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Brno, Czech Republic

Bednar D.,Masaryk University | Bednar D.,St Annes University Hospital Brno | Beerens K.,Masaryk University | Sebestova E.,Masaryk University | And 14 more authors.
PLoS Computational Biology | Year: 2015

There is great interest in increasing proteins’ stability to enhance their utility as biocatalysts, therapeutics, diagnostics and nanomaterials. Directed evolution is a powerful, but experimentally strenuous approach. Computational methods offer attractive alternatives. However, due to the limited reliability of predictions and potentially antagonistic effects of substitutions, only single-point mutations are usually predicted in silico, experimentally verified and then recombined in multiple-point mutants. Thus, substantial screening is still required. Here we present FireProt, a robust computational strategy for predicting highly stable multiple-point mutants that combines energy- and evolution-based approaches with smart filtering to identify additive stabilizing mutations. FireProt’s reliability and applicability was demonstrated by validating its predictions against 656 mutations from the ProTherm database. We demonstrate that thermostability of the model enzymes haloalkane dehalogenase DhaA and γ-hexachlorocyclohexane dehydrochlorinase LinA can be substantially increased (ΔTm = 24°C and 21°C) by constructing and characterizing only a handful of multiple-point mutants. FireProt can be applied to any protein for which a tertiary structure and homologous sequences are available, and will facilitate the rapid development of robust proteins for biomedical and biotechnological applications. © 2015 Bednar et al.

Koudelakova T.,Masaryk University | Bidmanova S.,Masaryk University | Dvorak P.,Masaryk University | Pavelka A.,Masaryk University | And 5 more authors.
Biotechnology Journal | Year: 2013

Haloalkane dehalogenases (EC, HLDs) are α/β-hydrolases which act to cleave carbon-halogen bonds. Due to their unique catalytic mechanism, broad substrate specificity and high robustness, the members of this enzyme family have been employed in several practical applications: (i) biocatalytic preparation of optically pure building-blocks for organic synthesis; (ii) recycling of by-products from chemical processes; (iii) bioremediation of toxic environmental pollutants; (iv) decontamination of warfare agents; (v) biosensing of environmental pollutants; and (vi) protein tagging for cell imaging and protein analysis. This review discusses the application of HLDs in the context of the biochemical properties of individual enzymes. Further extension of HLD uses within the field of biotechnology will require currently limiting factors - such as low expression, product inhibition, insufficient enzyme selectivity, low affinity and catalytic efficiency towards selected substrates, and instability in the presence of organic co-solvents - to be overcome. We propose that strategies based on protein engineering and isolation of novel HLDs from extremophilic microorganisms may offer solutions. © 2013.

Bidmanova S.,Masaryk University | Hlavacek A.,Masaryk University | Damborsky J.,Masaryk University | Damborsky J.,Enantis Ltd. | And 2 more authors.
Sensors and Actuators, B: Chemical | Year: 2012

An approach for the immobilization of a pH indicator in optical pH sensors and biosensors was developed. Fluorescent dyes 5(6)-carboxyfluorescein and 5(6)-carboxynaphthofluorescein were conjugated with bovine serum albumin as a non-enzymatic scaffold protein and the conjugation procedure was optimized. Fluorescent properties, sensitivity to temperature and photostability of conjugates have been studied and characterized into details. The conjugates were immobilized on glass support by: (i) glutaraldehyde cross-linking or (ii) entrapment in sol-gel matrix ORMOCER with subsequent glutaraldehyde cross-linking. The response to pH and leaching potential were evaluated. The sensor layer, based on immobilized conjugates of 5(6)-carboxyfluorescein and bovine serum albumin, displayed rapid response over the pH range 4.0-9.0, making it compatible with a range of applications such as bioprocessing, clinical diagnostics or environmental monitoring. Immobilized conjugates of 5(6)-carboxynaphthofluorescein and bovine serum albumin showed an enhanced sensitivity in alkaline pH levels, response for glutaraldehyde cross-linking ranged from pH 9.1 to 11.0 and from 8.3 to 10.0 for the ORMOCER entrapment, however, control of the indicator leaching was essential. © 2011 Elsevier B.V. All rights reserved.

Stepankova V.,Masaryk University | Stepankova V.,St Annes University Hospital | Damborsky J.,Masaryk University | Damborsky J.,St Annes University Hospital | And 3 more authors.
Biotechnology Journal | Year: 2013

Haloalkane dehalogenases are microbial enzymes with a wide range of biotechnological applications, including biocatalysis. The use of organic co-solvents to solubilize their hydrophobic substrates is often necessary. In order to choose the most compatible co-solvent, the effects of 14 co-solvents on activity, stability and enantioselectivity of three model enzymes, DbjA, DhaA, and LinB, were evaluated. All co-solvents caused at high concentration loss of activity and conformational changes. The highest inactivation was induced by tetrahydrofuran, while more hydrophilic co-solvents, such as ethylene glycol and dimethyl sulfoxide, were better tolerated. The effects of co-solvents at low concentration were different for each enzyme-solvent pair. An increase in DbjA activity was induced by the majority of organic co-solvents tested, while activities of DhaA and LinB decreased at comparable concentrations of the same co-solvent. Moreover, a high increase of DbjA enantioselectivity was observed. Ethylene glycol and 1,4-dioxane were shown to have the most positive impact on the enantioselectivity. The favorable influence of these co-solvents on both activity and enantioselectivity makes DbjA suitable for biocatalytic applications. This study represents the first investigation of the effects of organic co-solvents on the biocatalytic performance of haloalkane dehalogenases and will pave the way for their broader use in industrial processes. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Bidmanova S.,Masaryk University | Bidmanova S.,Enantis Ltd. | Pohanka M.,University of Hradec Kralove | Cabal J.,University of Hradec Kralove | And 4 more authors.
Chemicke Listy | Year: 2010

Chemical warfare agents are toxic compounds showing negative effects on living organisms. This review focuses on detection of nerve and blister agents, which are two most important classes of chemical warfare agents. Main attention is paid to the construction and use of biosensors. Acetylcholinesterase, butyrylcholinesterase and phosphotriesterase are presented as convenient biorecognition components of biosensors for detection of nerve agents, while dehalogenases are useful for detection of mustard agents.

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