Classifications

• • A—HUMAN NECESSITIES
  • A62—LIFE-SAVING; FIRE-FIGHTING
  • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
  • A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
  • A62D3/02—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances by biological methods, i.e. processes using enzymes or microorganisms
• • A—HUMAN NECESSITIES
  • A62—LIFE-SAVING; FIRE-FIGHTING
  • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
  • A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
  • A62D2101/04—Pesticides, e.g. insecticides, herbicides, fungicides or nematocides
• • A—HUMAN NECESSITIES
  • A62—LIFE-SAVING; FIRE-FIGHTING
  • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
  • A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
  • A62D2101/20—Organic substances
• • A—HUMAN NECESSITIES
  • A62—LIFE-SAVING; FIRE-FIGHTING
  • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
  • A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
  • A62D2101/20—Organic substances
  • A62D2101/22—Organic substances containing halogen
• • A—HUMAN NECESSITIES
  • A62—LIFE-SAVING; FIRE-FIGHTING
  • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
  • A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
  • A62D2101/20—Organic substances
  • A62D2101/26—Organic substances containing nitrogen or phosphorus
• • A—HUMAN NECESSITIES
  • A62—LIFE-SAVING; FIRE-FIGHTING
  • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
  • A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
  • A62D2101/20—Organic substances
  • A62D2101/28—Organic substances containing oxygen, sulfur, selenium or tellurium, i.e. chalcogen

Definitions

• This invention relates to a biocatalytic process and also to compositions for use in such processes.
• 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.
• 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.
• aliphatic compounds including aliphatic alcohols and halogenated aliphatics, e.g.
• transition metals either alone or complexed
• haem, its derivatives and haem-containing enzymes have proved very versatile.
• these reactions generally take place only under harsh conditions: in the presence of a strong oxidant, at high temperature, or both.
• 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.
• 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.
• 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.
• Metals andmetal complexes 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.
• the degradation of ivermectin can be carried out under mild conditions.
• the oxidant may be molecular oxygen.
• 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.
• the peroxide can be supplied directly as, for instance, t-butyl hydroperoxide or another organic peroxide. Any suitable metal may be used.
• 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.
• results can be improved by adding the catalyst gradually over a period of time rather than having it all present at the start.
• concentration of, say, hemin can be increased from 0 to 10 ⁇ M, or from 10 to 20 ⁇ M, 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.
• 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.
• 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 ⁇ M 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
• Example 3 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 ⁇ g/ml purified soybean lipoxygenase was agitated at 23°C in an orbital incubator.
• 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.
• 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.
• 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) :
• 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.
• Ivermectin degradation also occurred, at various rates and with a variable lag time, when co-substrates other than linoleic acid were included.
• 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.
• 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.

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• 1992
  • 1992-04-01 GB GB929207181A patent/GB9207181D0/en active Pending
• 1993
  • 1993-03-29 AU AU38955/93A patent/AU3895593A/en not_active Abandoned
  • 1993-03-29 EP EP93907944A patent/EP0633801A1/en not_active Withdrawn
  • 1993-03-29 JP JP5517222A patent/JPH07505332A/ja active Pending
  • 1993-03-29 WO PCT/GB1993/000651 patent/WO1993019811A1/en not_active Application Discontinuation
• 1994
  • 1994-09-30 FI FI944570A patent/FI944570A/sv unknown

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