Alexander Makower

Purification and characterization of the constitutive form of laccase from the basidiomycete Coriolus hirsutus and effect of inducers on laccase synthesis

An isolate of Coriolus hirsutus constitutively expresses substantial amounts of extracellular laccase on a defined growth medium. The most efficient inducer of extracellular laccase synthesis was syringaldazine, which increased the enzyme yield by 1000% at a concentration of 0·11 μM. The constitutive form of the enzyme was purified 312‐fold. Laccase from C. hirsutus, with an estimated molecular mass of 55 kDa and pI of 4·0, is a monomeric glycoprotein containing 12% carbohydrate consisting of mannose and N‐acetylglucosamine. The laccase was found to contain 3·9–4·1 copper atoms per molecule. The absorption spectrum shows a maximum at 610 nm and a shoulder at 330 nm, which is typical of laccase possessing type 1 and type 3 copper atoms. The parameters of the first type of copper were determined by EPR as g⊥=2·046 and g ∥=2·200, A∥ =8·103×10−3 cm−1. Laccase was found to be a pH‐stable and thermostable enzyme. With organic substrates it exhibits a pH optimum of 4·5, but with the inorganic substrate K4[Fe(CN)6] this decreased to 3·5. The highest efficiency of catalysis was observed with sinapinic acid as the substrate. The kinetic constants kcat and Km of this reaction were 578 s −1 and 24 μM respectively. It was established that the kinetics of the assayed reaction shows a Ping Pong mechanism.

Catecholamine detection using enzymatic amplification.

Different amplification sensors based on the substrate recycling principle were investigated with respect to their applicability to catecholamine detection. In the bioelectrocatalytic approach, glassy carbon electrodes were modified by laccase or a PQQ-dependent glucose dehydrogenase. Substrate recycling occurs and the detection limit is in the lower nanomolar concentration range (e.g. 10 nM dopamine and 1 nM noradrenaline for the laccase- and glucose dehydrogenase-modified electrodes, respectively). Combinations of glucose dehydrogenase with laccase or tyrosinase were investigated as bienzymatic probes. Among the systems we studied, the laccase/glucose dehydrogenase sensor is the most sensitive (detection limit: 0.5 nM adrenaline). The selectivities of the different sensor systems are discussed. Application of the laccase/glucose dehydrogenase electrode in different media (i.e. brain homogenate, heart effluate) was successfully shown. For samples with high concentrations of interfering substances (uric and ascorbic acid), the interferences can be effectively removed using enzymatic methods.

Determination of p-aminophenol and catecholamines at picomolar concentrations based on recycling enzyme amplification

A bienzyme electrode based on the amplification of a signal has been developed which allows the determination of picoto nanomolar concentrations of p-aminophenol. The active element of the sensor comprised of coimmobilised laccase and glucose dehydrogenase enzymes coupled with an oxygen electrode. Laccase catalyzes p-aminophenol oxidation by oxygen to give p-iminoquinone. The latter is reduced by excess of glucose in the presence of glucose dehydrogenase and results in recycling of the substrate. The detection is realized by measuring the decrease in oxygen concentration. The detection limit for p-aminophenol is 100 pM. The feasibility of the determination of a number of other substrates (polyphenols, polyamines, ferrocene derivatives) in the nanomolar range has been demonstrated. A significant background signal has been found for p-aminophenylphosphate. This background is probably caused by the ability of laccase to catalyze the oxidative dephosphorylation. In the presence of phosphate ions this background is practically completely eliminated. 50 pM of alkaline phosphatase could be determined after a 2 min incubation in p-aminophenylphosphate solution by determination of the p-aminophenol formed as the result of hydrolysis. The whole analysis time does not exceed 5 min. The new technique is suitable for application in alkaline phosphatase based enzyme immunoassays.

Highly sensitive detection of cocaine using a piezoelectric immunosensor

This paper describes the development of a highly sensitive competitive immunoassay with the piezoelectric sensor. The immobilized derivative of cocaine was benzoylecgonine-1,8-diamino-3,4-dioxaoctane (BZE-DADOO). For the immobilization of BZE-DADOO, the conjugate BZE-DADOO with 11-mercaptomonoundecanoic acid (MUA) was synthesized via 2-(5-norbornen-2,3-dicarboximide)-1,1,3,3-tetramethyluronium-tetrafluoroborate (TNTU), followed by the creation of the conjugate monolayer on the piezosensor electrodes. For the optimization of the competitive assay we used electrodes with rough or smooth gold areas and for the interaction with immobilized antigen different anti-cocaine sheep polyclonal (pAb, either whole IgG or Fab fragment) and mouse monoclonal (mAb, whole IgG) antibodies. The assay of cocaine developed achieved a detection limit (LOD) of 100 pmol/l (34 ng/l) using the sheep antibody (IgG) and piezoelectric sensors with a smooth gold surface. The total time of one analysis was 15 min and the measuring area of the sensor could be used more than 40 times without losing its sensitivity.

Ultrasensitive bienzyme sensor for adrenaline

A biosensor consisting of an analyte-recycling two-enzyme system using laccase (Coriolus hirsutus) and PQQ-dependent glucose dehydrogenase in combination with the electrochemical detection of oxygen depletion at a platinum electrode was used for adrenaline determination in the nano- and subnanomolar concentration range. Measurements were performed in a flow cell providing excellent baseline stability and fast recovery of the sensor. Improved design of the polymer matrix resulted in a lower detection limit of 200 pmol/l for adrenaline. The sensor has successfully been applied to the analysis of adrenaline in effluate of isolated rabbit hearts.

Tyrosinase-glucose dehydrogenase substrate-recycling biosensor: A highly-sensitive measurement of phenolic compounds

Mushroom tyrosinase and glucose dehydrogenase from Acinetobacter calcoaceticus were immobilized in poly(vinyl)alcohol membranes and coupled with a Clark-type oxygen electrode to give a substrate (analyte) regenerating cycle for monitoring of nanomolar concentrations of phenolic compounds. In this way the response for catechol, phenol, p-cresol, p-chlorophenol and p-acetamidophenol was amplified by a factor of 450, 300, 240, 150, and 140, respectively. The resulting detection limit for catechol and phenol is 0.6 nmol dm -3 and 0.9 nmol dm -3 , respectively. The measuring linear range for phenol obtained by the amplified electrode extends from 1 to 400 nmol dm -3 . The comparison with the chemical (ascorbic acid) regeneration of the phenolic compounds demonstrates the efficiency of the enzymatic procedure. The biosensor can be used for monitoring of phenolic compounds in environmental or industrial samples.

Application of a sensitive catechol detector for determination of tyrosinase inhibitors

Abstract A substrate regenerating bienzyme sensor was used to measure a variety of organic and inorganic inhibitors with high sensitivity. The bienzyme system consists of the two cooperating enzymes, cytosolic quinoprotein glucose dehydrogenase and mushroom tyrosinase. The cooperation takes place on the substrate/product level. Under kinetic control, compounds that affect one of the enzymes can be detected with high sensitivity. For the construction of the sensor, the enzymes were immobilized in polyvinyl alcohol, coupled to a Clark-type oxygen electrode and the oxygen consumption was monitored during catechol conversion. In this way, carboxylic acids, kojic acids, inorganic ions and thiourea derivatives were studied. Theoretical considerations reveal the relationship between amplification gain and inhibitor concentration.

New principle of direct real-time monitoring of the interaction of cholinesterase and its inhibitors by piezolectric biosensor.

This paper describes a new method for the sensitive detection of cholinesterase inhibitors based on real-time monitoring using a piezoelectric biosensor. The cholinesterase inhibitor paraoxon was immobilized on the sensing surface via a chelate complex as the recognition element. At first, the conjugate of N-mercaptoundecanoic acid (MUA) with Nalpha,Nalpha-bis (carboxymethyl)-L-lysine (NTA-Lys) was chemisorbed to form a self-assembled monolayer on the surface of the gold electrode of the piezosensor. In the next step, paraoxon-spacer-hexahistidine conjugate was linked to the MUA-Lys-NTA layer via the chelate complex with Ni2+. The paraoxon-modified surface thus obtained was applied for the binding of human butyrylcholinesterase (BChE). Regeneration of the sensing surface was achieved by splitting the chelate complex with EDTA and depositing a fresh layer of Ni2+ followed by addition of the paraoxon-spacer-hexahistidine. In the presence of free inhibitors like diisopropylfluorophosphate (DFP), binding of BChE to the surface-bound paraoxon was decreased. In this way, a competitive affinity assay for organophosphorus compounds was developed. The limit of detection for DFP as a model compound was 10 nmol/l (ca. 2 microg/l). This new concept seems suitable for constructing biosensors for the group-specific detection of cholinesterase-inhibiting substances like insecticides in the field.

Electron transfer between cytochrome c and copper enzymes

Abstract The electron transfer between cytochrome c and ascorbate oxidase or laccase from Coriolus hirsutus was investigated using both an electrochemical and a spectrophotometric method. A quasi-reversible cyclic volammogram of cytochrome c was observed on a gold electrode modified with 4,4′-dithiodipyridine. The addition of laccase resulted in the appearance of a catalytic current due to the regeneration of ferricytochrome c by laccase in the presence of oxygen. The second-order rate constant of the reaction between cytochrome c and laccase is calculated to be 9.2 × 103 M−1 s−1 in 50 mM phosphate buffer of pH 5.8. The reaction rate with ascorbate oxidase is almost three orders of magnitude slower. The difference in the redox potential is considered to be the driving force of the reaction between cytochrome c and the copper proteins investigated.

Piezoelectric affinity sensors for cocaine and cholinesterase inhibitors

We report here the development of piezoelectric affinity sensors for cocaine and cholinesterase inhibitors based on the formation of affinity complexes between an immobilized cocaine derivative and an anti-cocaine antibody or cholinesterase. For both binding reactions benzoylecgonine-1,8-diamino-3,4-dioxaoctane (BZE-DADOO) was immobilized on the surface of the sensor. For immobilization, pre-conjugated BZE-DADOO with 11-mercaptomonoundecanoic acid (MUA) via 2-(5-norbornen-2,3-dicarboximide)-1,1,3,3-tetramethyluronium-tetrafluoroborate (TNTU) allowed the formation of a chemisorbed monolayer on the piezosensor surface. The detection of cocaine was based on a competitive assay. The change of frequency measured after 300s of the binding reaction was used as the signal. The maximum binding of the antibody resulted in a frequency decrease of 35Hz (with an imprecision 3%, n = 3) while the presence of 100pmoll(-1) cocaine decreased the binding by 11%. The limit of detection was consequently below 100pmoll(-1) for cocaine. The total time of one analysis was 15min. This BZE-DADOO-modified sensor was adapted for the detection of organophosphates. BZE-DADOO - a competitive inhibitor - served as binding element for cholinesterase in a competitive assay.