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- Griss, Patrick, et al.
(författare)
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Spiked biopotential electrodes
- 2000
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Ingår i: IEEE International Workshop on Micro Electromechanical Systems (MEMS2000), Miyazaki, Japan, Jan. 23-27. ; , s. 323-328
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Konferensbidrag (refereegranskat)
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| 6. |
- Hansson, Therese
(författare)
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Beta-Glycosidases from Hyperthermophiles as Biocatalysts
- 2002
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The thesis concerns glycosyl transfer with use of b-glycosidases, most of them from hyperthermophilic organisms. Both optimisation of the reaction conditions and protein engineering in terms of site-directed mutagenesis were used to improve b-glycosidase-catalysed reactions. The first part of the work deals with conversion of lactose into valuable products such as galacto-oligosaccharides (GOS) and alkyl-glycosides, the activity and selectivity of b-glycosidases in model reactions being investigated in the second part. b-glycosidases from the hyperthermophilic Archaea Sulfolobus solfataricus (LacS) and Pyrococcus furiosus (CelB) were shown to be very well suited for GOS synthesis from lactose. For both enzymes the maximum yield of GOS increased with increasing lactose concentration. In an optimally designed reaction involving LacS, a combined yield of 37% for the tri- and tetrasaccharides was attained and a maximum yield of 40% for CelB. Use of site-directed mutagenesis of CelB resulted in a further improvement in GOS yield. An exchange of phenylalanine for tyrosine (F426Y) increased the GOS yield (45%) compared with wild-type CelB (40%). Furthermore, a double mutant, M424K/F426Y, showed much better glycosyl transfer properties at low lactose concentrations than the wild-type enzyme did. At a lactose concentration of 10%, the GOS yield for the mutant was 40% as compared with 18% for the wild-type. In the synthesis of hexyl-b-glycoside from lactose in hexanol, these two b-glycosidases from hyperthermophiles showed higher activity than other b-glycosidases did, and produced high yields of both hexyl-b-galactoside (LacS: 41%, CelB: 63%) and hexyl-b-glucoside (LacS: 29%, CelB: 28%). Adding SDS to the reaction increased both the initial reaction rate of LacS and the hexyl-b-galactoside yield (from 41% to 51%). The competition between b-glucosidase-catalysed transglucosylation and hydrolysis was studied using wild-type CelB and active site mutants (M424K, F426Y, M424K/F426Y) in the conversion of pentyl-b-glucoside to hexyl-b-glucoside in hexanol as a model transglucosylation reaction. Hydrolysis to glucose was a side reaction to this. Each of the mutants showed lower activity but higher selectivity than the wild-type enzyme. The largest increase in selectivity (2.6-fold) was attained by the F426Y mutant, for which the hexyl-b-glucoside yield increased from 56% to 69%. In contrast to the wild-type CelB, the F426Y mutant had a transferase activity as low as aw 0.29. Surprisingly, the selectivity of CelB towards transglucosylation increased as the water activity increased, up to aw 0.92. To investigate the influence of the water activity on the selectivity of b-glycosidases further five different enzymes (P. furiosus b-glucosidase, S. solfataricus b-galactosidase, Caldocellum saccharolyticum b-glucosidase, almond b-glucosidase and Escherichia coli b-galactosidase) were evaluated as transglycosylation catalysts in hexanol containing various amounts of water. For all the enzymes tested, the selectivity for alcohol increased with an increase in water activity. On the other hand, in a hexanol/water two-phase system, hydrolysis was by far the most dominant reaction, despite the total activity for all the enzymes increasing. It was also shown that b-glycosidases of differing origin differs markedly in their substrate specificity and in the extent to which the temperature influences their selectivity for the glycon part of the donor substrate.
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- Persson, Mattias
(författare)
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Lipases as Biocatalysts in Organic Media. Influence of Enzyme Preparation and Reaction Conditions on Activity and Selectivity
- 2001
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The discovery that enzymes can function in organic solvents has broadened the scope of biocatalysis, making enzymes highly useful tools for organic chemists. The widespread use of enzymes as catalysts in organic solvents has, however, been limited by the low activity obtained for enzymes in organic solvents compared to aqueous solutions. The increasing demand for optically pure enantiomers in pharamaceuticals and in agrochemicals has further broadened the interest in using enzymes as catalysts for the production of enantiopure compounds. In the present study an evaluation of different methods of enzyme preparation for increasing the activity of lipases in organic solvent is performed. The influence of different reaction conditions on enantioselectivity in lipase catalysed kinetic resolution reactions is also investigated. Furthermore the importance of controlling the water activity to obtain high catalytic activity is emphasised. By changing the solvent composition and the water activity the enantioselectivity can be altered but the effects upon a change of the reaction medium is difficult to rationalise and predict. The temperature was found to influence enantioselectivity in all of the lipase catalysed reactions investigated in the present work and a thermodynamic model describing the effects of temperature on enantioselectivity enabled such effects to be rationalised. Addition of different additives such as KCl, crown ether and phosphate buffer during the lyophilisation of Humicola lanuginosa lipase increases the esterification activity up to 46 fold compared to the crude powder. The increase in activity for preparations lyophilised in the presence of KCl or phoshate buffer is probably due to the additives serving as an immobilisation matrix for the enzyme. The higher activity obtained for the crown ether preparation is probably due to a molecular imprinting effect. The crown ether supposedly binds to the active site during the dehydration step, keeping the active site of the enzyme in a conformation that is catalytically active. Immobilisation by adsorption onto a porous polypropylene support (Accurel EP-100) proved to be the most efficient method for using lipases in organic solvents. The specific activity was 400 fold higher as compared with the crude powder when lipase from Humicola lanuginosa was immobilised by this adsorption technique. Active site titration revealed that part of the explanation for the high activity obtained when lipases are immobilised onto Accurel EP-100 is due to a higher accessibility of the enzyme molecules as compared with lyophilised preparations. At optimal enzyme loading 93 % of the enzyme molecules were titrated at a high rate indicating that this adsorption on a hydrophobic surface was a very efficent way for reducing mass transfer limitations and to immobilise the enzyme in active conformation for use in organic solvents. A further advantage with the immobilisation technique is that a rather high protein loading (100 mg/g carrier) can be combined with the high specific activity, making this enzyme preparation interesting for industrial applications.
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- Skirnisdottir, Sigurlaug
(författare)
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Phylogenetic characterization of microbial mats and isolation of Thermus spp. and sulfur-oxidizing bacteria from Icelandic hot springs
- 2001
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Until recently, the only way to describe microbial communities was by cultivation. This is a major obstacle because typically only a small fraction of the microbes can be cultivated by standard techniques. However, the use of rRNA and molecular phylogenetic techniques has allowed us to bypass this limitation. In this thesis, both of these approaches were used to describe the bacterial diversity of a few Icelandic hot springs. One of these hot springs was a sulfide rich hot spring (sulfur-mat), which was of special interest because it had a great biomass above the photosynthetic border. In order to evaluate the abundance and stability of cultivable Thermus spp. as a function of seasonal changes in this hot spring, fifty strains of Thermus were isolated during one and a half-year period. Multilocus enzyme electrophoresis (MEE) was used to identify the Thermus strains and to examine the genetic structure of the population. The majority of the strains were T. oshimai and that population showed temporal variation. Furthermore, estimation of the association index indicated that recombination events were frequent within the population. A similar study of the population structure of the genus Thermus, within and between geothermal areas in Iceland, indicated that there were some geographical variations and some physiological factors like pH that affect the population structure of Thermus in Iceland. A new sulfur-oxidizing Thermus strain was isolated and characterized from this same hot spring. A new Hydrogenobacter strain that was able to grow on thiosulfate but could not utilize hydrogen was also isolated. These metabolic features were previously unknown for these genera. An examination of the microbial diversity of the sulfur-mat, a Chloroflexus-mat and a bacterial mat at 88°C by sequencing of SSU rRNA genes obtained by PCR and cloning from the microbial mats, showed that the population structure was quite different in these three ecosystems. Furthermore, the overall diversity of the sulfur-mat and the 88°C mat was lower than in the Chloroflexus-mat, which may be explained by more extreme physiochemical features in the sulfur-mat and the 88°C mat. Although, representatives of novel divisions were found, the majority of the sequences were >95% related to currently known sequences. Comparison of the present results to published data, indicated that there was a relationship between mat type and composition of Aquificales on one hand, and temperature and sulfide concentration on the other hand.
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