| 1. |
- Grey, Carl, et al.
(författare)
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Improved operational stability of chloroperoxidase through use of antioxidants.
- 2008
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Ingår i: Journal of Biotechnology. - : Elsevier BV. - 1873-4863 .- 0168-1656. ; 135:2, s. 196-201
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Tidskriftsartikel (refereegranskat)abstract
- Chloroperoxidase (CPO) from Caldariomyces fumago is a potentially very useful enzyme due to its ability to catalyze a large variety of stereoselective oxidation reactions, but poor operational stability is a main limitation for commercial use. In the present study, the possibility of increasing the operational stability by use of antioxidants was investigated using the oxidation of indole as model reaction. Caffeic acid was the antioxidant showing the strongest positive effects, reaching a total turnover number (TTN) of 135,000 at pH 4 and 4mM hydrogen peroxide, compared to 28,700 in the absence of antioxidant. Portion-wise addition of hydrogen peroxide in the presence of caffeic acid caused a further increase in TTN to 171,000. An alternative way to reach high TTN was to use tert-butyl hydroperoxide as oxidant instead of hydrogen peroxide: a TTN of 600,000 was achieved although the reaction was quite slow. In this case, antioxidants did not have any positive effect. Possible mechanisms for the observed inactivation of CPO are discussed.
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| 2. |
- Gräber, Martin, et al.
(författare)
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A novel direct screening method for alkyl glucoside production by glucosidases expressed in E. coli in 96-well plates.
- 2010
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Ingår i: Journal of Biotechnology. - : Elsevier BV. - 1873-4863 .- 0168-1656. ; 145, s. 186-192
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Tidskriftsartikel (refereegranskat)abstract
- The present work describes the development of a novel direct screening method, assayed in 96-well format, for evaluation of enzymatic alkyl glycoside production in a hexanol water two-phase system. Alkyl glycosides are surfactants with a range of applications and with good biodegradability and low toxicity. Enzymatic synthesis makes it possible to prepare beta-D-glucopyranosides with high purity. In the developed screening assay, hexyl-ss-D-glucopyranoside was chosen as a model product to be synthesised by reversed hydrolysis in a water-hexanol two-phase system. In a first step the model product is produced by glucosidases expressed in E. coli cells in 96 deep well plates. After phase separation, the hexyl-ss-D-glucopyranoside in the organic phase is degraded enzymatically and the released glucose detected spectrophotometrically at 405nm utilizing peroxidase/glucose oxidase, and the reagent 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS). The aqueous phase is used to monitor hydrolysis of p-NPG at 405nm, allowing use of a ratio of the two assays to compensate for expression differences. The complete method was used for comparison of two different ss-glucosidases, classified under glycoside hydrolase family 1 and 3, respectively, showing a significant difference in their ability to synthesise hexyl-ss-D-glucopyranoside by reversed hydrolysis.
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| 3. |
- Rundbäck, Fabian
(författare)
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Biocatalysed redox reactions - Possibilities and limitations
- 2012
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The use of enzymes for synthetic applications is an area of increasing interest. Enzymes have been pointed out to be an environmentally friendly alternative to existing chemical methods, as well as making completely new synthetic routes possible. Over the last decades, much focus has been put on the enzyme group oxidoreductases, which can selectively introduce oxygen and hydrogen into molecules. These reactions can generate optically active molecules that are highly useful building blocks in pharmaceutical production. The limitations for a broader application of oxidoreductases are their need for expensive cofactors and limited operational stability. This thesis is based on research with the objective to improve the understanding of and develop methodology for enzymatically catalysed reduction and oxidation reactions. The problem of cofactor dependency was addressed in a study where hydrogenase and hydrogen gas were used to regenerate the NADH cofactor. A new application of hydrogenase containing Ralstonia eutropha was demonstrated, where these cells were used in combination with another whole cell catalyst for asymmetric ketone reduction. The two-cell system showed a higher specific initial reaction rate and yield, but a slightly lower enantiomeric purity of the product, in comparison with a system where a cosubstrate was used for cofactor regeneration.
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| 4. |
- Rundbäck, Fabian, et al.
(författare)
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Coupling of permeabilized cells of Gluconobacter oxydans and Ralstonia eutropha for asymmetric ketone reduction using H(2) as reductant.
- 2012
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Ingår i: Journal of Biotechnology. - : Elsevier BV. - 1873-4863 .- 0168-1656. ; 157, s. 154-158
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Tidskriftsartikel (refereegranskat)abstract
- A combined two-cell reaction system containing Gluconobacter oxydans and Ralstonia eutropha was evaluated with regard to asymmetric ketone reduction using H(2) as the reductant. Whole cells permeabilized by EDTA/toluene were used, and synthesis was performed in a biphasic aqueous/organic reaction medium. The two-cell system was compared with a system in which G. oxydans alone was used for both ketone reduction and cofactor regeneration, using an alcohol as co-substrate. The two-cell system exhibited almost twice the initial reaction rate of the single-cell system, a higher yield (75% vs. 48%) but slightly lower enantiomeric purity (93% vs. 98%) of the product (S)-2-octanol. The permeabilized R. eutropha cells are worth evaluating for byproduct-free NADH regeneration in combination with other whole cell catalysts.
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