WO1993019811A1

WO1993019811A1 - Biocatalysis

Info

Publication number: WO1993019811A1
Application number: PCT/GB1993/000651
Authority: WO
Inventors: Stephen Philip Mann; Nicholas Peter Martin Foote
Original assignee: Biotal Ltd.
Priority date: 1992-04-01
Filing date: 1993-03-29
Publication date: 1993-10-14
Also published as: FI944570A; FI944570A0; EP0633801A1; AU3895593A; JPH07505332A; GB9207181D0

Abstract

Environmental contaminants such as ivermectins and chlorophenols are degraded by the use of a hydroperoxide and a metal catalyst. The metal catalyst may be a naturally-occurring complex such as a metalloprotein. The hydroperoxide may be generated in situ, e.g. by the oxidation of linoleic acid.

Description

BIOCATALYSIS Field of the Invention

This invention relates to a biocatalytic process and also to compositions for use in such processes. Background of the Invention

Many complex molecules are found in nature that are toxic to humans and animals alike. Some in addition are manufactured by man to control pests and undesirable parasites. Examples of these naturally-occurring materials are the ergotamines in rye flour and the aflatoxins in maize; both can cause severe toxicological problems when consumed. Of the various synthetic types, many are related to naturally-occurring compounds. Many are also slow to be degraded in the environment. Classical amongst these latter, synthetic compounds are the avermectins, of which ivermectins have found wide application in the treatment of internal and external parasitic infections.

Organic compounds in general, some naturally-occurring and others synthetic, are in general slow to degrade in the environment, especially those which are predominantly hydrocarbon or halohydrocarbon. Such compounds include aromatic compounds such as halogenated phenols, e.g. polychlorophenols such as tetra- and pentachlorophenol, permethrin, substituted benzenes, phenols, e.g. cresol, polynuclear compounds such as polyaromatic hydrocarbons and similar structures with a cyclic formation. There are also many such aliphatic compounds, including aliphatic alcohols and halogenated aliphatics, e.g. unsaturated halogenated aliphatics. Many applications have been reported where transition metals, either alone or complexed, have been used as relatively non-specific catalysts of oxidative reactions. In particular, haem, its derivatives and haem-containing enzymes (especially peroxidases) have proved very versatile. However, these reactions generally take place only under harsh conditions: in the presence of a strong oxidant, at high temperature, or both. For example, US-A—4773966 describes the degradation of lignin model compounds using transition metal ions, but the reactions reguired the presence of persulphate and were carried out by refluxing in 83% acetic acid. O-A-8807988 describes the synthesis of a series of water-soluble tetraphenyl porphyrins which can oxidise lignin, lignin model compounds and kraft pulp, but again these reactions required the addition of a strong oxidant such as a peroxide, peracid or hypochlorite. Paszczynski et al. Appl. Environ. Microbiol. 5 (1) , 62-68 (1988) , used hemin and a synthetic iron porphyrin to bleach kraft pulp, but the reactions required hydrogen peroxide or tert-butyl hydroperoxide and were performed under reflux. Summary of the Invention One or both of the problems described above, i.e. harsh conditions and compounds difficult to degrade, have been met by the discovery of a suitable oxidising system, intended to provide biomimetic catalysis, comprising a hydroperoxide (or the means to provide a hydroperoxide in situ, and a metal catalyst, which is active at ambient temperatures and which does not contain a powerful' oxidant. The compound to be degraded is usually organic, and is one susceptible to reaction with a free radical, and will therefore usually include an unsaturated or conjugated bond structure, and/or a suitable substituent.

The reagent therefore has potential in the degradation of environmental pollutants, being both efficient and safe to use in the field. In industrial processes, such as the bleaching of pulp, it may offer economic advantages, if the reaction can be carried out at lower temperatures and with less toxic by-products than hitherto. Description of the Invention

Metals andmetal complexes, includingmetalloproteins, may be used as catalysts in the oxidative degradation of target substances at room temperature. They have been shown to work in a test system, using ivermectin, an anti- parasitic agent used in veterinary medicine which is known to be destroyed only slowly in the environment. More generally, the material to be degraded may be one of those named above. It may be, for example, a complex glycoside antibiotic or similar multi-ring structure, e.g. having anti-parasitic properties.

By way of example only, the degradation of ivermectin can be carried out under mild conditions. The oxidant may be molecular oxygen. In addition, a co-substrate (reductant) is usually required: particularly effective (and non-hazardous) are unsaturated fatty acids, especially linoleic acid, and their derivatives. The mechanism appears to involve an initial metal-catalysed oxidation of the linoleic acid to linoleic acid hydroperoxide (i.e. a lipoxygenase or lipoxidase activity) in a reaction that consumes molecular oxygen. The linoleic acid hydroperoxide thus formed is then used to attack the target substance. Alternatively the peroxide can be supplied directly as, for instance, t-butyl hydroperoxide or another organic peroxide. Any suitable metal may be used. As shown for instance in Example 5, the catalytic species may be formed jLn situ, where glucose is a reducing agent with respect to Fe(III) or Cu(II) which is respectively converted to Fe(II) or Cu(I) . The optimum pH for the reaction can be determined experimentally. A suitable buffer may be used, in order to minimise pH shifts.

In some circumstances, results can be improved by adding the catalyst gradually over a period of time rather than having it all present at the start. For instance, the concentration of, say, hemin can be increased from 0 to 10 μM, or from 10 to 20 μM, by continuous addition over 24 hours using a pump. This method may counteract loss of catalyst by over-oxidation during the course of the reaction.

A further aspect of the invention lies in the discovery that ivermectin and other such toxic compounds can be made safe by the oxidative properties of a naturally-occurring biocatalyst which may be extracellular or, if necessary, extracted and purified from living organisms. The mechanism of action is apparently stimulated by Mn ions and detergents such as Tween 80. The detergent may itself be broken down.

This invention is based in part on the identification and, if necessary, extraction and purification of biocatalysts from organisms, which may be used as the reaction medium to break down complex molecules of the type described above, either in isolation or in the open environment.

Suitable biocatalysts have been identified in Phanaerochaete and Streptomvces; other microorganism sources may include Pseudomonas and other bacteria. Other potential sources are Coriolus. Coprimus _f Thoma. Phlebia. Humicola. Actinomycetes. snow-mould fungi, wood-rotting and ligninase-containing and, especially, white-rot fungi.

The microorganisms may be found, inter alia, in sheep- dips, fruit-washes, cowpats etc., i.e. where they are present in the environment after use of the toxin. Careful isolation, enrichment procedures etc. may be necessary to avoid the masking effect of other enzymatic activities, but whole cells may be used in some cases. The fact that these organisms may be present in environments including the target compound and also a naturally-occurring metal complex catalysts means that, for its degradation, only a substrate need be added.

The following Examples illustrate the invention. Example 1 Hemin + linoleic acid

A solution containing ivermectin with 50 mM Na acetate pH 4.0, 0.1% (w/v) Tween 20 (a non-ionic detergent), 0.25 g/1 linoleic acid and 10 μM hemin was agitated at 23°C in an orbital incubator. Ivermectin concentration was monitored by HPLC; the results were: Time (hr) Ivermectin concentration (ppm) 0 24 72

In the presence of 0.1 mM manganese sulphate the extent of ivermectin breakdown was generally slightly greater, as shown by the following results:

Time (hr) Ivermectin concentration (ppm) 0 25 50 100 250 500 24 6.0 7.4 8.6 11.5 20.7 72 1.6 4.6 0.2 <1.0 <1.0

Example 2 Haemoglobin + linoleic acid

A solution containing ivermectin with 50 mM Na acetate pH 4.0, 0.1% (w/v) Tween 20, 0.25 g/1 linoleic acid and 20 μg/ml haemoglobin was agitated at 23°C in an orbital incubator.

Time (hr) Ivermectin concentration (ppm) 0 25 50 100 250 500 24 15.0 24.1 45.0 128 359 72 4.2 5.9 11.8 27.5 152

In the presence of 0.1 mM manganese sulphate the results were:

Time (hr) Ivermectin concentration (ppm) 0 25 50 100 250 500 24 9.3 13.8 21.2 45.4 154 72 3.8 4.3 4.7 9.1 54.0

Example 3 Hemin + t-butyl hydroperoxide A solution containing ivermectin with 50 mM K phosphate pH 7.0, 0.1% (w/v) Tween 20, 10 mM t-butyl hydroperoxide and 50 μM hemin was agitated at 23°C in an orbital incubator. Time (hr) Ivermectin concentration (ppm) 0 25 50 100 250 500 24 1.3 2.1 4.0 11.6 121 72 0.5 1.4 2.6 8.5 74.4

The procedure of Example 3 was repeated, but in the presence of tetrachlorophenol or pentachlorophenol, respectively, rather than ivermectin.

A solution containing ivermectin with 50 mM Na borate pH 9.0, 0.1% (w/v) Tween 20, 0.25 g/1 linoleic acid, 0.1 mM manganese sulphate and 20 μg/ml purified soybean lipoxygenase was agitated at 23°C in an orbital incubator.

Time (hr) Ivermectin concentration (ppm) 0 25 50 100 250 500 24 3.6 8.9 19.2 68.2 193 72 5.4 11.3 22.2 60.9 190

In this case there seems to be no further breakdown of ivermectin after 24 hours (increases in concentration are caused by sample evaporation) . Example 5 Metal ions + linoleic acid

A mixture containing 50 mM K phosphate pH 7.0, 0.1% (w/v) Tween 20 or Brij 35, 0.25 g/1 linoleic acid, 10 mM fructose and 10 μM ferric chloride or copper sulphate has been shown to degrade ivermectin. The reaction is slow at room temperature but considerably faster at 37°C. Example 6 Sheep wash + linoleic acid

A "dummy" sheep wash was used as an example of a situation in which ivermectin might be found in the environment. The "sheep wash" was simply water in which sheep had been dipped, and it contained a large amount of faecal matter. To a 10-fold dilution of the wash was added 50 mM Na acetate pH 4.0, 0.1% (w/v) Tween 20, 0.25 g/1 linoleic acid and ivermectin.

Time (hr) Ivermectin concentration (ppm)

0 25 50 100 250 500

24 16.7 29.5 59.3 145 246 72 10.4 19.0 30.0 44.8 117

In this case the catalyst is provided by the test matrix itself, presumably in the form of free or complexed metal ions (such as haem breakdown products) or as microbial metalloproteins. Example 7

Phanaerochaete chrvsoporium was grown in a shake flask in the following defined medium (based on P. Bonnarme & T. . Jeffries, Appl. Environ. Technol. 5j6 (1), 210-217, 1990) :

Glucose 10 g/1

Diammonium D-tartrate 0.2 g/1

Na₂tartrate.2H₂0 4.6 g/1

KH₂P0₄ 2.0 g/1 MgS0₄.7H₂0 0.5 g/1

CaCl₂.2H₂0 0.1 g/1

Tween 80 0.5 g/1

Thiamine.HCl 1.0 mg/1

Veratryl alcohol 2.5 mM Trace element salts 70 ml/1

(pH adjusted to 5.0 with H₃P0₄) The trace element salt solution comprised:

MgS0₄.7H₂0 3.0 g/1

NaCl 1.0 g/1

FeS0₄.7H₂0 0.1 g/1 CθCl₂.6H₂0 0.15 g/1

CaCl₂.2H₂0 0.1 g/1

ZnS0₄.7H₂0 0.1 g/1

CuS0₄.5H₂0 0.01 g/1

H₃BO₃ 0.01 g/1 Na₂Mθ0₄.2H₂0 0.01 g/1

MnCl₂.4H₂0 0.5 g/1

A1K(S0₄)₂.12H₂0 0.02 g/1

Nitriloacetic acid 1.5 g/1

100 ml of medium in an unbaffled 250 ml flask was inoculated with 0.2 ml of a Phanaerochaete chrvsosporium (ATCC 24725) spore suspension (approx. 2 x 10⁷ spores) and shaken at 150 rpm at 30°C. After 48 hr, and subsequently every 24 hr, the flask was flushed for 1 minute with oxygen. After 4 days' growth, the contents of the flask were filtered through a double layer of muslin.

A reaction mixture was prepared containing 250 ppm ivermectin, 50 mM sodium acetate + 10 mM potassium phosphate pH 5.0, 0.1 mM manganese (II) sulphate, 0.1% (w/v) Tween 20 and 0.1 g/1 linoleic acid. 0.5 ml aliquots of this reaction mixture were placed in plastic microcentrifuge tubes, and 0.0145 ml of the clarified culture fluid was added to each. The tubes were capped and incubated at 25°C. At intervals the contents of an entire tube were assayed by HPLC.

Incubation time (hr) [IVM] (g/1)

0 250.0

1.5 223.5

3.5 121.3 5.5 63.8

21.0 1.1 Ivermectin degradation also occurred, at various rates and with a variable lag time, when co-substrates other than linoleic acid were included. For example, the linoleic acid could be replaced by oleic acid or linoleic acid, or both Tween 20 and linoleic acid could be replaced by a detergent containing unsaturated fatty acid chains, such as Tween 80 or Brij 99. Example 8

Lipoxygenase (lipoxidase) was purified from the clarified culture fluid by application to a column of DE-Sepharose in 10 mM sodium acetate pH 6.0, followed by elution with a linear gradient of sodium acetate (10-500 mM in 25 column volumes) . Fractions containing lipoxygenase activity were pooled and concentrated by ultrafiltration. The lipoxidase activity was found to co-purify with manganese peroxidase activity (oxidation of vanillyl alcohol in the presence of hydrogen peroxide and manganese (II) ions) .

The partially-purified enzyme (0.016 U/ml manganese peroxidase activity) was added to the reaction mixture of Example 7 instead of the clarified culture fluid.

Time (hr) Ivermectin concentration (ppm)

0 25 50 100 250 500 24 0.3 0.6 0.5 5.1 3.7

72 <0.2 <0.2 0.7 <1.0 <1.0

Claims

1. Use of a hydroperoxide and a metal complex catalyst, to degrade an environmental contaminant susceptible to reaction with a free radical.

2. Use according to claim 1, wherein the catalyst is a biological metal complex.

3. Use according to claim 2, wherein the complex is a metalloporphyrin, metalloprotein, haem or a derivative thereof.

4. Use according to any preceding claim, wherein the hydroperoxide is generated .in situ by reaction of a suitable substrate and an oxidising system.

5. Use according to claim 4, wherein the substrate is linoleic acid or another unsaturated long-chain fatty acid.

6. Use according to claim 4 or claim 5, wherein the oxidising system utilises ambient oxygen.

7. Use according to claim 4, wherein the reaction is by means of the metal catalyst.

8. Use according to any of claims 4 to 7, wherein the oxidising system comprises an enzyme.

9. Use according to claim 8, wherein the enzyme is a peroxidase, lipoxidase or lipoxygenase.

10. Use according to any claim 9, wherein the enzyme is as found in Phanaerochaete.

11. Use according to any preceding claim, wherein the degradation is conducted under ambient conditions.

12. Use according to any preceding claim, wherein the degradation is conducted in the presence of a surfactant.

13. Use according to any preceding claim, wherein the degradation is conducted in the presence of Mn ion.

14. Use according to any preceding claim, wherein the contaminant is selected from halohydrocarbons, substituted benzenes, polynuclear compounds and alcohols.

15. Use according to claim 14, wherein the contaminant is selected from tetrachlorophenol, pentachlorophenol, cresol and permethrin.

16. Use according to any of claims l to 13, wherein the contaminant is selected from avermectins, overtoxins, aflatoxins and glycoside antibiotics.

17. Use according to claim 16, to degrade ivermectin.

18. A mixture of, or a kit of containers respectively containing, a catalyst as defined in any of claims 1 to 3 and either a hydroperoxide or a substrate as defined in claim 4 or claim 5.

19. A mixture or kit according to claim 18, comprising the catalyst, the substrate and, optionally, an enzyme as defined in any of claims 8 to 10.