MXPA97003737A - Oxidasas containing copper useful as oxidasasde yod - Google Patents

Abstract

A method for the oxidation of iodide is described, which comprises contacting, in an aqueous solution, an oxidase enzyme containing copper and a source of ionic iodide (I ') for a time and under conditions sufficient to allow conversion of iodine iodide to iodine by the enzyme. Enzymes containing copper can be, for example, a laccase oxidase or bilirubin

Description

OXIDASE CONTAINING USEFUL COPPER AS YODIDE OXIDASE Field of the invention The present invention relates to a method for the oxidation of iodide. More specifically, the invention relates to the use of copper containing oxidases, particularly laccases, in the oxidation of iodide.

BACKGROUND OF THE INVENTION Iodine (12) has been widely used for many years as a disinfectant for many types of situations. Skin cleansers, wound disinfection, contact lens cleaning and water hygiene are only a few of the uses to which iodine has been applied. In addition, iodine is useful in catalysts, an additive for animal feed, in pharmaceuticals and as precursor additives for polymers. Although the system of disinfection based on is extremely effective, several factors limit the scope of directly applying the. In particular, production, storage, transportation and handling are extremely dangerous, due to the chemical compounds involved and also due to the toxicity of the same I2 even at moderate concentrations. In general, I2 is obtained from natural sources, such as brine, through processes that use strong inorganic acids, chlorine gas and other hazardous chemical compounds REF: 24793. Iodophors have been developed as carriers of I2 to replace simple l2 solutions for industrial and domestic disinfection. In addition, binary systems capable of generating l2 from a salt of I "and a chemical oxidant are also available, both of which create the need to dispose of potentially toxic large amounts of byproducts. scale and apply I2 as a disinfectant has been found in the generation of l2 based on peroxidase (US patents Nos. 4,282,324, 4,617,190, 4,588,586, 4,937,072, 5,055,287, 5,227,161, 5,169,455, 4,996,146, 4,576,817). of enzymatic peroxidase, the oxidizing agent H2O2 and a source of iodide icr-iao Unfortunately, this method has the disadvantage of requiring hazardous and volatile peroxide or perishing, which has to be either transported or generated in situ by enzymatic steps or additional chemicals, to make the system more complex and expensive .There is thus a need for a method for the production of iodine ion which avoids the need to deal with hazardous chemical compounds either in the plant or in situ and still efficiently produce sufficient quantities of the desired product. The present invention now provides a means to obtain this.

SUMMARY OF THE INVENTION The present invention relates to a method for the production of iodine. The method comprises contacting, in an aqueous solution, a copper-containing oxidase enzyme and a source of ionic iodide (I ") for a time and under conditions sufficient to allow the conversion of the ionic iodide to iodine by the enzyme. a preferred embodiment, the enzyme is either a laccase or bilirubin oxidase.In another preferred embodiment, the method is also carried out in the presence of a substrate with high affinity for the copper-containing enzyme.The substrate acts as a mediator, which it releases electrons in the reaction, thereby improving the rate at which the oxidation of I 'is carried out.When using laccase or oxidized bilirubin, the ABTS substrate is a particularly useful mediator in the oxidation process.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates double reciprocal plots for oxidation of the I 'catalyzed by the laccase Myceliophthora thermophila recombinante (r-MtL). (A) Correlations between initial velocities and concentrations of I- at various pH with 14 μM r-MtL. (B) Dependencies of Km and Vmax (extracted from Figure 1A) on the pH. Figure 2 illustrates the oxidation of I 'catalyzed by rMtL, aided by ABTS. (A) The dependence of the oxidation rates of the initial I "on ABTS and r-MtL concentrations (nM). (B) The pH profile of the oxidation rate of the initial I" with r-MtL 2.4 nM and ABTS 10 μM.

DETAILED DESCRIPTION OF THE INVENTION It has been well documented that certain halide molecules are capable of inhibiting laccase. Specifically, the inhibition of tree and fungal laccases has been described by fluoride (F ") (Koudelka and Ettinger, J. Biol. Chem. 263: 2698-3705, 1988), as well as the inhibition of fungal laccases by bromide (Br ") and chloride (CI") (Naki and Varfolomeev, Biokhimya 46: 1694-1702, 1981). These halides are supposed to interact with the type II copper site of the laccase to result in the interruption of the Transfer of electrons from the site of type I to the dioxygen site The interaction of the laccase with the iodide (I ') has not been previously reported, since the halide inhibition potency is inversely related to the size of the ionic radius, it would be expected that the I ", with the larger ionic radius, would have a weak inhibitory effect on the laccase. However, it is quite surprising to find that I "acts in effect as a substrate by yielding an electron to type I copper. The observation that I" can act as a laccase substrate has led to the development of a method by which I 'is oxidized to elemental iodine by the use of a laccase or other oxidase enzymes containing copper. In an aqueous solution, in which a source of ionic iodide is provided, the laccase slowly converts the I "to I2.The conversion does not require volatile or hazardous chemical compounds such as chlorine.The source of ionic iodide can be any of the currently known sources, such as alkali metal salts in binary iodine disinfectants, raw brine solutions or initially separated from iodine, mother liquor (salines), ionic iodide solutions in which the caliche iodate (sodium nitrate) is reduced to iodine or algae In the case in which the chloride is inhibitory to the enzyme used, the residual chlorine in the starting material must first be reduced to less than the inhibition constant.In the case of Myceliophthora laccase, the constant of inhibition is about 70 nM The enzyme employed in the present process can be any of a variety of oxidase enzymes containing Cu. As the following examples show , although the laccase is the most active in the oxidation of I ', there are other similar enzymes which also provide a qualitatively similar activity, although in many cases at considerably lower levels than the laccase. In particular, the bilirubin oxidase exhibits an activity which is essentially equivalent to that of the laccase. However, other copper-containing enzymes tested, such as tyrosinase and ceruloplasmin, also show some level of activity, indicating that utility is not limited to laccase. Copper containing oxidases are obtainable from a wide variety of plant, fungal, bacterial and animal sources and many are commercially available. In addition to those enzymes listed above, these also include polyphenol oxidase, ferroxidase II, phenoxazinone synthase, glycerol oxidase, and cytochrome oxidase. The preferred oxidase, laccase, is available from a variety of species, particularly the fungal species, for example Aspergillus, Neurospora, Podospora, Botrytis, Collybia, Fomes, Lentinus, Pleurotus, Trametes, Rhizoctonia (US patent application Serial No. 08) / 172,331, incorporated herein by reference), Coprinus, Psatyrella, Myceliophthora (U.S. Patent Application Serial No. 08 / 253,781, incorporated herein by reference), Scytalidium (U.S. Patent Application Serial No. 08 / 253,784 , incorporated herein by reference), Polyporus (U.S. Patent Application Serial No. 08 / 265,534, incorporated herein by reference), Phlebia (WO 92/01046) and Coriolus (JP 2-238885). Additionally, bilirubin oxidase is readily available from Myrothecium verrucaria and Trachyderma tsunodae. The reaction is carried out in aqueous solution. The enzyme can be free or in pure or crude form or immobilized on an organic or inorganic support by any means known in the art., for example polyacrylamide, cellulose, glass beads, agarose, dextran, methacrylic polymers or ethylenemalic acid copolymers. Reactions with immobilized enzymes can be carried out in tanks or in columns. The reaction can also be carried out with microbial cells which produce the enzyme. The aqueous solution is regulated to a pH which provides an optimal activity for the enzyme that is used and thus the pH of the solution will vary depending on the enzyme used. The pH is maintained in the preferred range by the use of an appropriate pH regulating agent. Such pH regulators include buffer solutions of phosphate, gluconate, citrate, formate or sodium or potassium acetate. The amount of enzyme used will also vary depending on the identity of the enzyme used, but in general it will be in the range of nM to μM. The amount of I 'used in the reaction mixture should preferably be at mM or higher levels. The reaction can be conducted at a temperature between about 15-50 ° C, but is preferably carried out at a temperature of about 20-30 ° C. In a preferred embodiment of the present method, the reaction as described above is carried out in the presence of a mediator which accelerates the conversion rate of I 'to I2. Although copper-containing enzymes such as laccase oxidize I-, production is usually slow due to low affinity. Accordingly, a compound which has high affinity for the enzyme and which can act as an intermediate in releasing electrons between the enzyme and I ', can improve the efficiency of the reaction. For example, as shown in the examples below, the compound 2,2'-az-rα ± -Ls- (3-ethylbenzthiazoline-6-sulfonic acid) (ATBS), which is a known and excellent substrate for laccase and bilirubin oxidase, greatly improves the efficiency of oxidation of 17 Although ABTS may not be useful for improving the reaction rate with other copper-containing enzymes, such as tyrosinase and ceruloplasmin, since it is not an excellent substrate for these enzymes, alternative mediators can be used which are more appropriate substrates with these enzymes. The important factors in the selection of alternative mediators for any given enzyme is that the mediator is a good substrate for the enzyme and has a redox potential similar or higher than I. "Examples of additional mediators which should be useful for reactions using enzymes other than laccase and bilirubin oxidase are phenolic compounds, heterocyclic hydroquinones and free transition metal ions or coordinated chelates (ie, Fe2 *, Fe (bipyridyl) 22+, Ru (CN) 6 * ", and the like The amount of the mediator will differ depending on the identity of the compound used and its convenience based on the favorable characteristics affirmed; for ABTS, which is an ideal mediator for laccase and bilirubin oxidase when judged by the stated criteria, only a small amount, approximately 0.1 mM, is required to improve the reaction 103 times over the results observed in the absence of ABTS. For any given mediator, the concentration used depends on its Km for the enzyme that is used, which value can be determined systematically by the one skilled in the art. The iodine produced in the course of the reaction can be recovered by any appropriate means, such as filtration / centrifugation, blowing with inert gas and extraction by immiscible organic solvents. The invention is further illustrated by the following non-limiting examples.

EXAMPLES I Materials and Methods A. Materials The chemical compounds used as buffer solutions and substrates are commercial products of at least reactive grade. The ascorbate oxidase (Cucurbita species), ceruloplasmin (porcine plasma), bilirubin oxidase (Myrothecium verrucaria) and tyrosinase (mushrooms) are purchased from Sigma and used as received. The content of the lyophilized powder enzymes or solution is confirmed by UV-visible absorption and published extinction coefficients. The claimed activity of ascorbate oxidase is confirmed with ascorbic acid.

B. Methods The recombinantly produced Myceliophthora thermophila laccase (r-MtL, described in copending patent application Serial No. 08 / 278,473, the content of which is incorporated herein by reference) is purified and the activity is determined by oxidations with syringaldazine and 2,2'-azinobis- (3-ethylbenzthiazo! in-6-sulfonic acid) (ABTS) as follows: Oxidation of syringaldazine is carried out at 30 ° C in a quartz cuvette of 1 cm. 60 μl of concentrated syringaldazine solution (0.28 mM in 50% methanol) and 20 μl of sample are mixed with 0.8 ml of pre-warmed pH buffer. Oxidation is verified at 530 nm for 5 minutes. The ABTS oxidation analyzes are made by using 0.4 mM ABTS, pH regulating solution of B & R at various pH, at room temperature when verifying the absorption change at 418 nm. The extinction coefficient of 36 mM "1cm" 1 is used to calculate the velocity. Spectroscopic analyzes are carried out either on a spectrophotometer (Shimadzu UV160) or a microplate reader (Molecular Devices). PH buffer solutions of Britton & Robinson according to the standard protocol (Quelle, Biochemisches Taschenbuch, H.M. Raven, II, Teil, S.93, 102, 1964). The enzymatic assays for the determination of I 'are carried out in B & R buffer which contains 1-20 nM of MtL (concentration in subunits) and 10 to 200 mM of Nal at 20 ° C for 5 10 minutes. The extinction coefficient of 26 mM "1cm" 1 is used to calculate the velocity. The oxidation of I 'is verified spectrally at 340 or 353 nm and the spectra of the oxidation product is identical to that of l3"(Hosoya, Biochem. (Tokyo) 53: 381, 1963) .The kinetic parameters are extracted by a linear regression adjustment (Prism, GraphPad) of velocity and concentration data to the velocity equation = Vma? * [substrate] / Km + [substrate] .Ascorbate oxidase, ceruloplasmin, bilirubin oxidase and tyrosinase at concentrations up to μM level are also tested under similar conditions for oxidation catalysis of I '.

II Results Catalysts for oxidation of I "by rMtL and other enzymes containing copper In aerated solution, I" can be oxidized slowly to I2. When the I "(in the form of Nal) is in excess, the I3" is formed irreversibly from I "and l2 to give a characteristic spectrum with a band centered around 353 (Hosoya, supra). -MtL can accelerate the process, at a pH of about 3, the oxidation of catalyzed I can be many orders faster than non-enzymatic oxidation. The reciprocals of the initial velocities of the formation of l2 and the concentration of I "exhibit a classical linear correlation (Figure 1A) Over the proven range of 1.15 μM, the maximum initial velocities are proportional to the concentration of r-MtL. activity is optimal at pH 3.4 At a pH higher than 6, the reaction is minimal, as a substrate the I "shows an apparent Km dependent on the pH. The smallest Km, 0.16 ± 0.02 M, is at pH 3.4. The pH dependence of Vma is less significant (figure 1 B). At a pH of 3.4, a Vma of 2.7 ± 0.2 cycles per minute is found. I "is not as good substrate for r-MtL as syringaidazine, whose Km and Vmax at optimum pH (7) are approximately 10 μM and production levels of approximately 400 cycles per minute respectively., ascorbate oxidase at concentrations up to 1.5 μM shows an activity of less than 0.1 cycles per minute. At the μM level, ceruloplasmin shows negligible catalysis in the oxidation of I 'with 0.1 M Nal at pH 6 (approximately 0.1 cycles per minute). As an oxidase of I ", tyrosinase has a pH-activity profile in which the relative activity at pH 4.1, 5.2, 6.0 and >; 7 is -58, 33, 10 and < 5% of that at pH 2.7 respectively. At its optimum pH of 2.7, tyrosinase catalyses the oxidation of I "with a Km of -0.1 M and a Vmax of -0.1 cycles per minute.The initial oxidation rates of I" are proportional to the concentration of tyrosinase over a range from 2 to 20 μM. As an oxidase of I ', the bilirubin oxidase has a pH activity profile in which the relative activity at pH 2.7, 5.2, 6.0, 7.0, 8.2 and > 9 is -95, 91, 80, 59, 32 and < 6% of that at pH 4.1 respectively. At its optimum pH of 4.1, the bilirubin oxidase catalyses the I "with a Km of -0.15 ± 0.03 M and a Vmax of 1.7 ± 0.1 cycles per minute.The initial velocities are proportional to the concentration of bilirubin oxidase over the range of 0.5-5 μM.

Catalysis for oxidation by ABTS. At pH 5, the ascorbate oxidase at concentrations up to 1.5 μM shows no activity in the ABTS oxidation. The oxidation rates of ABTS catalyzed by tyrosinase are proportional to the concentration of tyrosine over the 0.2-2 μM range tested. Tyrosinase has an optimal activity at pH 2.7, of which one Km is 0.18 ± 0.0 1 mM and a Vmax of 3.1 ± 0.1 cycles per minute. The initial rates of oxidation of ABTS catalyzed by ceruloplasmin are proportional to a concentration of ceruloplasmin over the range of 0.1 to 1 μM tested. Ceruloplasmin has an optimal ABTS oxidase activity at pH 4.1 of which is one Km of 0.11 ± 0.04 mM and a Vmax of 9.3 ± 1.3 cycles per minute. The initial oxidation rates of the ABTS catalyzed by the bilirnjbin oxidase are proportional to the oxidase concentration over the 1-10 nM range tested. The pH profile shows an optimum pH of 4.1, of which one Km is 0.12 ± 0.01 mM and one Vma? of (1310 ± 40) cycles per minute. RMtL has an activity pH profile with an optimum pH < 2.7. At pH 4.1, the ABTS has a Km of 5.6 ± 0.5 μM and a Vmax of 1460 ± 31 cycles per minute is found.

Catalysis aided by ABTS. The oxidized ABTS (ABTS +), prepared by pre-incubation with laccase, easily oxidizes I. "In the presence of small amounts of ABTS, the oxidation of I" catalyzed by laccase can be greatly improved (Figure 2A). Kinetic analysis shows that the step that limits speed lies in the oxidation of ABTS through laccase. When the ABTS and I 'are present at sufficient levels, only the spectrum of l3 ~ is detected while the I "remains in excess with respect to the ABTS.At the time the I" is consumed, the spectrum of the oxidized ABTS arises. In the enzymatic catalysis assisted by ABTS of the oxidation of I ", the pH-activity profile (Figure 2B) is similar to that for the oxidation of ABTS catalyzed by rMtL (optimum pH displaced from 3.4 to <2.7). apparent and the Vma (6.4 ± 0.8 μM and 860 ± 40 cycles per minute) extracted from the dependence of oxidation velocities of I- on the concentration of ABTS are close to those of oxidation catalyzed by r-MtL of ABTS itself. Due to its poor activities of ABTS oxidase, ascorbate oxidase, ceruloplasmin and tyrosinase all show a negligible ABTS-assisted catalysis for oxidation of I ". When ABTS is included in the oxidation of I 'catalyzed by bilirubin oxidase, an optimal activity at pH 4.1 is observed. At this pH, an apparent Km of 0.18 ± 0.02 mM and a Vma? of 600 ± 40 cycles per minute for the oxidation of I. "These kinetic parameters, which are close to those for the oxidation of ABTS catalyzed by oxidase itself, indicate that the stage that limits the speed in the catalysis is the oxidation of the ABTS .

Discussion. There have been no previous reports describing the oxidase activity of I 'of the laccase or oxidation of I' catalyzed by oxidase. Although it is known that F "and probably also IC" and Br "are linked to Cu type II in the laccase, the observed catalysis of the laccase in the oxidation of I" implies that I "must interact with copper, I. The redox potentials (against the Normal Hydrogen Electrode (NHE)) of Cu type I are in the range of 480 to 530 mV against the NKE for the r-MtL and the bilirubin oxidase, and approximately 8Q0 mV for the Lacasa Polyporus (Reinhammar, Biochim, Biophys, Acta 275: 245-259, 1972) The potential for the ABTS + / ABTS pair is approximately 7QQ-raV .. These potentials are close to or greater than the l37l pair "(540). mV) to do so to the oxidation of I "by Cu type I or ABTS * thermodynamically feasible.Although I" could be a potentially stronger reducer than ABTS, kinetic factors make the former much less reactive towards the laccase. As a substrate, the ABTS has a Km -103 times smaller and a Vmax -103 times larger compared to the I. "Because the oxidized ABTS freely exchanges electrons with the I" in solution, the electron exchange by the ABTS and the Lacasa greatly improves the oxidation of I "catalyzed by laccase Since the step that limits the speed is the oxidation of ABTS, it is possible to regulate the catalysis for the oxidation of I" by the concentration of ABTS or other appropriate mediators. Among the copper-containing enzymes tested, bilirubin oxidase functions quantitatively similar to laccase, while the other enzymes tested function to direct oxidation of I 'less efficiently. Since ABTS itself is not an excellent substrate for ascorbate oxidase, ceruplasmia and tyrosine, only a minor effect of ABTS to aid oxidation of I 'catalyzed by these enzymes would be expected. However, other materials which are good substrates for these enzymes, such as phenolic compounds, hydroquinones, heterocyclics and chelate or free transition metal ions can be used as alternative mediators with these enzymes for I oxidation.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following

Claims (18)

1. Claims 1. A method for the oxidation of iodide, characterized in that it comprises contacting, in an aqueous solution, an oxidase enzyme containing copper and a source of ionic iodide (D. for a time and under conditions sufficient to allow conversion of iodine iodide to iodine by the enzyme.
2. 2. The method according to claim 1, characterized in that the enzyme is laccase or bilirubin oxidase.
3. 3. The method according to claim 2, characterized in that the laccase is a fungal laccase or bilirubin oxidase.
4. 4. The method according to claim 3, characterized in that the enzyme is a laccase.
5. 5. The method according to claim 3, characterized in that the enzyme is a laccase selected from the group consisting of Myceliophthora laccase, a Scytalidium laccase, a Polyporus laccase and a Rhizoctonia laccase, an Aspergillus laccase, a Neurospora laccase, a Podospora laccase, an laccase Botrytis, a laccase Collybia, laccase Fomes, laccase Lentinus, laccase Pleurotus, laccase Trametes, laccase Coprinus, laccase Psatryella, laccase Phlebia and laccase Coriolus.
6. 6. The method according to claim 1, characterized in that the enzyme is a Myceliophthora laccase, a Scytalidium laccase, a Polyporus laccase or a Rhizoctonia laccase.
7. 7. The method according to claim 1, characterized in that the enzyme is a Myceliophthora laccase.
8. 8. The method according to claim 3, characterized in that the enzyme is a bilirubin oxidase.
9. 9. The method according to claim 3, characterized in that the enzyme is a bilirubin oxidase Myrothecium or Trachyderma.
10. 10. The method according to claim 3, characterized in that the enzyme is Myrothecium bilirubin oxidase.
11. 11. The method according to claim 1, characterized in that it is carried out in the presence of a mediator.
12. 12. The method according to claim 1, characterized in that the mediator is ABTS.
13. 13. A method for the oxidation of iodide, characterized in that it comprises contacting, in an aqueous solution, a fungal laccase or a bilirubin oxidase and a source of ionic iodide (I ") in the presence of a mediator, for a time and under conditions sufficient to allow the conversion of ionic iodide to iodine by the enzyme.
14. 14. The method according to claim 13, characterized in that the mediator is ABTS.
15. 15. The method according to claim 14, characterized in that the enzyme is a laccase.
16. 16. The method according to claim 15, characterized in that the enzyme is a Myceliophthora laccase.
17. 17. The method according to claim 14, characterized in that the enzyme is a bilirubin oxidase.
18. 18. The method according to claim 17, characterized in that the bilirubin oxidase is a bilirubin oxidase Myrothecium.