Process for oxidising aromatic compounds

Details Process for oxidising aromatic compounds Process for oxidising aromatic compounds

2002-04-04

2004-09-21

Saidha, Tekchand (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Oxidoreductase

C435S132000, C435S069100, C530S402000

Reexamination Certificate

active

06794168

ABSTRACT:

The invention relates to a process for enzymatically oxidizing halogenated aromatic compounds.
Chlorinated aromatic compounds such as the chlorobenzene and polychlorinated biphenyls (PCBs) are among the most wide-spread organic contaminants in the environment due to their common application as solvents, biocides, and in the heavy electrical industry. They are also some of the most problematic environmental pollutant, not only because of the health hazards (lipid solubility and hence accumulation in fatty tissues, toxicity and carcinogenicity) but also because of their slow degradation in the environment.
Whilst microorganisms have shown extraordinary abilities to adapt and evolve to degrade most of the organic chemicals released into the environment, the most chemically inert compounds such as PCBs do persist for two main reasons. First, these compounds have very low solubility in water and therefore their bioavailability is low. Research into this problem has focussed on the use of detergents and other surfactants to enhance their solubility and bioavailability. Second, these compounds require activation by enzymatic oxidation or reduction, and it can take a long time for the necessary genetic adaptations by microorganisms to occur, and even then the organisms may not be stable and viable.
We have now found, according to the present invention, that a monoxygenase, in particular P450
cam
and its physiological electron transfer partners putidaretoxin and putidaretoxin reductase, can be used to oxidise halogenated aromatic compounds. Also mutants of the monoxygenase with substitutions in the active site have enhanced oxidation activity. Thus suitable monoxygenases can be expressed in microorganisms, animals and plants which are going to be used to oxidise the halogenated aromatic compounds.
Accordingly the present invention provides a process for oxidizing a substrate which is a halo aromatic compound, which process comprises oxidizing said substrate with a monooxygenase enzyme.
The process may be carried out in a cell that expresses:
(a) the enzyme
(b) an electron transfer reductase; and
(c) an electron transfer redoxin
The halo aromatic compound is typically a benzene or biphenyl compound. The benzene ring is optionally fused and can be substituted. The halogen is typically chlorine. In many cases there is more than one halogen atom in the molecule, typically 2 to 5 or 6, for example 3. Generally 2 of the halogen atoms will be ortho or para to one another. The compound may or may not contain an oxygen atom such as a hydroxy group, an aryloxy group or a carboxy group. The compound may or may not be chlorophenol or a chlorophenoxyacetic compound.
Specific compounds which can be oxidised by the process of the present invention include 1,2; 1,3- and 1,4-dichlorobenzene, 1,2,4; 1,2,3- and 1,3,5-trichlorobenzene, 1,2,4,5- and 1,2,3,5-tetrachlorobenzene, pentachlorobenzene, hexachlorobenzene, 3,3′-dichlorobiphenyl and 2,3,4,5,6- and 2,2′,4,5,5′-pentachlorobiphenyl.
Other compounds which can be oxidised by the process include recalcitrant halo aromatic compounds, especially dioxins and halogenated dibenzofurans, and the corresponding compounds where one or both oxygen atoms is/are replaced by sulphur, in particular compounds of the formula:
which possess at least one halo substituent, such as dioxin itself, 2,3,7,8-tetrachlorodibenzioxin.
The oxidation typically gives rise to 1,2 or more oxidation products. These oxidation products will generally comprise 1 or more hydroxyl groups. Generally, therefore, the oxidation products are phenols which can readily be degraded. It is particularly noteworthy that pentachlorobenzene and hexachlorobenzene can be oxidised in this way since they are very difficult to degrade. In contrast the corresponding phenols can be readily degraded by a variety of Pseudomonas and other bacteria. The atom which is oxidized is generally a ring carbon.
The enzyme is typically a natural monooxygenase or a mutant thereof. The natural monooxygenase is generally a prokaryotic or eukaryotic enzyme. Typically it is a haem-containing enzyme and/or a P450 enzyme. The monooxygenase may or may not be a TfdA (2,4-dichlorophenoxy) acetate/&agr;-KG dioxygenase. The monooxygenase is generally of microorganism (e.g. bacterial), fungal, yeast, plant or animal origin, typically of a bacterium of the genus Pseudomonas. These organisms are typically soil, fresh water or salt water dwelling. In the case of a mutant monooxygenase the non-mutant form may or may not be able to oxidize the substrate.
The monooxygenase typically has a coupling efficiency of at least 1%, such as at least 2%, 4%, 6% or more. The monooxygenase typically has a product formation rate of at least 5 min
−1
, such as at least 8, 10, 15, 20, 25, 50, 100, 150 min
−1
or more. The coupling efficiency or product formation rate is typically measured using any of the substrates or conditions mentioned herein. Thus they are typically measured in the in vitro conditions described in Example 2, in which case the relevant monooxygenase, reductase and redoxin would be present instead of, but at the same concentration as, P450
cam
, putidaretoxin reductase and putidaretoxin.
The mutant typically has at least one mutation in the active site. A preferred mutant comprises a substitution of an amino acid in the active site by an amino acid with a less polar side chain. Thus the amino acid is typically substituted with an amino acid which is above it in Table 1.
TABLE 1
HYDROPATHY SCALE FOR AMINO ACID SIDE CHAINS
Side Chain
Hydropathy
Ile
4.5
Val
4.2
Leu
3.8
Phe
2.8
Cys
2.5
Met
1.9
Ala
1.8
Gly
−0.4
Thr
−0.7
Ser
−0.8
Trp
−0.9
Tyr
−1.3
Pro
−1.6
His
−3.2
Glu
−3.5
Gln
−3.5
Asp
−3.5
Asn
−3.5
Lys
−3.9
Arg
−4.5
An amino acid ‘in the active site’ is one which lines or defines the site in which the substrate is bound during catalysis or one which lines or defines a site through which the substrate must pass before reaching the catalytic site. Therefore such an amino acid typically inateracts with the substrate during entry to the catalytic site or during catalysis. Such an interaction typically occurs through an electrostatic interaction (between charged or polar groups), hydrophobic interaction, hydrogen bonding or van der Waals forces.
The amino acids in the active site can be identified by routine methods to those skilled in the art. These methods include labelling studies in which the enzyme is allowed to bind a substrate which modifies (‘labels’) amino acids which contact the substrate. Alternatively the crystal structure of the enzyme with bound substrate can be obtained in order to deduce the amino acids in the active site.
The monooxygenase typically has 1, 2, 3, 4 or more other mutations, such as substitutions, insertions or deletions. The other mutations may be in the active site or outside the active site. Typically the mutations are in the ‘second sphere’ residues which affect or contact the position or orientation of one or more of the amino acids in the active site. The insertion is typically at the N and/or C terminal and thus the enzyme may be part of a fusion protein. The deletion typically comprises the deletion of amino acids which are not involved in catalysis, such as those outside the active site. The monooxygenase may thus comprise only those amino acids which are required for oxidation activity.
The other mutations in the active site typically alter the position and/or conformation of the substrate when it is bound in the active site. The mutation may make the site on the substrate which is to be oxidized more accessible to the haem group. Thus the mutation may be a substitution to an amino acid which has a smaller or larger, or more or less polar, side chain.
The other mutations typically increase the stability of the protein, or make it easier to purify the protein. They typically prevent the dimerisation of the protein, typically by removing cysteine residues from the protein (e.g. by substitution of cy

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