Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 53
Filter
Add more filters










Publication year range
1.
Int J Mol Sci ; 24(8)2023 Apr 15.
Article in English | MEDLINE | ID: mdl-37108499

ABSTRACT

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.


Subject(s)
Epoxide Hydrolases , Epoxy Compounds , Epoxide Hydrolases/genetics , Epoxide Hydrolases/chemistry , Catalysis , Epoxy Compounds/chemistry , Hydrolysis , Genetic Techniques , Stereoisomerism
2.
Methods Mol Biol ; 2460: 207-222, 2022.
Article in English | MEDLINE | ID: mdl-34972939

ABSTRACT

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.


Subject(s)
Colorectal Neoplasms , Somatomedins , Biomarkers/metabolism , Biomarkers, Tumor/metabolism , Colorectal Neoplasms/diagnosis , Colorectal Neoplasms/metabolism , Glycosylation , Humans , Lectins/metabolism , Microarray Analysis/methods , Polysaccharides/analysis , Protein Array Analysis/methods , Somatomedins/metabolism
3.
Ultramicroscopy ; 211: 112954, 2020 04.
Article in English | MEDLINE | ID: mdl-32018072

ABSTRACT

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.

4.
Biotechnol Lett ; 39(5): 667-683, 2017 May.
Article in English | MEDLINE | ID: mdl-28181062

ABSTRACT

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.


Subject(s)
Biocatalysis , Bioengineering , Bioreactors , Cells, Immobilized , Animals , Humans
5.
Appl Microbiol Biotechnol ; 100(15): 6585-6599, 2016 Aug.
Article in English | MEDLINE | ID: mdl-27328941

ABSTRACT

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.


Subject(s)
Biocatalysis , Bioreactors , Mixed Function Oxygenases/metabolism , Biotechnology , Oxidation-Reduction , Stereoisomerism , Substrate Specificity
6.
Glycoconj J ; 33(5): 717-24, 2016 10.
Article in English | MEDLINE | ID: mdl-27085877

ABSTRACT

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.


Subject(s)
Glycocalyx/metabolism , Granulosa Cells/metabolism , Lectins/chemistry , Tissue Array Analysis/instrumentation , Tissue Array Analysis/methods , Animals , Female , Granulosa Cells/cytology , Horses
7.
Biotechnol Appl Biochem ; 63(4): 457-64, 2016 Jul.
Article in English | MEDLINE | ID: mdl-26075587

ABSTRACT

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.


Subject(s)
Colorectal Neoplasms/blood , Colorectal Neoplasms/metabolism , Lectins/metabolism , Protein Array Analysis/methods , alpha-Macroglobulins/metabolism , Aged , Case-Control Studies , Female , Glycosylation , Humans , Male , Middle Aged
8.
Anal Chim Acta ; 854: 140-4, 2015 Jan 07.
Article in English | MEDLINE | ID: mdl-25479877

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 7h 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.


Subject(s)
Biosensing Techniques , Gluconobacter oxydans/metabolism , Phenylethyl Alcohol/metabolism , Oxidation-Reduction
9.
Methods Mol Biol ; 1200: 421-45, 2014.
Article in English | MEDLINE | ID: mdl-25117256

ABSTRACT

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. Besides already established concepts of glycoprofiling several novel aspects, which we think will be taken into account for future, more robust glycan analysis, are described including modified lectins, peptide lectin aptamers, and DNA aptamers with lectin-like specificity introduced by modified nucleotides. The last part of the chapter describes a novel concept of a glycocodon, which can lead to a better understanding of glycan-lectin interaction and for design of novel lectins with unknown specificities and/or better affinities toward glycan target or for rational design of peptide lectin aptamers or DNA aptamers.


Subject(s)
Genetic Engineering/methods , Glycomics/methods , Lectins/metabolism , Aptamers, Peptide/metabolism , Humans , Immobilized Proteins/chemistry , Immobilized Proteins/genetics , Immobilized Proteins/metabolism , Lectins/chemistry , Lectins/genetics , Polysaccharides/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
10.
Appl Biochem Biotechnol ; 174(5): 1834-49, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25149462

ABSTRACT

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 0.82 mm; RSD = 5.6 %), lens-shaped LentiKats® are characterised by wider size distribution (diameter of 3.65 mm; RSD = 10.3 % and height of 0.341 mm; RSD = 32.3 %) and showed the same porous structure throughout their whole volume at the mesoscopic (micrometre) level. Despite differences in their inner structure and surface properties, the pore diameter of ∼ 2.75 nm for regular polyelectrolyte complex microcapsules and ∼ 1.89 nm for LentiKats® were similar. These results were used for mathematical modelling, which provided the estimates of the effective diffusion coefficient of sucrose. This value was 1.67 × 10(-10) m(2) s(-1) for polyelectrolyte complex microcapsules and 0.36 × 10(-10) m(2) s(-1) for LentiKats®. Recombinant cells Escherichia coli-overexpressing enzyme cyclopentanone monooxygenase were immobilised in polyelectrolyte complex microcapsules and LentiKats® for comparison of their operational stability using model Baeyer-Villiger oxidation of (±)-cis-bicyclo [3.2.0] hept-2-en-6-one to regioisomeric lactones as important chiral synthons for potential pharmaceuticals. Both immobilisation matrices rendered high operational stability for whole-cell biocatalyst with no reduction in the biooxidation rate over 18 repeated reaction cycles.


Subject(s)
Enzymes, Immobilized/chemistry , Escherichia coli/enzymology , Oxygenases/chemistry , Polyvinyl Alcohol/chemistry , Capsules , Electrolytes/chemistry , Enzyme Activation , Materials Testing , Oxidation-Reduction
11.
Biosens Bioelectron ; 50: 235-8, 2013 Dec 15.
Article in English | MEDLINE | ID: mdl-23871870

ABSTRACT

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. coli overexpressing either cyclopentanone monooxygenase or cyclohexanone monooxygenase was immobilised in the form of solid polyelectrolyte complex gel membrane made of cellulose sulphate, sodium alginate and poly(methylene-co-guanidine) and attached to the surface of miniaturised oxygen electrode. The time response of the biosensor was 30s, the linear range of the calibration curve (R(2)=0.9993) was 8-130 µM and the sensitivity was 1.8 nA µM(-1) (RSD=5.0%) for substrate of Baeyer-Villiger oxidation (±)-cis-bicyclo[3.2.0]hept-2-en-6-one as analyte. The biosensor sensitivity was assessed for two other commercially available substrates, 4-methylcyclohexanone and 3-methylcyclohexanone. No interferences from ampicillin, citric acid, acetic acid, ethanol, methanol, glucose and products of Baeyer-Villiger oxidation (1R, 5S)-3-oxabicyclo[3.3.0]oct-6-en-2-one and (1S, 5R)-2-oxabicyclo[3.3.0]oct-6-en-3-one were detected. After 1 week of storage at 4°C the biosensor sensitivity was without changes. The biosensor was employed for monitoring of Baeyer-Villiger biotransformation and the results were correlated with gas chromatography. Till now, this is the first described biosensor based on Baeyer-Villiger monooxygenase and the first reported application of biosensor for monitoring of biotransformation based on Baeyer-Villiger oxidation.


Subject(s)
Biosensing Techniques/methods , Escherichia coli/enzymology , Oxygenases/metabolism , Biotransformation , Escherichia coli/genetics , Guanidines/chemistry , Oxidation-Reduction , Oxygenases/genetics , Polyamines/chemistry , Up-Regulation
12.
Macromol Rapid Commun ; 34(8): 635-9, 2013 Apr 25.
Article in English | MEDLINE | ID: mdl-23401120

ABSTRACT

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.


Subject(s)
DNA/metabolism , Escherichia coli/metabolism , Light , Polymers/chemistry , Acrylamides/chemistry , Cations/chemistry , DNA/chemistry , Escherichia coli/drug effects , Free Radicals/chemistry , Photolysis , Polymers/chemical synthesis , Polymers/pharmacology , Quaternary Ammonium Compounds/chemistry , Surface Plasmon Resonance
13.
Mikrochim Acta ; 180(1): 151-159, 2013 Jan.
Article in English | MEDLINE | ID: mdl-27231402

ABSTRACT

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 Sambucus nigra 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. The sensor layer was characterised by atomic force microscopy, electrochemical impedance spectroscopy and X-ray photoelectron spectroscopy. The biosensor exhibits a linear range that spans 7 orders of magnitude for both glycoproteins, with a detection limit as low as 0.33 fM for fetuin and 0.54 fM for asialofetuin. We also show, by making control experiments with oxidised asialofetuin, that the biosensor is capable of quantitatively detecting changes in the fraction of sialic acid on glycoproteins. We conclude that this work lays a solid foundation for future applications of such a biosensor in terms of the diagnosis of diseases such as chronic inflammatory rheumatoid arthritis, genetic disorders and cancer, all of which are associated with aberrant glycosylation of protein biomarkers.

14.
Mikrochim Acta ; 180(1): 1-13, 2013 Jan.
Article in English | MEDLINE | ID: mdl-27239071

ABSTRACT

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.e., unique sequences) from the same number of "letters" (building blocks) than DNA or proteins. Glycans are physicochemically similar and it is not a trivial task to identify their sequence, or - even more challenging - to link a given glycan to a particular physiological or pathological process. Lectins can recognise differences in glycan compositions even in their bound state and therefore are most useful tools in the task to decipher the "glycocode". Thus, lectin-based biosensors working in a label-free mode can effectively complement the current weaponry of analytical tools in glycomics. This review gives an introduction into the area of glycomics and then focuses on the design, analytical performance, and practical utility of lectin-based electrochemical label-free biosensors for the detection of isolated glycoproteins or intact cells.

15.
Biotechnol Lett ; 34(6): 1033-9, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22350332

ABSTRACT

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.


Subject(s)
Biosensing Techniques/methods , Ethanol/analysis , Gluconobacter oxydans/metabolism , Nanocomposites/chemistry , Fermentation , Ferricyanides/chemistry , Nanotubes, Carbon/chemistry , Sensitivity and Specificity
16.
Biotechnol Lett ; 34(2): 309-14, 2012 Feb.
Article in English | MEDLINE | ID: mdl-21983971

ABSTRACT

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.


Subject(s)
Escherichia coli/enzymology , Escherichia coli/physiology , Gene Expression , Microbial Viability , Oxygenases/metabolism , Cyclohexanones/metabolism , Escherichia coli/metabolism , Fluorescent Dyes/metabolism , Microscopy, Confocal/methods , Oxidation-Reduction , Oxygenases/genetics , Propidium/metabolism , Staining and Labeling/methods
17.
Enzyme Microb Technol ; 49(3): 284-8, 2011 Aug 10.
Article in English | MEDLINE | ID: mdl-22112513

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.


Subject(s)
Acinetobacter calcoaceticus/enzymology , Bacterial Proteins/metabolism , Bioreactors , Escherichia coli/enzymology , Industrial Microbiology/methods , Oxygenases/metabolism , Acinetobacter calcoaceticus/genetics , Calorimetry , Drug Compounding , Enzyme Stability , Equipment Design , Industrial Microbiology/instrumentation , Ketones/metabolism , Kinetics , Lactones/metabolism , Microchemistry/instrumentation , Oxidation-Reduction , Recombinant Fusion Proteins/metabolism , Stereoisomerism , Substrate Specificity , Temperature
18.
Talanta ; 84(2): 355-61, 2011 Apr 15.
Article in English | MEDLINE | ID: mdl-21376957

ABSTRACT

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. Finally the performance of the sensor modified by CNT-HA was tested in a batch and flow injection analysis (FIA) mode of operation with basic characteristics revealed. The NADH sensor exhibits a good long-term operational stability (95% of the original sensitivity after 22 h of continuous operation). Subsequently a d-sorbitol biosensor based on such a nanoscale built interface was prepared and characterised with a d-sorbitol dehydrogenase used as a biocatalyst.


Subject(s)
Biosensing Techniques , Electrodes , Hyaluronic Acid/chemistry , NAD/analysis , Nanotubes, Carbon , Microscopy, Electron, Scanning , Spectroscopy, Fourier Transform Infrared
19.
Article in English | MEDLINE | ID: mdl-20222845

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.


Subject(s)
Capsules/metabolism , Drug Compounding , Gluconates/metabolism , Glucose Oxidase/metabolism , Lactones/metabolism , Alginates/chemistry , Alginates/metabolism , Alkanes/metabolism , Biocatalysis , Capsules/chemistry , Cellulose/analogs & derivatives , Cellulose/chemistry , Cellulose/metabolism , Dimethylpolysiloxanes/chemistry , Dimethylpolysiloxanes/metabolism , Fluorocarbons/metabolism , Gluconates/chemistry , Glucose Oxidase/chemistry , Glucuronic Acid/chemistry , Glucuronic Acid/metabolism , Guanine/analogs & derivatives , Guanine/chemistry , Hexuronic Acids/chemistry , Hexuronic Acids/metabolism , Manganese Compounds/metabolism , Nylons/chemistry , Nylons/metabolism , Organophosphonates/chemistry , Oxides/metabolism , Oxygen/metabolism , Solubility
20.
Biotechnol Lett ; 32(5): 675-80, 2010 May.
Article in English | MEDLINE | ID: mdl-20111981

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.


Subject(s)
Bridged Bicyclo Compounds, Heterocyclic/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Oxygenases/metabolism , Biopolymers , Capsules/chemistry , Cells, Immobilized , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Oxidation-Reduction , Oxygenases/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL
...