| 1. |
- Gaber, Yasser, et al.
(författare)
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Enzymatic synthesis of N-alkanoyl-N-methylglucamide surfactants: solvent-free production and environmental assessment
- 2010
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Ingår i: Green Chemistry. - : Royal Society of Chemistry (RSC). - 1463-9270 .- 1463-9262. ; 12:10, s. 1817-1825
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Tidskriftsartikel (refereegranskat)abstract
- A biocatalysis based method for the solvent-free production of N-alkanoyl-N-methylglucamide (MEGA) surfactants was developed and used as a case study for the evaluation of different environmental assessment tools, such as the freeware package EATOS (Environmental Assessment Tool for Organic Synthesis). In order to also consider energy usage and process facilities, e. g. heating, stirring and vacuum, a complementary tool was needed; hence the EcoScale method and the use of an energy monitoring socket were also exploited. The solvent-free method followed by a simple hydrolysis step gave a final amide yield of 99% and a product essentially free of remaining substrate, N-methylglucamine (MEG). The latter is important since MEG can potentially be converted to carcinogenic nitrosamines. The absence of solvent in the reaction medium was also found to result in a significantly reduced potential environmental impact. The environmental tools used in this study were further scrutinized, and even if they represent some of the best freely available tools for evaluation of early stage process development, some points for further improvements are suggested.
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| 2. |
- Gaber, Yasser, et al.
(författare)
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HPLC-EAT (Environmental Assessment Tool): A tool for profiling safety, health and environmental impacts of liquid chromatography methods
- 2011
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Ingår i: Green Chemistry. - : Royal Society of Chemistry (RSC). - 1463-9270 .- 1463-9262. ; 13:8, s. 2021-2025
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Tidskriftsartikel (refereegranskat)abstract
- A simple and efficient approach for profiling the greenness of high performance liquid chromatography (HPLC) methods is presented. This environmental assessment tool (EAT) takes into consideration the environmental, health and safety issues for all solvents involved in the chromatographic method, and calculates a total score that can be used for comparison of the greenness of different methods. A software, HPLC-EAT, has been designed to facilitate the calculation and can be downloaded free of charge at http://www.biotek.lu.se/hplc-eat/. HPLC-EAT was successfully applied for a set of different HPLC methods from the literature, including both analytical and preparative chromatography. The performance of the tool was validated and it was further combined with another free software Eco-solvent tool to perform life cycle assessments of waste disposal options of distillation or incineration. HPLC-EAT can be routinely used in method development to calculate the greenness beside the conventional standards of accuracy, robustness and reproducibility.
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| 3. |
- Hagström, Anna, et al.
(författare)
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Chemo-enzymatic epoxidation-process options for improving biocatalytic productivity.
- 2011
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Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 27:1, s. 67-76
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Tidskriftsartikel (refereegranskat)abstract
- The reactor choice is crucial when designing a process where inactivation of the biocatalyst is a problem. The main bottleneck for the chemo-enzymatic epoxidation has been found to be enzyme inactivation by the hydrogen peroxide, H(2)O(2), substrate. In the work reported here, the effect of reaction parameters on the reaction performance have been investigated and used to establish suitable operating strategies to minimize the inactivation of the enzyme, using rapeseed methyl ester (RME) as a substrate in a solvent-free system. The use of a controlled fed-batch reactor for maintaining H(2)O(2) concentration at 1.5 M resulted in increased productivity, up to 76 grams of product per gram of biocatalyst with higher retention of enzyme activity. Further investigation included a multistage design that separated the enzymatic reaction and the saturation of the RME substrate with H(2)O(2) into different vessels. This setup showed that the reaction rate as well as enzyme inactivation is strongly dependent on the H(2)O(2) concentration. A 20-fold improvement in enzymatic efficiency is required for reaching an economically feasible process. This will require a combination of enzyme modification and careful process design. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010.
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| 4. |
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| 5. |
- Hatti-Kaul, Rajni, et al.
(författare)
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Industrial biotechnology for the production of bio-based chemicals - a cradle-to-grave perspective
- 2007
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Ingår i: Trends in Biotechnology. - : Elsevier BV. - 0167-7799. ; 25:3, s. 119-124
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Forskningsöversikt (refereegranskat)abstract
- Shifting the resource base for chemical production from fossil feedstocks to renewable raw materials provides exciting possibilities for the use of industrial biotechnology-based process tools. This review gives an indication of the current developments in the transition to bio-based production, with a focus on the production of chemicals, and points out some of the challenges that exist in the large-scale implementation of industrial biotechnology. Furthermore, the importance of evaluating the environmental impact of bio-based products with respect to their entire life cycle is highlighted, demonstrating that the choice of the raw material often turns out to be an important parameter influencing the life cycle performance.
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| 6. |
- Irani, Mehdi, et al.
(författare)
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Amino Acid Oxidation of Candida antarctica Lipase B Studied by Molecular Dynamics Simulations and Site-Directed Mutagenesis
- 2013
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 52:7, s. 1280-1289
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Tidskriftsartikel (refereegranskat)abstract
- Molecular dynamics simulations have been performed on lipase B from Candida antarctica (CalB) in its native form and with one or two oxidized residues, either methionine oxidized to methionine sulfoxide, tryptophan oxidized to 5-hydroxytryptophan, or cystine oxidized to a pair of cysteic acid residues. We have analyzed how these oxidations affect the general structure of the protein as well as the local structure around the oxidized amino acid and the active site. The results indicate that the methionine and tryptophan oxidations led to rather restricted changes in the structure, whereas the oxidation of cystines, which also caused cleavage of the cystine S-S linkage, gave rise to larger changes in the protein structure. Only two oxidized residues caused significant changes in the structure of the active site, viz., those of the Cys-22/64 and Cys-216/258 pairs. Site-directed mutagenesis studies were also performed. Two variants showed a behavior similar to that of native CalB,(M83I and M129L), whereas W155Q and M72S had severely decreased specific activity. M83I had a slightly higher thermostability than native CalB. No significant increase in stability toward hydrogen peroxide was observed. The same mutants were also studied by molecular dynamics. Even though no significant increase in stability toward hydrogen peroxide was observed, the results from simulations and site-directed mutagenesis give some clues about the direction of further work on stabilization.
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| 7. |
- Orellana-Coca, C, et al.
(författare)
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Chemo-enzymatic epoxidation of oleic acid and methyl oleate in solvent-free medium
- 2005
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Ingår i: Biocatalysis and Biotransformation. - : Informa UK Limited. - 1024-2422 .- 1029-2446. ; 23:6, s. 431-437
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Tidskriftsartikel (refereegranskat)abstract
- Chemo-enzymatic epoxidation of oleic acid (OA) and its methyl ester has been performed using hydrogen peroxide and immobilized lipase from Candida antarctica (Novozym(R) 435). The purpose of the study was to characterize the reaction under solvent-free conditions. The reaction temperature had a significant impact on epoxidation of OA. At lower temperatures, the substrate conversion was hindered by the formation of solid epoxystearic acid product. Nearly 90% conversion of OA to the epoxide product was obtained after 6 h at 50 degrees C. Longer reaction times at 40 degrees C and above resulted in by-product formation and eventually lowered the product yield. In contrast, the reaction with methyl oleate (MO) was less influenced by temperature. Almost complete epoxidation was achieved at 40-60 degrees C, the higher the temperature the shorter was the reaction time. The main epoxidation product obtained was epoxystearic acid methyl ester (EME), and the remaining was epoxystearic acid (EA) formed by the hydrolytic action of the lipase. Recycling of the lipase for epoxidation of MO at 50 degrees C indicated that the immobilized enzyme was prone to activity loss.
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| 8. |
- Tufvesson, Pär, et al.
(författare)
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Towards a cost-effective immobilized lipase for specialty chemicals
- 2011
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Ingår i: Journal of Molecular Catalysis B: Enzymatic. - : Elsevier BV. - 1873-3158 .- 1381-1177. ; 68:2, s. 200-205
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Tidskriftsartikel (refereegranskat)abstract
- Biocatalysis has the potential to provide the chemical industry with several advantages for the production of chemicals; but the use of this technology for the production of speciality and bulk chemicals is unfortunately limited due to the high costs related to the production of the biocatalyst. We have immobilized Candida antarctica lipase B (CALB) on different resins in order to obtain a more cost-effective biocatalyst, i.e. to find the cheapest preparation per catalytic activity, for one esterification and one amidation reaction. It was found that lipase immobilized on macroporous polypropylene (Accurel MP1000) and polymethylmethacrylate (Lewatit) provided biocatalyst preparations where the cost of enzyme and carrier was significantly less than the cost of commercially available Novozym (R) 435, indicating a potential for decreased cost. Also the stability of Accurel MP1000 bound enzyme during repeated use matched that of Novozym (R) 435. Finally the in-house biocatalyst was used in a packed-bed set-up, showing an excellent stability in repeated batches at 70 degrees C. 2010 Elsevier B.V. All rights reserved.
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| 9. |
- Törnvall, Ulrika, et al.
(författare)
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Biocatalytic production of fatty epoxides from rapeseed and tall oil derivatives: Process and environmental evaluation
- 2009
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Ingår i: Industrial Biotechnology. - 1550-9087. ; 5:3, s. 184-192
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Tidskriftsartikel (refereegranskat)abstract
- Rapeseed and tall oil are important renewable feedstocks for chemicals and energy — the former, an agricultural crop, and the latter, is a by-product of the pulp and paper industry. This report presents a study on the production of fatty epoxides from rapeseed methyl ester, tall oil methyl ester, and tall oil fatty acids by a solvent-free lipase-mediated process. Fatty epoxides are useful reactive building blocks for production of lubricants, plasticizers, and surfactants. Epoxidation of the different raw materials exhibited similar reaction speeds, energy requirements, and final conversions, and the epoxide products were biodegradable and nontoxic up to high concentrations. From environmental and economic perspectives, however, the use of tall oil as raw material is preferable to rapeseed. For the chemo-enzymatic process of epoxidation to be economically feasible, improvements in biocatalyst cost and/or stability are still needed.
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| 10. |
- Törnvall, Ulrika
(författare)
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Chemo-enzymatic epoxidation: Activity and stability of Candida antarctica lipase B
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Growing concerns about climate change and depleting fossil feedstocks are driving a shift within the chemical industry from energy-intensive processes to more environmentally sound and sustainable processing using renewable feedstocks. One such process is the utilization of lipases for the chemo-enzymatic epoxidation of vegetable oils and fatty acids. The lipase mediated route for production of fatty epoxides is a two-step process in which the enzyme catalyzes the first step; the formation of a peracid from a carboxylic acid and hydrogen peroxide. This thesis is based on research with the objective to develop a cost-effective, environmentally friendly enzyme-based industrial process for the production of fatty epoxides. A solvent-free process was first established using immobilized Candida antarctica lipase B (Novozym 435). Both rapeseed methyl ester and tall oil derivatives gave biodegradable epoxide products with high degree of epoxidation, but being a by-product of the pulp and paper industry, tall oil scored much better as a raw material for chemical production from an environmental point of view. The enzyme was prone to deactivation under the given conditions and hydrogen peroxide was found to cause by far the most severe deactivation of the enzyme. In order to prolong the lifetime of the enzyme, the hydrogen peroxide was added to the process continuously rather than all at once. It was found that maintaining the peroxide concentration at a low level in the reactor was an even better alternative for extending the enzyme lifetime. A thorough study of the enzyme was also performed, to shed light on the mechanism behind the deactivation. Since an industrial process would involve the immobilized enzyme, a method was developed to evaluate the changes occurring in Novozym 435 at the amino acid level upon treatment with hydrogen peroxide without detaching the enzyme from the carrier. All four methionines and two tryptophans present in the enzyme were oxidized. Moreover, cystine was oxidized to cysteic acid which implied the disruption of all three disulfide bridges in CalB. Circular dichroism and dynamic light scattering investigations verified that profound changes in the secondary structure occurred upon exposure to hydrogen peroxide. These results are laying the basis for mutagenesis experiments, recently initiated, with the aim of developing an improved variant of this enzyme.
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| 11. |
- Törnvall, Ulrika, et al.
(författare)
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Mass spectrometric analysis of peptides from an immobilized lipase: focus on oxidative modifications.
- 2009
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Ingår i: Rapid Communications in Mass Spectrometry. - : Wiley. - 1097-0231 .- 0951-4198. ; 23:18, s. 2959-2964
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Tidskriftsartikel (refereegranskat)abstract
- Liquid chromatography/tandem mass spectrometry (LC/MS/MS) was used to study the primary structure of immobilized Candida antarctica lipase B (Novozym(R)435) without detaching the enzyme from the carrier. The immobilized enzyme packed in a miniature column was subjected to proteolysis and the peptides released were injected into the mass spectrometer for analysis. The set-up was utilized to determine amino acid oxidation after treatment of the biocatalyst with hydrogen peroxide. In total, sequence coverage of more than 90% was obtained, containing almost all of the amino acids sensitive to oxidation. Oxidation of methionine, tryptophan and cystine residues was observed. The flow system also allowed evaluation of the enzyme activity prior to peptide analysis. The developed method is general and should be applicable to other immobilized enzyme systems and to different treatments.
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| 12. |
- Törnvall, Ulrika
(författare)
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Pinpointing oxidative modifications in proteins-recent advances in analytical methods
- 2010
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Ingår i: Analytical Methods. - : Royal Society of Chemistry (RSC). - 1759-9660 .- 1759-9679. ; 2:11, s. 1638-1650
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Forskningsöversikt (refereegranskat)abstract
- Oxidation is one of the key degradation pathways in proteins and is of relevance to analyze in a wide variety of research disciplines. The types of different modifications occurring during oxidation by reactive oxygen species (ROS) are almost as many as the number of available analytical methods. Protein oxidation in biological samples has traditionally been analyzed by various protein carbonyl assays, sometimes combined with mass spectrometry (MS) for identification of the carbonylated proteins. MS is now increasingly used also for the determination of the exact position of the oxidation in the amino acid sequence, an approach that is aided by recent developments within the field of proteomics. The following review summarizes the effects of ROS on proteins, describes methods for labeling and separation of oxidized proteins in complex mixtures, and provides insight into various MS-based methods to localize the modifications within the protein primary structure. Pitfalls with the different techniques are given, as well as examples from various applications within biological studies, protein therapeutics, plant science, the food industry and industrial biotechnology.
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| 13. |
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| 14. |
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| 15. |
- Törnvall, Ulrika, et al.
(författare)
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Stability of immobilized Candida antarctica lipase B during chemo-enzymatic epoxidation of fatty acids
- 2007
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Ingår i: Enzyme and Microbial Technology. - : Elsevier BV. - 0141-0229. ; 40:3, s. 447-451
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Tidskriftsartikel (refereegranskat)abstract
- The parameters affecting the lipase activity and operational lifetime during chemo-enzymatic epoxidation of fatty acids were investigated. Immobilized Candida antarctica lipase B (Novozym (R) 435) was incubated in the presence of various reaction components (i.e. toluene, water, H2O2, oleic acid, perpalmitic acid, and epoxystearic acid, respectively) at temperatures between 20 and 60 degrees C followed by measurement of residual enzyme activity. Epoxystearic acid was shown to slightly inactivate the enzyme at 50 degrees C, while oleic acid and perpalmitic acid did not. No deactivation of the enzyme was observed in presence of toluene/water mixture within 48 h at 20-60 degrees C. In the presence of 6-12 M hydrogen peroxide, the enzyme was rather stable at 20 degrees C, while at 60 degrees C the enzyme lost activity rapidly, with the rate of deactivation increasing with increasing hydrogen peroxide concentration. These results imply that temperature control and careful dosage of hydrogen peroxide would be essential in an industrial chemo-enzymatic process. (c) 2006 Elsevier Inc. All rights reserved.
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| 16. |
- Törnvall, Ulrika, et al.
(författare)
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Structural, functional and chemical changes in Pseudozyma antarctica lipase B on exposure to hydrogen peroxide.
- 2010
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Ingår i: Biochimie. - : Elsevier BV. - 1638-6183 .- 0300-9084. ; 92:Online 21 July 2010, s. 1867-1875
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Tidskriftsartikel (refereegranskat)abstract
- The effect on primary, secondary, tertiary and quaternary structure of Pseudozyma (formerly Candida) antarctica lipase B (PalB) on exposure to hydrogen peroxide was investigated using nano-electrospray ionization-mass spectrometry (nano-ESI-MS), liquid chromatography tandem mass spectrometry (LC/MS/MS), circular dichroism (CD), and dynamic light scattering (DLS). Treatment with hydrogen peroxide generated heavier protein variants, with a mass gain that increased with increasing incubation time. Furthermore, elevated concentration of H(2)O(2) was shown to result in partial fragmentation of the protein. Proteolytic digestion of the enzyme gave primary sequence coverage of more than 90%, revealing oxidation of methionine, tryptophan and cystine residues. The active site histidine was not observed in oxidized form in any of the experiments. However, oxidation of cystine to cysteic acid indicated disruption of disulphide bridges, and CD evaluations confirmed that severe changes to the secondary structure towards random coil had occurred. The structural changes could be an effect of the observed amino acid side chain oxidations, and was correlated with deactivation of the lipase. From DLS experiments, it was seen that the lipase exposed to both high temperature and H(2)O(2) formed large and intermediate sized aggregates, not observed for the heat treated enzyme. The findings reported here could lay the basis for developing enzyme variants with higher oxidative stability.
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