Browsing by Author "Eiben, Sabine"

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Open Access
Catalytic hydroxylation in biphasic systems using CYP102A1 mutants
(2005) Maurer, Steffen Christian; Kühnel, Katja; Kaysser, Leonard A.; Eiben, Sabine; Schmid, Rolf D.; Urlacher, Vlada B.
Cytochrome P450 monooxygenases are biocatalysts that hydroxylate or epoxidise a wide range of hydrophobic organic substrates. To date their technical application is limited to a small number of whole-cell biooxidations. The use of the isolated enzymes is believed to be impractical due to the low stability of this enzyme class, to the stochiometric need of the expensive cofactor NADPH, and due to the low solubility of most substrates in aqueous media. To overcome these problems we have investigated the application of a bacterial monooxygenase (mutants of CYP102A1) in a biphasic reaction system supported by cofactor recycling with NADP+-dependent formate dehydrogenase from Pseudomonas sp 101. Using this experimental setup, cyclohexane, octane and myristic acid were hydroxylated. To reduce the process costs a novel NADH-dependent double mutant of CYP102A1 was designed. For recycling of NADH during myristic acid hydroxylation in a biphasic system NAD+-dependent FDH was used. Stability of the monooxygenase under the reaction conditions is quite high as revealed by total turnover numbers of up to 12850 in NADPH-dependent cyclohexane hydroxylation and up to 30000 in NADH-dependent myristic acid oxidation.
Open Access
Covalent incorporation of tobacco mosaic virus increases the stiffness of poly(ethylene glycol) diacrylate hydrogels
(2018) Southan, Alexander; Lang, Tina; Schweikert, Michael; Tovar, Günter E. M.; Wege, Christina; Eiben, Sabine
Hydrogels are versatile materials, finding applications as adsorbers, supports for biosensors and biocatalysts or as scaffolds for tissue engineering. A frequently used building block for chemically cross-linked hydrogels is poly(ethylene glycol) diacrylate (PEG-DA). However, after curing, PEG-DA hydrogels cannot be functionalized easily. In this contribution, the stiff, rod-like tobacco mosaic virus (TMV) is investigated as a functional additive to PEG-DA hydrogels. TMV consists of more than 2000 identical coat proteins and can therefore present more than 2000 functional sites per TMV available for coupling, and thus has been used as a template or building block for nano-scaled hybrid materials for many years. Here, PEG-DA (Mn = 700 g/mol) hydrogels are combined with a thiol-group presenting TMV mutant (TMVCys). By covalent coupling of TMVCys into the hydrogel matrix via the thiol-Michael reaction, the storage modulus of the hydrogels is increased compared to pure PEG-DA hydrogels and to hydrogels containing wildtype TMV (wt-TMV) which is not coupled covalently into the hydrogel matrix. In contrast, the swelling behaviour of the hydrogels is not altered by TMVCys or wt-TMV. Transmission electron microscopy reveals that the TMV particles are well dispersed in the hydrogels without any large aggregates. These findings give rise to the conclusion that well-defined hydrogels were obtained which offer the possibility to use the incorporated TMV as multivalent carrier templates e.g. for enzymes in future studies.
Open Access
CYP102A P450 monooxygenases: comparative analysis and construction of cytochrome P450 chimera
(2007) Eiben, Sabine; Schmid, Rolf D. (Prof. Dr.)
The members of the CYP102A subfamily are in several ways unique. They are natural fusion proteins of approximately 117-119 kDa comprised of the N terminal monooxygenase domain and a FAD and FMN containing diflavin reductase domain. These fatty acid hydroxylases exhibit extraordinary activities of 3000-10000 min-1 in comparison to other P450 monooxygenases making them promising candidates for industrial applications. Up to now, cloning and characterisation of only three members of this subfamily have been published: CYP102A1 from Bacillus megaterium and CYP102A2 and CYP102A3 from Bacillus subtilis strain 168. We have currently cloned and characterised another member of this family, CYP102A7, and there are at least eight more genes for CYP102A monooxygenases, which have been identified in several genome sequencing projects. Multiple sequence alignment of all amino acid sequences revealed that the similarity between different members is between 50 and 90 % according to which these enzymes can be divided into four groups. The four cloned enzymes belonging to three different groups but with 65 to 70 % similarity were used for further investigation. Despite their appearance to be similar, they often exhibit different properties. For example, the new isolated CYP102A7 catalyses the deethylation of 7-ethoxycoumarin which is not accepted as substrate by CYP102A1, CYP102A2 and CYP102A3. The hydroxylation patterns produced by these four monooxygenases are also different. Additionally we investigated solvent and thermal stability of these P450 monooxygenases. For example, the monooxygenase domain of CYP102A1 is much more stable than those of CYP102A2and CYP102A3. On the other hand, its reductase domain is less stable than the corresponding ones of CYP102A2 and CYP102A3. Therefore we exchanged the natural more unstable reductase domain of CYP102A1 with the more stable reductase domain of CYP102A3, using the natural linker of CYP102A1. The new chimera was able to hydroxylate substrates within a wider temperature range (up to 50°C) compared to the parental enzymes, showed higher process stability and has a half-life at 50°C more than ten times longer than CYP102A1. Further we substituted the reductase domain of CYP102A1 by a thermostable analogue. In the genome of the thermophilic Geobacillus stearothermophilus a gene was found exhibiting 50 % protein similarity to the reductase domain of CYP102A1. The gene encodes a hypothetical alpha-subunit of a sulfite reductase, but also belongs to the diflavin reductases. This reductase was cloned downstream of the monooxygenase domain of CYP102A1. Activity of the foreign reductase within the chimera A1GR was confirmed by cytochrome c reduction. Next to the ability to transfer electrons to the heme iron, oxidation of C14-C18 fatty acids was proven. While the hydroxylation activity of CYP102A1 towards myristic and palmitic acid decreased to about 5 % of the initial rate after incubation at 49°C, the chimera exhibited no loss of activity under the same conditions.
Open Access
Modified TMV particles as beneficial scaffolds to present sensor enzymes
(2015) Koch, Claudia; Wabbel, Katrin; Eber, Fabian J.; Krolla-Sidenstein, Peter; Azucena, Carlos; Gliemann, Hartmut; Eiben, Sabine; Geiger, Fania; Wege, Christina
Tobacco mosaic virus (TMV) is a robust nanotubular nucleoprotein scaffold increasingly employed for the high density presentation of functional molecules such as peptides, fluorescent dyes, and antibodies. We report on its use as advantageous carrier for sensor enzymes. A TMV mutant with a cysteine residue exposed on every coat protein (CP) subunit (TMVCys) enabled the coupling of bifunctional maleimide-polyethylene glycol (PEG)-biotin linkers (TMVCys/Bio). Its surface was equipped with two streptavidin [SA]-conjugated enzymes: glucose oxidase ([SA]-GOx) and horseradish peroxidase ([SA]-HRP). At least 50% of the CPs were decorated with a linker molecule, and all thereof with active enzymes. Upon use as adapter scaffolds in conventional “high-binding” microtiter plates, TMV sticks allowed the immobilization of up to 45-fold higher catalytic activities than control samples with the same input of enzymes. Moreover, they increased storage stability and reusability in relation to enzymes applied directly to microtiter plate wells. The functionalized TMV adsorbed to solid supports showed a homogeneous distribution of the conjugated enzymes and structural integrity of the nanorods upon transmission electron and atomic force microscopy. The high surface-increase and steric accessibility of the viral scaffolds in combination with the biochemical environment provided by the plant viral coat may explain the beneficial effects. TMV can, thus, serve as a favorable multivalent nanoscale platform for the ordered presentation of bioactive proteins.
Open Access
Piezoelectric templates - new views on biomineralization and biomimetics
(2016) Stitz, Nina; Eiben, Sabine; Atanasova, Petia; Domingo, Neus; Leineweber, Andreas; Burghard, Zaklina; Bill, Joachim
Biomineralization in general is based on electrostatic interactions and molecular recognition of organic and inorganic phases. These principles of biomineralization have also been utilized and transferred to bio-inspired synthesis of functional materials during the past decades. Proteins involved in both, biomineralization and bio-inspired processes, are often piezoelectric due to their dipolar character hinting to the impact of a template’s piezoelectricity on mineralization processes. However, the piezoelectric contribution on the mineralization process and especially the interaction of organic and inorganic phases is hardly considered so far. We herein report the successful use of the intrinsic piezoelectric properties of tobacco mosaic virus (TMV) to synthesize piezoelectric ZnO. Such films show a two-fold increase of the piezoelectric coefficient up to 7.2 pm V−1 compared to films synthesized on non-piezoelectric templates. By utilizing the intrinsic piezoelectricity of a biotemplate, we thus established a novel synthesis pathway towards functional materials, which sheds light on the whole field of biomimetics. The obtained results are of even broader and general interest since they are providing a new, more comprehensive insight into the mechanisms involved into biomineralization in living nature.