Construction of a Xylanase A Variant Capable of Polymerization

The aim of our work is to furnish enzymes with polymerization ability by creating fusion constructs with the polymerizable protein, flagellin, the main component of bacterial flagellar filaments. The D3 domain of flagellin, exposed on the surface of flagellar filaments, is formed by the hypervariable central portion of the polypeptide chain. D3 is not essential for filament formation. The concept in this project is to replace the D3 domain with suitable monomeric enzymes without adversely affecting polymerization ability, and to assemble these chimeric flagellins into tubular nanostructures. To test the feasibility of this approach, xylanase A (XynA) from B. subtilis was chosen as a model enzyme for insertion into the central part of flagellin. With the help of genetic engineering, a fusion construct was created in which the D3 domain was replaced by XynA. The flagellin-XynA chimera exhibited catalytic activity as well as polymerization ability. These results demonstrate that polymerization ability can be introduced into various proteins, and building blocks for rationally designed assembly of filamentous nanostructures can be created

Synthesis of an antiviral drug precursor from chitin using a saprophyte as a whole-cell catalyst

Background: Recent incidents, such as the SARS and influenza epidemics, have highlighted the need for readily available antiviral drugs. One important precursor currently used for the production of Relenza, an antiviral product from GlaxoSmithKline, is N-acetylneuraminic acid (NeuNAc). This substance has a considerably high market price despite efforts to develop cost-reducing (biotechnological) production processes. Hypocrea jecorina (Trichoderma reesei) is a saprophyte noted for its abundant secretion of hydrolytic enzymes and its potential to degrade chitin to its monomer N-acetylglucosamine (GlcNAc). Chitin is considered the second most abundant biomass available on earth and therefore an attractive raw material. Results: In this study, we introduced two enzymes from bacterial origin into Hypocrea, which convert GlcNAc into NeuNAc via N-acetylmannosamine. This enabled the fungus to produce NeuNAc from the cheap starting material chitin in liquid culture. Furthermore, we expressed the two recombinant enzymes as GST-fusion proteins and developed an enzyme assay for monitoring their enzymatic functionality. Finally, we demonstrated that Hypocrea does not metabolize NeuNAc and that no NeuNAc-uptake by the fungus occurs, which are important prerequisites for a potential production strategy. Conclusions: This study is a proof of concept for the possibility to engineer in a filamentous fungus a bacterial enzyme cascade, which is fully functional. Furthermore, it provides the basis for the development of a process for NeuNAc production as well as a general prospective design for production processes that use saprophytes as whole-cell catalysts

Quantitative comparison of chiral catalysts selectivity and performance: A generic concept illustrated with cyclododecanone monooxygenase as baeyer-villiger biocatalyst

Contribución equivalente como primer autor: Fink, Michael J. y Rial, Daniela V. Within this work a generic tool for chiral catalyst evaluation is established based on the application-oriented properties activity and selectivity; the concept aims at quantitatively comparing catalyst performance in general on a multitude of substrates. It is designed and intended to serve as decision guidance for challenges in catalysis and comprehensible information extraction from already recorded but unrefined data sets. The underlying algorithm assigns function points to catalytic entities via a statistically solid model possessing high flexibility and generates a relative ranking. This is coupled to an automated iterative refinement process towards maximum information content of results employing Shannon entropy optimization. Consequently, the developed workflow facilitates high distinguishability between catalysts even in low-scattering data sets. The numerical ranking is complemented by a clearly arranged graphic representation permitting facile and reliable visual interpretation of generality or niche capabilities of catalysts. Usefulness of the title concept is demonstrated by the performance evaluation of cyclododecanone monooxygenase, a highly versatile Baeyer-Villiger enzyme. To retain broad applicability, an open-source MATLAB® script is provided in electronic form.Fil: Fink, Michael J.. Vienna University of Technology; AustriaFil: Rial, Daniela Veronica. Consejo Nacional de Investigaciones Científicas y Técnicas Centro Científico Tecnológico - CONICET -Rosario. Instituto de Biologia Molecular y Celular de Rosario; ArgentinaFil: Kapitanova, Petra. Vienna University of Technology; AustriaFil: Lengar, Alenka. Institute Of Applied Synthetic Chemistry; AustriaFil: Rehdorf, Jessica. ERNST MORITZ ARNDT UNIVERSITÄT GREIFSWALD (UG);Fil: Cheng, Qiong. No especifíca;Fil: Rudroff, Florian. Vienna University of Technology; AustriaFil: Mihovilovic, Marko D.. Vienna University of Technology; Austri

Stereochemistry of phase-1 metabolites of mephedrone determines their effectiveness as releasers at the serotonin transporter

Mephedrone (4-methyl-N-methylcathinone) is a psychostimulant that promotes release of monoamines via the high affinity transporters for dopamine (DAT), norepinephrine (NET) and serotonin (SERT). Metabolic breakdown of mephedrone results in bioactive metabolites that act as substrate-type releasers at monoamine transporters and stereospecific metabolism of mephedrone has been reported. This study compared the effects of the enantiomers of the phase-1 metabolites nor-mephedrone, 4-hydroxytolyl-mephedrone (4-OH-mephedrone) and dihydro-mephedrone on (i) DAT, NET and SERT mediated substrate fluxes, (ii) determined their binding affinities towards a battery of monoamine receptors and (iii) examined the relative abundance of the enantiomers in human urine. Each of the enantiomers tested inhibited uptake mediated by DAT, NET and SERT. No marked differences were detected at DAT and NET. However, at SERT, the S-enantiomers of nor-mephedrone and 4-OH-mephedrone were several times more potent than the corresponding R-enantiomers. Moreover, the R-enantiomers were markedly less effective as releasers at SERT. S-nor-mephedrone displayed moderate affinities towards human alpha; 1A; , human 5-HT; 2A; and rat and mouse trace amine-associated receptor 1. These results demonstrate that stereochemistry dictates the pharmacodynamics of the phase-1 metabolites of mephedrone at SERT, but not at DAT and NET, which manifests in marked differences in their relative potencies, i.e. DAT/SERT ratios. Chiral analysis of urine samples demonstrated that nor-mephedrone predominantly exists as the S-enantiomer. Given the asymmetric abundance of the enantiomers in biological samples, these findings may add to our understanding of the subjective effects of administered mephedrone, which indicate pronounced effects on the serotonergic system