GB2024205A - Biotransformations - Google Patents

Abstract

A process for the oxidation of organic compounds including C5-16 alkanes, C3-16 aliphatic alcohols, C3-8 alkenes and cyclic compounds uses as oxidising agent methane-utilizing bacteria of the species Methylosinus trichosporium or enzyme extracts thereof. A wide range of oxidations can be performed. Preferably a carbon - 1 compound such as methane or methanol is present as an electron donor. Living cells, immobilised whole cells or enzyme extracts may be used.

Description

SPECIFICATION Biotransformations This invention relates to biotransformations in which certain organic molecules are oxidised in the presence of bacteria of the species Methylosinus trichosporium or extracts thereof.

Bacteria capable of utilizing methane as a source of carbon and energy have been known for many years.

Such bacteria include strains of the species Methylosinus trichosporium. Whilst such bacteria cannot usually utilize more complex carbon sources such as ethane for growth, it has been found that they are capable of oxidising a limited number of simple organic molecules for example ethane and propane. These oxidations have been discussed in papers by R. Whittenbury, K.C. Phillips and J.F. Wilkinson (J. Gen. Microbiol., (1970), 61,205 - 218) and by A.W. Thomson, J.G. O'Neill and J.F. Wilkinson (Arch. Microbiol., (1976), 109,243 - 246).

Surprisingly it has now been found that methane-utilizing strains of the species Methylosinus trichosporium and enzyme extracts thereof are capable of oxidising more complex organic compounds than had previously been considered possible.

According to the present invention there is provided a process for the oxidation of an alkane having from 5 to 16 carbon atoms, an aliphatic alcohol having from 3 to 16 carbon atoms, an alkene having from 3 to 8 carbon atoms or a cyclic organic compound wherein a culture of a methane-utilizing bacterium of the species Methylosinus trichosporium or an extract thereof containing a methane oxidising system is used as an oxidising agent.

Alkanes which may be oxidised include branched and straight-chain alkanes, particularly pentane, hexane heptane and octane and their branched-chain analogues. Substituted alkanes may also be oxidised by the process of the present invention. Alkenes which may be oxidised include alkenes having terminal and internal double bonds and also substituted alkenes. The process of the invention is very suitable for the oxidation of propene.

The process of the invention is also applicable to the oxidation of cyclic organic compounds. Cyclic compounds which may be oxidised include alicyclic hydrocarbons such as cycloalkanes, and in particular cyclohexane, aromatic compounds such as benzene and substituted benzenes, for example having alkyl - or alkenyl - substituents containing up to 12 carbon atoms and/or having substituted hydroxyl groups, heterocyclic compounds such as pyridine, and compounds containing a plurality of aromatic rings, in particular naphthalene and derivatives thereof such as methyl - substituted naphthalenes.

Any methane-utilizing strain of Methylosinus trichosporium may be used in the process. A very suitable strain is the known strain OB 3b deposited at the National Collection of Industrial Bacteria, Torry Research Station, Aberdeen, Scotland as NCIB 11131. Cultures of this strain are maintained at a number of universities, particularly the Biological Laboratory, The University of Kent, Canterbury, Kent, England.

The process may be performed by contacting the compound to be oxidised with living bacterial cells suitably in a suspension, e.g. in aqueous suspension. Flocculated whole cells produced by the process of UK Specification No. 1368650 may also be used. The cells may also be immobilised using a suitable support material. In place of whole cells appropriate enzyme extracts of Methylosinus trichosporium may be used.

These may be either membrane associated or soluble extracts. Preferably the enzyme extracts are immobilised using a suitable support for example as described in UK Patent Application No. 33374/78.

During the process of the invention using whole cells it may be necessary for an additional compound to be present to supply reducing power to the system. The additional compound is suitably a C1 - compound such as a formate, methane or methanol. When enzyme extracts are used co-factors such as NADH are usually necessary for oxidation to proceed. Thus processes using enzyme extracts usually feature the supply or the regeneration of co-factors or other biochemical species to drive the reaction. Regeneration of suitable species may be achieved by any suitable means, for example formate, formaldehyde or other electron donors may be used together with a catalytic amount of NAD+ to regenerate NAD.

Oxidation products produced during the process of the invention may be separated and recovered using suitable conventional means.

The Methylosinus trichosporium species has an electron recycling system and its methane oxygenase, which is responsible for the initial oxidative attack to convert methane to methanol, is not NAD(P)H-linked.

Methane (mono)oxygenase is an enzyme which oxidises methane to methanol and belongs to the class mono-oxygenase. In the oxidation to produce methanol, one atom of a molecule of diatomic oxygen is incorporated into the organic substrate whilst the other atom of oxygen is reduced to water. This oxidation can be generally represented by the formula: R-H + 2 + XH2 R-OH + H2O + X wherein: XH2 = the mono-oxygenase, R-H = compound to be oxidised, and X = a reducing agent.

As indicated above, the methane mono-oxygenase is not NAD(P)H-linked but there is a feedback of "H2" from the next enzyme (methanol dehydrogenase) in the sequence of enzymes that effect the oxidation of CH4to CO2 in the bacterium.

The above mentioned feedback also occurs when other compounds are oxidized. In a bacterium that does not have the feedback loop, but uses NAD(P)H, NAD(P)H would not be generated.

The above discussion with respect to the use of the bacterium species itself is also generally applicable to the embodiments of the present invention wherein an enzyme extract from the bacterium species is utilized to effect the oxidation.

The enzyme extract may be used either as a crude extract or as a purified enzyme system.

Before use in the process cultures of Methylosinus trichosporium OB 3b can be grown under batch or continuous conditions with methane as a carbon source. The cells from the cultures can be harvested during the late logarithmic phase (batch culture) or during the steady state (continuous culture) by centrifuging at 3000 g for 45 minutes. The cells are then washed twice with 20 mM sodium phosphate buffer (eg at pH 7.0) and, after resuspending in the same buffer, they may be stored at a low temperature, ie 0 C or less, until required for use.

The above is an example ofthe mode of producing suitable cells of one strain. Workers skilled in the art will know how to vary these conditions in particular instances or when using other strains.

Atypical transformation using cells of Methylosinus trichosporium strain OB 3b prepared as described above may be performed in the following manner. A washed suspension, containing 70 - 80 mg dry weight of cells in 20 ml of 20 mM sodium phosphate buffer (pH 7.0) is shaken in a 250 ml conical flask for 12 hours at 30"C. The flask is sealed when containing an atmosphere of air or 50% V/v air/methane (or containing the substrate if gaseous). In the case of liquid substrates, 3 ml volumes of these are contained in centre wells from which they can diffuse to contact the bacterium. Products obtained may be identified by combined gas chromatography and mass spectrometry.

The invention is illustrated by the following examples: Example 1 The oxidations listed in the Table below were performed using cells of Methylosinus trichosporium strain OB 3b prepared as described above. A washed suspension, containing 70 - 80 mg dry weight of cells in 20 ml of 20 mM sodium phosphate buffer (pH 7.0) was shaken in a 250 ml conical flask for 12 hours at30"C. The flask was sealed when containing an atmosphere of air or 50% V/v air/methane (or containing the substrate if gaseous). With liquid substrates, 3 ml volumes were contained in a centre well from which they could diffuse to contact the bacterium. The products obtained were identified by combined gas chromatography and mass spectrometry.Conversion efficiencies based upon the amount of substrate entering the process, e.g. from the centre well, ranged between 80% and 90%.

Table Conversion Substrate Oxidation Products) No.

1 Benzene Phenol 2 Benzyl alcohol Benzaldehyde + p-hydroxybenzyl alcohol 3 o-Cresol 5-methyl, 1.3 benzene diol 4 Hexane Hexan-1-ol 5 Hexadecane Hexadecane-1-ol 6 Cyclohexane Cylcohexanol + 3-hydroxycyclo hexanone 7 Cyclohexanol 3-hydroxycyclohexanone 8 Ethylbenzene Benzoic acid + Benzyl alcohol + Phenylacetic acid + p-hydro xyethylbenzene 9 Propylene Propylene oxide 10 Octane Octan-1-ol 11 Phenol Catechol + 1,4-dihydroxybenzene 12 Pyridene Pyridene-N-oxide 13 Toiuene Benzoic acid + p-hydroxytoiuene 14 Styrene Styrene epoxide 15 Naphthalene 1-Naphthol 16 iso Propyl benzene p-hydroxy isopropyl benzene 17 p-xylene 4-methyl benzoic acid Example 2 The procedure of Example 1 was repeated using propylene as the substrate and with methanol present in the reaction mixture as an electron donor. In this instance however an enzyme extract was used in place of whole cells. The extract was prepared from cells of Methylosinus trichosporium strain OB 3b prepared as described above. The cells were broken down either using a French pressure cell or ultrasonication and any unbroken cells were separated by centrifuging for 15 minutes at 12,000 g and removed. The resultant extract was used in amounts of 10 mgs in 3 ml of the reaction mixture. The oxidation product was propylene oxide.

Example 3 The procedure of Example 1 was repeated using propylene as the substrate and using whole cells immobilised on to silanated glass beads, the cells being attached using glutaraldehyde. The product was propylene oxide.

Claims (8)

1. A process for the oxidation of an organic compound selected from the group consisting of an alkane having from 5 to 16 carbon atoms, an aliphatic alcohol having from 3 to 16 carbon atoms, an alkene having from 3 to 8 carbon atoms and a cyclic organic compound wherein a culture of a methane-utilising bacteria of the species Methylosinus trichosporium or an extract thereof containing a methane oxidising system is used as an oxidising agent.

2. A process according to claim 1 wherein a compound containing one carbon atom is present as an electron donor.

3. A process according to claim 2 wherein the compound containing one carbon stom is selected from the group consisting of methane and methanol.

4. A process according to claim 1 for the oxidation of an aromatic compound.

5. A process according to claim 4for the oxidation of an aromatic compound selected from the group consisting of naphthalene and derivatives thereof.

6. A process for the oxidation of an alicyclic hydrocarbon wherein a culture of a methane-utilizing bacteria of the species Methylosinus trichosporium or an extract thereof containing a methane oxidizing system is used as an oxidizing agent.

7. A process for the oxidation of cyclohexane wherein a culture of a methane-utilizing bacteria of the species Methylosinus trichosporium or an extract thereof containing a methane oxidizing system is used as an oxidizing agent.

8. A process according to claim 1 wherein the bacteria is Methylosinus trichosporium strain OB 3b.