Tyrosine hydroxylase activity of immobilized tyrosinase on enzacryl-AA and CPG-AA supports: Stabilization and properties

Frog epidermis tyrosinase has been immobilized on Enzacryl-AA (a polyacrylamide-based support) and CPG(zirclad)-Arylamine (a controlled pore glass support) in order to stabilize the tyrosine hydroxylase activity of the enzyme; in this way, the immobilized enzyme could be used to synthesize L-dopa from L-tyrosine. The activity immobilization yield Y(IME) (act) (higher than 86%), coupling efficiency (up to 90%), storage stability (no loss in 120 days), and reaction stability (t(1/2) was higher than 20 h in column reactors) were measured for tyrosinase after its immobilization. The results showed a noticeable improvement (in immobilization yield, coupling efficiency, and storage and operational stabilities) over previous reports in which tyrosinase was immobilized for L-dopa production. The activity and stability of immobilized enzyme preparations working in three different reactor types have been compared when used in equivalent conditions with respect to a new proposed parameter of the reactor (R(p)), which allows different reactor configurations to be related to the productivity of the reactor during its useful life time. The characteristic reaction inactivation which soluble tyrosinase shows after a short reaction time has been avoided by immobilization, and the stabilization was enhanced by the presence of ascorbate. However, another inactivation process appeared after a prolonged use of the immobilized enzyme. The effects of reactor type and operating conditions on immobilized enzyme activity and stability are discussed.     

Hydroxylating activity of frog epidermis tyrosinase.

Trypsin activated in a similar way both the tyrosine hydroxylase and the dopa-oxidasa activities of frog epidermis tyrosinase. Several electron donors reduced or eliminated the lag period for the hydroxylating enzyme. 4 x 10(-5) M dopa was particularly effective, but without affecting the stationary activity after lag period. Tyrosine hydroxylase had KM = 2.6 X 10(-3) M for tyrosine and 2 x 10(-3) M dopa was a competitive inhibitor with Ki = 5 x 10(-4) M. The enzyme was inactivated during its actuation. Data on thermal denaturation were similar to other obtained from dopa oxidase. Our results tend to confirm our previous hypothesis that the activatory process of the enzyme is accompanied by a spatial unfolding of the enzyme molecule.     

Dithiothreitol inactivation of frog epidermis tyrosinase.

Frog epidermis tyrosinase inactivation by dithiothreitol (DTT), both in the proenzyme and active forms, have been studied. Upon increasing DTT:enzyme-up to 1o(6):1 ratios and depending on the incubation period, two inactivation steps both in proenzyme and enzyme were observed. Enzyme lost its activity faster than proenzyme. Oxygen favoured inactivation. After dialysis of the DTT:protein (10(6):1) incubation medium, 20% of the original enzyme activity was recovered. However it decreased to 15% if the enzyme had been incubated with substrate. Conformational changes due to loss of activity were not shown on the fluorescence spectra.     

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.     

Immobilized pectolytic enzymes on acrylic supports

The paper deals with the pectolytic enzymes immobilization on different acrylic supports, and the application of immobilized preparations in the apple juice pectinization process. The correlation between the protein content (C_p) and specific catalytic activities of immobilized enyzme preparations suggest a specific immobilization process only in the case of PONILEX ASH type acrylic supports. The active immobilization degree on PONILEX ASH type supports of Ultrazym 100 G and technical pectinase extract ranged from 99.80 to 296.40% for the Pectinesterase (PE) activity, and from 101.85 to 252.94% for the chain splitting (CS) activity, proving that the ionic immobilization process is a selective one. The simulated operational stability of the immobilized pectolytic enzymes tested by the PE and CS activity values proves the preservation of enzyme catalytic activity.     

A hydrostatic Michell framework supports frog lungs

A braced framework of tubular struts, in the walls and air spaces of frog lungs, suspends the respiratory surface and holds the lung open at zero transmural pressure withstanding imploding forces created by abdominal viscera, much as would the supports of a bell tent. The struts are tubes, having a larger second moment of area than do solid struts of the same cross-sectional area, and so are stronger, and contain pulmonary vessels within a flexible wall. The orthogonal arrangement of the struts in the framework, explained in part by Maxwell’s Lemma and Michell’s Theorem, strengthens the framework and minimizes its weight; orthogonality is maintained as the lungs change size. A model is presented, in which a frog might control pre-and post-pulmonary vascular resistances and, hence, blood volume in the struts, without compromising pulmonary perfusion. Such adjustments could vary the area of lung and the extent of perfused capillaries exposed to pulmonary gas, helping match the lung’s surface area, weight and metabolic load to activity.     

Immobilized electric eel acetylcholinesterasemii. II. Flow kinetics of acetylcholinesterase chemically attached to nylon tubing.

Acetylcholinesterase has been attached covalently to the inner surface of nylon tubing. An experimental study has been carried out on the flow kinetics; solutions of acetylthiocholine at various concentrations were passed through tubing at various flow rates, and measurements made of the rates of formation of product. The results were analyzed in the light of the theoretical treatment of Kobayashi and Laidler, four different methods of analysis being employed. It is found that at lower substrate concentrations and flow rates the reactions are largely diffusion controlled. The Km(app) values are substantially higher than the Km value for diffusion-free conditions, but approach it as the flow rate is increased, when the diffusion layer becomes less important. The results are entirely consistent with the Kobayaski-Laidler theory, and provide guidelines for the design of open tubular heterogeneous enzyme reactors, both for industrial and analytic purposes.     

Immobilized artificial membrane chromatography: supports composed of membrane lipids

Cell membranes provide an environment for several types of molecular processes and we are attempting to mimic the cell membranes' environment on a chromatography solid support. Chromatography solid supports utilizing lecithin as the bonded phase were synthesized and the HPLC behavior of hydrophilic peptides evaluated. A diC14 lecithin containing a terminal carboxy group on the C2 fatty acid chain was amidated with the surface amines of Nucleosil-300 (7NH2) silica particles. Based on elemental analysis, lecithin was coupled to Nucleosil-300 (7NH2) at a surface density near that of lecithin found in biological membranes and this novel chromatographic support material is denoted as Nucleosil-lecithin, the prototype immobilized artificial membrane. Infrared difference spectra of Nucleosil-lecithin minus Nucleosil-300 (7NH2) clearly showed amide I (1653.1 cm-1) and amide II (1550.9 cm-1) bands, giving direct spectroscopic evidence for the amide linkage. Spectral deconvolution resolved two peaks for the amide I band, and three peaks for the amide II band. This demonstrates lecithin interchain amide hydrogen bonding and/or hydrogen bonds between the lecithin amide link and unreacted silica surface amines. Nucleosil-lecithin as a solid phase mimics membranes and can be used to study the interactions of biomolecules with membranes. Our primary objective is to develop HPLC methods for studying the interaction between cell membranes and peptide sequences found near the interfaces of cell membranes. A frequency distribution of amino acids bracketing approximately 400 transmembrane peptide sequences showed Cys to be the least frequently occurring amino acid at this putative interfacial membrane region. Hydrophilic peptide analogs bearing Cys were used as model compounds to test Nucleosil-lecithin solid supports. Small peptides, six to eight amino acids in length, containing Cys bind approximately 2X tighter to Nucleosil-lecithin compared to identical peptides without the Cys residue. Thus, Cys at the interface of cells may stabilize protein-lipid interactions.     

Enzymatic oxidation by frog epidermis tyrosinase of 4-methylcatechol and p-cresol. Influence of L-serine

A study has been made of the kinetics of cresolase and catecholase activities of tyrosinase on the p-cresol/4-methyl-catechol pair in the presence of L-serine. For this, a spectrophotometer assay for both activities based on the spectrophotometric and stoichiometric characteristics of the chemical reactions in the evolution of 4-methyl-o-benzoquinone is proposed. The presence of L-serine in the reaction medium caused a modification in the lag period and the steady-state rate expressed during the cresolase activity of tyrosinase, but no modification was observed during the expression of catecholase activity. These results can be explained taking into account the complex mechanism proposed for tyrosinase which included the chemical steps involved in the process. Furthermore, a singular form of regulation of enzymatic activity by L-serine has been clarified, not by any direct interaction between the protein molecule and the nucleophile, but by modification of the chemical reactions involved in the mechanism of tyrosinase.     

Kinetic study of the interaction between frog epidermis tyrosinase and chloride

The effect of halide ions on frog epidermis tyrosinase has been characterized with the trypsin-activated enzyme. At pH 7, the order of inhibition is I- greater than Br- greater than Cl- greater than F-. Chloride, the most extensively studied halide, shows a competitive pattern with respect to the substrate, L-DOPA. Inhibition is strongly pH-dependent, with a pKa of 6.12 for the responsible protonatable group. Other kinetic constants are also calculated using a novel approach. The mechanism of interaction between chloride and the enzyme is discussed, and a model is proposed in which chloride interferes the tyrosinase activity by displacing a catalytically important ligand, probably a histidine residue of the side-chain, from the copper at the enzyme-active site.     

Immobilized glucose oxidase on different supports for biotransformation removal of glucose from oligosaccharide mixtures

Four immobilized forms of glucose oxidase (GOD) were used for biotransformation removal of glucose from its mixture with dextran oligosaccharides. GOD was biospecifically bound to Concanavalin A-bead cellulose (GOD-ConA-TBC) and covalently to triazine-bead cellulose (GOD-TBC). Eupergit C and Eupergit CM were used for preparation of other two forms of immobilized GOD: GOD-EupC and GOD-EupCM. GOD-ConA-TBC and GOD-EupC exhibited the best operational and storage stabilities. pH and temperature optima of these two immobilized enzyme forms were broadened and shifted to higher values (pH 7 and 35 °C) in comparison with those of free GOD. The decrease of V_max values after immobilization was observed, from 256.8 ± 7.0 μmol min⁻¹ mg_GOD⁻¹ for free enzyme to 63.8 ± 4.2 μmol min⁻¹ mg_GOD⁻¹ for GOD-ConA-TBC and 45 ± 2.7 μmol min⁻¹ mg_GOD⁻¹ for GOD-EupC, respectively. Depending on the immobilization mode, the immobilized GODs were able to decrease the glucose content in solution to 3.8–15.6% of its initial amount The best glucose conversion, was achieved by an action of GOD-EupCM on a mixture of 100 g dextran with 9 g of glucose (i.e. 98.7% removal of glucose).     

Stabilization of enzymes by multipoint attachment via reversible immobilization on phenylboronic activated supports

In this work, we have used supports activated with m-amino-phenylboronic groups to “reversibly” immobilize proteins under very mild conditions. Most of the proteins contained in a crude extract from E. coli could be immobilized on Eupergit C-250 L activated with phenylboronic and then fully desorbed from the support by using mannitol or SDS. This suggested that the immobilization of the proteins on these supports was not only via sugars interaction, but also by other interaction/s, quite unspecific, that might be playing a key role in the immobilization of the proteins. Penicillin acylase from E. coli (PGA) was also immobilized in Eupergit C activated with m-amino-phenylboronic groups. The enzyme could be fully desorbed with mannitol immediately after being immobilized on the support. However, longer incubation times of the immobilized preparation caused a reduction of protein elution from the boronate support in presence of mannitol. Moreover, these immobilized preparations showed a higher stability in the presence of organic solvents than the soluble enzyme; the stability also improved when the incubation time was increased (to a factor of 100). By desorbing the weakest bound enzyme molecules, it was possible to correlate adsorption strength with stabilization; therefore, it seems that this effect was due to the rigidification of the enzyme via multipoint attachment on the support.     

Stabilization of enzymes by multipoint immobilization of thiolated proteins on new epoxy-thiol supports

The controlled and partial modification of epoxy groups of Eupergit C and EP-Sepabeads with sodium sulfide has permitted the preparation of thiol-epoxy supports. Their use allowed not only the specific immobilization of enzymes through their thiol groups via thiol-disulfide interchange, but also enzyme stabilization via multipoint covalent attachment. Penicillin G acylase (PGA) from Escherichia coli and lipase from Rhizomucor miehei were used as model enzymes. Both enzymes lacked exposed cysteine residues, but were introduced via chemical modification under very mild conditions. In the first moments of the immobilization, a certain percentage of immobilized protein could be released from the support by incubation with DTT; this confirms that the first step was via a thiol-disulfide interchange. Moreover, the promotion of some further epoxy-enzyme bonds was confirmed because no enzyme release was detected after some immobilization time by incubation with DTT. In the case of the heterodimeric PGA, it was possible to demonstrate the formation of at least one epoxy bond per enzyme subunit by analyzing with SDS-PAGE the supernatants obtained after boiling the enzyme derivatives in the presence of mercaptoethanol and SDS. Thermal inactivation studies showed that these multipoint enzyme-support attachments promoted an increase in the stability of the immobilized enzymes. In both cases, the stabilization factor was around 12-15-fold comparing optimal derivatives with their just-thiol immobilized counterparts.     

Stabilization of a formate dehydrogenase by covalent immobilization on highly activated glyoxyl-agarose supports

Formate dehydrogenase (FDH) is a stable enzyme that may be readily inactivated by the interaction with hydrophobic interfaces (e.g., due to strong stirring). This may be avoided by immobilizing the enzyme on a porous support by any technique. Thus, even if the enzyme is going to be used in an ultra-membrane reactor, the immobilization presents some advantages. Immobilization on supports activated with bromocianogen, polyethylenimine, glutaraldehyde, etc., did not promote any stabilization of the enzyme under thermal inactivation. However, the immobilization of FDH on highly activated glyoxyl agarose has permitted increasing the enzyme stability against any distorting agent: pH, T, organic solvent, etc. The time of support-enzyme reaction, the temperature of immobilization, and the activation of the support need to be optimized to get the optimal stability-activity properties. Optimized biocatalyst retained 50% of the offered activity and became 50 times more stable at high temperature and neutral pH. Moreover, the quaternary structure of this dimeric enzyme becomes stabilized by immobilization under optimized conditions. Thus, at acidic pH (conditions where the subunit dissociation is the first step in the enzyme inactivation), the immobilization of both subunits of the enzyme on glyoxyl-agarose has allowed the enzyme to be stabilized by hundreds of times. Moreover, the optimal temperature of the enzyme has been increased (even by 10 degrees C at pH 4.5). Very interestingly, the activity with NAD(+)-dextran was around 60% of that observed with free cofactor.     

Cloning and sequencing of the cDNA encoding tyrosinase of the Japanese pond frog, Rana nigromaculata.

We cloned and sequenced the cDNA encoding tyrosinase (TYN) of the Japanese pond frog, Rana nigromaculata. The 3511-bp cDNA contained a 54-bp 5'-noncoding region, a 1596-bp open reading frame encoding TYN of 532 amino acids (aa), and a 1861-bp 3'-noncoding region. The aa sequence of frog TYN predicted from the cDNA sequence was homologous to that of mouse and human TYNs. The aa sequence including the copper-binding domain, which is likely the active center of TYN, was highly conserved among these three species and Neurospora crassa, Streptomyces antibioticus, and S. glaucescens. The frog TYN also contains possible glycosylation sites and conserved Cys at sites similar to those in the mouse and human TYNs. There are two hydrophobic regions at the N-terminus and near the C-terminus, which are likely the signal (leader) peptide and a transmembrane domain, respectively.     

The synthesis and activity of tyrosinase during development of the frog Rana pipiens

We have established by radioimmunoprecipitation that tyrosine-DOPA oxidase (TDO, tyrosinase) [EC 1.14.18.1] is first synthesized by frog embryos at the early neurula stage soon after embryonic induction of the neural plate by the underlying chordamesoderm. The DOPA moiety of the enzyme, at the time of its first appearance, is almost inactive enzymatically and can be activated by mild proteolysis (with trypsin). A very large increase in the amount of active DOPA oxidizing enzyme (without trypsinization) is observed at hatching (stage 21), and this is accompanied by melanin deposition in pigment cells. The tyrosine moiety of the enzyme is also partially inactive at the time of first synthesis, but the ratio of active to inactive enzyme remains approximately constant throughout early development. DOPA decarboxylase enzymatic activity is first detected at neurula stage, and this activity is accompanied by the first appearance of catechol amines.     

Kinetic study in the transient phase of the suicide inactivation of frog epidermis tyrosinase

This paper deals with the kinetic study of a multisubstrate mechanism with enzyme inactivation induced by a suicide substrate. A transient phase approach has been developed that enables the deduction of explicit equations of product concentration vs. time. From these equations kinetic constants which characterize the suicide substrate can be obtained. This study with tyrosinase enzyme, which acts on L-dopa and catechol allowed us to determine the corresponding kinetic parameters, indicating that catechol is about 8-times more powerful as a suicide substrate than is L-dopa.