Epoxide Hydrolases: Multipotential Biocatalysts.

Epoxide hydrolases are attractive and industrially important biocatalysts. They can catalyze the enantioselective hydrolysis of epoxides to the corresponding diols as chiral building blocks for bioactive compounds and drugs. In this review article, we discuss the state of the art and development potential of epoxide hydrolases as biocatalysts based on the most recent approaches and techniques.

Lectin-Based Protein Microarray for the Glycan Analysis of Colorectal Cancer Biomarkers: The Insulin-Like Growth Factor System.

Lectin-based protein microarrays are used for glycoprofiling of various kinds of biological samples. Here we describe lectin-based microarray assay in the reverse-phase format where glycoprotein samples are spotted onto microarray slide and then are incubated with set of lectins. This configuration allows high-throughput screening of a large cohort of samples by a set of lectins without need of separation of glycans from glycoproteins.

Simulation-based optimisation of thermodynamic conditions in the ESEM for dynamical in-situ study of spherical polyelectrolyte complex particles in their native state.

We present a complex analysis and optimisation of dynamic conditions in the environmental scanning electron microscope (ESEM) to allow in-situ observation of extremely delicate wet bio-polymeric spherical particles in their native state. According to the results of gas flow and heat transfer simulations, we were able to develop an improved procedure leading to thermodynamic equilibrium between the sample and chamber environment. To quantify and hence minimise the extent of any sample deformation during specimen chamber pumping, a strength-stress analysis is used.

Progress in biocatalysis with immobilized viable whole cells: systems development, reaction engineering and applications.

Viable microbial cells are important biocatalysts in the production of fine chemicals and biofuels, in environmental applications and also in emerging applications such as biosensors or medicine. Their increasing significance is driven mainly by the intensive development of high performance recombinant strains supplying multienzyme cascade reaction pathways, and by advances in preservation of the native state and stability of whole-cell biocatalysts throughout their application. In many cases, the stability and performance of whole-cell biocatalysts can be highly improved by controlled immobilization techniques.

Baeyer-Villiger oxidations: biotechnological approach.

Baeyer-Villiger monooxygenases (BVMOs) are a very well-known and intensively studied class of flavin-dependent enzymes. Their substrate promiscuity, high chemo-, regio-, and enantioselectivity are prerequisites for the use in synthetic chemistry and should pave the way for successful industrial processes. Nonetheless, only a very limited number of industrial relevant transformations are known, mainly due to the lack of BVMOs stability and cofactor dependency.

A lectin-based cell microarray approach to analyze the mammalian granulosa cell surface glycosylation profile.

The high complexity of glycome, the repertoire of glycans expressed in a cell or in an organism, is difficult to analyze and the use of new technologies has accelerated the progress of glycomics analysis. In the last decade, the microarray approaches, and in particular glycan and lectin microarrays, have provided new insights into evaluation of cell glycosylation status. Here we present a cell microarray method based on cell printing on microarray slides for the analysis of the glycosylation pattern of the cell glycocalyx.

Lectin-based protein microarray analysis of differences in serum alpha-2-macroglobulin glycosylation between patients with colorectal cancer and persons without cancer.

Glycosylation is co- and posttranslational modifications affecting proteins. The glycopattern changes are associated with changes in biological function and are involved in many diseases including cancer. We present the lectin-based protein microarray method enabling determination of differences in protein glycosylation.

Whole-cell Gluconobacter oxydans biosensor for 2-phenylethanol biooxidation monitoring.

A microbial biosensor for 2-phenylethanol (2-PE) based on the bacteria Gluconobacter oxydans was developed and applied in monitoring of a biotechnological process. The cells of G. oxydans were immobilized within a disposable polyelectrolyte complex gel membrane consisting of sodium alginate, cellulose sulphate and poly(methylene-co-guanidine) attached onto a miniaturized Clark oxygen electrode, forming whole cell amperometric biosensor.

Physical and bioengineering properties of polyvinyl alcohol lens-shaped particles versus spherical polyelectrolyte complex microcapsules as immobilisation matrices for a whole-cell Baeyer-Villiger monooxygenase.

Direct comparison of key physical and chemical-engineering properties of two representative matrices for multipurpose immobilisations was performed for the first time. Polyvinyl alcohol lens-shaped particles LentiKats® and polyelectrolyte complex microcapsules were characterised by advanced techniques with respect to the size distribution of the particles, their inner morphology as revealed by fluorescent probe staining, mechanical resistance, size-exclusion properties, determination of effective diffusion coefficient and environmental scanning electron microscope imaging. While spherical polyelectrolyte complex microcapsules composed of a rigid semipermeable membrane and a liquid core are almost uniform in shape and size (diameter of 0.

Perspectives in glycomics and lectin engineering.

This chapter would like to provide a short survey of the most promising concepts applied recently in analysis of glycoproteins based on lectins. The first part describes the most exciting analytical approaches used in the field of glycoprofiling based on integration of nanoparticles, nanowires, nanotubes, or nanochannels or using novel transducing platforms allowing to detect very low levels of glycoproteins in a label-free mode of operation. The second part describes application of recombinant lectins containing several tags applied for oriented and ordered immobilization of lectins.

Microbial monooxygenase amperometric biosensor for monitoring of Baeyer-Villiger biotransformation.

A whole-cell amperometric biosensor consisting of genetically engineered Escherichia coli immobilised in polyelectrolyte membrane onto a miniaturised oxygen electrode was developed and used for monitoring of biotransformation based on Baeyer-Villiger oxidation. Baeyer-Villiger oxidation is commonly performed using microorganisms overexpressing Baeyer-Villiger monooxygenase enabling the production of enantiopure lactones or esters used in pharmaceutical industry. The biorecognition element, genetically modified E.

Light-switchable polymer from cationic to zwitterionic form: synthesis, characterization, and interactions with DNA and bacterial cells.

A novel cationic polymer poly(N,N-dimethyl-N-[3-(methacroylamino) propyl]-N-[2-[(2-nitrophenyl)methoxy]-2-oxo-ethyl]ammonium chloride) is synthesized by free-radical polymerization of N-[3-(dimethylamino)propyl] methacrylamide and subsequent quaternization with o-nitrobenzyl 2-chloroacetate. The photolabile o-nitrobenzyl carboxymethyl pendant moiety is transformed to the zwitterionic carboxybetaine form upon the irradiation at 365 nm. This feature is used to condense and, upon the light irradiation, to release double-strand DNA tested by gel electrophoresis and surface plasmon resonance experiments as well as to switch the antibacterial activity to non-toxic character demonstrated for Escherichia coli bacterial cells in solution and at the surface using the self-assembled monolayers.

Electrochemical lectin based biosensors as a label-free tool in glycomics.

Glycans and other saccharide moieties attached to proteins and lipids, or present on the surface of a cell, are actively involved in numerous physiological or pathological processes. Their structural flexibility (that is based on the formation of various kinds of linkages between saccharides) is making glycans superb "identity cards". In fact, glycans can form more "words" or "codes" (i.

Ultrasensitive impedimetric lectin based biosensor for glycoproteins containing sialic acid.

We report on an ultrasensitive label-free lectin-based impedimetric biosensor for the determination of the sialylated glycoproteins fetuin and asialofetuin. A sialic acid binding agglutinin from I was covalently immobilised on a mixed self-assembled monolayer (SAM) consisting of 11-mercaptoundecanoic acid and 6-mercaptohexanol. Poly(vinyl alcohol) was used as a blocking agent.

Analysis of ethanol in fermentation samples by a robust nanocomposite-based microbial biosensor.

A robust microbial biosensor was constructed from a bionanocomposite prepared by a direct mixing of bacterial cells of Gluconobacter oxydans and carbon nanotubes with ferricyanide employed as a mediator for enhanced sensitivity of ethanol oxidation. A successful integration of the device into flow injection analysis mode of operation provided a high sensitivity of detection of (74 ± 2.7) μA mM(-1) cm(-2), a low detection limit of 5 μM and a linear range from 10 μM up to 1 mM.

Continuous testing system for Baeyer-Villiger biooxidation using recombinant Escherichia coli expressing cyclohexanone monooxygenase encapsulated in polyelectrolyte complex capsules.

An original strategy for universal laboratory testing of Baeyer-Villiger monooxygenases based on continuous packed-bed minireactor connected with flow calorimeter and integrated with bubble-free oxygenation is reported. Model enantioselective Baeyer-Villiger biooxidations of rac-bicyclo[3.2.

Viability of free and encapsulated Escherichia coli overexpressing cyclopentanone monooxygenase monitored during model Baeyer-Villiger biooxidation by confocal laser scanning microscopy.

Baeyer-Villiger biooxidation of 4-methylcyclohexanone-5-methyloxepane-2-one catalysed by recombinant Escherichia coli overexpressing cyclopentanone monooxygenase encapsulated in polyelectrolyte complex capsules was used to investigate effect of substrate conversion on the viability of cells. Confocal laser scanning microscopy (CLSM) was used to assess cell viability using propidium iodide fluorescence marker for necrosis, and flavin autofluorescence to identify living bacteria. Viability of encapsulated cells decreased with increasing substrate concentration from 99 ± 1 to 83 ± 4%, while substrate conversions from decreased 100 to 6 ± 1%.

A hyaluronic acid dispersed carbon nanotube electrode used for a mediatorless NADH sensing and biosensing.

A biocompatible nanocomposite consisting of single-walled carbon nanotubes (CNTs) dispersed in a hyaluronic acid (HA) was investigated as a sensing platform for a mediatorless electrochemical detection of NADH. The device was characterised by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and extensively by electrochemistry. CNT-HA bionanocomposite showed more reversible electrochemistry, higher short-term stability of NADH sensing and higher selectivity of NADH detection compared to frequently used CNT-CHI (chitosan) modified GCE.

Protective effects of manganese(II) chloride on hyaluronan degradation by oxidative system ascorbate plus cupric chloride.

The degradation of several high-molar-mass hyaluronan samples was investigated in the presence of ascorbic acid itself and further by an oxidative system composed of ascorbic acid plus transition metal ions, i.e. Fe(II) or Cu(II) ions.

Coencapsulation of oxygen carriers and glucose oxidase in polyelectrolyte complex capsules for the enhancement of D-gluconic acid and delta-gluconolactone production.

A novel encapsulated oxidative biocatalyst comprising glucose oxidase (GOD) coencapsulated with oxygen carriers within polyelectrolyte complex capsules was developed for the production of D-gluconic acid and delta-gluconolactone. The capsules containing immobilized GOD were produced by polyelectrolyte complexation with sodium alginate (SA) and cellulose sulfate (CS) as polyanions, poly(methylene-co-guanidine) (PMCG) as the polycation, CaCl(2) as the gelling agent and NaCl as the antigelling agent (GOD-SA-CS/PMCG capsules). Poly(dimethylsiloxane) (PDMS) and an emulsion of n-dodecane (DOD) or perfluorodecaline (PFD) with PDMS were used as the oxygen carriers and MnO(2) was used as a hydrogen peroxide decomposition catalyst.

Encapsulation of recombinant E. coli expressing cyclopentanone monooxygenase in polyelectrolyte complex capsules for Baeyer-Villiger biooxidation of 8-oxabicyclo[3.2.1]oct-6-en-3-one.

Recombinant Escherichia coli cells, over-expressing cyclopentanone monooxygenase activity, were immobilized in polyelectrolyte complex capsules, made of sodium alginate, cellulose sulfate, poly(methylene-co-guanidine), CaCl(2) and NaCl. More than 90% of the cell viability was preserved during the encapsulation process. Moreover, the initial enzyme activity was fully maintained within encapsulated cells while it halved in free cells.

Glycan and lectin microarrays for glycomics and medicinal applications.

Three different array formats to study a challenging field of glycomics are presented here, based on the use of a panel of immobilized glycan or lectins, and on in silico computational approach. Glycan and lectin arrays are routinely used in combination with other analytical tools to decipher a complex nature of glycan-mediated recognition responsible for signal transduction of a broad range of biological processes. Fundamental aspects of the glycan and lectin array technology are discussed, with the focus on the choice and availability of the biorecognition elements, fabrication protocols, and detection platforms involved.

Off-line FIA monitoring of D-sorbitol consumption during L-sorbose production using a sorbitol biosensor.

A ferricyanide mediated amperometric biosensor system implementing D-sorbitol dehydrogenase together with diaphorase for sensitive detection of D-sorbitol was used. The biosensor system was successfully integrated into an off-line FIA system with a throughput of detection of 10 h(-1). The device exhibited limit of detection of 20 microM with an average relative standard deviation of analysis of samples of 2.

Membrane-bound dehydrogenases from Gluconobacter sp.: interfacial electrochemistry and direct bioelectrocatalysis.

Although membrane-bound dehydrogenases isolated from Gluconobacter sp. (mainly PQQ-dependent alcohol and fructose dehydrogenase) have been used for preparing diverse forms of bioelectronic interfaces for almost 2 decades, it is not an easy task to interpret an electrochemical behaviour correctly. Recent discoveries regarding redox properties of membrane-bound dehydrogenases along with extensive investigations of direct electron transfer (DET) or direct bioelectrocatalysis with these enzymes are summarized in this review.

Degradation of high-molar-mass hyaluronan by ascorbate plus cupric ions: effects of D-penicillamine addition.

Pro- and anti-oxidative effects of an anti-rheumatoid drug, D-penicillamine (D-PN), on the kinetics of high-molar-mass hyaluronan (HA) degradation were monitored using the method of rotational viscometry. The degradation of the dissolved HA macromolecules was attained by applying the Weissberger's system comprising ascorbic acid plus cupric ions. Electron paramagnetic resonance (EPR) spectroscopy was used to identify the generated free radicals.