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. The review covers new approaches to discover epoxide hydrolases using genome mining and enzyme metagenomics, as well as improving enzyme activity, enantioselectivity, enantioconvergence, and thermostability by directed evolution and a rational design. Further improvements in operational and storage stabilization, reusability, pH stabilization, and thermal stabilization by immobilization techniques are discussed in this study. New possibilities for expanding the synthetic capabilities of epoxide hydrolases by their involvement in non-natural enzyme cascade reactions are described.

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. This review summarizes the current progress in the development of immobilized whole-cell biocatalysts, the immobilization methods as well as in the bioreaction engineering aspects and economical aspects of their biocatalytic applications.

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. In order to demonstrate the reliability of the developed method, the glycome profiles of equine native uncultured mural granulosa cells (uGCs) and in vitro cultured mural granulosa cells (cGCs) were determined and compared. The method consists in the isolation of GCs, cell printing into arrays on microarray slide, incubation with a panel of biotinylated lectins, reaction with fluorescent streptavidin and signal intensity detection by a microarray scanner. Cell microarray technology revealed that glycocalyx of both uGCs and cGCs contains N-glycans, sialic acid terminating glycans, N-acetylglucosamine and O-glycans. The comparison of uGCs and cGCs glycan signals indicated an increase in the expression of sialic acids, N-acetylglucosamine, and N-glycans in cGCs. Glycan profiles determined by cell microarray agreed with those revealed by lectin histochemistry. The described cell microarray method represents a simple and sensitive procedure to analyze cell surface glycome in mammalian cells.

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. In this review, we focus on novel BVMO-mediated transformations, BVMOs in cascade type reactions, potential industrial applications, and how limitations have been tackled by the community. Special attention will be put on whole-cell immobilization strategies. We emphasize to bridge recent developments in fundamental research to industrial applications.

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. The method involves isolation of targeted protein from samples by immunoprecipitation, spotting of protein from multiple samples into arrays on a microarray slide, incubation with set of biotinylated lectins, the reaction with fluorescent conjugate of streptavidin, and detection of fluorescent intensities by microarray scanner. Lectin-based protein microarray was applied in investigation of differences in alpha-2-macroglobulin (α2M) glycosylation isolated from sera samples of healthy persons and patients with colorectal cancer (CC). From 14 lectins used in analysis, statistically significant differences (Student's t-test, P < 0.05) between two groups of samples (persons without cancer and CC patients) were found for 5 of them. α2M molecules isolated from sera of CC patients have higher content of α2,6 sialic acid, N-acetylglucosamine and mannose residues, and tri-/tetraantennary complex type high-mannose N-glycans. A novel lectin-based protein microarray developed and described can serve as a suitable analytical technique for sensitive, simple, fast, and high-throughput determination of differences in protein glycosylation isolated from serum or other samples.

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.

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%. Storage stabilization of encapsulated cells was observed by increased substrate conversion form 68 ± 2 to 96 ± 3%. Measurements by CLSM with standard deviations up to 5% may be regarded as powerful tool for recombinant cell viability determination during Baeyer-Villiger biooxidations.

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. A short response time of the biosensor allowed a sample throughput of 67 h(-1) at 0.3 ml min(-1). The biosensor exhibited high operational stability with a decrease in the biosensor response of 1.7% during 43 h of continuous operation. The device was used to analyse ethanol in fermentation samples with a good agreement with a HPLC method.

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. We applied the described method for glycan analysis of glycoprotein biomarkers of colorectal cancer associated with the insulin-like growth factor system.

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. Moreover, practical applications of both technologies especially in the field of clinical diagnostics are provided. The future potential of a complementary in silico array technology to reveal details of the protein-glycan-binding profiles is discussed here.

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. Both encapsulated and free cells reached the end point of the Baeyer-Villiger biooxidation of 8-oxabicyclo[3.2.1]oct-6-en-3-one to 4,9-dioxabicyclo[4.2.1]non-7-en-3-one at the same time (48 h). Similarly, the enantiomeric excess above 94% was identical for encapsulated and free cells.

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. Water-soluble PDMS was found to act as both an oxygen carrier and an emulsifier of water-insoluble DOD and PFD. Stable microcapsules could be produced with concentrations of up to 4% (w/w) of PDMS, 10% (w/w) of DOD and PFD, and 25% (w/w) of MnO(2) in the polyanion solution of SA and CS. Roughly a two-fold increase in the GOD activity from 21.0+/-1.1 to 38.4+/-2.0 U*g(-1) and product space-time yields (STY) from 44.3+/-2.0 to 83.4+/-3.4 g*H*day(-1) could be achieved utilizing coencapsulated oxygen carriers compared to GOD encapsulated in the absence of oxygen carriers. This enhanced production does not significantly depend on the selected oxygen carrier under the conditions used in this study.

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. Monte Carlo simulations of beam-gas, -water, and -sample interactions describe beam scattering, absorbed energy, interaction volume and the emission of signal electrons in the ESEM. Finally, we discuss sample damage as a result of drying and the production of beam-induced free radicals. Based on all experimental and simulation results we introduce a Delicate Sample Observation Strategy for the ESEM. We show how this strategy can be applied to the characterization of polyelectrolyte complex spherical particles containing immobilized recombinant cells E. coli overexpressing cyclohexanone monooxygenase, used as a model biocatalyst. We present the first native-state electron microscopy images of the viscous core of a halved polyelectrolyte complex capsule containing living cells.

High-molar-mass hyaluronan behavior during testing its radical scavenging capacity in organic and aqueous media: effects of the presence of manganese(II) ions.

This study compares the radical scavenging capacity of high-molar-mass hyaluronan (HA) using standardized methods applying 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals and 2,2'-azinobis[3-ethylbenzthiazoline sulfonate] (ABTS) radical cations as oxidants. Additionally, spin-trapping technique combined with electron paramagnetic resonance (EPR) was used to evaluate the ability of HA to scavenge reactive radicals. The thermal decomposition of K2S2O8 in pure H2O or in a H2O/dimethyl sulfoxide (DMSO) mixture at 333 K was used as a source of reactive paramagnetic species. We found that HA does not exhibit radical-scavenging activity when DPPH radicals or ABTS(.+) radical cations are used as oxidant, but that HA is an effective radical scavenger at low concentrations, if the oxidation reactions are initiated by the decomposition of K2S2O8. At higher HA concentrations, a more complex behavior and prooxidant HA action was observed. The influence of Mn(II) ions on the reaction mechanisms of radical generation and termination in the K2S2O8/H2O/DMSO system in the presence of HA was studied in detail.

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. The results obtained indicate that the initial anti-oxidative action of D-PN is followed by induction of pro-oxidative conditions due to the generation of reactive free radicals. It is speculated, however, that the latter situation may be considered as an advantageous property of D-PN. Hydroxyl radicals formed in this way may participate in decomposition of proteinases, which are believed to be responsible for the destruction of joint cartilage under rheumatoid arthritic conditions.

Study of pro- and anti-oxidative properties of D-penicillamine in a system comprising high-molar-mass hyaluronan, ascorbate, and cupric ions.

OBJECTIVES: The study presents results of pro- and anti-oxidative effects of D-penicillamine on hyaluronan degradation by ascorbate plus cupric ions. METHODS: The well established degradative system comprising high-molar-mass hyaluronan and ascorbate plus Cu(II) ions was used. Primarily, the effects of replacement of ascorbic acid in this system by D-penicillamine were investigated. Then, D-penicillamine was added into the above degradative system before reaction onset or 1h after the reaction had started. To monitor hyaluronan degradation kinetics, rotational viscometry was applied. RESULTS: No hyaluronan degradation occurred when ascorbate was replaced by D-penicillamine. The drug addition into the complete degradative system at the reaction onset caused a marked inhibition of hyaluronan degradation. However, the inhibitory effect turned to a pro-oxidative one within appr. 1 h. CONCLUSION: The dual behavior of D-penicillamine on hyaluronan degradation can relate to: (i) the drug completely traps *OH radicals generated from ascorbate plus Cu(II) ions under aerobic conditions; (ii) thiyl radicals generated from D-penicillamine react with D-penicillamine anions resulting in novel radical reactive species, which e.g. by reducing dioxygen molecules can generate further *OH radicals.

Oligosaccharides, neoglycoproteins and humanized plastics: their biocatalytic synthesis and possible medical applications.

Glycobiology has become one of the fastest growing branches of the biological sciences. Glycomics, which is the study of an organism's entire array of oligosaccharides, is now emerging as the third informatics wave after genomics and proteomics. For example, it is possible to see this progress in the KEGG (Kyoto Encyclopedia of Genes and Genomes) database (http://www.genome.jp/kegg/pathway/map/map01110.html). The interest in this area stems from the realization that carbohydrates, especially oligosaccharides, and their interactions with proteins, play diverse informative roles in all organisms, and that more than half of all proteins are glycosylated. When the biological and pharmaceutical importance of glycoconjugates is considered, it is surprising how little glycobiotechnology has developed. This review reports the latest developments in the biocatalytic synthesis of oligosaccharides and glycoconjugates, with special attention paid to the glycosyltransferase approach. The second part of the review takes the 'conceptual approach' and covers possible medical applications of synthesized glycoconjugates. Various new examples of the conjugation of glyco-informative saccharide sequence to known pharmaceuticals or biomaterials are cited.

Solution properties of high-molar-mass hyaluronans: the biopolymer degradation by ascorbate.

An accurate molecular characterization, molar mass and size distributions, of 10 hyaluronan (HA) samples was performed by using a multi-angle light scattering detector connected on-line to a size exclusion chromatographic system. The dynamic viscosity eta of the HA solutions was investigated using a rotational viscometer. On monitoring the sample dynamic viscosity for up to 5h, a small however constant increase of the eta value was observed, indicating rheopectic behavior of all 10 HA solutions. Addition of ascorbic acid to the HA solutions caused significant changes in the rheological properties of the samples investigated. The change of eta values in the course of time was explained by the redox reactions (caused by the added ascorbate) that occur during the dynamic viscosity monitoring.

Hyaluronic acid: a natural biopolymer with a broad range of biomedical and industrial applications.

Hyaluronic acid (hyaluronan, HA) is a linear polysaccharide formed from disaccharide units containing N-acetyl-D-glucosamine and glucuronic acid. It has a high molecular mass, usually in the order of millions of Daltons, and interesting viscoelastic properties influenced by its polymeric and polyelectrolyte characteristics. HA is present in almost all biological fluids and tissues. In clinical medicine, it is used as a diagnostic marker for many diseases including cancer, rheumatoid arthritis and liver pathologies, as well as for supplementation of impaired synovial fluid in arthritic patients by means of intra-articular injections. It is also used in certain ophthalmological and otological surgeries and cosmetic regeneration and reconstruction of soft tissue. Herein we present an overview of the occurrence and physiological properties of HA, as well as of the recent advances in production biotechnology and preparation of the HA-based materials for medical application.

Thermoswitched immobilization-a novel approach in reversible immobilization.

The present work is based on the finding that the mesophilic carbohydrate-binding domain from Clostridium cellulovorans fused with thermophilic enzymes from Pyrococcus furiosus can be reversibly denaturated and renaturated by a simple switch of temperature. Modular recombinant enzymes are active and free in the reaction mixture at 80-90 degrees C and deactivated and immobilized by affinity adsorption on cellulose at 40-30 degrees C. The temperature transition between both modes is rather sharp and occurs within the range of 40-50 degrees C. Due to the elevated temperature, there is no limitation by a diffusion step, and contamination does not occur during the reaction. After the reaction, the enzymes are quickly deactivated, adsorbed on the affinity matrix, removed from the reaction mixture, and ready for use in another reaction cycle.