reaction systems are presented together with developedmodelling protocols for explanation, prediction or enhancement ofselectivity. Geometrical as well as energy-based methodology were used,and provided an understanding of the structural basis of enzymeselectivity. In total, the protocols were successful in making qualitative explanationsand predictions of stereoselectivity, although quantitative determinationswere not achieved.
|
|
| 33. |
|
|
| 34. |
- Gardossi, Lucia, et al.
(författare)
-
Guidelines for reporting of biocatalytic reactions
- 2010
-
Ingår i: Trends in Biotechnology. - : Elsevier BV. - 0167-7799 .- 1879-3096. ; 28:4, s. 171-180
-
Forskningsöversikt (refereegranskat)abstract
- Enzymes and whole cells are being increasingly applied in research and industry, but the adoption of biocatalysis relies strongly on useful scientific literature. Unfortunately, too many published papers lack essential information needed to reproduce and understand the results. Here, members of the scientific committee of the European Federation of Biotechnology Section on Applied Biocatalysis (ESAB) provide practical guidelines for reporting experiments. The document embraces the recommendations of the STRENDA initiative (Standards for Reporting Enzymology Data) in the context of pure enzymology and provides further guidelines and explanations on topics of crucial relevance for biocatalysis. In particular, guidelines are given on issues such as the selectivity, specificity, productivity and stability of biocatalysts, as well as on methodological problems related to reactions in multiphase systems. We believe that adoption and use of these guidelines could greatly increase the value and impact of published work in biocatalysis, and hence promote the further growth of applications.
|
|
| 35. |
- Graber, Marianne, et al.
(författare)
-
Solvent as a competitive inhibitor for Candida antarctica lipase B
- 2007
-
Ingår i: Biochimica et Biophysica Acta - Proteins and Proteomics. - : Elsevier BV. - 1570-9639 .- 1878-1454. ; 1774:8, s. 1052-1057
-
Tidskriftsartikel (refereegranskat)abstract
- In enzyme-catalyzed reactions, the choice of solvent often has a marked effect on the reaction outcome. In this paper, it is shown that solvent effects could be explained by the ability of the solvent to act as a competitive inhibitor to the substrate. Experimentally, the effect of six solvents, 2-pentanone, 3-pentanone, 2-methyl-2-pentanol, 3-methyl-3-pentanot, 2-methylpentane and 3-methylpentane, was studied in a solid/gas reactor. As a model reaction, the CALB-catalyzed transacylation between methyl propanoate and I -propanol, was studied. It was shown that both ketones inhibited the enzyme activity whereas the tertiary alcohols and the hydrocarbons did not. Alcohol inhibition constants, K-il were changed to "K-i", determined in presence of 2-pentanone, 3-pentanone, and 3-methyl-3-pentanol, confirmed the marked inhibitory character of the ketones and an absence of inhibition of 3-methyl-3-pentanol. The molecular modeling study was performed on three solvents, 2-pentanone, 2-methyl-2-pentanol and 2-methyl pentane. It showed a clear inhibitory effect for the ketone and the tertiary alcohol, but no effect for the hydrocarbon. No change in enzyme conformation was seen during the simulations. The study led to the conclusion that the effect of added organic component on lipase catalyzed transacylation could be explained by the competitive inhibitory character of solvents towards the first binding substrate methyl propanoate.
|
|
| 36. |
- Gustavsson, M., et al.
(författare)
-
Stable linker peptides for a cellulose-binding domain-lipase fusion protein expressed in Pichia pastoris
- 2001
-
Ingår i: Protein Engineering. - : Oxford University Press (OUP). - 0269-2139 .- 1460-213X. ; 14:9, s. 711-715
-
Tidskriftsartikel (refereegranskat)abstract
- Fusion proteins composed of a cellulose-binding domain from Neocallimastix patriciarum cellulase A and Candida antarctica lipase B were constructed using different linker peptides. The aim was to create proteolytically stable linkers that were able to join the functional modules without disrupting their function. Six fusion variants containing linkers of 4-44 residues were expressed in Pichia pastoris and analysed. Three variants were found to be stable throughout 7-day cultivations. The cellulose-binding capacities of fusion proteins containing short linkers were slightly lower compared with those containing long linkers. The lipase-specific activities of all variants, in solution or immobilized on to cellulose, were equal to that of the wildtype lipase.
|
|
| 37. |
- Gustavsson, M. T., et al.
(författare)
-
Modification of cellulose fiber surfaces by use of a lipase and a xyloglucan endotransglycosylase
- 2005
-
Ingår i: Biomacromolecules. - : American Chemical Society (ACS). - 1525-7797 .- 1526-4602. ; 6:1, s. 196-203
-
Tidskriftsartikel (refereegranskat)abstract
- A strategy for the modification of cellulose fiber surfaces was developed that used the ability of Candida antarctica lipase B (CALB) to acylate carbohydrates with high regioselectivity, combined with the transglycosylating activity of the Populus tremula x P. tremuloides xyloglucan endotransglycosylase 16A (PttXET16A). Xyloglucan oligosaccharides (XGOs) prepared from tamarind xyloglucan were acylated with CALB as a catalyst and vinyl stearate or gamma-thiobutyrolactone as acyl donors to produce carbohydrate molecules with hydrophobic alkyl chains or reactive sulfhydryl groups, respectively. The modified XGOs were shown to act as glycosyl acceptors in the transglycosylation reaction catalyzed by PttXET16A and could therefore be incorporated into high M-r xyloglucan chains. The resulting xyloglucan molecules exhibited a high affinity for cellulose surfaces, which enabled the essentially irreversible introduction of fatty acid esters or thiol groups to cellulose fibers.
|
|
| 38. |
- Gustavsson, Malin T., et al.
(författare)
-
Polyester coating of cellulose fiber surfaces catalyzed by a cellulose-binding module-Candida antarctica lipase B fusion protein
- 2004
-
Ingår i: Biomacromolecules. - : American Chemical Society (ACS). - 1525-7797 .- 1526-4602. ; 5:1, s. 106-112
-
Tidskriftsartikel (refereegranskat)abstract
- A new approach to introduce polymers to cellulosic materials was developed by using the ability of a cellulose-binding module-Candida antarctica lipase B conjugate to catalyze ring-opening polymerization of epsilon-caprolactone in close proximity to cellulose fiber surfaces. The epsilon-caprolactone was introduced to the cellulose surfaces either by simple addition of liquid monomer or through gas phase. The effects of water activity and temperature on the lipase-catalyzed polymerization process were investigated. Analysis showed that the water content in the system primarily regulated the obtained polymer molecular weight, whereas the temperature influenced the reaction rate. The hydrophobicity of the obtained surfaces did not arise from covalent attachment of the poly(epsilon-caprolactone) to the surface hydroxyl groups but rather from surface-deposited polymers which could be readily extracted. The degree of lipase-catalyzed hydrolysis through introduction of water to the polymer-coated cellulose fiber surfaces was also investigated and shown to be significant.
|
|
| 39. |
- Hamberg, Anders, et al.
(författare)
-
C-terminal ladder sequencing of peptides using an alternative nucleophile in carboxypeptidase Y digests
- 2006
-
Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 357:2, s. 167-172
-
Tidskriftsartikel (refereegranskat)abstract
- A method for improved sequence coverage in C-terminal sequencing of peptides, based on carboxypeptidase digestion, is described. In conventional carboxypeptidase digestions, the peptide substrate is usually extensively degraded and a full amino acid sequence cannot be obtained due to the lack of a complete peptide ladder. In the presented method, a protecting group is introduced at the C terminus of a fraction of the peptide fragments formed in the digest, and thereby further degradation of the C-terminally modified peptides are slowed down. The protecting group was attached to the C-terminal amino acid through a carboxypeptidase-catalyzed reaction with an alternative nucleophile, 2-pyridylmethylamine, added to the aqueous digestion buffer. Six peptides were digested by carboxypeptidase Y with and without 2-pyridylmethylamine present in the digest buffer, and the resulting fragments subsequently were analyzed with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Comparison of the two digestion methods showed that the probability of successful ladder sequencing increased, by more than 50% using 2-pyridylmethylamine as a competing nucleophile in carboxypeptidase Y digests.
|
|
| 40. |
- Hamberg, Anders, 1974-
(författare)
-
Enzyme selectivity as a tool in analytical chemistry
- 2007
-
Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Enzymes are useful tools as specific analytical reagents. Two different analysis methods were developed for use in the separate fields of protein science and organic synthesis. Both methods rely on the substrate specificity of enzymes. Enzyme catalysis and substrate specificity is described and put in context with each of the two developed methods. In paper I a method for C-terminal peptide sequencing was developed based on conventional Carboxypeptidase Y digestion combined with matrix assisted laser desorption/ionization mass spectrometry. An alternative nucleophile was used to obtain a stable peptide ladder and improve sequence coverage. In paper II and III, three different enzymes were used for rapid analysis of enantiomeric excess and conversion of O-acylated cyanohydrins synthesized by a defined protocol. Horse liver alcohol dehydrogenase, Candida antarctica lipase B and pig liver esterase were sequentially added to a solution containing the O-acylated cyanohydrin. Each enzyme caused a drop in absorbance from oxidation of NADH to NAD+. The conversion and enantiomeric excess of the sample could be calculated from the relative differences in absorbance.
|
|
| 41. |
|
|
| 42. |
- Hamberg, Anders, et al.
(författare)
-
High Throughput Synthesis and Analysis of Acylated Cyanohydrins
- 2007
-
Ingår i: Chemistry - A European Journal. - : Wiley. - 0947-6539 .- 1521-3765. ; 13:15, s. 4334-4341
-
Tidskriftsartikel (refereegranskat)abstract
- The yields and optical purities of products obtained from chiral Lewis acid/Lewis base-catalysed additions of alpha-ketonitriles to prochiral aldehydes could be accurately determined by an enzymatic method. The amount of remaining aldehyde was determined after its reduction to an alcohol, whilst the two product enantiomers were analysed after subsequent hydrolysis first by the (S)-selective Candida antarctica lipase B and then by the unselective pig liver esterase. The method could be used for analysis of products obtained from a number of aromatic aldehydes and aliphatic ketonitriles. Microreactor technology was successfully combined with high-throughput analysis for efficient catalyst optimization.
|
|
| 43. |
|
|
| 44. |
- Hamberg, Anders, et al.
(författare)
-
Rational engineering of Candida antarctica lipase B for selective monoacylation of diols
- 2012
-
Ingår i: Chemical Communications. - : Royal Society of Chemistry (RSC). - 1359-7345 .- 1364-548X. ; 48:80, s. 10013-10015
-
Tidskriftsartikel (refereegranskat)abstract
- The enzyme Candida antarctica lipase B was subjected to site directed mutagenesis suggested by molecular modelling. The selectivity for the enzyme increased towards a range of diols over their corresponding monoesters as an effect of the mutations.
|
|
| 45. |
|
|
| 46. |
- Hamberg, Anders, et al.
(författare)
-
Selective Monoacylation of Diols by Substrate Assisted Catalysis in T40A Candida antarctica Lipase B
- 2013
-
Ingår i: ChemCatChem. - : Wiley. - 1867-3880 .- 1867-3899. ; 5:3, s. 743-747
-
Tidskriftsartikel (refereegranskat)abstract
- The selectivity towards diols over monoesters in the esterification of diols catalysed by lipase B from Candida antarctica (CALB) was improved by the single point mutation T40A in the enzyme's oxyanion hole. Substrate-assisted catalysis was suggested from molecular modelling of the tetrahedral intermediate in esterification of 1,2-ethanediol catalysed by T40A CALB. The non-reacting hydroxyl group of the diol forms a hydrogen bond to the oxyanion in the transition state, replacing that deleted in mutation. Monoester yields in transacylation reactions were monitored over time to compare the selectivities for wild-type and T40A CALB. The results showed increased selectivities towards the diols tested over their corresponding monoesters as a result of the T40A mutation with substrate-assisted catalysis as a plausible explanation.
|
|
| 47. |
- Hamberg, Anders, 1974-
(författare)
-
Serine Hydrolase Selectivity : Kinetics and applications in organic and analytical chemistry
- 2010
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The substrate selectivities for different serine hydrolases were utilized in various applications, presented in papers I-VI. The articles are discussed in the thesis in view of the kinetics of the enzyme catalysis involved. In paper I the enantioselectivities towards a range of secondary alcohols were reversed for Candida antarctica lipase B by site directed mutagenesis. The thermodynamic components of the enantioselectivity were determined for the mutated variant of the lipase. In papers II-III Candida antarctica lipase B was engineered for selective monoacylation using two different approaches. A variant of the lipase created for substrate assisted catalysis (paper II) and three different variants with mutations which decreased the volume of the active site (paper III) were evaluated. Enzyme kinetics for the different variants were measured and translated into activation energies for comparison of the approaches. In papers IV and V three different enzymes were used for rapid analysis of enantiomeric excess and conversion of O-acylated cyanohydrins synthesized by a defined protocol. Horse liver alcohol dehydrogenase, Candida antarctica lipase B and pig liver esterase were sequentially added to a solution containing the O-acylated cyanohydrin. Each enzyme caused a drop in absorbance from oxidation of NADH to NAD+. The product yield and enantiomeric excess was calculated from the relative differences in absorbance. In paper VI a method for C-terminal peptide sequencing was developed based on conventional Carboxypeptidase Y digestion combined with matrix assisted laser desorption/ionization mass spectrometry. An alternative nucleophile was used to obtain a stable peptide ladder and improve sequence coverage.
|
|
| 48. |
|
|
| 49. |
- Hedfors, Cecilia, 1978-
(författare)
-
Lipase chemoselectivity - kinetics and applications
- 2009
-
Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- A chemoselective catalyst is preferred in a chemical reaction where protecting groups otherwise are needed. The two lipases Candida antarctica lipase B and Rhizomucor miehei lipase showed large chemoselectivity ratios, defined as (kcat/KM)OH / (kcat/KM)SH, in a transacylation reaction with ethyl octanoate as acyl donor and hexanol or hexanethiol as acyl acceptor (paper I). The chemoselectivity ratio of the uncatalyzed reaction was 120 in favour of the alcohol. Compared to the uncatalyzed reaction, the chemoselectivity was 730 times higher for Candida antarctica lipase B and ten times higher for Rhizomucor miehei lipase. The KM towards the thiol was more than two orders of magnitude higher than the KM towards the corresponding alcohol. This was the dominating contribution to the high chemoselectivity displayed by the two lipases. In a novel approach, Candida antarctica lipase B was used as catalyst for enzymatic synthesis of thiol-functionalized polyesters in a one-pot reaction without using protecting groups (paper II). Poly(e-caprolactone) with a free thiol at one of the ends was synthesized in an enzymatic ring-opening polymerization initiated with mercaptoethanol or terminated with either 3-mercaptopropionic acid or g-thiobutyrolactone.
|
|
| 50. |
- Hedfors, Cecilia, et al.
(författare)
-
Lipase chemoselectivity towards alcohol and thiol acyl acceptors in a transacylation reaction
- 2010
-
Ingår i: Journal of Molecular Catalysis B. - : Elsevier BV. - 1381-1177 .- 1873-3158. ; 66:1-2, s. 120-123
-
Tidskriftsartikel (refereegranskat)abstract
- The lipase chemoselectivity towards an alcohol and a thiol was investigated for the two lipases Candida antarctica lipase B (CalB) and Rhizomucor miehei lipase (Rml). Hexanol and hexanethiol were used as acyl acceptors in a transacylation reaction with ethyl octanoate in cyclohexane. CalB showed the highest chemoselectivity ratio (k(cat)/K-M)(OH)/(k(cat)/K-M)(SH), of 88,000 while the ratio for Rml was 1200. That could be compared with the ratio, k(OH)/k(SH), of 120 for the non-catalyzed reaction. Thus, the enzyme contribution to the chemoselectivity between hexanol and hexanethiol was 730 for CalB and 10 for Rml. High K-M values displayed towards hexanethiol (above 1.8 M) were the largest contribution to the selectivity. No saturation was achieved. The K-M values were more than two orders of magnitude higher than those of hexanol.
|
|
