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PubMed2003

Biosensors based on enzyme field-effect transistors for determination of some substrates and inhibitors.

This paper is a review of the authors' publications concerning the development of biosensors based on enzyme field-effect transistors (ENFETs) for direct substrates or inhibitors analysis. Such biosensors were designed by using immobilised enzymes and ion-selective field-effect transistors (ISFETs). Highly specific, sensitive, simple, fast and cheap determination of different substances renders them as promising tools in medicine, biotechnology, environmental control, agriculture and the food industry. The biosensors based on ENFETs and direct enzyme analysis for determination of concentrations of different substrates (glucose, urea, penicillin, formaldehyde, creatinine, etc.) have been developed and their laboratory prototypes were fabricated. Improvement of the analytical characteristics of such biosensors may be achieved by using a differential mode of measurement, working solutions with different buffer concentrations and specific agents, negatively or positively charged additional membranes, or genetically modified enzymes. These approaches allow one to decrease the effect of the buffer capacity influence on the sensor response in an aim to increase the sensitivity of the biosensors and to extend their dynamic ranges. Biosensors for the determination of concentrations of different toxic substances (organophosphorous pesticides, heavy metal ions, hypochlorite, glycoalkaloids, etc.) were designed on the basis of reversible and/or irreversible enzyme inhibition effect(s). The conception of an enzymatic multibiosensor for the determination of different toxic substances based on the enzyme inhibition effect is also described. We will discuss the respective advantages and disadvantages of biosensors based on the ENFETs developed and also demonstrate their practical application.

Biosensing Techniques↗
PubMed2003

Flow-injection spectrophotometric determination of cyanate in bioremediation processes by use of immobilised inducible cyanase.

A new flow injection (FI) method for photometric monitoring of cyanate in bioremediation processes using immobilised native cyanase is described. The method is based on the catalytic reaction between cyanate and bicarbonate to produce ammonia and carbon dioxide in the presence of an inducible native cyanase, immobilised in a reactor packed with glass beads. Two degrees of purification of the biocatalyst were used-heated cell-free extract and purified extract of cyanase from Pseudomonas pseudoalcaligenes CECT 5344. The ammonia produced by the enzymatic reaction is finally monitored photometrically at 700 nm using a modification of the conventional Berthelot method. The method furnishes different calibration curves depending on the degree of purification of the cyanase, with linear ranges between 1.23 and 616.50 micromol L(-1) ( r(2)=0.9979, n=7) and between 1.07 and 308.25 micro mol L(-1) ( r(2)= 0.9992, n=7) for the heated cell-free extract and the purified cyanase extract, respectively. No statistically significant differences between the samples were found in the precision study evaluated at two cyanate concentration levels using one-way analysis of variance. A sampling frequency of 15 h(-1) was achieved. The method was used to monitor cyanate consumption in a cyanate bioremediation tank inoculated with Pseudomonas pseudoalcaligenes CECT 5344 strain. The correlation between cyanate degradation and ammonia production was tested using a conventional method. Finally, the method was applied to different samples collected from the bioremediation tank using the standard addition method; recoveries between 85.9 and 97.4% were obtained.

Biodegradation, Environmental↗
PubMed2004

Insertion of a glycosylphosphatidylinositol-anchored enzyme into liposomes.

Incorporation of alkaline phosphatase (AP), a glycosylphosphatidylinositol (GPI)-anchored protein, into liposomes containing detergent, followed by detergent removal with hydrophobic resin was performed. Incorporation media were collected during different steps of detergent removal and were analyzed by flotation in sucrose gradient. The presence of protein was checked by measuring enzymatic activity, while the presence of (3)H-radio-labelled liposomes was followed by determination of the radioactivity. The incorporation yield of the protein into liposomes increased with incubation time in presence of hydrophobic resin. Protein was also incorporated at different protein/lipid ratios. At the highest protein lipid ratio, our data showed that 260 molecules of GPI-linked AP (AP-GPI) could be associated with one liposome, corresponding to 65% vesicle coverage. Finally, observations by electron cryomicroscopy indicated (i) that the protein seemed exclusively associated with the lipid bilayer via the GPI-anchor, as shown by the distance-about 2.5 nm-between the protein core and the liposome membrane; (ii) that the AP-GPI distribution was heterogeneous on the liposome surface, forming clusters of protein.

Alkaline Phosphatase↗
PubMed2002

Enzymatic (R)-phenylacetylcarbinol production in benzaldehyde emulsions.

(R)-Phenylacetylcarbinol [(R)-PAC)] is the chiral precursor for the production of the pharmaceuticals ephedrine and pseudoephedrine. Reaction conditions were improved to achieve increased (R)-PAC levels in a simple batch biotransformation of benzaldehyde emulsions and pyruvate, using partially purified pyruvate decarboxylase (PDC) from the filamentous fungus Rhizopus javanicus NRRL 13161 as the catalyst. Lowering the temperature from 23 degrees C to 6 degrees C decreased initial rates but increased final (R)-PAC concentrations. Addition of ethanol, which increases benzaldehyde solubility, was not beneficial for (R)-PAC production. It was established that proton uptake during biotransformation increases the pH above 7 thereby limiting (R)-PAC production. For small-scale studies, biotransformations were buffered with 2-2.5 M MOPS (initial pH 6.5). High concentrations of MOPS as well as some alcohols and KCl stabilised PDC. A balance between PDC and substrate concentrations was determined with regards to ( R)-PAC production and yields on enzyme and substrates. R. javanicus PDC (7.4 U/ml) produced 50.6 g/l (337 mM) ( R)-PAC in 29 h at 6 degrees C with initial 400 mM benzaldehyde and 600 mM pyruvate. Molar yields on consumed benzaldehyde and pyruvate were 97% and 59%, respectively, with 17% pyruvate degraded and 24% converted into acetaldehyde and acetoin; 43% PDC activity remained, indicating reasonable enzyme stability at high substrate and product concentrations.

Acetone↗
PubMed2005

Effect of additives on gas-phase catalysis with immobilised Thermoanaerobacter species alcohol dehydrogenase (ADH T).

This paper presents a strategy for preparing an efficient immobilised alcohol dehydrogenase preparation for a gas-phase reaction. The effects of additives such as buffers and sucrose on the immobilisation efficiency (residual activity and protein loading) and on the gas-phase reaction efficiency (initial reaction rate and half-life) of Thermoanaerobacter sp. alcohol dehydrogenase were studied. The reduction of acetophenone to 1-phenylethanol under in situ cofactor regeneration using isopropanol as co-substrate was used as a model reaction at fixed reaction conditions (temperature and thermodynamic activities). A strongly enhanced thermostability of the enzyme in the gas-phase reaction was achieved when the enzyme was immobilised with 50 mM phosphate buffer (pH 7) containing sucrose five times the protein amount (on weight/weight basis). This resulted in a remarkable productivity of 200 g L(-1) day(-1) even at non-optimised reaction conditions. The interaction of additives with the enzyme and water affects the immobilisation and gas-phase efficiencies of the enzyme. However, it was not possible to predict the effect of additives on the gas-phase reaction efficiency even after knowing their effect on the immobilisation efficiency.

Acetophenones↗
PubMed1996

News & Notes: Effect of Surfactants on Cellulase Production by Nectria catalinensis

The effect of different nonionic surfactants (Tween 80, Tween 20, Triton X-100) and polyethylene glycol (PEG 6000) was tested on cellulolytic enzyme system production. Tween 80 gave the highest yield of endoglucanase, exoglucanase, and cellobiase at the 20th day of growth, presumably by causing increased permeability of cell membranes and/or by promoting the release of cell-bound enzymes. Maximal yield of endoglucanase was achieved with 1.7 mM Tween 80, whereas exoglucanase and cellobiase were at 0.85 mM. In the same way, this compound increased fungal growth. On the other hand, Tween 20 and Triton X-100 inhibited growth and cellulolytic enzyme production. High yields of endoglucanase and exoglucanase were achieved with PEG 6000 in comparison with the control, presumably by increasing enzyme stability.

Journal Article↗
PubMed1999

Pk-cdcA encodes a CDC48/VCP homolog in the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1: transcriptional and enzymatic characterization.

A gene encoding a cell division control protein from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1, Pk-cdcA, was cloned and sequenced. The Pk-cdcA gene is composed of 2508 nucleotides, encoding a protein of 835 amino acids with a molecular mass of 93,666 Da. Pk-CdcA has a typical Walker-type ATPase motif and was classified as a new member of the CDC48/VCP subfamily of so-called AAA proteins. In addition, Pk-CdcA possesses a unique region composed of charged amino acids, which is not observed in other homologs from Archaea. Transcription of the gene was analyzed by primer extension and Northern analyses, revealing that Pk-cdcA is transcribed from a site 77 bases upstream of the initiation codon. Pk-CdcA and its deletion mutant Pk-CdcAdelta63, which lacks the unique inserted region, were expressed in Escherichia coli cells as His-tagged fusion proteins and purified. Both Pk-CdcA and Pk-CdcAdelta63 possess an ATPase activity, as do other CDC48/VCP proteins. However, Pk-CdcAdelta63 showed a higher level of ATPase activity and greater thermostability than Pk-CdcA. Furthermore, Pk-CdcAdelta63 has a higher Vmax value than wild type, even though the Km was unchanged. These observations indicated that the inserted region affects enzyme stability and activity. In order to investigate intracellular expression levels of Pk-CdcA, Western analysis was performed using anti-Pk-CdcA antisera obtained from immunized BALB/C mice. Equal levels of Pk-CdcA expression were observed during exponential and stationary phases. Growth phase-specific fragmentation of Pk-CdcA was found in stationary-phase cells.

Adenosine Triphosphatases↗
PubMed2006

Studies of reaction parameters on synthesis of Citronellyl laurate ester via immobilized Candida rugosa lipase in organic media.

Immobilized Candida rugosa lipase was used for the synthesis of citronellyl laurate from citronellol and lauric acid. Screening of different types of support (Amberlite MB-1 and Celite) for immobilization of lipase and solvent (n-hexane, n-heptane, and iso-octane) and optimization of reaction conditions, such as catalyst loading, effect of substrates molar ratio and temperature, have been studied. The maximum enzyme activity was obtained at 310 K. The immobilized C. rugosa lipase onto Amberlite MB-1 support was found to be the best support with a conversion of 89% of citronellyl laurate ester in iso-octane compared to Celite 545. Deactivation of C. rugosa lipase at 313, 318 and 323 K were observed. Ordered bi bi mechanism with dead end complex of lauric acid was found to fit the initial rate data and the kinetic parameters were obtained by non-linear regression analysis.

Acyclic Monoterpenes↗
PubMed2006

Enzymatic synthesis of cinnamic acid derivatives.

Using Novozym 435 as catalyst, the syntheses of ethyl ferulate (EF) from ferulic acid (4-hydroxy 3-methoxy cinnamic acid) and ethanol, and octyl methoxycinnamate (OMC) from p-methoxycinnamic acid and 2-ethyl hexanol were successfully carried out in this study. A conversion of 87% was obtained within 2 days at 75 degrees C for the synthesis of EF. For the synthesis of OMC at 80 degrees C, 90% conversion can be obtained within 1 day. The use of solvent and high reaction temperature resulted in better conversion for the synthesis of cinnamic acid derivatives. Some cinnamic acid esters could also be obtained with higher conversion and shorter reaction times in comparison to other methods reported in the literature. The enzyme can be reused several times before significant activity loss was observed.

Acrylic Resins↗
PubMed2006

Lipase-mediated transformation of vegetable oils into biodiesel using propan-2-ol as acyl acceptor.

Propan-2-ol was used as an acyl acceptor for immobilized lipase-catalyzed preparation of biodiesel. The optimum conditions for transesterification of crude jatropha (Jatropha curcas), karanj (Pongamia pinnata) and sunflower (Helianthus annuus) oils were 10% Novozym-435 (immobilized Candida antarctica lipase B) based on oil weight, alcohol to oil molar ratio of 4:1 at 50 degrees C for 8 h. The maximum conversions achieved using propan-2-ol were 92.8, 91.7 and 93.4% from crude jatropha, karanj and sunflower oils, respectively. Reusability of the lipase was maintained over 12 repeated cycles with propan-2-ol while it reached to zero by 7(th) cycle when methanol was used as an acyl acceptor, under standard reaction conditions.

2-Propanol↗
PubMed1988

Immobilization of linamarase and its use in the determination of bound cyanide in cassava using flow injection analysis.

Extracts from the tubers (cortex and parenchyma) and leaves of Manihot esculenta Crantz (cassava) were analyzed for their releasable cyanide content using flow injection analysis incorporating an immobilized linamarase bioreactor. Linamarase was immobilized under very mild conditions to an activated 2-fluoro-N-methylpyridinium Fractogel support. The released cyanide, which was monitored spectrophotometrically at 525 nm using an alkaline picrate reagent, was found to be highest in the cortex and lowest in the parenchyma.

Calibration↗
PubMed1991

Flow injection analysis of lactose using covalently immobilized beta-galactosidase, mutarotase, and glucose oxidase/peroxidase on a 2-fluoro-1-methylpyridinium salt-activated Fractogel support.

Milk samples were analyzed for their lactose content using flow injection analysis and incorporating immobilized beta-galactosidase or beta-galactosidase/mutarotase and glucose oxidase/peroxidase bioreactors. These enzymes were immobilized, under mild conditions, on to a 2-fluoro-1-methylpyridinium salt-activated Fractogel support. The use of a phosphate buffer (0.15 M) was found to facilitate the rapid mutarotation of alpha-D-glucose and hence could obviate the need for the more expensive mutarotase. The chromogenic agents of choice for monitoring the reaction were 3-methyl-2-benzothiazolinone hydrazone and 3-dimethylaminobenzoic acid. Linearity was observed over the concentration range 16-160 micrograms/ml using lactose standards (r = 0.996). Between 30 and 40 milk samples/h can be analyzed. Comparisons are made with existing HPLC and alkaline methylamine methods for a range of milk matrices. The FIA method consistently gives the lowest standard deviations and coefficient of variation for the various milk matrices analyzed.

Animals↗
PubMed1983

Factors influencing pyrroline 5-carboxylate synthesis from glutamate by rat intestinal mucosa mitochondria.

Factors influencing pyrroline 5-carboxylate (P5C) synthesis from glutamate by a subcellular fraction enriched in mitochondria of rat small intestinal mucosa have been studied. P5C synthesis decreased rapidly if this subcellular fraction was preincubated at 20 degrees C in the absence of substrates; this effect suggests that the enzyme(s) catalyzing P5C synthesis from glutamate (P5C synthase) is unstable in the absence of substrates. In the presence of substrates P5C synthesis increased linearly for the first 30 min of incubation, suggesting that the substrates promote enzyme stability. Pyridoxal 5'-phosphate is an effective inhibitor of P5C synthase whereas pyridoxamine 5'-phosphate and pyridoxal are not inhibitory. Potassium phosphate, KCl, and KBr each inhibited P5C synthase but potassium-Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) did not. Potassium phosphate was the most potent inhibitor followed by KBr, and then KCl. These results suggest P5C synthase is sensitive to anion inhibition. Both L-ornithine and D-ornithine inhibited P5C synthase; L-proline did not inhibit. Several analogs of ornithine and proline were also tested and none was found to inhibit P5C synthase; the inhibition by ornithine is, therefore, rather specific and it may prove to contribute to the regulation of metabolism of these amino acids.

Animals↗
PubMed1988

Purification of a manganese-containing superoxide dismutase from Halobacterium halobium.

An oxygen-induced superoxide dismutase was purified from the halophilic bacterium, Halobacterium halobium, strain NRL. Due to the high salt requirement for enzyme stability, the purification had to be performed in the presence of 2 M NaCl. The pI of the protein was 4.95. The approximate Mr was 38,500. The subunit size as determined by sodium dodecyl sulfate-electrophoresis was approximately 19,000. Metal analysis showed 1.5 atoms of manganese per dimer, 0.5 atom zinc, and 1.54 atoms copper. The N-terminal sequence of amino acids was determined, and based upon the first 26 amino acids significant homology to other manganese- and iron-containing superoxide dismutases was revealed.

Amino Acid Sequence↗
PubMed1979

Effect of salts on D-glycerate dehydrogenase kinetic behavior.

Bovine liver D-glycerate dehydrogenase (D-glycerate:NAD (NADP) oxidoreductase, EC 1.1.1.29) adapts its kinetic behaviour to a sequential mechanism. The presence of NaCl causes an appreciable variation in the Km and V values. relative to the both substrates in the hydroxypyruvate/D-glycerate dehydrogenase/NADH system, which does not happen in the D-glycerate/D-glycerate dehydrogenase/NAD system. The former system is inhibited by high concentrations of NaCl and activated by low salt concentrations. The hydroxypyruvate concentration causing substrate inhibition increases as the concentration of NaCl increases; excess NADH inhibition is independent of the salt concentration. The variation of the initial rates of both systems, in the presence of chlorides having monovalent and divalent cations, or sodium halides, Na2SO4 and NaNO3 (at constant ionic strength) suggests that the anions have a specific action on the enzyme. An increase in the NaCl concentration causes a displacement of the optimum D-glycerate dehydrogenase pH (with hydroxypyruvate and NADH as substrates) towards the acid area. The enzyme stability, at varying pH, varies with the salt concentration.

Animals↗
PubMed1982

On the pH dependency of lipoprotein lipase activity.

The relation between pH and activity for lipoprotein lipase against emulsions of long-chain triacylglycerols has previously been studied in several laboratories and found to be a bell-shaped curve with optimum activity between pH 8 and 9. In contrast, using short-chain triacylglycerols or monoacylglycerols as substrates we had found that the activity rises continuously with pH to at least pH 10.5. This suggested that some factor other than the active site mechanism limited the activity at high pH in traditional assay systems. We, therefore, reinvestigated the activity against long-chain triacylglycerols under conditions where binding of the enzyme to the emulsion droplets and enzyme stability was not limiting. Under these conditions the activity continued to rise from pH 8 to pH 10, and the degree of stimulation by apolipoprotein C-II was found to be the same over the whole range studied (pH 6.5-10.5).

Animals↗
PubMed1978

Acquired enzymopathy of erythrocyte glucose-6-phosphate dehydrogenase in acute viral hepatitis.

Erythrocyte glucose-6-phosphate dehydrogenase from patients with acute viral hepatitis has been purified and characterized. The enzyme showed decreased activity (relative to protein), reduced affinity for glucose 6-phosphate and was inactivated at 45 degrees C or at low pH values. The activity and properties of normal erythrocyte enzyme, incubated in vitro with blood plasma of patients, showed a similar pattern of modifications. Incubation with bilirubin affected the enzyme stability, but not its activity or affinity for substrate.

Bilirubin↗