Analysis of phosphorothionate pesticides using a chloroperoxidase pretreatment and acetylcholinesterase biosensor detection.

Acetylcholinesterase (AChE) is responsible for the hydrolysis of acetylcholine in the nervous system. It is inhibited by organophosphate and carbamate pesticides. However, this enzyme is only slightly inhibited by organophosphorothionates, which makes the detection of these pesticides analytically very difficult. A new enzymatic method for the activation and detection of phosphorothionates was developed with the capability to be used directly in food samples without the need of laborious solvent extraction steps. Chloroperoxidase (CPO) from Caldariomyces fumago was combined with tert-butyl hydroperoxide and two halides. Chlorpyrifos and triazophos were completely oxidized. Fenitrothion, methidathion and parathion methyl showed conversion rates between 54 and 61%. Furthermore, the oxidized solution was submitted to an AChE biosensor assay. Chlorpyrifos spiked in organic orange juice was oxidized, where its oxon product was detected in concentrations down to 5 microg/L (final concentration food sample: 25 microg/L). The complete duration of the method takes about 2 h.

Miniaturized on-line digestion system for the sequential identification and characterization of protein analytes.

A miniaturized on-column digestion system constructed for the sequential analysis of semi-purified protein analytes is presented. By utilizing fused silica capillary (diameter 150microm) packed with a zone of trypsin-modified Eupergit C beads and a second zone of reversed-phase C18 material, a linear column set-up was constructed. The protein analytes (pmol amounts) were first digested in the 600nl trypsin reactor portion of the system. Next, the generated peptides were trapped in the C18 column shaped as an electrospray emitter. Finally, after washing the matrix free from salts and other hydrophilic impurities present in the sample, peptides were eluted. A stepwise increased concentration profile of organic solvent, created by a dual syringe pump system, promoted the release of bound peptides, which were identified by electrospray ionization MS/MS. This approach proved to be very efficient, achieving almost complete digestion of the proteins studied, with suitable operational stability maintained for more than 1 week. Further, a small nebulizer was designed and fitted to the electrospray emitter. A significant improvement of the spray stability was observed and droplet build-up on the capillary was avoided, even at flow rates well above 1500nl/min. The proteins chloroperoxidase, staphylococcal enterotoxin B and protein A (injection volume 0.3microl, salt concentration 0.2-1M) were sequentially digested, desalted, eluted, detected and conclusively identified by bioinformatics web tools with an analytical cycle time of 10min.

Chloroperoxidase from Caldariomyces fumago is active in the presence of an ionic liquid as co-solvent.

Chloroperoxidase from Caldariomyces fumago catalyses the oxidation of 1,2-dihydronaphthalene to (1R,2R)-(+)-dihydroxytetrahydronaphthalene in homogenous citrate buffer/ionic liquid mixtures, using t-butyl hydroperoxide as O2 donor. It tolerates up to 30 (v/v) 1,3-dimethylimidazolium methylsulfate or 1-butyl-3-methylimidazolium methylsulfate. The enzyme activity in these ionic liquid co-solvent systems is retained for 24 h, but it falls to 3 h using non-ionic organic solvents such as t-BuOH or acetone.

Presence of a vanadium-dependent haloperoxidase in Botrytis cinerea.

The presence of a haloperoxidase in the mycelium of Botrytis cinerea, extractable with buffer, is demonstrated. A low level of extracellular enzyme activity was also detected. The haloperoxidase from the fungus is a vanadium-dependent glycoprotein, with a pH optimum of about 5.5. Native gel electrophoresis indicates that it is a high molecular mass protein. It appears to react with antibodies against haloperoxidase from Caldariomyces fumago. Enzyme activity is increased 3.5-fold and 15-fold by culture of the fungus in the presence of NaCl or vanadium, respectively. Activity is partly reduced by removal of vanadium and activity can be restored by the addition of vanadium to the enzyme. The possible function of this haloperoxidase is discussed.

A kinetic epoxidation assay for chloroperoxidase.

Chloroperoxidase exhibits a wide variety of enantioselective epoxidation reactions. Until now, the epoxidation activities have been mainly evaluated using elaborate gas chromatographic methods. This paper reports a rapid and convenient spectrophotometric assay for CPO. The disappearance of indene by catalytic epoxidation is monitored at 250 nm and this is used as an index of enzyme activity. This method will prove to be highly useful in large-scale screening of mutants.

The regulation of the vanadium chloroperoxidase from Curvularia inaequalis.

The effects of carbon and nitrogen source on the regulation of the vanadium chloroperoxidase secreted by the fungus Curnularia inaequalis were investigated. The addition of glucose showed a repressing effect on both the observed messenger RNA level and the measured enzyme activities, whereas the addition of glutamate as nitrogen source and the addition of both glutamate and glycerol had no effect. Addition of vanadate had no effect on the level of mRNA. Eight hundred base pairs of the upstream promoter region of vCPO were sequenced and various features of interest are highlighted. Closer inspection of the mycelium revealed that once secreted, vCPO probably remains tightly associated with the hyphae in two forms, one of which may be a proform of the enzyme. A possible cleavage event at the C-terminus may lower its potential for hyphal association and permit its disassociation into the growth medium. A putative role for the vanadium chloroperoxidase is put forward.

Isolation and characterization of a novel nonheme chloroperoxidase.

Chloroperoxidase, purified from the fermentation of Curvularia inaequalis, had a molecular weight of approximately 240,000 and was composed of 4 subunits of identical molecular weight (Mr 66,000). The enzyme was specific for I-, Br- and Cl-, and inactive toward F-. The optimum pH of the enzyme was centered around 5.0. X-ray fluorescence revealed that the enzyme contained 2.2 atoms of zinc and 0.7 atom of Fe per molecule of protein. The enzyme had no heme-like compound as a prosthetic group, making it the first nonheme chloroperoxidase to be reported. Under oxidative conditions that rapidly inactivated other haloperoxidases, this enzyme was remarkably stable.

A kinetic and spectral study of the alkaline transitions of chloroperoxidase.

The optical spectrum of chloroperoxidase in the near ultraviolet and visible region was studied from pH 6 to 12. Chloroperoxidase undergoes a first transition which is irreversible at pH 7 and a second transition near pH 11. The second transition is reversible provided the incubation period above pH 11 is kept as short as possible. The spectral properties of the intermediates were studied in the Soret region by means of a rapid scan apparatus. The rates of the transitions were measured in a stopped-flow apparatus. The pH dependence of both the spectra and the rate constants indicate that at least three ionizations are involved in the first alkaline transition.