L-DOPA production from tyrosinase immobilized on nylon 6,6

The production of L-DOPA immobilized on chemically modified nylon 6,6 membranes was studied in a batch reactor. Tyrosinase was immobilized on nylon using glutaraldehyde as a crosslinking agent. The effects of membrane pore size and glutaraldehyde concentration upon enzyme uptake and L-DOPA production were investigated. Enzyme uptake was unaffected by glutaraldehyde concentration; approximately 70% uptake was observed when 25% w/v (group 1), 5% (group 2), and 3% (group 3) glutaraldehyde were used, indicating that glutaraldehyde was in excess. Similarly, uptake was the same for membranes with 0.20 and 10 mum pore sizes.Membranes produced using different levels of glutaraldehyde exhibited dramatically different capacities for L-DOPA production, despite the fact that enzyme uptake was equivalent. Membranes from groups 2 and 3 (5% and 3% glutaraldehyde) produced L-DOPA at a rate of 1.70 mg L(-1) h(-1) over 170 h in a 500-mL batch reactor. However, no free L-DOPA was detected when group 1 membranes were used. Experimental evidence suggests that L-DOPA was produced, but remained bound to these membranes via excess glutaraldehyde left over from the immobilization process. Membrane pore size also effected L-DOPA production; less production was observed when 10-mum membranes were used, despite equivalent enzyme uptake. The observed difference in production may be due to differences in the pore density on the two types of membranes which could affect the access of the substrate to the immobilized enzyme.The results of these studies indicate that tyrosinase can be effectively immobilized on nylon 6,6. L-DOPA production was optimal when 0.20-mum-pore-size membranes were activated with 3-5% glutaraldehyde. Stability studies indicated a 20% reduction in activity over 14 days when the immobilized enzyme was used under turnover conditions. (c) 1996 John Wiley & Sons, Inc.     

Electrochemically modulated optrode for glucose.

One of the problems with fibre-optic sensors is the difficulty of finding reversible indicator reagents. This is a particular problem for fibre-optic glucose sensors. The development of an electrochemically modulated fibre-optic probe or optrode has been proposed as a convenient solution. Here the indicator reagent is regenerated electrochemically. In this work a design is proposed that offers considerable advantages in practical applications. In particular, it can be used in the same way as conventional optrodes. The optimization of working parameters and the application of the optrode to flow analysis under steady-state and flow-injection conditions is described.     

l-Dopa synthesis using tyrosinase immobilized on magnetic beads

Magnetic beads were prepared via suspension polymerization of glycidyl methacrylate (GMA) and methyl methacrylate (MMA) in the presence of ferric ions. Following polymerization, thermal co-precipitation of the Fe(III) ions in the beads with Fe(II) ions under alkaline condition resulted in encapsulation of Fe₃O₄ nano-crystals within the polymer matrix. The magnetic beads were activated with glutaraldehyde, and tyrosinase enzyme was covalently immobilized on the support via reaction of amino groups under mild conditions. The immobilized enzyme was used for the synthesis of l-Dopa (1-3,4-dihydroxy phenylalanine) which is a precursor of dopamine. The immobilized enzyme was characterized by temperature, pH, operational and storage stability experiments. Kinetic parameters, maximum velocity of the enzyme (V_max) and Michaelis–Menten constant (K_m) values were determined as 1.05 U/mg protein and 1.0 mM for 50–75 μm and 2.00 U/mg protein and 4.0 mM for 75–150 μm beads fractions, respectively. Efficiency factor and catalytic efficiency were found to be 1.39 and 0.91 for 75–150 μm beads and 0.73 and 0.75 for 50–75 μm beads fractions, respectively. The catalytic efficiency of the soluble tyrosinase was 0.37. The amounts of immobilized protein were on the 50–75 μm and 75–150 μm fractions were 2.7 and 2.8 mg protein/g magnetic beads, respectively.     

Multi‐wavelength light emitting diode‐based disposable optrode array for in vivo optogenetic modulation

We present a light emitting diode (LED)‐based optical waveguide array that can optogenetically modulate genetically targeted neurons in the brain. The reusable part of the system consists of control electronics and conventional multi‐wavelength LED. The disposable part comprises optical fibers assembled with microlens array fabricated on a silicon die. Both parts can be easily assembled and separated by snap fit structure. Measured light intensity is 3.35 mW/mm² at 469 nm and 0.29 mW/mm² at 590 nm when the applied current is 80 mA. In all the tested conditions, the light‐induced temperature rise is under 0.5°C and over 90% of the relative light intensity is maintained at 2 mm‐distance from the fiber tips. We further tested the efficiency of the optical array in vivo at 469 nm. When the optical array delivers light stimulation on to the visual cortex of a mouse expressing channelrhodopsin‐2, the neural activity is significantly increased. The light‐driven neural activity is successfully transformed into a percept of the mouse, showing significant learning of the task detecting the cortical stimulation. Our results demonstrate that the proposed optical array interfaces well with the neural circuits in vivo and the system is applicable to guide animal behaviors.      

Removal of phenols from wastewater by soluble and immobilized tyrosinase

An enzymatic method for removal of phenols from industrial wastewater was investigated. Phenols in an aqueous solution were removed after treatment with mushroom tyrosinase. The reduction order of substituted phenols is catechol > p-cresol > p-chlorophenol > phenol > p-methoxyphenol. In the treatment of tyrosinase alone, no precipitate was formed but a color change from colorless to dark-brown was observed. The colored products were removed by chitin and chitosan which are available abundantly as shellfish waste. In addition, the reduction rate of phenols was observed to be accelerated in the presence of chitosan. Tyrosinase, immobilized by using amino groups in the enzyme on cation exchange resins, can be used repeatedly. By treatment with immobilized tyrosinase, 100% of phenol was removed after 2 h, and the activity was reduced very little even after 10 repeat treatments. (c) 1993 John Wiley & Sons, Inc.     

Biodegradation of bisphenols with immobilized laccase or tyrosinase on polyacrylonitrile beads

The biodegradation of waters polluted by some bisphenols, endowed with endocrine activity, has been studied by means of laccase or tyrosinase immobilized on polyacrylonitrile (PAN) beads. Bisphenol A (BPA), Bisphenol B (BPB), Bisphenol F (BPF) and Tetrachlorobisphenol A (TCBPA) have been used. The laccase-PAN beads system has been characterized as a function of pH, temperature and substrate concentration. The biochemical parameters so obtained have been compared with those of the free enzyme to evidence the modification induced by the immobilization process. Once characterized, the laccase-PAN beads have been employed in a fluidized bed reactor to determine for each of the four bisphenols the degradation rate constant (k); the τ(50), i.e., the time to obtain the 50% of degradation, and the removal efficiency (RE(90)) after 90 min of enzyme treatment. The same parameters have been measured for each of the four pollutants with the same fluidized bed bioreactor loaded with tyrosinase-PAN beads. The internal comparison, i.e., in each of the two catalytic systems, has shown that both enzymes exhibit a removal efficiency in the following order BPF>BPA>BPB>TCBPA. The external comparison, i.e., the comparison between the two catalytic system, has shown that the catalytic power of laccase were higher than that of tyrosinase. The operational stability of both catalytic systems resulted excellent, since they maintained more than 80% of the initial activity after 30 days of work.     

Amperometric biosensor based on tyrosinase immobilized on a boron-doped diamond electrode

A novel method has been developed to immobilize tyrosinase onto the surface of boron-doped diamond (BDD) electrode. The hydrogen-terminated BDD (HBDD) surface was first functionalized by photochemically linking vinyl groups of allylamine, producing covalently linked amine-terminated active BDD (ABDD) surface. Then the tyrosinase was immobilized onto the ABDD surface by carbodiimide coupling reaction. The amperometric response was measured as a function of concentration of phenolic compounds in 0.1M phosphate buffer solution (pH 6.5). The tyrosinase-modified ABDD electrode gave a linear response range of 1-175, 1-200 and 1-200 microM and sensitivity of 80.0, 181.4 and 110.0 mA M(-1)cm(-2) for phenol, p-cresol, 4-chlorophenol, respectively. Moreover, selective detection of dopamine (DA) in the presence of ascorbic acid (AA) has been demonstrated with the tyrosinase-modified ABDD electrode. Linearity was observed within the range of 5-120 microM. The above enzyme electrode could maintain 90% of its original activity after intermittent use for 1 month when storing in a dry state at 4 degrees C.     

Glucose determination using immobilized polyphosphate glucokinase.

Polyphosphate glucokinase (EC 2.7.1.63, polyphosphate:glucose phosphotransferase) was covalently coupled to collagen-coated silica gel beads. The immobilized enzyme, as a packed-bed reactor, was used to determine glucose in serum and other samples. The method was based on a spectrophotometric measurement of NADPH produced by two consecutive reactions, similar to the hexokinase method. The described approach takes advantage of the greater stability of polyphosphate compared to that of ATP, the greater specificity of polyphosphate glucokinase versus that of hexokinase, and the reusability of the immobilized enzyme. Linearity, precision, and accuracy of the method were tested and found to be very good. The results were linear between 10 and 50 nmol of glucose in a 50-microliter sample and the coefficient of variation was less than 4% in five successive determinations. The recovery of glucose was about 100% after calibration of the method. The results of the measurements correlated well with those obtained with soluble polyphosphate glucokinase (r = 0.997, y = 1.036x - 0.016). The immobilized-enzyme reactor showed good operational stability during a month of use, losing about 12% of its initial activity.     

Aspartate aminotransferase immobilized on collagen films. Activity of dissociated subunits.

Aspartate aminotransferase has been covalently bound to insoluble films of collagen. The immobilized subunits of holoenzyme obtained after alkaline dissociation were inactive but their activity was recovered by incubation with either active enzyme, reduced holoenzyme, or apoenzyme. It was also possible to reduce the bound subunits by sodium borohydride and to reassociate them with native subunits of holoenzyme.     

Sequential conversion by catalytically active MIP and immobilized tyrosinase in a thermistor

To amplify the heat-signal generated by MIP catalyzed solvolysis of phenylacetate the reaction has been combined for the first time in a reactor with the subsequent oxidation by immobilized tyrosinase. The polymer cleaves the substrate and the released phenol is afterwards converted to o-benzoquinone by the tyrosinase. The separated and sequentially coupled reactions are characterized by the heat generated in a thermistor. The sequential substrate conversion results in a combined heat generation which results a five times higher signal than compared to the polymer alone.     

A simple and disposable sweat collector.

Apart from in cystic fibrosis, where sweat analysis provides valuable diagnostic information, sweat yields remain an overlooked biological fluid. Technical problems (dilution, condensation, contamination, evaporation, etc.) linked to currently available collection procedures are of concern and thwart their use. To overcome some of these technical difficulties, an original sweat-collection technique is described. A collection capsule is created inside a flexible, adhesive and disposable anchoring membrane pasted onto the skin. A fluid-tight window is positioned in the upper part of the pocket and gives access to its content. Through the collection window, complete emptying of the sweat collector can be achieved repeatedly by suction using a vacutainer tube inserted in a tube holder equipped with a long dull needle. With prior addition of a suitable marker, fractional samplings can also be performed using a precision micropipette. This collecting method allows for kinetic studies on sweat rate and sweat content. The limited bias-inducing manipulations linked to the described technique, coupled with the ease of performing kinetic studies on sweat volume and content, make this original tool a reliable and accurate sweat-collection technique.     

Development of electrochemical biosensor based on tyrosinase immobilized in composite biopolymeric film

An electrochemical enzyme electrode for dopa and dopamine was developed via an easy and effective immobilization method. The enzyme tyrosinase was extracted from a plant source Amorphophallus companulatus and immobilized in a novel composite of two biopolymers: agarose and guar gum. This composite matrix-containing enzyme forms a self-adhering layer on the active surface of glassy carbon electrode, making it a selective and sensitive phenol sensor. Dopa and dopamine were determined by the direct reduction of biocatalytically liberated quinone species at -0.18V versus Ag/AgCl (3M KCl). The analytical characteristics of this sensor, including linear range, lower detection limit, pH, and storage stability, are described. It has reusability up to 15 cycles and a shelf life of more than 2 months.     

A disposable bio-nano-chip using agarose beads for high performance immunoassays

This article reports on the fabrication of a disposable bio-nano-chip (BNC), a microfluidic device composed of polydimethylsiloxane (PDMS) and thiolene-based optical epoxy which is both cost-effective and suitable for high performance immunoassays. A novel room temperature (RT) bonding technique was utilized so as to achieve irreversible covalent bonding between PDMS and thiolene-based epoxy layers, while at the same time being compatible with the insertion of agarose bead sensors, selectively arranged in an array of pyramidal microcavities replicated in the thiolene thin film layer. In the sealed device, the bead-supporting epoxy film is sandwiched between two PDMS layers comprising of fluidic injection and drain channels. The agarose bead sensors used in the device are sensitized with anti-C-reactive protein (CRP) antibody, and a fluorescent sandwich-type immunoassay was run to characterize the performance of this device. Computational fluid dynamics (CFD) was used based on the device specifications to model the bead penetration. Experimental data revealed analyte penetration of the immunocomplex to 100 μm into the 280 μm diameter agarose beads, which correlated well with the simulation. A dose-response curve was obtained and the linear dynamic range of the assay was established over 1 ng/mL to 50 ng/mL with a limit of detection less than 1 ng/mL.     

Effects of the immobilization supports on the catalytic properties of immobilized mushroom tyrosinase: a comparative study using several substrates

Mushroom tyrosinase was immobilized from an extract onto glass beads covered with one of the following compounds: the crosslinked totally cinnamoylated derivatives of glycerine, D-sorbitol, D-manitol, 1,2-O-isopropylidene-alpha-D-glucofuranose, D-glucuronic acid, D-gulonic acid, sucrose, D-glucosone, D-arabinose, D-fructose, D-glucose, ethyl-D-glucopyranoside, inuline, dextrine, dextrane or starch, or the partially cinnamoylated derivative 3,5,6-tricinnamoyl-D-glucofuranose which was obtained by the acid hydrolysis of 1,2-O-isopropylidene-alpha-d-glucofuranose. The enzyme was immobilized by direct adsorption onto the support and the quantity of tyrosinase immobilized was found to increase with the hydrophobicity of the supports. The kinetic constants of immobilized tyrosinase acting on the substrates, 4-tert-butylcatechol, dopamine and DL-dopa, were studied. When immobilized tyrosinase acted on 4-tert-butylcatechol, the values of K(m)(app) were lower than these obtained for tyrosinase in solution while, when dopamine and DL-dopa were used, the K(m)(app) were higher. The order of the substrates as regards their ionizable groups, DL-dopa (two ionizable groups)>dopamine (one ionizable group)>4-tert-butylcatechol (no ionizable group) coincided with the order of the K(m)(app) values shown by tyrosinase immobilized on the hydrophobic supports, and was the inverse of that observed for tyrosinase in solution. The K(m)(app) values of immobilized tyrosinase were in all cases higher than those of soluble tyrosinase and depended on the nature of the support and the hydrophobicity of the substrate, meaning that it is possible to design supports with different degrees of selectivity towards a mixture of enzyme substrates in the reaction medium.     

Steroid transformation using magnetically immobilized Mycobacterium sp.

Mycobacterium sp. (NRRL-B 3683) has been immobilized by adhesion of magnetic materials of submicron size to the bacterial surface. Preparations based on laboratory-prepared magnetic oxide that had been derivatized with hydrophobic octyltrichlorosilane showed the best properties. The magnetically immobilized bacteria were used for side-chain degradation of cholesterol into androsta-1,4-diene-3,17-dione. The magnetic bacteria behaved as free cells in the transformation media and no mass transfer limitations were observed. The magnetic bacteria could be used repeatedly without any cell loss, the cells being retrieved at the end of each transformation cycle by a magnet.     

Bioaffinity chromatography of thyrotropin using immobilized concanavalin A

The behavior of crude preparations of whale and bovine thyrotropins was studied on an affinity column packed with concanavalin A-Sepharose. Only a small portion of the proteins applied was adsorbed to the column and eluted quantitatively with 0.5 M methyl-alpha-D-glucoside or -mannoside. Immunoreactive as well as hormonally active thyrotropin was recovered exclusively in the absorbed fraction. The usefulness of this chromatographic procedure for the group separation of pituitary glycoprotein hormones is discussed.     

Enzymatic determination of BPA by means of tyrosinase immobilized on different carbon carriers

Different tyrosinase carbon paste modified electrodes to determine bisphenol A (BPA) concentration in aqueous solutions have been constructed. Variables examined were in the carbon paste composition and in particular: (i) the immobilized enzyme amount; (ii) the carbon type (powder, single or multi-walled nanotubes); (iii) the nature of the pasting oil (mineral oil, hexadecane and dodecane). For each biosensor type the amperometric response was evaluated with reference to the linear range and sensitivity. Constant reference has been made to the amperometric signals obtained, under the same experimental conditions, towards the catechol, a specific phenolic substrate for tyrosinase. The most efficient biosensors were those constructed by using the following composition for the carbon paste: 10% of tyrosinase, 45% of single wall carbon nanotubes (SWCN) and 45% of mineral oil. This biosensor formulation displayed the following electrochemical characteristics: a sensitivity equal to 138 microA/mM, LOD of 0.02 microM (based on three times the S/N ratio), linear range of 0.1-12 microM and response time of 6 min. This experimental work represents a first attempt at construction of a new carbon nanotube-tyrosinase based biosensor able to determine the concentration of BPA, one of the most ubiquitous and hazardous endocrine disruptors which can pollute the drinking and surface water, as well as many products of the food chain.     

Kinetic cooperativity of tyrosinase. A general mechanism

Tyrosinase shows kinetic cooperativity in its action on o-diphenols, but not when it acts on monophenols, confirming that the slow step is the hydroxylation of monophenols to o-diphenols. This model can be generalised to a wide range of substrates; for example, type S(A) substrates, which give rise to a stable product as the o-quinone evolves by means of a first or pseudo first order reaction (α-methyl dopa, dopa methyl ester, dopamine, 3,4-dihydroxyphenylpropionic acid, 3,4-dihydroxyphenylacetic acid, α-methyl-tyrosine, tyrosine methyl ester, tyramine, 4-hydroxyphenylpropionic acid and 4-hydroxyphenylacetic acid), type S(B) substrates, which include those whose o-quinone evolves with no clear stoichiometry (catechol, 4-methylcatechol, phenol and p-cresol) and, lastly, type S(C) substrates, which give rise to stable o-quinones (4-tert-butylcatechol/4-tert-butylphenol).     

Antifouling potential of Subtilisin A immobilized onto maleic anhydride copolymer thin films

The proteinaceous nature of the adhesives used by most fouling organisms to attach to surfaces suggests that coatings incorporating proteolytic enzymes may provide a technology for the control of biofouling. In the present article, the antifouling (AF) and fouling release potential of model coatings incorporating the surface-immobilized protease, Subtilisin A, have been investigated. The enzyme was covalently attached to maleic anhydride copolymer thin films; the characteristics of the bioactive coatings obtained were adjusted through variation of the type of copolymer and the concentration of the enzyme solution used for immobilization. The bioactive coatings were tested for their effect on the settlement and adhesion strength of two major fouling species: the green alga Ulva linza and the diatom Navicula perminuta. The results show that the immobilized enzyme effectively reduced the settlement and adhesion strength of zoospores of Ulva and the adhesion strength of Navicula cells. The AF efficacy of the bioactive coatings increased with increasing enzyme surface concentration and activity, and was found to be superior to the equivalent amount of enzyme in solution. The results provide a rigorous analysis of one approach to the use of immobilized proteases to reduce the adhesion of marine fouling organisms and are of interest to those investigating enzyme-containing coating technologies for practical biofouling control.