Optical peroxide biosensor using the electrically controlled-release technique

An optical biosensor using an electrically controlled-release system was developed for the measurement of peroxide concentration. The electrically controlled-release system consisted of a current-supplying system and a polymer complex by hydrogen bonding between the carboxylic and oxazoline group. The polymer complex was formed below pH 5.0 and was degraded above pH 5.4. The local pH change near the surface of the polymer complex could be controlled by applying the electric current to release an enzyme reaction reagent, 4-hydroxyphenylacetic acid (HPA), in the polymer complex. The releasing rate of HPA was proportional to the electric current applied to the polymer complex. The model of the controlled-release system was proposed to predict the degradation velocity of the polymer complex, which is equivalent to the releasing rate of HPA. The released HPA and analyte, peroxide, flowed into the reactor with the immobilized enzyme and then reacted with the enzyme. The peroxide concentration was measured based on the fluorescence detection of enzyme reaction product, 6,6'-dihydroxy (1,1'-biphenyl) 3,3'-diacetic acid (DBDA). The proposed biosensor had the linear analytical range of 0.025 approximately 1.0 mM with a response time of 20 min, good repeatability, and reproducibility.     

The development and application of a new affinity partitioning system for enzyme isolation and purification.

A reactive water-soluble polymer was synthesized by copolymerizing N-isopropylacrylamide and glycidyl acrylate. The reactive polymer could react with the amino groups of enzymes/proteins or other ligands to form an affinity polymer. As a model, the reactive polymer was allowed to react with paraaminobenzamidine, a strong trypsin inhibitor. The affinity polymer could easily form an aqueous two-phase system with either dextran or pullulan, and the phase diagram was compared favorably to that of the well-known polyethylene glycol-dextran system. Once trypsin was attracted to the affinity polymer dominant phase, the enzyme could be dissociated from the polymer at low pH. Owing to the N-isopropylacrylamide units, the affinity polymer could be isolated from the solution by precipitation at a low level of ammonium sulfate. The enzyme recovery was always greater than 50%, and the affinity polymer could be reused in several cycles of affinity partitioning and recovery.     

Extracellular enzyme loss during polyelectrolyte flocculation of cells from fermentation broth

Association of extracellular protein product with flocculated cells reduces product yield. Here, partitioning of the enzyme subtilisin between the liquid and polyelectrolyte-flocculated and sedimented Bacillus increased as the polymer dosage was increased beyond that necessary to obtain optimum floc character (brain floc) for cell removal by centrifugation. Partitioning to the cell floc is partly physical entrapment at all polymer dosages; however, at higher levels there is also direct interaction between the polyelectrolyte and enzyme. Enzyme loss was not likely due to pH denaturation during the flocculation process because conditions were within the stable pH range of the enzyme. The direct interaction between polyelectrolyte and enzyme was characterized through turbidimetric titrations and partitioning studies. Neither changes in the polymer feed concentration nor the method of polymer addition reduced the enzyme loss at dosages optimal for cell removal.     

Simultaneous purification and immobilization of Aspergillus niger xylanase on the reversibly soluble polymer Eudragit(TM) L-100

The non-covalent immobilization of a commercial preparation of xylanase from A. niger was carried out on a reversibly soluble-insoluble enteric polymer Eudragit(TM) L-100. The immobilization of the xylanase activity by adsorption was simultaneously accompanied by removal of cellulase activity since the latter did not bind to the polymer. Thus, the soluble enzyme derivative may be useful for treatment of paper pulp bleaching in paper industry. The immobilized xylanase retained 60% of its activity toward xylan as the substrate. No change was observed in the pH optimum (5.5) of the enzyme upon immobilization. Only marginal increase in the K(m) of the free enzyme (3.6 mg ml(-1) to 5.0 mg ml(-1)) upon immobilization on the soluble polymer reflected that the enzyme-substrate binding continues to be efficient in spite of the macromolecular nature of the substrate. Fluorescence spectroscopy and UV difference spectroscopy were used to probe the change(s) in the enzyme structure upon immobilization. This change in structure was correlated with the "effectiveness factor" of the enzyme activity. CD spectra also showed that the enzyme undergoes drastic changes in the structure.     

Biosensor based on enzyme-catalysed degradation of thin polymer films

A biosensor based on the enzyme-catalysed dissolution of biodegradable polymer films has been developed. Three polymer-enzyme systems were investigated for use in the sensor: a poly(ester amide), which is degraded by the proteolytic enzyme alpha-chymotrypsin; a dextran hydrogel, which is degraded by dextranase; and poly(trimethylene) succinate, which is degraded by a lipase. Dissolution of the polymer films was monitored by Surface Plasmon Resonance (SPR). The rate of degradation was directly related to enzyme concentration for each polymer/enzyme couple. The poly(ester amide)/alpha-chymotrypsin couple proved to be the most sensitive over a concentration range from 4 x 10(-11) to 4 x 10(-7) mol l(-1) of enzyme. The rate of degradation was shown to be independent of the thickness of the poly(ester amide) films. The dextran hydrogel/dextranase couple was less sensitive than the poly(ester amide)/alpha-chymotrypsin couple but showed greater degradation rates at low enzyme concentrations. Enzyme concentrations as low as 2 x 10(-11) mol l(-1) were detected in less than 20 min. Potential fields of application of such a sensor system are the detection of enzyme concentrations and the construction of disposable enzyme based immunosensors, which employ the polymer-degrading enzyme as an enzyme label.     

Temperature-induced switching of enzyme activity with smart polymer-enzyme conjugates

A method for thermally induced switching of enzyme activity has been developed, based on the site-directed conjugation of end-reactive temperature-responsive polymers to a unique cysteine (Cys) residue positioned near the enzyme active site. The reversible temperature-induced collapse of N,N-dimethylacrylamide (DMA)/N-4-phenylazo-phenylacrylamide (AZAAm) copolymers (DMAAm) has been used as a molecular switch to control the catalytic activity of endoglucanase 12A (EG 12A). The polymer was conjugated to the EG 12A site-directed mutant N55C, directly adjacent to the cellulose binding cleft, and to the S25C mutant, where the conjugation site is more distant. The N55C conjugate displayed a larger activity shutoff efficiency in the collapsed polymer state than the S25C conjugate. Increasing the polymer molecular weight was also shown to increase the shutoff efficiency of the switch. Related to these effects of conjugation site and polymer size, the switching efficiency was found to be strongly dependent on substrate size. With a small substrate, o-nitrophenyl-beta-d-cellobioside (ONPC), there was minimal blocking of enzyme activity when the polymer was in the expanded state. With a large substrate, hydroxyethyl cellulose (HEC), there was a large reduction of enzyme activity in the polymer expanded state, even with relatively small polymer chains, and a further reduction when the polymer was collapsed. Similar general trends for the interactive effects of conjugation site, polymer size, and substrate size were observed for immobilized conjugates. Kinetic studies demonstrated that the switching activity was due to the blocking of substrate association by the collapsed polymers. These investigations provide mechanistic insight that can be utilized to design molecular switches for a variety of stimuli-responsive polymer-protein conjugates.     

A selective molecularly imprinted polymer for immobilization of acetylcholinesterase (AChE): an active enzyme targeted and efficient method

In the present study, we immobilized acetylcholinesterase (AChE) enzyme onto acetylcholine removed imprinted polymer and acetylcholine containing polymer. First, the polymers were produced with acetylcholine, substrate of AChE, by dispersion polymerization. Then, the enzyme was immobilized onto the polymers by using two different methods: In the first method (method A), acetylcholine was removed from the polymer, and then AChE was immobilized onto this polymer (acetylcholine removed imprinted polymer). In the second method (method B), AChE was immobilized onto acetylcholine containing polymer by affinity. In method A, enzyme‐specific species (binding sites) occurred by removing acetylcholine from the polymer. The immobilized AChE reached 240% relative specific activity comparison with free AChE because the active enzyme molecules bounded onto the polymer. Transmission electron microscopy results were taken before and after immobilization of AChE for the assessment of morphological structure of polymer. Also, the experiments, which include optimum temperature (25–65°C), optimum pH (3–10), thermal stability (4–70°C), kinetic parameters, operational stability and reusability, were performed to determine the characteristic of the immobilized AChE. Copyright © 2015 John Wiley & Sons, Ltd.     

Purification of xylanase from Trichoderma viride by precipitation with an anionic polymer Eudragit S 100

Summary Endo-xylanase from T. viride has been purified 4.2 fold by precipitation with a commercially available enteric polymer Eudragit S-100. Electrophoretic analysis also indicated removal of contaminant proteins. The enzyme could be recovered in more than 89% yield. The binding of the enzyme to the polymer was predominantly by electrostatic interaction.     

Recovery of Clostridium thermosulfurogenes produced beta-amylase by (hydroxypropyl)methylcellulose partition.

A procedure for recovering Clostridium thermosulfurogenes produced beta-amylase from fermentation broth by partition was developed. The partition was achieved by addition of ammonium sulfate to an aqueous solution of the enzyme with (hydroxypropyl)methylcellulose. The beta-amylase-containing pellet formed upon centrifugation could be redissolved and the polymer recovered by a second salt addition. The process was not dependent on polymer/enzyme solution pH, but it was affected by temperature, polymer nominal molecular weight and loading, and fermentation carbon source. Unlike more traditional aqueous-phase partitions, such as poly(ethylene glycol)/dextran, the current approach appeared to be biospecific.     

Simultaneous refolding/purification of xylanase with a microwave treated smart polymer

Affinity precipitation with a smart polymer, Eudragit S-100 (a methyl methacrylate polymer), was exploited for simultaneous refolding and purification of xylanase. Affinity precipitation consisted of this reversibly soluble-insoluble polymer-binding xylanase selectively. The complex was precipitated by lowering the pH and xylanase was eluted off the polymer using 1 M NaCl. For refolding experiments, the commercial preparation of Aspergillus niger xylanase was denatured with 8 M urea. Addition of microwave irradiated Eudragit S-100 and affinity precipitation led to recovery of 96% enzyme activity by refolding. Simultaneously, the enzyme was purified 45 times. Thermally inactivated preparation, when subjected to similar steps, led to 95% recovery of enzyme activity with 42-fold purification. The strategy has the potential for recovering pure proteins in active forms from overexpressed proteins, which generally form inclusion bodies in E. coli.     

Enzyme immobilization by radiation-induced polymerization of 2-hydroxyethyl methacrylate at low temperatures.

Enzyme immobilization by radiation-induced polymerization of hydrophilic glass-forming monomers, such as 2-hydroxyethyl methacrylate, was studied. Enzyme radiation damage could be sufficiently retarded at low temperatures. The immobilized enzyme activity yield was markedly higher at low temperature than at higher temperature polymerization. At low temperatures the polymerized composite had a porous structure owing to ice crystallization which depends on the monomer concentration. It was deduced that the enzyme was partially trapped on the polymer surface, partially isolated in the pore, and partially occluded inside the polymer matrix. A decrease in activity caused by enzyme leakage was observed with repeated use in enzyme reactions where the composites had a large porosity. The activity yield showed a maximum at certain optimum porosities, i.e., at optimum monomer concentrations. Continuous enzyme reaction was preferably carried out using immobilized enzyme columns.     

Synthesis and characterization of a water-soluble affinity polymer for trypsin purification

A specific ligand bound polymer has been synthesized for the purpose of purification and stabilization of trypsin, an easily autodigestible enzyme. The affinity polymer was formed by copolymerizing N-acryloyl-m-aminobenzamidine, a strong trypsin inhibitor, and acrylamide in the absence of oxygen. Kinetic studies on the trypsin inhibition revealed that there was a strong binding between this enzyme and the polymer and the mechanism was of a competitive manner with an inhibition constant of 0.6 x 10(-3)M. Such an affinity polymer was also very effective in preventing trypsin from auto-digestion at 4 degrees C.Based on this finding and the principle of cross flow filtration, a new process has been developed for purification of trypsin from a solution containing chymotrypsin. The experimental data indicated that trypsin was bound to the polymer (MW > 10(5)) and remained in the retentate while unbound chymotrypsin was collected in the filtrate. This purification process has a capability of recovering 98% pure trypsin at 90% yield.     

The mechanism of biosynthesis and direction of chain extension of a polyl-(N-acetylglucosamine 1-phosphate) from the walls of Staphylococcus lactis N.C.T.C. 2102

1. The synthesis of a polymer of N-acetylglucosamine 1-phosphate, occurring in the walls of Staphylococcus lactis N.C.T.C. 2102, was examined by using cell-free enzyme preparations. The enzyme system was particulate, and probably represents fragmented cytoplasmic membrane. 2. Uridine diphosphate N-acetylglucosamine was the only substrate required for polymer synthesis and labelled substrate was used to show that N-acetylglucosamine 1-phosphate is transferred as an intact unit from substrate to polymer. 3. The properties of the enzyme system were studied. A high concentration of Mg(2+) or Mn(2+) was required for optimum activity, and the pH optimum was about 8.5. 4. End-group analysis during synthesis in vitro showed that newly formed chains contain up to about 15 repeating units. Pulse-labelling indicated that chain extension occurs by transfer from the nucleotide to the ;sugar-end' of the chain, i.e. to the end that is not attached to peptidoglycan in the wall.     

One step purification of peanut phospholipase D by precipitation with alginate

A simple titrimetric assay with soybean lecithin has been used for screening phospholipase D activity from some plant sources, viz. peanut, wheat germ, cabbage and carrot. The enzyme from peanut has been purified by binding to alginate which is a water soluble polymer. The purification consisted of co-precipitation of enzyme with alginate upon addition of 0.06 M Ca⁺⁺. The enzyme was eluted from the polymer using 0.2 M sodium chloride. The activity recovery was 61% with 34 fold purification.     

Immobilization of xylan-degrading enzymes from<Emphasis Type="Italic"> Melanocarpus albomyces</Emphasis> IIS 68 on the smart polymer Eudragit L-100

Xylanase of Melanocarpus albomyces IIS 68 was immobilized on Eudragit L-100. The latter is a copolymer of methacrylic acid and methyl methacrylate and is a pH-sensitive smart polymer. The immobilization was carried out by gentle adsorption and an immobilization efficiency of 0.82 was obtained. The enzyme did not leach off the polymer even in the presence of 1 M NaCl and 50% ethylene glycol. The K(m) of the enzyme changed from 5.9 mg ml(-1) to 9.1 mg ml(-1) upon immobilization. The V(max) of the immobilized enzyme showed an increase from 90.9 micro mol ml(-1) min(-1) (for the free enzyme) to 111.1 micro mol ml(-1) min(-1). The immobilized enzyme could be reused up to ten times without impairment of the xylanolytic activity. The immobilized enzyme was also evaluated for its application in pre-bleaching of eucalyptus kraft pulp.     

Peptide Synthesis Catalyzed by Crosslinked α-Chymotrypsin in Water/Dimethylformamide Solvent System

-Chymotrypsin was crosslinked to give a water-insoluble polymer by treatment with the bifunctional reagent glutaraldehyde. The specific activity of the crosslinked enzyme in aqueous media was three orders of magnitude lower than for the native chymotrypsin. In a medium containing more than 50% (v/v) of dimethylformamide the specific activities of both enzymes were comparable. In addition, the insoluble polymer was more stable in the presence of 60% (v/v) dimethylformamide compared with the native enzyme. Therefore, in this medium enzymatic peptide synthesis could be successfully accomplished with the crosslinked enzyme, but not with the same amount of native chymotrypsin.     

Mechanism of polynucleotide phosphorylase

The de novo polymerization of RNA initiated by polynucleotide phosphorylase from nucleoside diphosphates was examined. End group analysis performed under conditions designed to specifically end label the polymer revealed no evidence for a 5'-pyrophosphate-terminated polymer. However, we observed preferential incorporation of the ADP alpha S(RP) diastereomer into the 5' end (Marlier & Benkovic, 1982) in chain initiation, suggesting that the enzyme incorporates a nucleoside diphosphate specifically into the 5' end of the product, with subsequent enzymatic removal of the polyphosphate linkage. No evidence could be obtained for a covalent adduct between the enzyme and the 5' end of the polymer chain, despite the high processivity of the polymerization reaction. Gel electrophoretic analysis showed the polymer to be highly disperse, varying from 1 to 30 kb. Scanning transmission electron microscopy supported this product analysis and further suggested that (i) each subunit can produce an RNA polymer and (ii) both 5' and 3' ends of the RNA can be bound simultaneously to the same or differing enzyme molecules.     

The action of gamma irradiation on terrilytin immobilized on cellulose-fiber materials

The effect of gamma-radiation on terrilytin, a proteolytic enzyme immobilized on modified and nonmodified cellulose materials was studied by EPR. Dialdehyde cellulose and graft copolymer of cellulose and polyacrylic acid were used as the modified cellulose materials. Dependence of the native and immobilized terrilytin activity and the content of free radicals in the irradiated samples on the irradiation dose was observed. It was shown that immobilization of the enzyme led to increasing of its stability to the effect of the ionizing radiation. This was due to transfer of the free valency from terrilytin to the carrying polymer which prevented radiation and chemical destruction of the enzyme. The proteolytic activity of native terrilytin subjected to gamma-irradiation markedly decreased because of intramolecular and intermolecular interactions during reactions of the terrilytin free radicals, since in this case there was no polymer as an acceptor of the enzyme free valency.     

Expression of the Streptococcus pneumoniae type 3 synthase in Escherichia coli. Assembly of type 3 polysaccharide on a lipid primer.

Synthesis of the type 3 capsular polysaccharide of Streptococcus pneumoniae is catalyzed by the membrane-localized type 3 synthase, which utilizes UDP-Glc and UDP-GlcUA to form high molecular mass [3-beta-d-GlcUA-(1-->4)-beta-d-Glc-(1-->](n). Expression of the synthase in Escherichia coli resulted in synthesis of a 40-kDa protein that was reactive with antibody directed against the C terminus of the synthase and was the same size as the native enzyme. Membranes isolated from E. coli contained active synthase, as demonstrated by the ability to incorporate Glc and GlcUA into a high molecular mass polymer that could be degraded by type 3 polysaccharide-specific depolymerase. As in S. pneumoniae, the membrane-bound synthase from E. coli catalyzed a rapid release of enzyme-bound polysaccharide when incubated with either UDP-Glc or UDP-GlcUA alone. The recombinant enzyme expressed in E. coli was capable of releasing all of the polysaccharide from the enzyme, although the chains remained associated with the membrane. The recombinant enzyme was also able to reinitiate polysaccharide synthesis following polymer release by utilizing a lipid primer present in the membranes. At low concentrations of UDP-Glc and UDP-GlcUA (1 microm in the presence of Mg(2+) and 0.2 microm in Mn(2+)), novel glycolipids composed of repeating disaccharides with linkages consistent with type 3 polysaccharide were synthesized. As the concentration of the UDP-sugars was increased, there was a marked transition from glycolipid to polymer formation. At UDP-sugar concentrations of either 5 microm (with Mg(2+)) or 1.5 microm (with Mn(2+)), 80% of the incorporated sugar was in polymer form, and the size of the polymer increased dramatically as the concentration of UDP-sugars was increased. These results suggest a cooperative interaction between the UDP-precursor-binding site(s) and the nascent polysaccharide-binding site, resulting in a non-processive addition of sugars at the lower UDP-sugar concentrations and a processive reaction as the substrate concentrations increase.     

Biocatalytic synthesis of fluorinated polyesters

The biocatalytic synthesis of fluorinated polyesters from activated diesters and fluorinated diols has been investigated. The effects of time, continuous enzyme addition, enzyme concentration, and diol chain length were studied to determine the factors that would limit chain extension, such as enzyme inactivation, enzyme specificity, the equilibrium position for the reaction, hydrolytic side reactions, and polymer precipitation. An enzyme screen demonstrated that only Novozym 435, an immobilized lipase from Candida antarctica, was effective in producing the fluorinated polyester. Molecular weight and polydispersity analyses were performed by means of gel permeation chromatography. End group analysis was accomplished through the use of matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy. Polymer molecular weight steadily increased and then leveled off after approximately 30 h, with a weight average molecular weight of approximately 1773. The majority of the polymer chains were terminated with either hydroxyl or vinyl groups. Polymers that were synthesized from bulk monomers had higher molecular weights, but high enzyme concentrations were required. Enzyme specificity toward shorter chain fluorinated diols appeared to be the governing factor in limiting chain growth. However, polymer molecular weight increased further (M(w) = 8094) when a fluorinated diol that contained an additional methylene spacer between the fluorine atoms and hydroxyl groups was used.