Polyaniline emeraldine salt-type products were synthesized under mild, environmentally friendly conditions using hemin as a cost-effective catalyst, p-aminodiphenylamine (PADPA) as a monomer, and micelles formed from SDBS as templates.
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Ferric heme b (= ferric protoporphyrin IX = hemin) is an important prosthetic group of different types of enzymes, including the intensively investigated and widely applied horseradish peroxidase (HRP). In HRP, hemin is present in... more
Ferric heme b (= ferric protoporphyrin IX = hemin) is an important prosthetic group of different types of enzymes, including the intensively investigated and widely applied horseradish peroxidase (HRP). In HRP, hemin is present in monomeric form in a hydrophobic pocket containing among other amino acid side chains the two imidazoyl groups of His170 and His42. Both amino acids are important for the peroxidase activity of HRP as an axial ligand of hemin (proximal His170) and as an acid/base catalyst (distal His42). A key feature of the peroxidase mechanism of HRP is the initial formation of compound I under heterolytic cleavage of added hydrogen peroxide as a terminal oxidant. Investigations of free hemin dispersed in aqueous solution showed that different types of hemin dimers can form, depending on the experimental conditions, possibly resulting in hemin crystallization. Although it has been recognized already in the 1970s that hemin aggregation can be prevented in aqueous solution ...
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The synthesis of the emeraldine salt form of polyaniline (PANI-ES) from aniline with Aspergillus sp. glucose oxidase (GOD), d-glucose, dissolved O2, and horseradish peroxidase isoenzyme C (HRPC) in the presence of large unilamellar... more
The synthesis of the emeraldine salt form of polyaniline (PANI-ES) from aniline with Aspergillus sp. glucose oxidase (GOD), d-glucose, dissolved O2, and horseradish peroxidase isoenzyme C (HRPC) in the presence of large unilamellar vesicles of AOT (sodium bis-(2-ethylhexyl)sulfosuccinate) as templates at pH = 4.3 and T ~ 25 °C was investigated in a systematic way. In this cascade reaction mixture, the oxidation of aniline is catalyzed by HRPC with H2O2 that is formed in situ as byproduct of the GOD-catalyzed oxidation of d-glucose with O2. Under the elaborated experimental conditions which we considered ideal, the formation of PANI-ES products is evident, as judged by UV/Vis/NIR and EPR measurements. Comparison was made with a reference reaction, which was run under similar conditions with added H2O2 instead of GOD and d-glucose. Although the reference reaction was found to be superior, with the cascade reaction, PANI-ES products can still be obtained with high aniline conversion (&...
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This article deals with artificial vesicles and their membranes as reaction promoters and regulators. Among the various molecular assemblies which can form in an aqueous medium from amphiphilic molecules, vesicle systems are unique.... more
This article deals with artificial vesicles and their membranes as reaction promoters and regulators. Among the various molecular assemblies which can form in an aqueous medium from amphiphilic molecules, vesicle systems are unique. Vesicles compartmentalize the aqueous solution in which they exist, independent on whether the vesicles are biological vesicles (existing in living systems) or whether they are artificial vesicles (formed in vitro from natural or synthetic amphiphiles). After the formation of artificial vesicles, their aqueous interior (the endovesicular volume) may become - or may be made - chemically different from the external medium (the exovesicular solution), depending on how the vesicles are prepared. The existence of differences between endo- and exovesicular composition is one of the features on the basis of which biological vesicles contribute to the complex functioning of living organisms. Furthermore, artificial vesicles can be formed from mixtures of amphiph...
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... Structural Aspects and Enzymology LINDA MAGID', PETER WALDE2, GIANNI ZAMPIERI2, EZIO BATTISTEL2 *, QIAOQIAN PENG2, EDOARDO TROTTA2,**, MARCO MAESTR02 ... myelin basic protein; wo= [ H20 ] / [ AOT ] ; wo,;: wo initial; wo,f:... more
... Structural Aspects and Enzymology LINDA MAGID', PETER WALDE2, GIANNI ZAMPIERI2, EZIO BATTISTEL2 *, QIAOQIAN PENG2, EDOARDO TROTTA2,**, MARCO MAESTR02 ... myelin basic protein; wo= [ H20 ] / [ AOT ] ; wo,;: wo initial; wo,f: wo filled micelles; wode: wo empty ...
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The kinetic properties of trypsin have been studied in reverse micelles formed by two surfactant systems, namely bis(2-ethylhexyl) sodium sulfosuccinate (AOT) in isooctane, and hexadecyltrimethyl ammonium bromide (CTAB) in... more
The kinetic properties of trypsin have been studied in reverse micelles formed by two surfactant systems, namely bis(2-ethylhexyl) sodium sulfosuccinate (AOT) in isooctane, and hexadecyltrimethyl ammonium bromide (CTAB) in chloroform/isooctane (1:1, by vol.). Three substrates have been used, namely N alpha-benzoyl-L-Arg ethyl ester, N alpha-benzoyl-L-Phe-L-Val-L-Arg p-nitroanilide (BzPheValArg-NH-Np) in AOT and N alpha-benzyloxycarbonyl-L-Lys p-nitrophenyl ester (ZLysO-Np) in CTAB. One of the main aims of the work was to compare the behaviour of trypsin in reverse micelles with that of alpha-chymotrypsin, for which an enhancement of kcat had been observed with respect to aqueous solutions. The pH profile is not significantly altered in reverse micelles with respect to water, however the kinetic parameters (kcat and Km) differ widely from one another, and are markedly affected by the micellar conditions, in particular by the water content wo (wo = [H2O]/[AOT]). Whereas in the case of BzPheValArg-NH-Np kcat is much smaller than in water, in the case of ZLysO-Np at pH 3.2 (but not at pH 6.0) a slight enhancement with respect to water is observed. On the basis of rapid kinetic spectrophotometry (stopped-flow) and solvent isotope effect studies, this enhancement is ascribed to a change in the rate-limiting step (acylation rather than hydrolysis). As in the case of alpha-chymotrypsin, the maximal activity is found for all substrates at rather small wo values (below 12), which is taken to suggest that the enzyme works better when is surrounded by only a few layers of tightly bound water. Spectroscopic studies [ultraviolet absorption, circular dichroism (CD) and fluorescence] have been carried out as a function of wo. Whereas the absorption properties are practically unchanged, the CD spectrum in AOT micelles has a lower intensity than in water, which is interpreted as a partial unfolding. The intensity is partly restored when Ca2+ ions are added, indicating that the micellar environment may cause a partial denaturation by depleting it of calcium ions. Fluorescence data show that the emission properties of the protein in reverse micelles match those in aqueous solution at around wo = 13 approx., whereas lambda max shifts towards the red by increasing wo, indicating an exposure of the tryptophan residues and probably an unfolding of the whole protein, at wo values above 15. Finally the reaction between trypsin and its specific macromolecular Kunitz inhibitor from soybeans is studied.(ABSTRACT TRUNCATED AT 400 WORDS)
Research Interests: Chemistry, Kinetics, Comparative Study, Medicine, Enzyme Kinetics, and 15 moreMicelle, Circular Dichroism, Trypsin, European, Emulsions, Acylation, Calcium Chloride, Structure activity Relationship, Aqueous Solution, Hydrolysis, Biochemistry and cell biology, Molecular Structure, Chymotrypsin, Chloroform, and Medical biochemistry and metabolomics
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Contemporary biological cells are highly sophisticated dynamic compartment systems which separate an internal volume from the external medium through a boundary, which controls, in complex ways, the exchange of matter and energy between... more
Contemporary biological cells are highly sophisticated dynamic compartment systems which separate an internal volume from the external medium through a boundary, which controls, in complex ways, the exchange of matter and energy between the cell's interior and the environment. Since such compartmentalization is a fundamental principle of all forms of life, scenarios have been elaborated about the emergence of prebiological compartments on early Earth, in particular about their likely structural characteristics and dynamic features. Chemical systems that consist of potentially prebiological compartments and chemical reaction networks have been designed to model pre-cellular systems. These systems are often referred to as "protocells". Past and current protocell model systems are presented and compared. Since the prebiotic formation of cell-like compartments is directly linked to the prebiotic availability of compartment building blocks, a few aspects on the likely chemi...
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ABSTRACT
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The two enzymes Aspergillus sp. glucose oxidase (GOD) and horseradish peroxidase (HRP) were co-immobilized on solid silica supports in a spatially controlled way by using mesoporous silica nanoparticles (Hiroshima Mesoporous Materials,... more
The two enzymes Aspergillus sp. glucose oxidase (GOD) and horseradish peroxidase (HRP) were co-immobilized on solid silica supports in a spatially controlled way by using mesoporous silica nanoparticles (Hiroshima Mesoporous Materials, HMM) and a polycationic dendronized polymer (denpol). The silica support was first coated with the denpol, followed by the deposition of the mesoporous silica nanoparticles into which – in a next step – GOD was adsorbed. Finally, the GOD-loaded silica nanoparticles were coated with a denpol-HRP conjugate constituting of several HRP molecules which were covalently bound to the denpol via bis-aryl hydrazone (BAH) bonds. The entire immobilization process was followed in real time with quartz crystal microbalance with dissipation monitoring (QCM-D). The activities and storage stabilities of the co-immobilized enzymes were determined by analyzing a two-step cascade reaction involving the two immobilized enzymes GOD and HRP. D-glucose and o-phenylenediamine...
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Lipid vesicles (liposomes) are a unique and fascinating type of polymolecular aggregates, obtained from bilayer-forming amphiphiles—or mixtures of amphiphiles—in an aqueous medium. Unilamellar vesicles consist of one single self-closed... more
Lipid vesicles (liposomes) are a unique and fascinating type of polymolecular aggregates, obtained from bilayer-forming amphiphiles—or mixtures of amphiphiles—in an aqueous medium. Unilamellar vesicles consist of one single self-closed bilayer membrane, constituted by the amphiphiles and an internal volume which is trapped by this bilayer, whereby the vesicle often is spherical with a typical desired average diameter of either about 100 nm or tens of micrometers. Functionalization of the external vesicle surface, basically achievable at will, and the possibilities of entrapping hydrophilic molecules inside the vesicles or/and embedding hydrophobic compounds within the membrane, resulted in various applications in different fields. This review highlights a few of the basic studies on the phase behavior of polar lipids, on some of the concepts for the controlled formation of lipid vesicles as dispersed lamellar phase, on some of the properties of vesicles, and on the challenges of eff...
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Most linear peptides directly interact with membranes, but the mechanisms of interaction are far from being completely understood. Here, we present an investigation of the membrane interactions of a designed peptide containing a... more
Most linear peptides directly interact with membranes, but the mechanisms of interaction are far from being completely understood. Here, we present an investigation of the membrane interactions of a designed peptide containing a non-natural, synthetic amino acid. We selected a nonapeptide that is reported to interact with phospholipid membranes, ALYLAIRKR, abbreviated as ALY. We designed a modified peptide (azoALY) by substituting the tyrosine residue of ALY with an antimicrobial azobenzene-bearing amino acid. Both of the peptides were examined for their ability to interact with model membranes, assessing the penetration of phospholipid monolayers, and leakage across the bilayer of large unilamellar vesicles (LUVs) and giant unilamellar vesicles (GUVs). The latter was performed in a microfluidic device in order to study the kinetics of leakage of entrapped calcein from the vesicles at the single vesicle level. Both types of vesicles were prepared from a 9:1 (mol/mol) mixture of POPC...
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Different types of templates consisting of sulfonate or sulfate groups were compared for the horseradish peroxidase/H2O2-catalysed synthesis of the emeraldine salt form of polyaniline from aniline at pH = 4.3.
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A laccase-catalysed oligomerisation of p-aminodiphenylamine was investigated in an aqueous medium containing 80–100 nm-sized anionic vesicles formed from AOT, the sodium salt of bis(2-ethylhexyl)sulfosuccinic acid.
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Microbial transglutaminase (MTG) has been stably solid-phase immobilized on glass microbeads using a second generation dendronized polymer. Immobilized MTG enabled the efficient generation of site-specifically conjugated proteins... more
Microbial transglutaminase (MTG) has been stably solid-phase immobilized on glass microbeads using a second generation dendronized polymer. Immobilized MTG enabled the efficient generation of site-specifically conjugated proteins including scFvs and Fab-fragments as well as whole antibodies via distinct glutamines and unprecedented, also via lysines with various bifunctional substrates with defined stoichiometry. With this method we generated dual site-specifically modified antibodies comprising a fluorescent probe and a metal chelator for radiolabeling - a strategy anticipated to design antibodies for imaging and therapy. Furthermore, we provide evidence that immobilized MTG features higher site-selectivity than soluble MTG.
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A spectrophotometric assay for the determination of horseradish peroxidase (HRP) in aqueous solution with p-phenylenediamine (PPD, benzene-1,4-diamine) as electron donor substrate and hydrogen peroxide (H2O2) as oxidant was developed. The... more
A spectrophotometric assay for the determination of horseradish peroxidase (HRP) in aqueous solution with p-phenylenediamine (PPD, benzene-1,4-diamine) as electron donor substrate and hydrogen peroxide (H2O2) as oxidant was developed. The oxidation of PPD by HRP/H2O2 leads to the formation of Bandrowski's base ((3E,6E)-3,6-bis[(4-aminophenyl)imino]cyclohexa-1,4-diene-1,4-diamine), which can be quantified by following the increase in absorbance at 500 nm. The assay was applied for monitoring the activity of HRP inside ≈180 nm-sized lipid vesicles (liposomes), prepared from POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and purified by size exclusion chromatography. Due to the high POPC bilayer permeability of PPD and H2O2, the HRP-catalyzed oxidation of PPD occurs inside the vesicles once PPD and H2O2 are added to the vesicle suspension. In contrast, if instead of PPD the bilayer-impermeable substrate ABTS(2-) (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate)) is u...
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The literature concerning the oxidative oligomerization and polymerization of various arylamines, e.g., aniline, substituted anilines, aminonaphthalene and its derivatives, catalyzed by oxidoreductases, such as laccases and peroxidases,... more
The literature concerning the oxidative oligomerization and polymerization of various arylamines, e.g., aniline, substituted anilines, aminonaphthalene and its derivatives, catalyzed by oxidoreductases, such as laccases and peroxidases, in aqueous, organic, and mixed aqueous organic monophasic or biphasic media, is reviewed. An overview of template-free as well as template-assisted enzymatic syntheses of oligomers and polymers of arylamines is given. Special attention is paid to mechanistic aspects of these biocatalytic processes. Because of the nontoxicity of oxidoreductases and their high catalytic efficiency, as well as high selectivity of enzymatic oligomerizations/polymerizations under mild conditions—using mainly water as a solvent and often resulting in minimal byproduct formation—enzymatic oligomerizations and polymerizations of arylamines are environmentally friendly and significantly contribute to a “green” chemistry of conducting and redox-active oligomers and polymers. C...
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The oxidation of the aniline dimer, p-aminodiphenylamine (PADPA), with Trametes versicolor laccase and O2 in an aqueous solution of pH 3.5 is controlled by negatively charged AOT (sodium bis(2-ethylhexyl) sulfosuccinate) vesicles. With... more
The oxidation of the aniline dimer, p-aminodiphenylamine (PADPA), with Trametes versicolor laccase and O2 in an aqueous solution of pH 3.5 is controlled by negatively charged AOT (sodium bis(2-ethylhexyl) sulfosuccinate) vesicles. With vesicles, a product resembling polyaniline in its emeraldine salt form (PANI-ES) is obtained, in contrast to the reaction without vesicles where no such product is formed. To understand this observation, the product distribution and structures from the reaction with and without vesicles were determined by using partially selectively deuterated PADPA as a starting material and analyzing the products with HPLC-MS. We found that in the presence of vesicles the main product is obtained in about 50% yield, which is the N-C-para-coupled PADPA dimer that has spectroscopic properties of PANI-ES, as determined by time-dependent density functional theory (TD-DFT) calculations. A secondary reaction route leads to longer PADPA oligomers that must contain a phenazine core. Without vesicles, PADPA and its products undergo partial hydrolysis, but in the presence of vesicles, hydrolysis does not occur. Because molecular dynamics (MD) simulations show that the main intermediate oxidation product is embedded within the vesicle membrane, where the water content is very low, we propose that the microenvironment of the vesicle membrane protects the oxidation products from unwanted hydrolysis.
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We report about the first Raman spectroscopy study of a vesicle-assisted enzyme-catalyzed oligomerization reaction. The aniline dimer N-phenyl-1,4-phenylenediamine (= p-aminodiphenylamine, PADPA) was oxidized and oligomerized with... more
We report about the first Raman spectroscopy study of a vesicle-assisted enzyme-catalyzed oligomerization reaction. The aniline dimer N-phenyl-1,4-phenylenediamine (= p-aminodiphenylamine, PADPA) was oxidized and oligomerized with Trametes versicolor laccase and dissolved O2 in the presence of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) vesicles (80–100 nm diameter) as templates. The conversion of PADPA into oligomeric products, poly(PADPA), was monitored during the reaction by in situ Raman spectroscopy. The results obtained are compared with UV/vis/NIR and EPR measurements. All three complementary methods indicate that at least some of the poly(PADPA) products, formed in the presence of AOT vesicles, resemble the conductive emeraldine salt form of polyaniline (PANI-ES). The Raman measurements also show that structural units different from those of “ordinary” PANI-ES are present too. Without vesicles PANI-ES-like products are not obtained. For the first time, the as-prepared stabl...
Bicelles composed of DMPC and phospholipids capable of chelating lanthanide ions, such as 1,2-dimyristoyl-sn-glycero-3-phospho-ethanolamine-diethylene triaminepentaacetate (DMPE-DTPA), are highly tunable magnetically responsive soft... more
Bicelles composed of DMPC and phospholipids capable of chelating lanthanide ions, such as 1,2-dimyristoyl-sn-glycero-3-phospho-ethanolamine-diethylene triaminepentaacetate (DMPE-DTPA), are highly tunable magnetically responsive soft materials. Further doping of these systems with cholesterol-DTPA conjugates complexed to a lanthanide ion considerably enhances the bicelle's size and magnetic alignability. The high value of these cholesterol conjugates for bicelle design remains largely unexplored. Herein, we examine how molecular structural alterations within the cholesterol-DTPA conjugates lead to contrasting self-assembled polymolecular aggregate structures when incorporated into DMPC/DMPE-DTPA/Tm(3+) bilayers. The nature of the linker connecting the DTPA-chelating moiety to the sterol backbone is examined by synthesizing conjugates of various linker lengths and polarities. The incorporation of these compounds within the bilayer results in polymolecular aggregate geometries of h...
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... Acknowledgment. We would like to thank Erich Sackmann and Walter Hack1 for helpful discussions and for allowing us to carry out some of the control experiments in their laboratory at the Technische Universitiit in Munchen. ...
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Within each biological cell, surface- and volume-confined enzymes control a highly complex network of chemical reactions. These reactions are efficient, timely, and spatially defined. Efforts to transfer such appealing features to in... more
Within each biological cell, surface- and volume-confined enzymes control a highly complex network of chemical reactions. These reactions are efficient, timely, and spatially defined. Efforts to transfer such appealing features to in vitro systems have led to several successful examples of chemical reactions catalysed by isolated and immobilized enzymes. In most cases, these enzymes are either bound or adsorbed to an insoluble support, physically trapped in a macromolecular network, or encapsulated within compartments. Advanced applications of enzymatic cascade reactions with immobilized enzymes include enzymatic fuel cells and enzymatic nanoreactors, both for in vitro and possible in vivo applications. In this Review, we discuss some of the general principles of enzymatic reactions confined on surfaces, at interfaces, and inside small volumes. We also highlight the similarities and differences between the in vivo and in vitro cases and attempt to critically evaluate some of the necessary future steps to improve our fundamental understanding of these systems.
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This paper describes the increase of antiproliferative activity toward tumor cell lines of liposome-delivered retinoids and aromatic polyamidines.
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The activity of porcine pancreatic elastase has been studied in reverse micelles formed by AOT (sodium bis(2-ethylhexyl) sulfosuccinate) in isooctane. For the two substrates succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide and... more
The activity of porcine pancreatic elastase has been studied in reverse micelles formed by AOT (sodium bis(2-ethylhexyl) sulfosuccinate) in isooctane. For the two substrates succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide and succinyl-L-Ala-L-Ala-L-Pro-L-Leu-p-nitroanilide, the catalytic constant, kcat, in reverse micelles increases with increasing wo until, at high wo, the value of kcat measured in bulk buffer solution is approached (wo = [H2O/]AOT]). In analogy to alpha-chymotrypsin--and in apparent contrast to many other enzymes--elastase does not show a maximum in the kcat-wo profile. Within the wo range of 8 to 35, for both substrates, the Michaelis constant Km (as expressed relative to the total volume of the solution, Km,overall) increases with increasing wo.
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ABSTRACT The hydrolytic activity of lipolytic enzymes in reverse micelles can be measured continuously with Fourier Transform infrared spectroscopy (FTIR) by following in the region of the OH-stretching band the water consumption during... more
ABSTRACT The hydrolytic activity of lipolytic enzymes in reverse micelles can be measured continuously with Fourier Transform infrared spectroscopy (FTIR) by following in the region of the OH-stretching band the water consumption during the reaction. This possibility is unique to reverse micellar solutions, because they are optically transparent and because they contain only a limited amount of water.
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ABSTRACT
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... c) Lentz, BR Chem. Phys. Lipids 1989, 50, 171−190. ... 213−304. (b) Cullis, PR; Fenske, DB; Hope, MJ In Biochemistry of Lipids, Lipoproteins and Membranes; Vance, DE,Vance, JE, Eds.; Elsevier: Amsterdam, 1996; pp 1−33. (16 ...
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Research Interests: Electron Microscopy, Kinetics, Medical Microbiology, Transmission Electron Microscopy, Cell Division, and 14 moreBiological Sciences, Cell line, Cell Differentiation, Mice, Animals, Physical sciences, Liposomes, Liposome, Encapsulation, Cell Proliferation, Biological activity, Biochemistry and cell biology, Deferoxamine, and Freeze-fracturing
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Engyodontium album proteinase K (proK) is widely used for degrading proteinaceous impurities during the isolation of nucleic acid from biological samples, or in proteomics and prion research. Towards applications of proK in flow reactors,... more
Engyodontium album proteinase K (proK) is widely used for degrading proteinaceous impurities during the isolation of nucleic acid from biological samples, or in proteomics and prion research. Towards applications of proK in flow reactors, a simple method for the stable immobilization of proK inside glass micropipette tubes was developed. The immobilization of the enzyme was achieved by adsorption of a dendronized polymer-enzyme conjugate from aqueous solution. This conjugate was first synthesized from a polycationic dendronized polymer (denpol) and proK and consisted, on average, of 2000 denpol repeating units and 140 proK molecules, which were attached along the denpol chain via stable bis-aryl hydrazone bonds. Although the immobilization of proK inside the tube was based on non-specific, non-covalent interactions only, the immobilized proK did not leak from the tube and remained active during prolonged storage at 4 °C and during continuous operation at 25 °C and pH = 7.0. The proc...
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... R. Green and JW Szostak, Science 258, 1910 (1992). G. von Kiedrowski, B. Wlotzka, J. Helbling, M. Matzen, and S. Jordan, Angew. Chem. 103, 456 (1991). ... 167, 181 (1992). S. Egelhaaf, P. Schurtenberger, E. Wehrli, M. Adrian, and PL... more
... R. Green and JW Szostak, Science 258, 1910 (1992). G. von Kiedrowski, B. Wlotzka, J. Helbling, M. Matzen, and S. Jordan, Angew. Chem. 103, 456 (1991). ... 167, 181 (1992). S. Egelhaaf, P. Schurtenberger, E. Wehrli, M. Adrian, and PL Luisi, Progr. Colloid Polym. Sci. ...