[Show abstract][Hide abstract] ABSTRACT: 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. Measured changes in oxygen concentration were proportional to changes in 2-PE concentration. The biosensor sensitivity was 864 nA mM−1 (RSD = 6%), a detection limit of 1 μM, and the biosensor response towards 2-PE was linear in the range 0.02–0.70 mM. The biosensor preserved 93% of its initial sensitivity after 7 h of continuous operation and exhibited excellent storage stability with loss of only 6% of initial sensitivity within two months, when stored at 4 °C. The developed system was designed and successfully used for an off-line monitoring of whole course of 2-PE biooxidation process producing phenylacetic acid (PA) as industrially valuable aromatic compound. The biosensor measurement did not require the use of hazardous organic solvent. The biosensor response to 2-PE was not affected by interferences from PA and phenylacetaldehyde at concentrations present in real samples during the biotransformation and the results were in a very good agreement with those obtained via gas chromatography.
[Show abstract][Hide abstract] ABSTRACT: Systemic sclerosis (SSc) is an autoimmune disease seriously affecting patient́s quality of life. The heterogeneity of the disease also means that identification and subsequent validation of biomarkers of the disease is quite challenging. A fully validated single biomarker for diagnosis, prognosis, disease activity and assessment of response to therapy is not yet available. The main aim of this study was to apply an alternative assay protocol to the immunoassay-based analysis of this disease by employment of sialic acid recognizing lectin Sambucus nigra agglutinin (SNA) to glycoprofile serum samples. To our best knowledge this is the first study describing direct lectin-based glycoprofiling of serum SSc samples. Three different analytical methods for glycoprofiling of serum samples relying on application of lectins are compared here from a bioanalytical point of view including traditional ELISA-like lectin-based method (ELLA), novel fluorescent lectin microarrays and ultrasensitive impedimetric lectin biosensors. Results obtained by all three bioanalytical methods consistently showed differences in the level of sialic acid present on glycoproteins, when serum from healthy people was compared to the one from patients having SSc. Thus, analysis of sialic acid content in human serum could be of a diagnostic value for future detection of SSc, but further work is needed to enhance selectivity of assays for example by glycoprofiling of a fraction of human serum enriched in antibodies for individual diagnostics.
[Show abstract][Hide abstract] ABSTRACT: We present here comparison of a build-up of two ultrasensitive lectin biosensors based on 2D or 3D
architecture. A 2D lectin biosensor was prepared by a covalent immobilisation of lectin Sambucus
nigra agglutinin (SNA) recognising sialic acid directly on a mixed self-assembled monolayer (SAM)
on planar gold surfaces. A 3D biosensor was prepared by covalent immobilisation of SNA lectin on a
mixed SAM layer formed on gold nanoparticles. Surface plasmon resonance technique allowed to
follow kinetics of a mixed SAM (1:1 mixture of 11-mercaptoundecanoic acid and 6-mercaptohexanol)
formation on a bare gold electrode and on an electrode modified by 5 nm and 20 nm gold nanoparticles
(AuNPs). Results from the study revealed that a mixed SAM formation is slower on surfaces with
increased curvature, the process of SAM formation on all surfaces is completed within 6 min, but a
density of thiols on such surfaces differs significantly. Quartz crystal microbalance experiments
showed that a surface density of immobilised lectin of (2.53 ± 0.01) pmol cm-2
was higher on planar
gold surface compared to the surface modified by 20 nm AuNPs with a surface density of (0.94 ± 0.01)
. Even though a larger amount of SNA lectin was immobilised on a surface of the 2D
biosensor compared to the 3D biosensor, lectin molecules immobilised on AuNPs were more
accessible for its analytes – glycoproteins fetuin and asialofetuin, containing different amount of sialic
acid on the protein surface. Most likely a better accessibility of lectin for its analytes on a 3D surface
and proper interfacial properties of a 3D surface are behind unprecedented detection limit down to aM
level for the lectin biosensor based on such a nanoscale tuned interface.
International journal of electrochemical science 01/2014; 9:890-900. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ultrasensitive impedimetric lectin biosensors recognising different glycan entities on serum glycoproteins were constructed. Lectins were immobilised on novel mixed self-assembled monolayer containing 11-mercaptoundecanoic acid for covalent immobilisation of lectins and betaine terminated thiol to resist non-specific interactions. Construction of biosensors based on Concanavalin A (Con A), Sambucus nigra agglutinin type I (SNA) and Ricinus communis agglutinin (RCA) on polycrystalline gold electrodes was optimised and characterised with a battery of tools including electrochemical impedance spectroscopy, various electrochemical techniques, QCM, FTIR spectroscopy, AFM, XPS and compared with a protein/lectin microarray. The lectin biosensors were able to detect glycoproteins from 1 fM (Con A), 10 fM (RCA) or 100 fM (SNA) with a linear range spanning 6 (SNA), 7 (RCA) or 8 (Con A) orders of magnitude. Furthermore, a detection limit for the Con A biosensor down to 1 aM was achieved in a sandwich configuration. A non-specific binding of proteins for the Con A biosensor was only 6.1% (probed with an oxidised invertase) of the signal towards its analyte invertase and a negligible non-specific interaction of the Con A biosensor was observed in diluted human sera (1000x), as well. The performance of the lectin biosensors was finally tested by glycoprofiling of human serum samples from healthy individuals and those having rheumatoid arthritis, which resulted in distinct glycan pattern between these two groups.
[Show abstract][Hide abstract] ABSTRACT: The genus Gluconobacter is frequently used for biotechnological and/or
nanotechnological applications. We studied endogenous fluorescence of
nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), indicator of
the oxidative metabolic state in mammalian cells, in Gluconobacter
oxydans (G. oxydans). Time-resolved measurements (excitation by 375nm
pulsed diode laser) were employed to record the bacterial fluorescence
intensity, as well as its modifications by metabolic modulation. Results
were gathered on fresh bacteria, on de-frozen ones, as well as on
bacteria encapsulated in alginate beads. NAD(P)H fluorescence increased
linearly with the concentration of bacteria. Freezing, which has little
effect on the viability of bacteria or the concentration-dependent
fluorescence rise, affected the temperature-dependence of NAD(P)H
fluorescence. Sodium cyanide (10 mM) provoked significant rise in the
NAD(P)H fluorescence, while dinitrophenol (200 μM) induced its
decrease, confirming the bacterial NAD(P)H fluorescence sensitivity to
modulators of electron transport chain. Gathered results demonstrate
that endogenous NAD(P)H fluorescence can be successfully recorded in the
bacterial strain G. oxydans using time-resolved measurements.
[Show abstract][Hide abstract] ABSTRACT: A novel bionanocomposite prepared by a direct mixing of bacterial cells of Gluconobacter oxydans and carbon nanotubes (CNTs) was applied for preparation of a mediated microbial bioanode for ethanol (EtOH) oxidation. The sensitivity of (162 ± 3) μA mM− 1 cm− 2 achieved is the highest among microbial based biosensor devices for EtOH oxidation published to date. Further, high performance of the oxidation process can be underlined by a short response time and a maximal current density of (261 ± 4) μA cm− 2.
[Show abstract][Hide abstract] ABSTRACT: 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[3.2.0]hept-2-en-6-one to corresponding lactones (1R,5S)-3-oxabicyclo-[3.3.0]oct-6-en-3-one and (1S,5R)-2-oxabicyclo-[3.3.0]oct-6-en-3-one as important chiral synthons for the synthesis of bioactive compounds were performed in the minireactor equipped with a column packed with encapsulated recombinant cells Escherichia coli overexpressing cyclohexanone monooxygenase. The cells were encapsulated in polyelectrolyte complex capsules formed by reaction of oppositely charged polymers utilizing highly reproducible and controlled encapsulation process. Encapsulated cells tested in minireactor exhibited high operational stability with 4 complete substrate conversions to products and 6 conversions above 80% within 14 repeated consecutive biooxidation tests. Moreover, encapsulated cells showed high enzyme stability during 91 days of storage with substrate conversions above 80% up to 60 days of storage. Furthermore, usable thermometric signal of Baeyer-Villiger biooxidation obtained by flow calorimetry using encapsulated cells was utilized for preparatory kinetic study in order to guarantee sub-inhibitory initial substrate concentration for biooxidation tests.
Enzyme and Microbial Technology 08/2011; 49(3):284-288. · 2.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A novel bionanocomposite prepared by a direct mixing of bacterial cells of Gluconobacter oxydans and carbon nanotubes (CNTs) was applied for preparation of a mediated microbial bioanode for ethanol (EtOH) oxidation. The sensitivity of (162±3)μAmM−1cm−2 achieved is the highest among microbial based biosensor devices for EtOH oxidation published to date. Further, high performance of the oxidation process can be underlined by a short response time and a maximal current density of (261±4)μAcm−2.
[Show abstract][Hide abstract] ABSTRACT: Glucose oxidase from Aspergillus niger, the specific enzyme for β-d-glucose oxidation, can also oxidize other related saccharides at very slow or negligible rates. The present study aimed to
compare the kinetics of d-glucose oxidation using immobilized glucose oxidase on bead cellulose for the oxidation of related saccharides using the
same biocatalyst. The significant differences were observed between the reaction rates for d-glucose and other saccharides examined. As a result, k
M ratio for d-glucose was determined to be 42 times higher than d-mannose, 61.6 times higher than d-galactose, 279 times higher than d-xylose, and 254 times higher than for d-fructose and d-cellobiose. On the basis of these differences, the ability of immobilized glucose oxidase to remove d-glucose from d-cellobiose, d-glucose from d-xylose, and d-xylose from d-lyxose was examined. Immobilized catalase on Eupergit and mixed with immobilized glucose oxidase on bead cellulose or co-immobilized
with glucose oxidase on bead cellulose was used for elimination of hydrogen peroxide from the reaction mixture. The accelerated
elimination of d-glucose and d-xylose in the presence of co-immobilized catalase was observed. The co-immobilized glucose oxidase and catalase were able
to decrease d-glucose or d-xylose content to 0–0.005% of their initial concentrations, while a minimum decrease of low oxidized saccharides d-xylose, d-cellobiose, and d-lyxose, respectively, was observed.
KeywordsGlucose oxidase-Catalase-Saccharides-Oxidation kinetics-Immobilized enzymes
Applied Biochemistry and Biotechnology 11/2010; 162(6):1669-1677. · 1.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Artificial Cells Blood Substitutes and Biotechnology (formerly known as Artificial Cells Blood Substitutes and Immobilization Bi 04/2010; 38(2):90-98. · 0.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Glucose oxidase from Aspergillus niger, the specific enzyme for beta-D-glucose oxidation, can also oxidize other related saccharides at very slow or negligible rates. The present study aimed to compare the kinetics of D-glucose oxidation using immobilized glucose oxidase on bead cellulose for the oxidation of related saccharides using the same biocatalyst. The significant differences were observed between the reaction rates for D-glucose and other saccharides examined. As a result, k (cat)/K (M) ratio for D-glucose was determined to be 42 times higher than D-mannose, 61.6 times higher than D-galactose, 279 times higher than D-xylose, and 254 times higher than for D-fructose and D-cellobiose. On the basis of these differences, the ability of immobilized glucose oxidase to remove D-glucose from D-cellobiose, D-glucose from D-xylose, and D-xylose from D-lyxose was examined. Immobilized catalase on Eupergit and mixed with immobilized glucose oxidase on bead cellulose or co-immobilized with glucose oxidase on bead cellulose was used for elimination of hydrogen peroxide from the reaction mixture. The accelerated elimination of D-glucose and D-xylose in the presence of co-immobilized catalase was observed. The co-immobilized glucose oxidase and catalase were able to decrease D-glucose or D-xylose content to 0-0.005% of their initial concentrations, while a minimum decrease of low oxidized saccharides D-xylose, D-cellobiose, and D-lyxose, respectively, was observed.
Applied biochemistry and biotechnology 03/2010; 162(6):1669-77. · 1.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Glucose oxidase (GOD) was immobilized on bead cellulose (BC) or Eupergit (Eup) to produce three types of GOD preparations: GOD directly immobilized on BC, GOD biospecifically immobilized on BC through Concanavalin A and GOD immobilized on Eup. These samples were used to remove d-glucose from a d-glucose–d-mannose solution obtained via the epimerization of glucose. The removal of d-glucose by immobilized GOD was markedly improved by the incorporation of an immobilized catalase, which converts hydrogen peroxide (an enzyme inactivator) produced by GOD to molecular oxygen and water. GOD covalently immobilized on bead cellulose (GOD-TBC) showed the best operational stability of the three types of immobilized GOD tested. It was found that GOD-TBC, supplemented with immobilized catalase, could be used up to 8 times without lowering its ability to exhaustively eliminate residual glucose. The removal of gluconic acid generated during the biotransformation of d-glucose was performed by precipitation with CaCl2. The final product contained d-mannose that was free of d-glucose, as confirmed by GC and HPLC tests.
Journal of Molecular Catalysis B Enzymatic 09/2009; 60(1):45-49. · 2.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 2.2%. The signal of the biosensor was linear up to 1.1 mM for D-sorbitol with sensitivity of (72 +/- 2) nA mM(-1), while a dynamic range was much wider up to 18 mM. The sorbitol biosensor gave reliable results even in the presence of a high molar excess of L-sorbose, a product of the biotransformation process, as judged from an excellent agreement with HPLC and GC.
[Show abstract][Hide abstract] ABSTRACT: Bioencapsulation involves the envelopment of tissues or biological active substances in semipermeable membranes. Bioencapsulation has been shown to be efficacious in mimicking the cell's natural environment and thereby improves the efficiency of production of different metabolites and therapeutic agents. The field of application is broad. It is being applied in bioindustry and biomedicine. It is clinically applied for the treatment of a wide variety of endocrine diseases. During the past decades many procedures to fabricate capsules have been described. Unfortunately, most of these procedures lack an adequate documentation of the characterization of the biocapsules. As a result many procedures show an extreme lab-to-lab variation and many results cannot be adequately reproduced. The characterization of capsules can no longer be neglected, especially since new clinical trials with bioencapsulated therapeutic cells have been initiated and the industrial application of bioencapsulation is growing. In the present review we discuss novel Approached to produce and characterize biocapsules in view of clinical and industrial application. A dominant factor in bioencapsulation is selection and characterization of suitable polymers. We present the adequacy of using high-resolution NMR for characterizing polymers. These polymers are applied for producing semipermeable membranes. We present the pitfalls of the currently applied methods and provide recommendations for standardization to avoid lab-to-lab variations. Also, we compare and present methodologies to produce biocompatible biocapsules for specific fields of applications and we demonstrate how physico-chemical technologies such as FT-IR, XPS, and TOF-SIMS contribute to reproducibility and standardization of the bioencapsulation process. During recent years it has become more and more clear that bioencapsulation requires a multidisciplinary approach in which biomedical, physical, and chemical technologies are combined. For adequate reproducibility and for understanding variations in outcome of biocapsules it is advisable if not mandatory to include the characterization processes presented in this review in future studies.