JPS6231918B2 - - Google Patents

Description

[Detailed description of the invention]

This invention relates to the conversion of lower alpha-olefins to epoxides. In particular, the invention relates to the production of propylene oxide from propylene or propylene-containing streams by the action of oxygen and Methylotrophic microorganisms or enzyme preparations derived therefrom. Epoxides are extremely useful products because of their ability to perform a number of chemical reactions, including the addition of nucleophilic reactants (eg, ammonia, organic acids, alcohols, water, etc.) with active hydrogen atoms. Epoxidation products (i.e. 1,2-epoxides, also known as α-epoxides and oxirane compounds) are polymerized under thermal, ionic and free radical catalysis to form epoxy homopolymers and copolymers. It is also important industrially because of its ability to produce
Ethylene oxide and propylene oxide are the two most important commercial epoxides. A widely used method is the silver-catalyzed "direct oxidation" method of Lefort [U.S. Pat. No. 1,998,878 (1935) and Reissue Pat.
No. 22241]. Dutch Patent No. 291163 (published June 25, 1965) to Shell International Research Corp. Inc. discloses that α-olefins are grown on and separated from hydrocarbons. A method for producing 1,2-epoxide by contacting oxygen with microorganisms capable of assimilating carbon is described. This patent states that it is preferred to grow the microorganism on a hydrocarbon having substantially the same number of carbon atoms as the alpha-olefin to be epoxidized.
Although the general description of this patent includes alpha-olefins having 2 to 30 carbon atoms, the only example in the patent is Pseudomonas aeruginosa (strain 473) grown in n-heptane. ) and air in the presence of 1-octene. Whittenbury, Dalton, Eccleston and Reed [Microbal Growth on C ₁ Compounds: International Symposium Abstracts on _{Microbial Growth on C 1 Compounds} ] Proceedings of the
International Symposium on Microbial Growth
on C ₁ Compounds), Society of Furmentation Technology (Society of
Fermentation Technology), pp.1-11 (1975)]
Methane-oxidizing bacteria have the interesting characteristic that they have the ability to oxidize but not to utilize substrates; for example, they do not grow on ethane, but they are growing on methane or have previously grown on methane. It has previously been reported that ethane oxidizes when grown in methane. DeBont (Antonie ven Hoek)
Leeuwenhoek, 42 , 59-71 (1976)] reported that ethylene is oxidized by Gram-positive bacteria that appear to belong to the genus Mycobacterium. Devont says that the strains he isolated do not grow in the presence of methane, deducing that these bacteria in the soil are not methane-oxidizing bacteria. Devont and Albers (Anthony Huang-Liuwenhoek, 42 , 73-80 (1976)) proposed that the oxidation product of Devont's (1976) ethylene-oxidizing strains was ethylene oxide. Hutchinson, Whittenbury and Dalton [J.
Theor. Biol., 58 , 325-335 (1976) A Possible Role of Free Radicals in the Oxidation of Methane by Methylococcus capsulatus.
Free Redicals in the Oxidation of Methane
by Methylococcs capsulatus] and Colby and Dalton [J.
Biochem. 157 , 495-597 (1976) "Some Properties of Soluble Methane Monooxygenase Obtained from Methylococcus capsulatus Bath Strain"
of a Soluble Methane Mono-Oxygenase
from Methylococcus capsulatus Strain
Bath)] is Methylococcus capsulatus Bath
reported that ethylene is oxidized by soluble methane monooxygenase derived from this strain. The latter researchers used “particulate membrane preparations” of Methylococcus capsulatus Bath strain.
Pseudomonas membrane preparations” have no methane oxygenase activity as measured by the brommethane disappearance test.
¹⁸ O from ¹⁸ O ₂ into the cellular components of
Based on the introduction of Leadbetter and Foster [Nature 184 , 1428
~1429 (1959)] suggested that oxygenases are involved in the initial oxidative attack on methane. Higgins and Quayle
Quayle) [J.Biochem., 118 , 201-208 (1970)]
is Pseudomonas methanica or Methanomonas methanoxydans.
CH ₃ ¹⁸ OH was isolated as a product of methane oxidation when a suspension of methano-oxidans) oxidized methane in an atmosphere rich in ¹⁸ O ₂ . Later Ribbons [J.Bacteriol., 122 , 1351-1363
(1975)] and Ribbons and Michalover [FEBS Lett. 11 , 41
-44 (1970)] by Methylococcus capsulatus or Ferenci (FEBS)
Lett., 41 , 94-98 (1974)] by Methylomonas capsulatus.
Observation of methane-stimulated NADH oxidation catalyzed by extracts of . These researchers measured methane stimulation by spectrophotometry.
Indirect enzymatic assays have been relied upon to measure NADH loss or methane-stimulated O ₂ loss by polarography. Recently, Methylosinus trichosporium OB3b (Methylosinus
trichosporium OB3b) [Tonge, Harrison and Higgins, J.
Biochem. 161 , 333-344 (1977) and Tonge, Harrison, Knowles and Higgins,
FEBG Lett., 58 , 293-299 (1975)] and Methylococcus capsulatus (Bath) [Colby and Dalton, J.
Biochem., 171 , 461-468 (1978) and Colby, Starling and Dalton.
Stirling and Dalton), J.Biochem., 165 , 395−
402 (1977)], a methane monooxygenase system was partially purified. The present inventors have now demonstrated that _C2 - _C4 n-alkenes or butadiene are brought into contact with oxygen in the presence of a microorganism cultured in a mineral nutrient medium containing methane or dimethyl ether or an enzyme preparation derived from this microorganism. C ₂
It has been discovered that epoxides of ~ _C4n -alkenes and butadiene, especially propylene, can be prepared. The microorganisms used in the method of the present invention are preferably obligative or facultative methylotrophs, more preferably Methylosinus, Methylocystis, Methylomonas, Methylobacter ( Methylobacter, Methylococcus and Methylobacterium. "Direct oxidation" of ethylene oxide with silver catalyst
Unlike the production method, methane-grown microorganisms (methane-grown microorganisms)
methylotrophic microorganisms) or enzyme preparations derived therefrom are capable of epoxidizing α-olefins with 2 to 4 carbon atoms and butadiene, but not capable of epoxidizing α-olefins of C ₅ or higher (at least not by conventional analysis). It was also discovered that there were no amounts readily detectable by this method. In one preferred embodiment, methaneinduced methylotrophic microorganisms (methaneinduced methylotrophic microorganisms)
methylotrophic microorganisms) or enzyme preparations derived therefrom are used for the oxidative conversion of propylene to propylene oxide. The term "microorganism" is used in a broad sense herein, and includes not only bacteria but also yeast, filamentous fungi,
Also includes actinomycetes and protozoa. Preferably, microorganisms mean bacteria, more preferably bacteria capable of oxidizing methane. The term "enzyme preparation" is used to mean a composition that exhibits the desired oxygenase enzyme activity. This term is used to refer to, for example, live whole cells, dried cells, cell extracts, and purified and concentrated preparations derived from cells. Enzyme preparations may be in dry or liquid form. The term enzyme preparation also includes immobilized forms of enzymes, such as covalent chemical bonds, adsorption, and gels with pores large enough to allow free passage of substrate and product molecules but small enough not to allow the enzyme to pass through. Also included are whole cells or enzyme extracts of methane-growing microorganisms bound, ie, immobilized, to an insoluble matrix by entrapment of the enzyme within the lattice. The term "enzyme preparation" refers to enzymes held within hollow fiber membranes [Rony, Biotechnology and Bioengineering].
Bioengineering), June 1971 issue]. The term "particulate fraction" is used with reference to the oxygenase enzyme activity in the precipitated or precipitated fraction of cell-free extracts of methane-growing microorganisms after centrifugation for 1 hour at 10000Xg to 80000Xg. It is being The invention includes the following features. ○B The isolates of methane-utilizing microorganisms of the present invention are obligate bacteria (
types and types) and facultative bacteria (facultative
bacteria) as well as methane-utilizing yeasts. ○B In addition to the ability to oxidize methane to methanol, quiescent cell suspensions of some specific types of methane-growing bacteria (e.g., obligate, obligate) and facultative bacteria can produce _C2 -C ₄ Oxidation of n-alkenes and butadiene to the corresponding 1,2-epoxides. ○C The product 1,2-epoxide is not further metabolized and accumulates outside the cell. ○D Methanol-grown cells have neither epoxidation nor hydroxylation activity. In the substrate gaseous alkene, propylene is oxidized at the highest rate. ○ Methane suppresses epoxidation of propylene. ○he The stoichiometry of consumption of propylene and oxygen and production of propylene oxide is 1:1:1. Results of inhibition studies indicate that the same monooxygenase system catalyzes both hydroxylation and epoxidation reactions. Both hydroxylation and epoxidation activities are present in the cell-free (enzyme extract) particulate fraction precipitated or precipitated by centrifugation at 10000Xg to 80000Xg for 1 hour. ○ Obligate and facultative methylotroph microorganisms
Cell-free particulate fractions from microorganisms) undergo hydroxylation of methane to methanol, _C2 - _C4 n-alkenes and dienes (e.g. ethylene, propylene, 1-butene and butadiene) and the hydroxylation of _C1 - _C4 n-alkanes (eg methane, ethane, propane and butane). ○nu Methane-growing methylotrophic bacteria (methane-
The hydroxylation and epoxidation activities of grown methylotrophs are simultaneously lost during storage and are strongly inhibited by various metal binders. ○ Substrate (propylene or methane), oxygen,
The stoichiometry of NADH consumption and product formation was found to be approximately 1:1:1:1. R. Whittenberry, KC Phillips and J.
F. Wilkinson (R. Whilttenbury, KC)
Phillips and JFWilkinson) [J.Gen.
Microbiology, 61 , 205-218 (1970)] (hereinafter referred to as Whittenberry et al.) proposed a classification system for methane-oxidizing bacteria that is the most widely recognized system in use today. In this classification system, methane-utilizing bacteria are divided into the following five groups based on their morphological characteristics. i.e. Methylosinus,
Methylocyshis, Methylomonas, Methylobacter and Methylococcus. These five groups of bacteria reported by Whittenberry et al. utilize methane, dimethyl ether, and methanol for growth energy, and all of these bacteria are strictly aerobic and gram-negative. These fungi are also characterized by non-endosporing, ie the ability to produce cysts and exospores with complex microstructure and internal structure. In one embodiment of the present invention, the microorganism described by Whittenberry et al. is capable of epoxidizing lower α-olefins, particularly propylene, in the presence of oxygen when cultured in the presence of methane. It's been found. These methane-utilizing microorganisms are generally called ``methylotrophs.''
known as. Although enzyme systems or preparations derived from these microorganisms are referred to herein as "epoxidizing enzyme systems," these enzyme systems may be "methane monooxygenases" and/or "methane hydroxylases." Therefore, the enzyme system or enzyme preparation referred to herein as "alkene epoxidase" or "propylene epoxidase" used to convert alpha-olefins to 1,2-epoxides is referred to herein as methane monooxygenase or It should be understood as an "epoxidizing enzyme system" which is believed to be a methane hydroxylase enzyme. In the practice of the present invention, the use of the methylotrophic microorganisms reported by Whittenberry et al., the disclosure of which is incorporated herein by reference, is contemplated. In particular, the methylotrophic microorganisms listed in Table 4 on page 214 of the paper by Whittenberry et al.
sporium), Methylocystis parvus, Methylomonas methanica, Methylomonas albus, Methylomonas streptobacterium
streptobacterium), Methylomonas agile, Methylomonas rubrum, Methylomonas rosaceus, Methylobacter chromococcum
chroococcum), Methylobacter bovis, Methylobacter capsulatus, Methylobacter vinelandii
vinelandii), Methylococcus capsulatus and Methylococcus capsulatus, Texas strain (Methylococcus
capsulatus Strain Texas) [DW Ribbons (D.
W. Ribbons), J. Bacteriol., 122 , 1351−1363
(1975)] and identified as Methylococcus minimus can be used. These methylotrophic microorganisms can be used in the form of whole cells or their enzymatic extracts, or
Immobilized formulations of whole cell or enzyme extracts such as by the use of DEAE cellulose or ion exchange resins or porous alumina carriers
It can also be used in the form of preparations. Subculturing of several methylotrophic microorganisms described by Whittenberry et al.
Regional Research Laboratory, Peoria;
Illinois 61604) public depositary institution (official
depository) by depositing each subculture in the
We received the NRRL strain from this depository. These subcultures were carried out in accordance with USDA procedures without any restrictions so that the progeny of these strains were made available to the public, including but not limited to U.S. and West German nationals. It has been deposited. The deposited strains of methylotrophic microorganisms are called by the following names:

【table】

[Table] The progeny of these strains are available to anyone who requests them without any restrictions on their use. The above strains were first subcultured by R. Whittenbury, Department of Biological Sciences, University of Wauwick, UK.
Biological Science, University of Warwick;
Warwickshire, Coventry, England). The morphological and taxonomic characteristics of the above methylotrophic bacterial strains are as follows. Methylocinus trichosporium OB3b NRRL
B-11, 196 produces white colonies on salt agar plates in the presence of methane or methanol. This bacterium is a motile, rod-shaped, Gram-negative, aerobic bacterium. Often forms rosettes. It has a type membrane structure. Metylocinus sporium 5 NRRL B-
11,197 Produces white colonies on salt agar plates in the presence of methane or methanol. This bacterium is a motile, rod-shaped, Gram-negative, aerobic bacterium. Often forms rosettes. This fungus produces heat-resistant exospores, and the spores isolated from the amastigote pole of the fungus take on a vibrio form. Organic compounds other than methane and methanol do not support growth. It has a type membrane structure. Methylocystis parbus OBBR NRRL B-
11,198 In the presence of methane or methanol, it produces slimy white colonies on saline agar plates. The bacterium is non-motile, coccobacillus-shaped, Gram-negative, aerobic. This fungus forms cysts that are drought resistant but not heat resistant. Grows using methane or methanol. Organic compounds other than methane and methanol do not support growth. It has a type membrane structure. Methylomonas metanica S ₁ NRRL B-11,
199 Produces pink colonies on salt agar plates in the presence of methane or methanol. This bacterium is a motile, rod-shaped, Gram-negative, aerobic bacterium. Generates slime-like capsules. This bacterium uses up methane and methanol to grow. Organic compounds other than methane and methanol do not support growth. It has a type membrane structure. Methylomonas albus BG8 NRRL B-11,
200 Gives white colonies on saline agar plates in the presence of methane or methanol. The bacterium is a motile, rod-shaped, Gram-negative, aerobic bacterium that produces slime-like capsules. Grows using methane and methanol. Organic compounds other than methane and methanol do not support growth. It has a type membrane structure. Methylobacter capsulatus Y NRRL B
−11,201 Produces white to brown colonies on salt agar plates in the presence of methane or methanol. The bacterium is a motile, rod-shaped, Gram-negative, aerobic bacterium that produces slime-like capsules. Grows using methane and methanol. Organic compounds other than methane and methanol do not support growth. It has a type membrane structure. Recently, Patt, Cole and Hanson (Patt,
Cole and Hanson)〔International J.Systmatic
Bacteriology, 27 , (2) 226-229 (1976)] states that methylotrophic bacteria can grow non-autotrophically using carbon compounds containing one or more carbon atoms but no carbon-carbon bonds. It was disclosed that it is a bacteria that can Patz et al. describe methylotrophic bacteria as "biased" when they can only utilize carbon compounds that do not contain carbon-carbon bonds (e.g., methane, methanol, dimethyl ether, methylamine, etc.) as their sole carbon and energy source. Facultative methylotrophs use compounds that do not contain carbon-carbon bonds and complex compounds that contain carbon-carbon bonds as their sole sources of carbon and energy. It is proposed that this is a bacterium that can be used. In the above-mentioned report, Patz et al. state that Methylobacterium organophilum sp nov.
(ATCC27886) is disclosed. This fungus probably differs from the genera and species of methane-oxidizing fungi mentioned above because of its ability to utilize various organic substrates with carbon-carbon bonds as a source of carbon and energy. In another embodiment of the present invention, this microorganism (Methylobacterium organophilum)
sp nov.ATCC27886) and other facultative methylotrophic microorganisms were also discovered to be capable of epoxidizing _C2 - _C4 alkenes. In other words, these microorganisms exhibit alkene epoxidase enzyme activity when cultured in the presence of methane. As discussed above with respect to the methylotrophic microorganisms of Whittenberry et al., facultative methylotrophic microorganisms, when used in the methods of the present invention, are
It can be used in the form of a supernatant (after centrifugation at 10000Xg for 30 minutes), or it can be in immobilized form, or it can be used in cell-bound form. Other known methylotrophic bacterial strains that can be used in the method of the invention include those cited in, for example, US Pat. Methylomonas sp.AJ−
3670 (FERM P-2400); and U.S. Pat.
4042458, NCIB
Methylococcus 999 with Accession No.11083 and NCIB Aecession
Methylomonas SM3 with No. 11084 (Netherlands Patent Application No.
74/16644). Mixtures of methylotrophic and non-methylotrophic microorganisms such as those described in US Pat. Nos. 3,996,105 and 4,042,458 can also be utilized. Commercial microbial propagation methods generally require a stepwise progression. These steps may be fewer or more, depending on the nature of the process and the characteristics of the microorganism. Propagation is usually initiated by inoculating the cells from a culture slant into a previously sterilized nutrient medium, usually contained in a flask. The growth of microorganisms in the flask is promoted by various methods, such as shaking for thorough ventilation and maintaining an appropriate temperature. This process or step is repeated one or more times in flasks or containers containing the same or larger amounts of nutrient medium. These stages can be conveniently referred to as culture development stages. The microorganism from the final stage of development, with or without a medium, is introduced or inoculated into a large scale fermentor to produce commercial quantities of the microorganism or to produce enzymes from the microorganism. There are various reasons why microorganisms grow in several stages, but the main ones are the growth of microorganisms and/or
It depends on the conditions necessary for the production of enzymes from it. These include microbial stability during fermentation, maintenance of appropriate nutrients, PH, osmotic pressure relations, aeration, temperature and pure culture conditions. For example, in order to obtain the highest yield of alkene epoxidase, the fermentation conditions in the final stage may have to be varied somewhat from the conditions carried out to obtain microbial growth in the culture development stage. Maintaining the purity of the medium is also a very important consideration, especially when the fermentation is carried out under aerobic conditions, as is the case with methylotrophic microorganisms. When starting fermentation for the first time in a large fermenter,
Relatively long times are required to obtain significant yields of microorganisms and/or alkene epoxidase enzymes from microorganisms. Of course, this increases the possibility of contamination of the medium as well as mutation of the microorganisms. The media used to grow methylotrophic microorganisms and induce oxygenase or epoxidizing enzyme systems contain inorganic salts such as phosphates, sulfates and nitrates and sources of oxygen and methane. Fermentation is generally carried out at a temperature of 5°C to about 55°C, preferably about 25°C to
It is carried out at a temperature of 50 ° C. The pH of the medium should be adjusted to a range of about 4-9, preferably about 5.5-8.5, more preferably 6.0-7.5. Fermentation can be carried out at normal pressure, but pressures up to about 5 atmospheres and higher can also be used. Typically, the methylotrophic microorganism is contacted with a medium that is in contact with a gas mixture containing methane and oxygen to grow the methylotrophic microorganism and induce the oxygenase or epoxidizing enzyme system. Methane can be supplied in the form of natural gas. continuous flow culture
Therefore, methylotrophic microorganisms can be grown in suitably adapted fermenters, e.g. stirred baffle tubes with internal cooling or external circulation cooling annular tubes.
baffled) fermenters or sparge column fermenters. Fresh medium 0.02 per hour
The culture can be continuously pumped into the culture at a rate equal to ~1 culture volume and removed at a rate such that the culture volume remains constant.
A gas mixture containing methane and oxygen, and possibly containing carbon dioxide or other gases, is contacted with the medium by constant bubbling, preferably from the bottom of the fermentor with a sparger.
The oxygen source for the culture may be air, oxygen, or oxygen-enriched air. Spent gas can be removed from the top of the fermenter. The spent gas may be circulated through an external annular tube or internally by a gas inducer impeller. Gas flow and circulation flow are set to obtain maximum growth of bacteria and maximum utilization of methane. The oxygenase enzyme system can be obtained as a crude extract, as described above, or as a cell-free particulate fraction, i.e., the supernatant after centrifuging the broken cells at 10,000Xg for 30 minutes, at 10,000Xg or more for 1 hour. It can be obtained as a precipitate or a substance that settles during centrifugation. Standard methods commonly used, such as flocculation, sedimentation and/or
After precipitation, microbial cells can be harvested from the growth medium by centrifugation and/or filtration. This biomass can also be dried by freezing or spray drying and used in this form for further use in epoxidation reactions. When using cell-free enzymes, NADH and metals (eg copper or iron) can be added to also enhance enzyme activity. To carry out the method of the invention, an oxygenase enzyme system is obtained which converts methane to methanol under oxidizing conditions, eg as described above. The source of the enzyme is not critical, but the microorganism can be prepared from one of the five genera of microorganisms described in the paper by Whittenberry et al. or from a facultative methylotroph (Methylobacterium). Preferably, such formulations are obtained by growth in a nutrient medium containing:
The nutrient medium may be that described by Whittenberry et al. or, more preferably, by Foster and Davis [J. Bacteriol.
91 , 1924-1931 (1966)] may be used. This enzyme preparation is then added in a buffer or nutrient medium (e.g. the same nutrient medium used for microbial production can be used, except that olefin is used instead of methane) in the presence of oxygen. , _C2 - _C4 alkenes such as ethylene, propylene, butene-1 or conjugated butadiene, or mixtures thereof, and the mixture is incubated until the desired conversion is obtained. After that, the usual method,
The epoxide is recovered, for example by distillation. To facilitate effective contact between the necessary oxygen and the enzyme (enzyme preparation or methylotrophic microorganism), for best results a solution containing water and a buffer and in which the enzyme preparation or microbial culture is suspended is generally recommended. Preferably, a strong fine air stream is introduced into the olefin dispersion in the epoxidation medium which is being vigorously stirred. The enzyme preparation can then be separated from the liquid medium, preferably by filtration or centrifugation. The produced epoxide can then generally be obtained. The process of the invention can be carried out batchwise or semi-continuously or continuously, or co-currently or counter-currently. Optionally, the suspension containing the enzyme preparation or methylotrophic microorganism and the buffer is flowed countercurrently into the tubular reactor with vigorous stirring against the ascending air flow.
The upper layer of the flowing suspension is removed and the culture and remaining buffer components (at least in part) are recycled with further olefin and optionally fresh enzyme preparation or methylotrophic microorganisms. The methylotrophic microorganism growth step and the epoxidation step can be conveniently combined by performing them simultaneously, but they can also be performed separately and each with a higher degree of aeration (e.g., at least twice the aeration required for growth) during the epoxidation step. It can also be carried out with an excess of air (preferably at least 5 times). Both the growth step and the epoxidation step can also be carried out in the same reactor in sequential or simultaneous operation by alternating normal and intensive aeration. The present invention will be further explained below with reference to Examples, but these Examples are not intended to limit the present invention. In the examples, all parts and percentages are by weight unless otherwise specified. In the following text, the microorganisms used in the present invention are described.
The correspondence between the NRRL number and the FERM P- number contracted by FERM is shown: NRRL FERM P- B-11202 5098 B-11208 5104 B-11219 5115 B-11222 5118 Example 1 Foster having the following composition (in 1) and Davis (Foster and Davis) [J. Bacteriol
91 , 1924-1931 (1966)] was prepared. Na ₂ HPO ₄ 0.21g NaH ₂ PO ₄ 0.09g NaNO ₃ 2.0g MgSO ₄・7H ₂ O 0.2g KCl 0.04g CaCl ₂ 0.015g FeSO ₄・7H ₂ O 1.0mg CuSO ₄・5H ₂ O 0.01mg H ₃ BO ₄ 0.02mg MnSO ₄・5H ₂ O 0.02mg ZnSO ₄ 0.14mg MoO ₃ 0.02mg Adjust the pH of the nutrient medium to 7.0 by adding acid or base, and shake each 50ml aliquot of this nutrient medium in multiple 300ml aliquots. I put it in a flask. One inoculation loop (an
inoculating loop) cells were inoculated into each shake flask. The isolates were kept on agar plates under an atmosphere of methane and air at a 1:1 v/v gas ratio and transferred every two cycles. Next, the gas phase of the inoculation flask was adjusted to 1:
It was replaced with a gas mixture consisting of methane and air in a 1v/v ratio. The inoculated flask was hermetically sealed and cultured on a rotary shaker with an orbital radius of 2.5 cm at 250 rpm at 30° C. for 2 days until turbidity appeared in the medium. Cells were harvested by centrifugation at 10,000×g for 30 minutes at 4°C. The cell pellet was washed twice with 0.15M phosphate buffer, pH 7.0 (containing _2M MgCl2).
Next, the washed cells were suspended in 0.15M phosphate buffer at pH 7.0. 0.5ml of each washed cell suspension (cells 2
mg) was placed in a 10 ml bottle at 4°C and sealed with a rubber stopper. After removing the gas phase from the bottle by reducing the pressure,
It was replaced with a gas mixture of alkene and oxygen in a 1:1 v/v ratio. Then, at 30°C in a rotary shaker at 300rpm.
It was cultured in Samples (3μ) of the product were periodically removed with a microsyringe and subjected to gas chromatography (ionization flame detector column).
Analyzed by. Table 1 shows the conversion of the hydroxylation of methane as well as the epoxylation of propylene with washed cell suspensions of several microbial strains grown on methane according to the experimental method described above. The data in the table shows that methane-growing microorganisms that are capable of hydroxylating methane to methanol are also capable of converting propylene to propylene oxide.

【table】

[Table] Table 2 shows the conversion rates of hydroxylation of methane and epoxidation of propylene (from Table 1) and the conversion of ethylene by washed cell suspensions of two microbial strains grown in methane by the above method.
Figure 2 shows the conversion of butene-1 and butadiene epoxidation. These methane-growing microorganisms grow pentene-1 and hexene-1 in the presence of air.
did not produce detectable epoxides when contacted with. The data in Table 2 also shows that for each methane growing microorganism, the conversion rate of propylene to propylene oxide is higher than the other conversion rates.

[Table] The above experimental procedures were performed on Methylocystis parbus.
Repeat for each strain of OBBP (NRRL B-11198), Methylomonas metanica S ₁ (NRRL B-11199), and Methylomonas albus BG8 (NRRL8-11200), and culture washed cell suspensions of these methane-growing microorganisms. It was successfully used for the conversion of methane to ethylene oxide at a dry weight of cells of 0.2 g/100 ml relative to the liquid standard, 0.9 and 0.9, respectively.
Conversion rates of 0.95 and 1.2 μmol/hr/mg protein were obtained. As indicated above, a novel method whereby propylene oxide is obtained by culturing propylene in the presence of microbial cells or cell-free extracts (or enzymes derived therefrom) grown in the presence of methane. was discovered. These microorganisms are also known to be able to hydroxylate short-chain alkanes (e.g. methane to methanol), and some researchers have suggested that these microorganisms may be capable of epoxidizing ethylene. It suggests that. This time, the inventors
It has been discovered that these methane growing microorganisms and their enzyme formulations are capable of epoxidizing propylene at relatively higher conversions than for ethylene, butene-1 and butadiene. In batch experiments using washed methane-grown cells, the epoxidation reaction proceeds linearly for at least 2 hours. No further oxidation of the epoxide product was detected. The epoxidizing enzyme system of methane-growing microorganisms is an inducible enzyme (by methane), and the epoxide product accumulates extracellularly (i.e., after the reaction, when the reaction mixture is centrifuged, the epoxide product is in the supernatant fraction). and not in the cell pellet). As a result of GLC analysis, there was no possibility of propanal as an oxidation product of propylene. In a comparative experiment, the microorganism Metylocinus trichosporium OB3b grown in methanol
(NRRL B-11196), Methylocystis parvus
Strains of OBBP (NRRL, B-11198), Methylomonas metanica S ₁ (NRRL B-11199), and Methylobacter capsulatus Y (NRRL B-11201) have the ability to hydroxylate methane and epoxidize C ₂ -C ₄ alkenes. I didn't even have the skills. These evidences indicate that only methane-growing microorganisms have methane hydroxylation and _C2 - _C4 alkene epoxidation abilities. As previously mentioned, whole cells of methane-growing methylotrophs and cell-free extracts containing oxygenase enzyme activity can be used in hydroxylation and epoxidation reactions in the presence of air.
When using cell-free or pure enzyme preparations, NADH and metals (iron or copper) may be added to increase activity. When using the cell-free enzyme system of the present invention, an enzyme preparation is prepared as follows. Preparation of Cell Fractions Example 1 with total carbon and methane as energy source (methane and air, 1:1 parts by volume)
The microorganisms were grown at 30° C. in 2.8 flasks containing 700 ml of the mineral salts medium described. During exponential growth,
Cells were harvested by centrifugation at 12,000×g for 15 minutes at 4°C. Cells were incubated at pH7.0 containing 5mM _MgCl2 .
Washed twice with 25mM potassium phosphate buffer. This cell suspension was transferred to a French Pressure cell (French Pressure cell) [1054.5Kg/
cm ² (15000lb/in ² ) once to disintegrate and remove non-destructive bacteria.
Centrifugation was performed at 5000Xg for 15 minutes. The supernatant (crude extract) was then centrifuged at 40,000×g for 30 minutes to obtain a particulate P(40) fraction and a soluble S(40) fraction. The S(40) fraction was then centrifuged at 8000Xg for 60 minutes to obtain a particulate P(80) fraction and a soluble S(80) fraction. Particulate fraction [P(40) and P(80)]
was suspended in 25mM potassium phosphate buffer of pH 7.0 containing 5mM MgCl ₂ and homogenized at 4°C. Enzyme assay To estimate the oxidation of methane and propylene by the particulate fraction [P(40) and P(80)] fraction and the soluble fraction [S(80)] fraction, and the amount of methanol and propylene oxide produced, respectively. Measured at 30°C. Reaction mixture contained in 1.0 ml: 150 mM at pH 7.0, containing 5 mM _MgCl2
0.6 ml potassium phosphate buffer; 10 μmol NADH and cell fraction. The reaction mixture was placed in a 10 ml bottle at 4°C. Each bottle was sealed with a rubber cap. The gas phase in each bottle was removed by vacuum and then replaced with methane or a mixture of propylene and oxygen in a 1:1 v/v ratio. Oxidation of other gaseous n-alkanes and n-alkenes was tested as described above. For liquid substrates, 10μ
substrate was used directly. Each bottle was then incubated at 30°C on a rotary shaker at 200 rpm. The products of n-alkene epoxidation and n-alkane hydroxylation were loaded onto 80/100 Chromosorb W and Porapak Q columns with 10% carbowax.
Stainless steel column packed with 20M [365.76cm
x 0.3175 cm (12′ x 1/8″)] by form ionization gas chromatography.
The column temperature was kept constant at 120°C. The carrier gas flow rate was 30 ml/min of helium. The various products were confirmed by comparison of retention times and simultaneous chromatography of authentic standards. Specific activity was expressed in μmol of product per mg protein per hour. The concentration of proteins in the various fractions was determined by Lowry et al. [J. Biol. Cham. 193 ,
265-275 (1951)]. Distribution of n-alkane oxidation activity and n-alkene oxidation activity in cell fractions Three completely different groups of methane-utilizing microorganisms were selected to investigate n-alkanes (C ₁ -C ₄ ) and n-alkenes (C 4 ) in cell-free systems. ₂ - _C4 ) oxidation test was conducted.
Obligate methane-utilizing microorganism, Methylomonas sp. (CRL-17, NRRL B-
11208) and Methylocsccus capsulatus (Texas,
ATCC19069); obligate methane-utilizing microorganisms,
Methylosinus trichasporium (OB3b, NRRL
B-11196) and Methylosinus sp. (CRL-15, NRRL B-
11202); and facultative methane-utilizing bacteria, Methylobacterium sp. (CRL-26, NRRL B
-11222). Table 3 shows the distribution of methane and propylene oxidation activities in various fractions derived from each of these microorganisms. Approximately 85-90% of the total activity is P
(40) fraction and 10 in the P(80) fraction.
% was detected. The soluble fraction S(80) contained no activity. The specific activities for methane and propylene oxidation of the P(40) and P(80) fractions were not significantly different for the various microorganisms tested (Table 4). Both propylene epoxidation and methane hydroxylation depended on the presence of oxygen and NADH. NADPH or ascorbate and other electronic carriers could also be utilized. For both reactions, the first 15
The minutes were linear.

【table】

[Table] Particulate fractions [P(40) and P(80)] from various microorganisms contain other n-alkenes, (ethylene, 1-
It also catalyzed the epoxidations of (butenes and 1,3-butadiene) to the corresponding 1,2-epoxides and the hydroxylation of methane and ethane to the corresponding alcohols. Table 5 shows methylocinus.
sp. (CRL-15, NRRL B-11202) P (40)
Figure 2 shows the oxidation rates of various n-alkanes and n-alkenes by the particulate fraction. To check for oxidation products,
P(40) fractions were incubated with various substrates at 30°C for 10 minutes and then analyzed by gas chromatography.

[Table] Sid

[Table] To further study the influence of various environmental factors on methane oxidation and propylene oxidation activities in a cell-free system, Methyrosinus sp.
15, NRRL B-11202) was selected. Effect of Particulate Fraction Concentration The effect of P(40) particulate fraction concentration on methane hydroxylation and propylene epoxidation was tested. In the range of 1-6 mg protein per ml, methanol and propylene oxide production was directly dependent on the particulate fraction concentration in that range. The reaction rate decreased when the particulate protein concentration was further increased to 8 mg/ml. Reaction time Methylocinus sp. (CRL-15, NRRL B-
The rate of production of methanol and propylene oxide by the P(40) particulate fraction of 11202) by hydroxylation of methane and epoxidation of propylene, respectively, was linear with time up to 15 minutes. Effect of PH Methylocinus sp. (CRL-15, NRRL B-
The effect of PH on the hydroxylation of methane and epoxidation of propylene by the P(40) particulate fraction of 11202) was investigated by estimating the amount of methanol and propylene oxide produced after incubating the reaction mixture for 10 min. Tested. The optimum pH for methane hydroxylation and propylene epoxidation was found to be 7.0. In carrying out these tests, reactions were carried out as described in Example 1. Approximate reaction products are obtained by heating the reaction mixture to 30°C.
After incubation on a rotary shaker for 5, 10 and 15 minutes, the cells were analyzed by gas chromatography. 100% activity per hour and 1 mg of protein
Equivalent to the production of 4.8 and 4.1 μmoles of methanol and propylene oxide. Effect of temperature Methylocinus sp. (CRL-15, NRRL B-
The effect of temperature on the production of methanol and propylene oxide by the P(40) particulate fraction of 11202) was tested after incubating the reaction mixture for 10 minutes at various temperatures. The optimum temperature for propylene epoxidation and methane hydroxylation was found to be 35°C. In conducting these tests,
The reaction was carried out as described in Example 1. Estimates of the reaction products were made by gas chromatography after incubating the reaction mixture for 5, 10 and 15 minutes on a rotary shaker at 30°C. 100% activity is equivalent to the production of 5.0 and 4.2 μmol of methanol and propylene oxide, respectively, per mg of protein per hour. Effect of storage Methylocinus sp. (CRL-15, NRRL B-
It was observed that both the methane hydroxylation activity and the propylene epoxidation activity by the P(40) particulate fraction of 11202) decreased simultaneously when stored at refrigerator temperatures (0-4°C). In carrying out these tests, reactions were carried out as described in Example 1. An estimate of the reaction products is given by adding the reaction mixture to
After incubation on a rotary shaker at 30°C for 5, 10 and 15 minutes, the results were analyzed by gas chromatography. 100% activity is equivalent to the production of 4.8 μmol and 4.1 μmol of methanol and propylene oxide, respectively, per mg of protein per hour. Effect of Inhibitors Methane oxidation by cell suspensions of methane-utilizing bacteria has been reported to be inhibited by various metal binding agents or metal chelating agents [Patel et al., J. Bacteriol. , 126 , 1017−1019
(1976)]. Therefore, Metylocinus sp. (CRL−
The effect of inhibitors on the methane oxidation activity and propylene oxidation activity of the P(40) particulate fraction of NRRL B-11202) was tested. The production of methanol and propylene oxide can be achieved using various ligand combinations as shown in Table 6: nitrogen-nitrogen (α·α-bipyridyl), oxygen-nitrogen (8-hydroxyquinoline) and sulfur-nitrogen (thiourea). , thiosemicarbazide). This suggests that metal ions are involved in the oxidation of both methane hydroxylation and propylene epoxidation. Similarly, as shown in Table 6a,
These compounds also inhibit methane hydroxylation and propylene epoxidation when using cell-containing enzyme preparations.

【table】

【table】

[Table] Effect of metals Methane monooxygenases from methane-utilizing bacteria are copper- or iron-containing proteins [Tonge et al., J.Biochem., 161 , 333-344
(1977)], Methylocinus sp. (CRL-15,
The effect of copper and iron salts on the oxidation of methane and propylene by the P(40) particulate fraction of NRRL B-11202) was tested. The rates of hydroxylation of methane to methanol and epoxidation of propylene to propylene oxide increased two-fold in the presence of added copper salts (Table 7).

[Table] Substrate Competition Experiment Hydroxylation of methane and epoxidation of propylene by the particulate fraction of methane-utilizing bacteria required oxygen and NADH. The question of whether the same or similar enzymes were involved in the oxidation of both substrates was tested by substrate competition experiments. The experiment consisted of determining the effect of methane on the oxidation of propylene to propylene oxide by the P(40) particulate fraction of Methylocinus sp. (CRL-15, NRRL B-11202). As shown in Table 8,
The presence of methane reduced the amount of propylene oxide produced. Therefore, methane inhibited the conversion of propylene to propylene oxide, probably by competing for available enzyme sites.

[Table] Similarly, as shown in Table 8a, methane is produced by Methylocinus trichosporium grown in methane.
Affects propylene epoxidation from cell suspensions of OB3b (NRRL B-11196).

[Table] Stoichiometry of propylene and methane oxidation Methylocinus sp. (CRL-15, NRRL B-
The stoichiometry of hydroxylation and epoxidation reactions was determined using the particulate fraction P(40) of 11202). The stoichiometry of methane- or propylene-dependent NADH oxidation, oxygen consumption and product formation was approximately 1:1:1 (Table 9). This is consistent with methane or propylene oxygenation being catalyzed by monooxygenases.

【table】

[Table] For comparison, Methylocinus trichosporium
The stoichiometry of propylene epoxidation by a cell suspension of OB3b (NRRL B-11196) was determined as follows. The reaction mixture (3.0ml) has a pH of 7.0.
0.05M sodium phosphate buffer and propylene
It contained 3.6 μmol. The reaction was initiated by injection of 0.1 ml of cell suspension (3.1 mg protein).
Corrections were made for endogenous oxygen consumption. The amount of oxygen consumed during the reaction (3 minutes) was measured by polarography with a Clark oxygen electrode. The amount of propylene consumed and the amount of propylene oxide produced were estimated by gas chromatography. Propylene consumption is 0.29 μmol, oxygen consumption is 0.30 μmol, propylene oxide product is 0.28
It was in μmol. Oxygen activity is in the particulate fraction (not in the supernatant)
To further demonstrate this, we conducted the following experiment. Methylococcus capsulatus (CRL M1, NRRL B11219) cells grown in methane were obtained by the method of Example 1. This crude extract was sonicated (3 x 50 seconds, Wave Energy Ultrasonic Oscillator,
W201 type (Wave Energy Ultrasonic
Oscillator, Model W201)] is 10000Xg
After centrifugation, it was found to exhibit neither epoxidation nor hydroxylation activity. However, the cells were transferred to a French pusher cell (1000Kg pressure) for 2 hours.
If it is destroyed by passing it twice,
Both activities were found in the crude extract after 10000Xg centrifugation. This crude extract was stored at 4°C.
All the activity in the crude extract was collected as particulate fraction by further centrifugation at 40000Xg for 90 min. As shown in Table 10, NADH stimulated both epoxidation and hydroxylation reactions.

[Table] Summary Van der Linden
[Biochem.Biophys Acta., 77 , 157-159
(1963)] and the system of Pseudomonas aeruginosa by Abbott and How [App.
Microbiol., 26 , 86-91 (1973)].
oleovorans) systems epoxidized _C6 - _C12 liquid 1-alkenes, but not gaseous alkenes. The present invention shows that ethylene, propylene, 1
- Provides for the epoxidation of butenes and butadiene. Epoxidation of alkenes and hydroxylation of methane were not found under anaerobic conditions or in methanol-grown cells. This suggests that the enzyme system is inducible. The product 1,2-epoxide accumulated extracellularly. The non-enzymatic degradation of propylene oxide in the disclosed assay format was not significant even after prolonged incubation. Juan der Linden (above) is a Pseudomonas
shows the production of 1,2-epoxyoctane from 1-octene by heptane-grown cells of sp. and also states that this epoxide is not further enzymatically oxidized. However, May and Abbott [Biochem.Biophys.Res.
Commun., 48 , 1230−1234 (1972) and J.
[ Biol . 1・
It was reported that both 2-ethoxyoctane were produced. Furthermore, Abbott and Hou (supra) found that the methyl group of the latter compound is also susceptible to hydroxylation. However, our results from studies of viable cell suspensions of methane-utilizing bacteria showed that propylene oxide is not further enzymatically metabolized. Juan der Linden (supra) has shown that epoxide accumulation from 1-octene by Pseudomonas aeruginosa involves the metabolism of large amounts of 1-octene by methyl group epoxidation. However, in propylene epoxidation using a cell suspension of methane-utilizing bacteria,
No formation of 3-hydroxypropene-1 was detected. Epoxidation of _C2 - _C41 -alkenes and hydroxylation of methane by cell suspensions are both suppressed with various metal binders and metal chelators;
This indicates that a metalloenzyme system is involved. The comparable inhibition of propylene and methane oxidation (Table 6a) indicates that the epoxidation and hydroxylation reactions may be catalyzed by the same or similar enzyme systems. Methane-growing bacterial strain, Methylococcus capsulatus (NRRL
Epoxidation of propylene to propylene oxide by cell suspensions of B-11219) was suppressed (50%) in the presence of the hydroxylation substrate methane (Table 10). This clearly suggests competition between hydroxylated and epoxidized substrates for a single enzyme system. The methane monooxygenase enzyme system appears to catalyze both the epoxidation of alkenes and the hydroxylation of methane.
May and Abbott (cited above) refer to Pseudomonas oleovorans.
reported that an ω-epoxidation system from A. oleovorans catalyzes both 1-octene epoxidation and n-octene hydroxylation. The optimal conditions for in vivo epoxidation of propylene by cell suspensions of three different methane-utilizing bacterial groups are quite similar. The optimum pH value is approximately 6
-7 and the optimum temperature was about 35°C. 40
The apparent decrease in epoxidation at temperatures higher than °C may be due to both the instability of the monooxygenase system and the volatility of the product propylene oxide (bp 35 °C). Both hydroxylation and epoxidation activities were in the cell-free particulate fraction that precipitated between 10,000Xg and 80,000Xg centrifugation. Tonge et al. [Biochem.J., 161 , 333-344
(1977) and FEBS Lett., 58 , 293−299.
(1975)] is Methylocinus trichosporium
Particulate fraction of OB3b (10000Xg centrifugation and 150000Xg
report on the purification of membrane-bound methane monooxygenase from the precipitated membrane during centrifugation. Recently, but after our discoveries, Colby et al. [Biochem.
J., 165 , 395-402 (1977)], n-alkanes,
A unique soluble methane monooxygenase from Methylococcus capsulatus (Bath strain) has been shown to catalyze the oxidation of n-alkenes, ethers, and alicyclic, aromatic, and heterocyclic compounds. Strains from three different methane-utilizing bacterial groups that we tested all catalyze the epoxidation of gaseous alkenes ( _C2 - _C4 ) and the hydroxylation of gaseous alkanes ( _C1 - _C4 ). . The present inventors also found that the enzyme activity is present in the particulate fraction (i.e., the substance that sediments when the supernatant after centrifuging disrupted cells at 10,000Xg for 30 minutes is centrifuged at 10,000Xg or higher for 1 hour). 80,000Xg of the soluble fraction (i.e. broken cells)
We also discovered the unexpected fact that it was not present in the supernatant (after centrifugation for 1 hour). Methylomonas sp. (CRL-17, NRRL B-
11208) and Methylococcus capsulatus (Texas strain ATCC 19069) (obligate methylotrophs); Methylocinus sp. (CRL-15, NRRL B-
11202) and Methylocinus trichosporium (OB3b, NRRL B-11196) (obligate methylotrophs); and Methylobacterium sp.
(CRL-26, NRRL B-11222) (facultative methylotrophic bacteria) catalyzes the hydroxylation of n-alkanes and the epoxidation of n-alkenes by differential centrifugation of disrupted cell suspensions. A cell-free particulate fraction was obtained. Both activities were primarily in the P(40) fraction and were dependent on the presence of oxygen as well as an electron carrier (eg NADH). Metylocinus sp. (CRL-15, NRRL B-
The hydroxylation of methane to methanol and the epoxidation of propylene to propylene oxide catalyzed by the P(40) particulate fraction of 11202) have similar PH and temperature optima. Both activities were simultaneously lost during storage of the P(40) particulate fraction at refrigerator temperatures. Hydroxylation of methane and epoxidation of propylene by cell-free extracts were strongly inhibited by various metal binders and metal chelators (Table 6); both reaction rates were significantly lower than those of copper or iron salts. (Table 7). This suggests that metalloenzyme systems are involved in the oxidation of both substrates. These results and the stoichiometry of the hydroxylation and epoxidation reactions indicate that both reactions can be catalyzed by the same metallized monooxygenase system. This is supported by the fact that the conversion of propylene to propylene oxide is inhibited by methane. Methylococcus caprusta (Teyas strain)
[Ribbons et al., J. Bacteriol., 122,
1351-1363 (1975)], Methylomonas metanica [Ferenci et al., J. Gen. Microbiol.
91 , 79-91 (1975)] and Methylocinus trichosporium ((OB3b) [Tonge et al.
Biochem .
It is known to catalyze NADH-dependent oxidation. Methane oxidation by these microbial particulate fractions was inhibited by various metal binders or metal chelators. However, epoxidation of n-alkenes by these microorganisms has not been reported. Metylocinus trichosporium (OB3b,
Methane monooxygenase from NRRL B-11196) has been purified and shown to consist of three components: soluble CO-binding cytochrome C, a copper-containing protein (methane monooxygenase), and a low molecular weight protein [ Tonge et al., 1977 (supra)]. Unlike the microorganisms mentioned above, Colby et al.
reported a unique soluble methane monooxygenase activity from Methylococcus capsulatus (Bath strain). Methane oxidation by the soluble fraction of this microorganism was not inhibited by various metal binders. Recently, Colby and Dalton [Biochem.J., 171 , 461-468 (1978)] divided the methane monooxygenase of Methylococcus capsulusta (Bath strain) into three components and found that one of the components was an iron-containing flavoprotein. confirmed. The methane oxidation activity from the above-mentioned methylotrophic bacteria is present in the particulate fraction and is different from the soluble activity of Methylococcus capruslasta (Bath strain) discovered by Colby et al. Juan der Linden (1963, supra) demonstrated the production of 1,2-epoxide from 1-octene by resting cells of Pseudomonas sp. grown in heptane. Epoxides have not been detected as products of alkane metabolism and are also found in Pseudomonas sp.
was not oxidized by Thus, the role of epoxides in alkane metabolism is unclear. Van der Linden speculated that the enzyme system that produces epoxides may be the same as the system that catalyzes the initial oxidation of alkanes. Cardini and Jurtshuk [J.Biol.
Chem., 245 , 2789-2796 (1970)] showed that cell-free extracts of Corynebacterium sp. oxidize 1-octene to epoxyoctane in addition to hydroxylating octane to octanol. I discovered that. Mc Kenna and Coon [J.Biol.Chem.,
245 , 3883-3889 (1970)] isolated an enzyme system from Pseudomonas oleovorans that catalyzes the hydroxylation of n-alkanes ( _C6 - _C12 ) and fatty acids. after that,
Abbott and Hou (supra) and May and Abbott (supra) reported that the enzyme system from Proseudomonas oleovorans catalyzes the epoxidation of 1-alkenes in addition to hydroxylation reactions. Enzyme systems from Pseudomonas and Corynebacterium sp. catalyzed the epoxidation of _C6 - _C12n -alkenes. Pseudomonas enzyme system
It did not catalyze the epoxidation of _C2 - _C5n -alkenes. The present inventors discovered the unexpected fact that three different groups of methane-oxidizing bacteria catalyze the hydroxylation of n-alkanes (C ₁ -C ₄ ) as well as the epoxidation of n-alkenes (C ₂ -C ₄ ). Indicated. Moreover, the hydroxylation and epoxidation reactions are catalyzed by the same or similar NADH-dependent monooxygenases. In addition to methylotrophs, other microorganisms can also be used to carry out the epoxidation of _C2 - _C4 alkenes. These microorganisms include bacteria, fungi, and yeasts that grow on short-chain alkanes.
Methylotrophic bacteria (obligate or facultative) or other microorganisms grown on methane as the sole carbon source or on other carbon compounds (in the presence of methane or another inducer) can Alternatively, enzymes derived from cells can be used in the methods of the invention.

Claims (1)

[Claims] 1. A method for epoxidizing a C ₂ -C ₄ n-alkene or diene selected from the group consisting of ethylene, propylene, butene-1 and butadiene, in which an epoxy-producing bacterium belonging to the genus Methylocinus is mixed with methane or dimethyl ether. A lower alpha-
How to epoxidize olefin. 2. The method of claim 1, wherein the n-alkene is propylene. 3. The method according to claim 1 or 2, in which epoxidation is carried out in batches. 4. The method according to claim 1 or 2, wherein the epoxidation is performed in a batch method and the enzyme preparation is immobilized. 5. The method according to claim 1 or 2, wherein the epoxidation is carried out in a continuous manner and the enzyme preparation is immobilized. 6. The method according to claim 1 or 2, wherein the enzyme preparation is derived from an enzyme extract of the microorganism. 7 In the propylene epoxidation method, bacteria having the ability to epoxidize propylene belonging to the genus Methylomonas, Methylococcus, or Methylobacterium are cultured in a nutrient medium containing methane or dimethyl ether and oxygen, and the granular fraction thereof or A method for epoxidizing propylene, which comprises bringing the propylene into contact with oxygen in the presence of an enzyme preparation derived from a bacterium. 8. The method according to claim 7, in which epoxidation is carried out in batches. 9. The method according to claim 7, wherein the epoxidation is carried out in a batch process and the enzyme preparation is immobilized. 10. The method according to claim 7, wherein the epoxidation is carried out in a continuous manner and the enzyme preparation is immobilized. 11. The method of claim 7, wherein the enzyme preparation is derived from an enzyme extract of the microorganism.