CA1148488A - Epoxidation of lower alpha-olefins - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A process is disclosed for the epoxidation of lower ?-olefins by contacting lower ?-olefins (e.g., propylene) with oxygen in the presence of microorgan-isms or enzyme preparations derived therefrom. The microorganisms or enzyme preparations derived therefrom are preferably those microorganisms which are known as methylotrophs particularly those which previously grown in a nutrient medium containing methane.

Description

EPOXIDATION OF LO~ER .~LP~ OLl;r NS

2 FIr._ D OF THE INVE~TION

3 The present invention relates to the conversion

4 of lower ~-olefins ~o epoxides, More par~icularl~ it re-lates to the formation of propylene o~cide from propylene 6 a~d streams c~ntaining the same, thx~ug~ t~e action of o~y-7 gen and methylotrophic microorganisms or e~zyme preparations 8 derived therefrom.
BAC~GROUND OF l~E INVENTION
Epoxides ha~e become extremely ~alu ble ~roducts 11 due to their ability to undergo a plurality of chemical 12 reactions such as ad~ition with tho active hydrogen atoms 13 of nucleophilic reagents (e.g., ammo~ia, organic acids, 14 alcohols, water, ete,). The products o epoxidati~n (i.e., 1,2-epoxides, also known as o<-epoxides a~d oxirane com-16 pounds) have also enjoyed industrial importance because of 17 their ability to polymerize under ~hermal, ionic, and free 18 radical catalysis ~o form epogy homopolymers and copolymers~
19 Ethylene oxide and propyl~ne oxide constitute the two mose 20 important commercial epoxides. A widely utilized process 21 is the silver-catalyzed 'ldirect oxidation" process of Lefort 22 (U.S . Patent No . 1, 998,878 (1935) and Reissue Patent Nos .
23 20, 370 and 22,241).

In recent years there have been several publica-26 tions relating to the microbiologicaL oxidation of hydrocar-27 bons including the epoxidation of ~-olefin~. These publ~
28 cations include:

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Ishikura asld Fos~er, Na~cur~, 192, 89Z-893 (1961) 2 "Illcorp~ration of Molecula:r Oxygen During Micr~bial Utili-3 zatio~ of Olefi~s" 9 4 van der LiTlde~a, ~, 77, 157 lS9 (1963) "Epo~idation of ~coolefins by ~leptane-~ro~
6 Pse~dom~nas Cells";
__ 7 Huybregtse and van der Lind~, A~
8 w~nhoe~, 30, 185-196 (1964) "The O~idati~ o~ olefins ~y __ _ 9 a Pseudomonas-Reactions Involving the Doub1e Bond";
van der Linden and Huybregtse, A t~ L~---11 wenhoe~, _ (4), 381^385 (1967~ "Inductioal of Alkane-Oxidiz~
12 ing and o~-Olefin-Epoxid~zing ~nzymes by ~ Nonhydr~carb~
13 a Pseudosllonacll; and 14 Cerniglia, BlevlIls a~d Pe~ ~ ~d mental Microbiolog~, 32 (6~ 764-768 (1976) '~Microblal O~ida~
16 tion and Assimilation of Propylene".
17 In these publications where ~he epo~dation o 18 o~-olefir~s is involved, it is shown t~at cer~ain microor-19 gas~isms which have beer~ wn on alkasles will epoxidize lo octe~e~
21 In Dutch Patent No. 291,163 t~ Shell Inter~ational 22 Re~earch Corp. Inc. ~laid open for ir~pe~tlon June 25, 1965) 23 there is disclo~ed a proce~ ~or preparirlg 1,2-epoxid s by 2~ contacting ~-ole~n~ with ox~gen a~ad mlcroorganis;ns capable of g;~owing on a hydrocarboll and assimilati~g car~on from ~t.
26 This patent teacheq that the m~croorganism i~ preferably 27 growEl on a hydrocar~n having substa~tially the same number ., , . .

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of carbon atoms as the o~olefin that is sub; ec~ed to e~oxi-2 da~ion. While ~he ge~eral d~cripti~ in ~he patent includes 3 o~-oleins having from 2 to 30 carbo~ atom~, the only exam-4 ple in the pat~nt shows the epoxidation o~ l-octerle in the

- 5 pre~ence of air and ~ tStrain 473

6 which had bee~ grown Oll n-hep~a~e.

7 Isl 'che paper by DeBont and Alber~ (Antonie van

8 Leeuwenhoek, 42 (1-2) 73-80 ~1976) "Microbial 2~etabolism o~ EthyleDe") it is disclosed ~ha~ ~he ethyle~e-oxidizing lû ~train (E 20~ was grown OTI different carbon source to ob-ll tain information on the me~abolism of ethylene. It is dis-12 closed in this paper that ethg~lene o$ide i~ a product of 13 ethylene catabolism and the bacte~ium W8S also able to gr~w : 14 on the ep oxide .
Leadbetter and Foster (Arcb Mi~roblol~E~, 30, 16 91-118 (1958) "Studie o~ Some Methane-Utilizing Bacteria") 17 repor~ed that methane-growrl Pseudomonas met anica 18 oxidized metha~ol" ethanol, n-propaslol, n-buta~ol and n-19 pentanol stoichiometrically to the corresponding carboxylic 20 acids but isopropa¢lol, ter~iary butyl alcohol a~d l-~ecanol 21 were not oxidized by this bacterium.
22 DeBont a~d Mhlder ( ~ , 83, 113-23 121 (1974) "~itroge~ Fixati~n a~d Co-Oxidati~n of Ethylene ~4 by a Methane-Utilizing Bac eriuml') reported that their methane-oxidizing ~train 41 (presumably a 26 Methylo~inus) co-o~idized ethylene in addition to fixing 27 nitrogen wh~n thi~ bac~erium was grown in t.he presence o ,:

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methane and acetylene. They did no~ indicate what oxida-2 tion prod~ct was forrr.ed by the oxidation of ethyl ~e. In 3 a more recent paper, however, (~y,~L~, 81, 4 119-121 (1975) "Oxidation of E~hylene by Bacteria") DeBont sp~culated that ethylene o~ide may be the micr~biolo~ical 6 oxidatio~ produc~ of e~hylene in this microbiological oxi~
7 datio~. Dalton a~d Whittenb~ry (Arch._Microbiol., 109, 8 147-151 (1976 repor~ed that irl their ele~:~r~de experimen~
g et~lene was very ~lowLy oxidized by ~u3pe~ of slllatus. Dalto~ ~d Whittenbury s~ated at page 149: "It 11 seems extremel~ u~lilcely that its oxidation by the cell 12 would account for its di~appe rance by n~troge~-f~cing 3 metha~e sxidizing cultures as suggested by DeBont and ~n~lder 14 ~1974) " .
Whi~tenbury, Dalton~ Ec~lesto~ and Reed (Micro~ial 16 Growth ~ Gl Gompotmds: Proceedings of the Intesnational 17 Symposium on Microbial Growth on Cl Compou~ds, Soeiety of 18 Fermentation Tec~nology~ pp. 1~ 1975~ "The Different 19 Types of Methane Oxidizing Bacteria and Some of Their More Unusual Propertie9") reported that methane-oxidizing bac-21 teria possess the interesting feature o having the ablli~y 22 to oxidize, but not to utilize s~bstrates, e.g~ ~hey will 23 not grow J~ ethane but will oxidize it if ~hey are growi~g 24 on methane or if previously grown on methane.
DeBont (Antonie van Leeuwe~hoek, 42, 59-71 (1976)) `
`: 26 reported that ethylene W8S o~cidized by certa~ gram-po~itive ~ 27 bacteria believet to belong to the genu~ ~ .

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: ; , , r DeBo~t repor~ed thaJc his isolated strai~s dld ~ot grow ~n I
2 the presence of methane and deduced that ~hese bacteria 3 loca~ed in the soil were not meth~ne-o~;ldizing bacter~a.
4 DeBo~t and Albers (A~tonie ~LD~b~9 429 73-80 (1976)) i theorized l:hat the oac~dation pro~uct of the et~le~e-o~idlz- `
6 ing 3~rains o DeBo~t (1976) was e~hyle~e o~ide.
7 ~utchi~son, Wh~tte~bury a~d Dalt~ (~
8 Biol., 58, 325-335 (1976) "A Possible ~.012 oi~ Free Radicals

9 ~ the Oxldatiorl of Metha~e by M~l~') and Colb~ and Dalto2~ (J. Blochem~, 157, 495-597 ~197~) "Some 11 Proper~ies of a Soluble MethaI~e Mo~o-Oxygenase fso~n ~ i 12 OCOCCU8 capsula~s Strai~ Ba~h") reported that ethyl~e is 13 o:Ridized by the solubla metharle mo~o-ox~genase der~ved from 14 Meth~1Ococc~s capsulatlls Strai~ Bath. The latter i~vest~ga- .
toss reported ~hat the "par~icu1ate membr~ne preparatio~ " .
16 of ~f~bh~Y~ S~airl 8ath dld ~ot ha7e methaale;;
17 oxyge~lase activlty as determined by the bromomethaDe disap-18 peara~ce test. ¦
19 May, Schwartz9 Abbott and Zaborsky (Biochimica et Bioph~rsi~a Acta, 4033 245-255 (1975) l'Stn~ctura1 Effects on 21 the Reac~ivitsr of Sul:~strates and Inhibitors i~ ~he Epo~:ida- ~¦
22 tiO~I S~stem of Pseudomona~ o1eo~orans"~ reported that it is 23 knowr~ that the en~me s~s~cem of Psaudomona~ cat-24 al~ze~ the epo2cidatioTl of terD~i~al oleins i~ addi~ion to the pre~ ly know~l methyl group hyd~oxylatio~ o~ a1kanes ~¦
26 and fa~cty acid8. These i~vestigator~ olmd that this ~nYy-27 matic epoxidation reaetion e~ibits a sub6trate speciici~y .

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1 far di~ere~t from that expected on the basis o chemical 2 reactivity in norl-enzymatic epoxidacion reacti~ns. These 3 investigat~rs folmd ~cha~ for this e~zyme sys~em, when the 4 carbon le~gth is decreased below C8 the epoxida~ioQ rate rapidly decreases whe~eas the hydroxylation rate increases 6 Their da~a show that propyl~ne a~d l-bu~ene are hydroæylated 7 tc the corresp~nding u~lsatura~ed alc~h~ls, but ~ot epo~i-8 dated by this enzyme s~stem~ Their evide~ce is a~ illu5-9 trati~n of the high degree of specificity and unpredictability of the oxida~ive ability of ~icroorganisms.
11 On the basis Of 180 incorporation from 180~ into 12 the cellular constituents of Pseudomonas me~hanica Leadbetter 13 and Fos~er (Nature9 184: 1428-1429 (1959) "Incorpora~ion of 14 Mole~:ular Oxyg n in Bacterial Cells Utilizing Hydrocarbons For Growth") suggested that the initial oxidati~e at~ac~ on 16 methane involves an oxygenase. Higgins and Quayle (J Biochem., 17 11~:201-208 (1970) "Oxygenation o~ Methane by Methane-Grown L8 Pseudomonas methanica and ethanomonas methanooxidans") 19 isolated CH3180H as the product of methan~ oxidation when 20 suspensions of Pseudomonas methanica or Methanomonas methano 21 oxidans were allowed to o~idize methane in 18O2~enriched 22 atmospheres. The subsequent observation of m~thane~-s~imulated 23 NADH oxidation catalyzed by extracts of MethYlococcus ca~su-24 latus by Ribbons (J. Bacteriol., 122:1351~1363 (1975) "Oxidation of Cl-Compounds by Particulate Fractions From 26 MethYlococcus ~ distribution and prop~rties o 27 Methane-Depentent Reduced Nicotinamide Adenine Dinucleotide -.~.

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1 Oxidase (methane hydroxylase)") and Ribbons and Michalover, 2 FEBSLett. 11:41-44 (1970) "~ethane Oxidation by Cell-Free 3 Extracts of Methylococcus capsulatus~7 or Methylomonas 4 capsulatus by Ferenci (FEBS Let~. 41:94-98 (1974) "Carbon S Monoxide-Stimulated Respiration in Methane-Utilizing Bacteria") 6 sugges~ed that the enzyme responsible for this oxygenation is 7 a monooxygenase. Thcse workers relied on indirect enzyme 8 assays9 measuring methane-stimulated NADH disappearance 9 spectrophotometrically or me~hane-s~imulat d 2 disappearance polarographically. Recently, methane mono-oxygenase system~
11 were partially purified from MethYlosinus ~richos~orium OB3b 12 (Tonge, Harrison and Higgins, J. Biochem, 161: 333-344 (1977) 13 "Purification and Properties of the Methane Mono-Oxygenase 14 Enzyme System From ~ LLy~L~L:~L~alc~El9~ OB3b"; and Tonge, Harrison, Knowles and Higgins, FEBS Lett., 58: 293-299 16 (1975) "Properties and Partial Purifîcation of the Methane-17 Oxidizing Enzyme System Erom MethYlosirlus trichosporium"~
18 and leob~ 'a~a~_~L~ ~e~ Y~ (Bath) (Colby and Dalton, J.
19 Biochem., 171: 461-468 (1978) "~esolution of the Metharle Mono-Oxygenase of Methylococcus ca~sulatus (Bath) Into Three 21 Components" and Colby~ Stirling and DaltoQ, J. Blochem., 165:
22 395-402 (1977) "The Soluble M. thane Mono-Oxygenase of 23 Meth~lococcus capsulatus (Bath), Its Abili~y to Oxygenate . ~
~`: 24 n-Alkanes, n-Alk~nes, Ethers, and Alicyclic, Aromatic and :" 25 He~ero-eyclic Compounds").
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'~ 27 It ha~ now been disco~ered that C2-~4 n-alXenes :` 28 and butadiene, es~ecially ~roDylene can be ~re~ared by a `

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1 Lcw energy intensive process comDrising contacting C2~C4 2 n-alkenes or butadiene r~ith oxygen 7 n the ~resenc~ of 3 microorganisms or enzyme preparations derived therefrom, 4 w~er~in said mic~oorganisms have bee~ cultivated in a mi~-eral nutrient medium containing methane or dimethylether. The 6 microorganisms used in the process are preferably obligate or 7 facultative me~hylotroph~ and more preferably deri~ed from 8 the genera~ Y~ E~ Methylocystis, ~ Y~EQ~
9 ethylobacter, Met~ylococcus and MethYlobacterium.
1~ Unlike the ~ilver-catalyzed "direct o~ida~ion"
11 process for prepaxing e~chylene oxide, i~ has been further 12 discovered that the methane~grGwn methylotro~ mlcroor~
3 ganism3 or an e~æyme preparation derived therefrcm are cap-able of epoxidizing o~Dolefins havi~lg two 'co four carbo~ atoms 15 and butadienesj but are nof capable of eooxidizi~&..Cs+ ~olefins 16 (a~ least in amounts whirh are easily detectable by ordin-17 ary analytical metho~s)O As a preferred embodiment the 18 methane-i~duced methylotrophic microorganisms or the enzym~
19 preparations derived there~rom are used to oxidatively con~
vert propylene to propyl~ne oxideJ
21 DETAILED DESCRIPTION OF ~HE INVENTION
22 The term ~Imicroorganism~ is used herein i~ its 23 broadest sense to include not only bacteria9 but also 24 yeasts, filamentous ~ungi~ actinomycetes and protozoa. Pref-erably, the microor~anisms will include bacteria~ and more 2~ preferably the bacteria capable o oxidizing me~hane.

The term "enzyme preparation" is used to refer to any composition of matter that exhibits the desired oxygenase enzy-matic activity. The term is used to refer, for example, to live whole cells, dried cells, cell extracts, and refined and con-centrated preparations derived from the cells. Enzyme prepara-tions may be either in dry or liquid form. The term also in-Cludes the immobilized form of the enzyme, e.g., the whole cells of the methane grown microorganism or enzyme extracts immobilized or bound to an insoluble matrix by covalent chemical linkages, adsorption and entrapment of the enzyme within a gel lattice having pores large enough to allow the molecules of the substrate and of the product to pass freely, but small enough to retain the enzyme. The term "enzyme preparation" also includes enzymes retained within hollow fiber membranes, e.g., as disclosed in :
Biotechnology and ~ioengineering, Vol. XIII, pp. 431-477(1971) ~` "Multiphase Catalysis II Hollow Fiber Catalyst" by P.R.Rony.
The term "particulate fraction" refers to the oxygenase enzyme activity in the precipitated or sedimented fraction of cell-free extracts of the methane grown microorganisms after centrifugation between 10,000 x g. and 80,000 x g. for 1 hour.
The instant invention includes the following features:

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- _ 9 _ -1 The isolates of methane-utilizing microbes 2 of th~ invention include obliga~e (Type I and Type II) and 3 facultative bacteria as well a~ new methane-utilizing yeasts.
4 o In addition to their ability to oxidize methane to methanol, resting cell suspensions o~ several distinct 6 tyPes of methane-grown bacteria (e.g., Type I, obligate;
7 Type II, obligate); andfacultati~e oxidize C2-C4 n-alkenes 8 and butadiene to their corresponding 1,2-epoxides~
9 ~ The product l,~-epoxides are not further me~abolized and accumulate e~tracellularly.
11 ~ Methanol-grown cells do not have either the 12 epoxidation or the hydroxylation activities. Among the 13 substrate gaseous alkenes, propylene is oxidized at the 14 highest rate ` 15 ~ M~thane inhibits the epo~idation of propylene.
16 ~ The stoichiometry of the consumption of propy-`` 17 lene and oxygen, and the production of propylene oxide is 18 1~
19 Results from in~ibition studies indicate that 20 the same mono-oxygenase system catalyzes both the hydroxyl-21 ation and the epoxidation reactions.
22 Both the hytroxylation and epoxidation activities 23 are located in the cell-ree (enzyme ex~ract~ par~iculat~ frac-24 tion precipitated or ~edi~e~ted between 10,000 x g. and 80,000 x g. centriugation for 1 hour.
26 ~ Cell-free particulate fractions from the obligate ` 27 and facultative methylotroph microorganisms catalyze ~he - - ~
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1 hydroxylation of methane to methanol and th~ epoxidation o~
2 C2-C4 n~alkenes and dienes (e.g.~ ethylene, propylene, 3 l-~utene and butadiene) in the prasence of oxygen and reduced . .
4 nicotinamide adenine dinucleotide (NADH) and the hydroxylation : 5 of Cl-C4 n-al~anes ~eOg., methane, ethane, propane and butane).
6 ~ The hydroxylation and epoxidation activities of 7 the methane-grown methylotrophs are lost simultaneously during . 8 storage and are strongly inhibited by various metal-binding :: 9 agents.
` 10 The s~oichiometry for the consumption of substrate

11 (propylene or methane), oxygen, NADH, and product formation

12 was found to be approximately 1:1:1:1.

13 The classification system of methane-oxidizing

14 bacteria proposed by R. Whit~enbury, K. C. PhilliDs and J. F. Wil~lQson [ ~ , 61~ 2~5~218 (1970) 16 (hereL~after Whittenbury et al.)~ is the mos~ widely recog-17 nized system used today In this system of classification, 18 the morpholo~ical characteristics of me~ha~e~utllizing bac-19 teria are divided into five groups. They are: Methylosinus, Meth~loc~stis, Methylomonas, ~ethYlobacter and ~
21 cusO Bacterla of the~e five groups repor~ed by Whi~te~bury 22 et al.utilize methane9 dimethylether9 and methanol for 23 growth energy and they wer2 all repor~ed as strictly ~ero-24 bic and gram~negatlveO They are also eharacterized as 25 being non-endosporlng~ i e,~ the ability ~o form cysts and 26 exospores with ccmplex flne structure and complex internal 2 7 s tructure O

As one e~nbodimenc of the present in~ention3 it 2 has been disco~rerPd tha~ microorganisms described by Whit 3 tenbury e~ alD when cultivated in the prese~ce of methane, are capable of epoxidi~in~ lower oc-oleflnsg particularly propylene,in the presence of oxyge~O The~e methane-6 utilizing microorganlsms are gener~lly known as "methylo-7 trophs", The enzyme s~stem or the preparatio~s derived 8 from these mlcroorga~lsms are referred to herein as an g "epoxidiæi~g enzyme system" which is believed to be a "methane m~no-oxygenase" ~nd/or "me~hane hydroxylase".
Ll Thus, it is to be under~ood ~hat the en~yme sys~em or 12 enzyme prepara~ions ~hereof referred to herei~ as the 3 "alkene epo~idase" or "prop~lene epogidase" used to co~-14 vert the c~-oleins to 192~epo~ides are the "epoxidlzing enzyme system" believed to be methane mono-o~yg~nase or 16 methane hydroxyla~e enzymesO
17 The methylotrophic microorgani~m reported by 18 Whittenb~ry e~ al.(the disclosure of which is incorporated 19 herein by refere~ce~ are c~nt~plated for use in the p~ac-tice of the present ln~ention. Speciically, one may u~e 21 those methylotroph~c microor~anisms mentioned in Table 4, 22 page 214 of the Whitt~nbury et al paper, i.e., those micro-23 organisms identified as: MethYlosinus trichosporium, Me~hyl-24 osinus spori~m, ~ , ~M~5~ a~ C~
25 ~ a~ c_~Leyc, Met~lom~na~ s~ccoD~c ,~
26 monas a~ile, M~:bvlo~onA~ rubrum, Mec~ r~--c~u~, 27 ~ ~ Ybb2YI ~YYYYYY_~YYIC~ 5~
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1 capsulatus, Methylobacter_vinelandii, Methy10coccus_~esu-2 latus (includ~ng MethYlococcus capsulatu~ Strain Ba~h ref-; 3 ferred ~o by J. CoL~ and H. Dal~on; J. Biochem., 157, 495-`' 4 497 (1976)~ and MethYlococcus c~sulatus Strai~ Texas ref-: S ferred to by D. W. Ribbo~s, JO Bacteriol., 122, 1351~1363 .
~` 6 (1975)), and Methyloco~cus ml mus. These methylotrophic 7 microorganisms may ~e used in the form o~ their whole cells, 8 enzyme extracts ~hereof or immobilized preparations of 9 those whole cells or enzyme e~tracts, such as by use of ~` 10 D~E cellulose or ion exchange resin or porous alumina 11 carriers~ .
12 Subcultures of some methylotrophic microorganisms -~ 13 described by Whittenbury et al have been deposited with ~he 14 offlcial depository of the United States Department of Agri~ ¦

15 culture, Agriculture Research Service, Northerr~ Regional

16 Research Laboratory, Peorla, Illinois 61604, by depositing

17 therein subcultures of each, and have received from the

18 depository the i~dividual ~L s~rai~ designations a~ indi- !

19 cated below. These subculture~ have bee~ deposited ~ ac~

20 cordaIlce with the procedures of the Departmerlt of Agricul-.
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3~3 ~ 13 -ture without any restriction such ~hat progen~ of these 2 strains are available to ~he public, includi~g but not 3 limited to those citizens i~ the Uni~ed State3 of America 4 and tho~e citizens i~ Wes~ &ermany~ S~rain~ of me~hylo-5 trophic microorganisms depo~ited are ldeIlti~ied as follows:
6 USDA Agricul~ral 7 Re~earch Service 8 _~S~ ~L8!~D
9 ~ OB3bNR~ B-11,196 ~ 5~RRL B-lL ,197 11 M~ OBBP~ L B-ll ,198 12 ~g~ S~L B~ 199 13 `I ~b~ BG8~RL B~ll, 200 14 M}~ Y~RRI. B-11,201 ProgeDy of these strains are available to a~yone 16 who requests the same without ang restrictioT~ as to avail~
17 abilitsr. Sl~bcul~ures of the aorementiotled strai~s were 18 orlgi~ally obtai~ed from R. t~hittenburg~ Department of Bio~
19 logical Scl~nce9 UTliversity of Warwick9 Warwic~cshire, Coven-2 0 try, ~gland .

21 The morphological ~n~ taæonomical characteristics

22 of the a~ove-mentioned me~hylotrophie strai~s are as follows:

23 ~9~ OB3b ~R~ B-ll ,196

24 Produces white colonies o~ salt agar pla~es i~

25 the presence of methane or methanol. The orgaslisms are

26 motile, rod-shaped, gram negative a~d aerobic. Rosettes

27 are frequently formed. Has a Type II menibrane structure.
2 8 Methylosi~us sporl~m 5 ~RL B -11,197 ' .

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, Produc~ white colo~ies on salt agar place~
-2 the prese~ce of methane or methanol. The organlsms are 3 motile, rod-shaped, gram-negative and aerobic. Rose~tes 4 ara frequently formed. Organism form e~ospo~es which are 5 heat-resist~t; spores budded of~ ~he non-flagellated poles 6 of the organisms which assumed a vlbrio shape. Organic com-7 potmds other ~han methane and metha~ol do no~c support grow~h.
8 Has a Type II mesibrane structure.
9 ~9~ OBBP ~R~L B-ll ,198 Prod~ces mucoid w~ite colanies o~ salt agar plate~
the presence of methalle or methanol. The organisms are 12 non-motile, c~c o~acillus ln shape, gram-negative asld aero-3 bic. Organisms ~orm cgsts which are dessicatios~-resistasl~, 4 but not heat resistantO Grows at the e~peIlse o methane or methaxlol. Org~ic compounds other tha;Zl metha~e and methanol 16 do no~ suppor~ growth. Has a Type II membrane structure.
17 ~5b~ 9~ 5~ 9~ Sl NRRL B~119199 18 Prod~ces piak col~aies on salt~agar plates in the 19 presence o~ methane or metha~ol. The orga~isms are motile, rod-shaped, gram-~eg~tive and aerobic. Produces slimy cap-21 sules. They grow at the eæpense of methane and methanol.
22 Org~nic compounds other than me~hane and methanol do not 23 suppor~ growthO Has a Type I membrane structure.
24 2b~59:o ~ lbu~ BG8 NR~L B-ll 9 200 Prod~ces white colonies o~ salt~Agar plates in 26 the presence of metha~e or methanol. The organisms are 27 motile, rod-~haped9 gram-negative and aerobic. Produce~

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1 slimy capsule. Grows at the expense of methane and meth~nol.
2 Organic comp~unds othe~ than methane and me~hanol do not 3 support grow~h. Has ~ Type I membsane ~ructure.
4 ~13~Lb~ eD:~:L~te~ Y ~RRL ~ 201 Produces white to br~w~ c~l~Qie~ al~-agar 6 pla~es in the pr~sence of ~ethane or wetha~ol. The orga~-7 ismR are motile, ro~shaped, gram-negati~e and aerobic.
8 Produces 91imy capsule. Grows at the e~pense of methane 9 and methanol. Orga~ic c~mpounds other than methane and methanol do ~o~ support growth. Has a Type I ~embrane 11 structure.
12 Recently, Patt, ~ole and Hanson (~ ~en~ ~ooal;
13 SYstematic Bacteriolo~3 27 (2) 226-229 (L976)) disclosed 14 that methylotrophlc bacteria are those bacteria that caa 15 grow non-auto~rophically usi~g car~o~ c~mpou~ds containing 16 one or m~re car~o~ atoms, but contai~ing Ilo carb~n-car~on 17 bo~ds. Pa~t et al ha~e proposed chat methylotrophs should 18 be considered "obligate" i they are capable of utilizing 19 only carbcn compolmds containi~g no car~ car~o~ bonds ~e.g., methane, methanol, dimethylether, methylamines, etc.) 21 as the sole sources of car~on a~d energy whereas ~Ifaoulta-22 tive" met~ylotrophs are those organisms that can u~e com~
23 pounds containing no carbon~car~o~ bonds and complex com-24 pounds con~aining carbon-carboQ bonds as the sole sources of car~on a~d e~ergy~ In their paper9 Patt et al.disclosed 26 a methane-ox~dizing bacterium9 which they id~ntified as 27 ~ sp novO (ATCC 27,886). Thi~

bacterium presumably differs from all previously described 2 genera and species of methane~oxidizi~g bacteria because of 3 i'cs ability to utlli2e a varlety of orga~ic, sub~trates with 4 carboa-carb~ bonds as ~ources of carbon and energ~.
As another embod~ment of the present in~tention"
6 it has been discovered that this microorga~ism 7 ~_ ~eb~ sp novO ATCC 27,886) a~d o~her fac~lta-8 tive meth~lotrophic microorga~isms are also capable of epoxidizing C2^C4 alkenes. In other words, they possess alkene epoxidase esszyme ac~ y when cul~ivated in the 11 presence of methane. As discussed above with respect to 12 the Whittenbury et al methylctr~phic microorga~isms, the 13 facultative Methylotrophs ~ay 'be u~ed in the form of their 14 crudP extract . (i-.e. supPrna~ent after centriugin~s broken ce~ls at 10; 000 x g. for 30 min . ) or to be placed In immo-16 bilized form or used in the cell-bound form when put to use.
17 in the process of the present invention.
18 Other ~nown me~hylotrophi~ strains may be used ia 19 ~e process of ~he present inventio~, e.g., n:-h-lo C~
AJ^3670 (FERM P-2400) referred to i~ U.S. Patent ~o.
21 3,930-947 as fre~ly a~ailable from the Fexmentati~Q Research 22 In~titute, Age~c7 of Industrial Science and Tech~ology, Min~
23 istry ~or Indu~trial Trade a~d Isldustry, Chiba" Japan; z~d 24 Methylococcus 999 referred to in U.S. Patent No. 4,042,458 25 as havi~g NCI3 Acces~i~ NoO 11083 as well as 26 SM3 having NCIB Acces~ioT~ No. 11084 (which has been tes-27 cribed in Netherland9 patent applica~ion No. 74/16644~.

28 Mixtures of methylotrophic and non-me~hylotrophic ~nicroor--: `~

- 17 ~

1 ganisms may be utilized, such as the sys~ems described in 2 U-S. Pate~t Nos. 3,996,105 and 4~042,458.
3 In commercial processes for ~he propaga~i~n of 4 microorganisms9 it is ge~erally necessary ~o proceed by stages. These stages may be few or m~g~, depending on the 6 nature of the procesg and the characterist~es of ~he r~icro~
7 orga~isms. Ordinarily, propagation i9 started by inoculat-8 ing cells from a slaslt of a culture into a pre-sterillzed g nutrie~t medium usually c~ntained in a flask. In the flask, 10 growth of the microorga~isms i~ encouraged by ~arious means, 11 e.g., shaking for ~horough aeration9 and mainte~a~ce of 12 suitable tempera~ure. This step or stage is repeated o~e 13 or more time~ i~ fl~sks or vessels c~nt~ining the same or 14 larger volumes of nutrienk medium. These stages may be co~enie~tly referred to as cult~re developme~t stages.
16 The microorga~isms with or without accompanying culture 17 medium, from t~e last developme~t stage, are lntr~duced or 18 inoculated into a large scale ferme~tor to produce e~ r-19 cial quantities of the microorganisrns or enzymes therefro~D.
Reaso~s for growi~g the microorganisms in stages 21 are manyold, but are primarily depe~dent upon the c~di-22 tions necessary for ~he growth of the microorgani~ms a~d/or 23 the production of enzymes therefrom. l~ese i~clude stabil-24 ity of ~he microorgaIIisms, proper nul:rients, pH, osmotic 25 rela~ionships, degree of aerati~n, temperature and the main-26 ten2slce of pure culture conditions ~uring fermentation. For 27 insta~ce, to obtaln maximum yields of the alkene epoxidase, 1 the conditions of fermentation in the final stage may have 2 to be changed somewhat from those practiced to obtain growth 3 of the microorganisms in the culture develo~ment stages.
4 Mai~tai~ing ~he purity of the medium, also, is an extr~mely importa~t considerati~n, ~specially where the fermentation 6 iQ performed under aerobic c~nditions as in the case of the 7 methylotrophic microorganisms~ If the ferme~tation is ini 8 tially s~arted in a large fermentor, a relatively l~ng period g of time will be needed to achieve an appreciable yield of microorganisms and/or ~lkene epoxidase enzyme t~erefrom.
11 ThiS, of c~urse, ~nhances the possibili~y of contamination 12 o~ the medium and mutation of the microorga~isms.
13 The culture media used or gxowlng the methylo-14 trophic microorga~isms a~d inducing the oxygenase or epox-idation enzyme system will be comprised of inorganic salts 16 ofphosphate,sulfates and nitrates as well as oxygen and a 17 source o~ methane. The fermentation will generally be con-18 ducted at temperatures ranging from 5 to about 55C., pref-19 erably at temperatures ranging from about 25 to about 50C.
The pH of the culture medium should be con~rolled at a pH
21 ranging from about 4 to 9 and preferably fr~m about 5.5 to 22 8.5 and more preferably from 6.0 to 7.5, The fermentation 23 may be conducted at atmospheric pressure although higher 24 pressures up to about 5 atmospheres and higher may be employed.
2S Typically, to grow the methylotrophic microorganisms ~6~; and to induce the oxygenase or epoxidation enzyme system the 27 microorganisms are inoculated into the medium which is contacted -1 with a gas mixture containing me~hane and oxygen. Methane 2 may be supplied i~ the form of natural gas. For continuous 3 flow cu~ture the microorganisms may be g~o~n in any suitably 4 adapted fenmentation vess21, for example, a stirred baf~led fermen~or or sparged tower fenmentor~ which is provided - 6 either with internal cooling or an external recycle cooling 7 loop. Fresh medium may be continuously pumped into the cul-8 ture at rates equivalent to 0.02 to 1 culture volume per 9 hour and the culture may be removed at a rate such that the 10 volume of culture remains constant. A gas mixture contain-11 ing methane and oxygen and possibly carbon dioxid~ or other 1~ gases is contacted wit~ the medium preferably by bubbling 13 continuously ~hrough a sparger at the base of the vessel.
14 The source o~ oxygen for the culture may be alr, oxygen or oxygen-enriched air. Spent gas may be removed from the 16 head o~ the vessel. The spent gas may be recycled either 17 through aQexternal loop or internally by means of a gas 18 inducer impeller. The gas flows and recycle should be ar-- 19 ranged to give maximum growth of microorganism and maximum 20 utili~ati~n of methane.
21 The o~ygenase enzyme system may be obtained, as des-22 cribed above, as a crude extract, or a cell-free particu~ te 23 fraction, i.e.J the material which precipitates or sediments 24 when the supernatant ater centrifuging broken cells at 25 10,000 x g.for 30 min. i~ centrifuged for 1 hour at 10,000 x 26 g.or greater.

. ~ 20 -1 The microbial cells may be harves~ed rom the growth medium 2 by any of the standard techni~ues commonly used, for exam-3 ~le, flooculation, sedimentation, and/or precipitation, 4 followed by centrifugation and/or filtration. The biomass 5 may also be dried, e.g., by freeze or spray drying and may be ~ ~ used in thi~ form for further use in the epoxidation reac-: 7 tion. I~hen using the cell-free enzyme, NADH and a metal ` 8 (e.g., copper or iron), may be added to enhance the enzyme . 9 activity.
10 To put the invention to prac~ice, an oxygenase 11 enzyme system is cbtained, such as, for example, in the 12 ma~ner described above, which will con~er~ methane to meth-13 anol under o~ida~ive condition~. The source of the enzyme 14 is ~ot critical, but it is pre~erred to obtain such a prepar~
ati~n from one of th~ five genera of microorganisms d~s-16 closet i~ the t~hittenbury et al paper or from the faculta-17 tive ~ethylotrophs ~Methylobacterium) and grow the micro-18 org2nism in a ~utrient ~edium c~ntaining meth2ne and oxygen 19 as described above. The ~utrie~t medium may be the one des-20 cribed by Whittenbury et al.or more preferably the cultu~e 21 medium described by Foster a~d Davis~ J. Bac~eri~l, 91, 2~ 1924-1931 (1966). The enzyme preparation is then brought 23 into coQtact with a C2-C4 alkene, e.g., ethylene, propylene3 24 butene-l or con~ugated but~diene cr mixtures thereo~ i~ t~e presence of oxygen in a buffer solution or in a nutrient medium 26 (e.g., the same nutrient medium used to produce the mlcroorganism 27 may be used except that ~he olefin has replaced the methane) and :, ~`1 t~e mixture is incubated un~il the desired degxee of conver-'2 si~n has been obtained. m ereafter, the epo~ide is recov~
`3 ered by c~nventional means~ e.g., distillation, etc.
To faeili~ate ~he ~ecessary effec~ive contact of oxygen and the e~zyme (whether it be an enzyme preparation :6 or met~ylotrophic microorganisms), it is pre~erred, for best 7 resul~s, to e~ploy a strong, ~lnely divided air stream into 8 a vigorously stirred dispersion of olefin in the epoxidatio~
9 mediu~ that generally contai~s water a~d a buffer, and in which the enzyme preparation or microorganism culture is 11 suspended. The enzyme preparation may then be separated from :12 the liquid medium, preferably by filtration or centri~ugation.
13 The resulting epoxide may then generally be obtained.
14 The process of the invention may be carried out ba~ch^
wise, semicontinuously9 continuously, concurrently or counter-16 c~rrently. Optionally, the suspçnsion containing ~he enzy~e 17 preparation or ~thylotrophic microorganis~s and a buffer 18 solution is passed downwardly with vigorous stirring counter 19 currently to an air stream risi~g in a tube reactor. The top layer is removed from the downflowing suspension, while culture 21 and remaining buffer solution constituents are recycled, at 22 least partly, with more olefin and addition of fresh enzyme 23 preparation or methylotrophic microorganism, as required.
~4 The growth of the methylotrophic microorganisms and the epoxidatlon process may be convenie~tly coupled by 26 conducting them simultane~usly, but separately and using 27 much higher aeration in the epoxidation process (e.g., an `~
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1 air excess of at least ~wice that required or growth, pref~
2 erably at least ive times as much aeration). Bo~h the grow~h 3 process and epo~idation process may be conducted in the 4 same reactor in sequential or simul~ane~us operati~s by 5 al~erna~e use of normal an~ strong aeration.
6 The inven~ion is illustra~ed fur~cher by the fol~
7 lowing examples which, h~wever, are ~o~ to ~e ~aken as lim-8 i~ing in any respect. All par~s and percentages, unless 9 expressly sta~ed otherwlse, are by w2ight.

11 A nu~rient medium as described by Foster and 12 Da~is, J. Bacteriol., 91, 1924-1931 (1966) having the fol~
13 lowing c~mposition pe~ liter was prepared.
a2HP04 0.21g NaHZP04 0.09g 16 NaN03 2.0g 17 MgS04~7H20 0,2g 18 KCl 0,04g 19 CaC12 0, OlSg FeS04 ~ 7H20 1 . ûmg 21 CuSO4~ 5H2O
22 H3BO4 0.02mg 23 ~fnSO~ 5H20 0 . 02mg 24 ZnSO4 0.14mg MoO3 0 O 02mg 26 The p~ of the nutrient medi~Lm was adjusted to 7.0 by the addition of acid or base and S0 ml aliquots of the nutrient 2 medium was charged into a plurality o~ 300 ml shake 1asks.
3 The shake flasks were inocula~ed with an inoculating loop 4 of cells ~rom an agar plate contai~ing homogeneous coloniPs of the mi~roorga~isms on the plate (the purity of ~che iso-lates were c~nfirmed by microscopic examisla~ion). The iso~
7 Lates had beesl maintained on agar plate~ under a~ atmosphe~:e 8 o methane and air having a 1~1 V/v gas ratio which had been 9 trarlsferred every two weelcs~ The g~se~u~ phase of the inoc~
ulated flasks was then replaced with a gas mix~ure comprised 11 of methane and air having a ra~io of 1~ a V/v basis.
12 The inoculated flasks were sealed air tight and were incu-13 bated on a rotary shaker of orbital radius 2.5 rm at 250 14 rpm and a~ 30C. for two days until the turbidity ~n 'che medium had developedO
16 The cells were harvested by centrifugatiora at 17 10 ,000 x g at 4C . for 30 minute~ . The cell pellet was 18 washed twice with a 0.15M phosphate buLfer at a pH of 7,0 19 (contai~ing û.002M MgC12~. The wa~hed cells were then sus-p ~ded in a 0 . i5~ phosphate buf~er at pH 7, O ~
21 A 0 . 5 ml aliquot of eac~ washed cell suspension 22 (2mg cells) was put into 10 mlv~als at 4C ~ich were sealed 23 with a 3~ubber cap. The gaseous phase of the vials was re-24 moved wi~h ~acuum and then was replaced with a gas mixture, of alk~e as~d oxygen at a 1:1 V/v ratlo~ ~c was 1hen ineu-26 bated at 30C. OR a rotary shaker at 300 rpm. Samples of 27 product (3,6~,1) were withdrawn periodieally wlth a microsy--- ^ -.

ringe a~d ~he products were analyzed by gas chromatography 2 (ioniæation 1ame detector eolumn).
3 Table I shows the conversion r~tes for the hydroxy~
4 lation of methane ~d the epoxidation of propylene by washed 5 cell susperlsions of several microorganis~sS,strai~s of which 6 had baen grown on metharle by ~che experimental procedure des~
7 cribed above. It car~ be seen from the~e d~ta that the methane-8 grown microorganisms which are capal:le of hydroxyla~ g 9 methane to methanol are also capable of converting propylene 10 to propylene oxide.

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. - 26 -1 Table II shows the conversion rates for the hydrox-2 ylation of meth~ne and the epoxida~ion of propylene (from 3 Table I) and the epoxidation of ethylene, butene-l and buta~
4 diene by washed cell suspensions of two microorganism ~trains which had been grown ~n methane by the ~perimental proceduse 6 described above, These methane-g~own microorganisms, whe~
7 contacted with pentene-l and hexene-l in the prese~ce of air 8 did not produce any detectable epoxide, Also, it can be 9 seen from the data that the conversion rates for propylesle to propylene oxide were higher for the respective me~hane-11 grown microorganism than for the other conversions.

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1 The experIm~ntal procedure desoribed above was 2 repeated 1~ th~ case of the strai~s ethylocy8tis Parvus 3 OBBP (NRRL B-11,198), Methylom~nas metha~ica S~ ~NRBL B-4 11,199) and ~5~1e~ y~ BG8 (NRRL B-11,200) a~d was~ed cell suspensi~s o~ these methane-grow~ microorgan-6 lsms were sucoess~ully used t~ convert ethylene to e~hylene 7 ox~de at con~ersi~n rates of 0.9, 0.95 a~d 1.2 ~mole/hr/m~
8 protein at a O.2g/100 ml dry weight of cells pe~ culture broth 9 basis, respectively.
As shown above, a novel method has been disco~ered 11 whereby propyle~e oxide is obtai~ed by incubating propylene 12 in the presence of cells or cell-ree extract~ of micro-13 organisms (or enzymes derived therefrom) which have been 14 grown in the presence of methane. These microorganisms are ~nown to be able to hydroxylate short chain alkan~s (e.g., 16 methane to methanol) ant some investigators have suggested 17 they may be capable of epoxidizing ethylene. It has now been 18 discovered that these methane-grown microorganisms and their 19 enzyme preparatisns have the ability to epoxidize propylene at relatively higher c~nversion rates than in the case of 21 ethylene, butene-l and butadie~e. I~ batch experiments 22 using washed~ metha~e~grown oells, the epogidati~ ~eaction 23 proceeds li~early for at least 2 hours~ No furt~er oxida-24 tion o~ the epoxide product was detectedO
The epoxida~ion enz~7me sy~tem of the methane-26 grown microorganisms is inducible (bg~ ~he meth~ae) aT~d the 27 epo~ide product accumulates e~tracellularly (i.e., after , .

.

- 29 -the reaction, the reaction mix~ure was centrifuged and the 2 epoxide pro~uct was olaly fouTld in the supe~natant fracti~n 3 a~d ~o in the cell pellet). The possibility o~ propanal 4 as an oxidation product of propylene was ruled out as a re-5 sult of g . l.c r analysis, 6 I~ comparative experiment~l ~ests, washed cell 7 susp~sions o the methanol-grown microorga~ism 5trai~15 8 ~ OB3b (NRRL B-11,196); 2~
g ~ystis~ar~7us OBBP (~RL B-11,198), Methylomonas methanica 10 Sl (~L B-ll,l99) ar~d ~g~ Y (NB~ B-11 11,2013 did ~ot po~s~ss the abilit r for eithe~ the hydrox3r-12 latio~ of metha~e or the abilit~ to epo~idize C2-C4 alkenes, 13 particulàrly propyle~e. From the eYidence shown, only the 14 me~ane-grown mic~oorgarl~sms posses~ bo~h met~a~e hydr~xy-la iosl a~d C2-C4 ep~xidatioll abilitie~. :
16 As previously indicated both the whole cells and 17 ~he cell- free extracts containing the oxygenase enzyme ac-18 tivity oi the methane grown met~ylotrophs may be used in the 19 hydroxylation and epoxidation reactions in the presence of 20 air. NADH and metal (iron or copper~ may be added to en-21 hance activity when the cell-free or pure enzyme preparations 22 are used. In utilizing the cell-~ree enz~me sy~tem of the 23 invention the enzyme preparation~ were prepared as follows.
24 eparation of Cellular Fractions Organisms were gr~wn at 30C. i~ 2.8 liter flasks 26 containing 700 ml m~neral salts medium as described in Ex-27 ample 1 with methane (methane ant air, 1:1 parts by volume) 1 1 4li4,8

- 30 -1 as th~sole carbon and energy source. Cells were harvested 2 during e~ponential ~rowth by centriugation at 12,000 x g.
3 for 15 min. at 4C. Cells were washed twice with 25 mM
4 potassium phosphate bu~fer, pH 7.0 containing 5 mM MgC12.
Cells were suspended in ~he same buffer. The cell susoen-6 sions at 4C. were disintegrated by a single passage ~hrough 7 a French Pressure cell (15,000 lb./in.2) and centrifuged a~
8 5000 ~ g.for 15 min. to remove unbroken bacteria. The super- ~¦
9 natant solution (crude extract) was then centrifuged at 40,000 x g.for 30 min., yielding particulate P(4~) and soluble S(40?
11 fractions. The S(4~) fraction wa~ subsequently centrifuged 12 at 80,000 ~ g. for 60 min., yielding particulate P(80) and -13 soluble S(80) fractions. The particulate fxactions [P(40j 14 and P(80)~ were suspended i~ 25 ~M potassium phosphate buffer, pH 7.0, containing 5 mM MgC12 and homogenized at 4~C.
16 Enz~e Assay 17 The oxidation oi methane and propylene by particulate 18 ~(P~40 and (P)80] fractions and soluble [$(80~ fraction was 19 measured at 30C. by estimating the productlon of methanol and 20 propylene o~ide, respectively. The reaction mixtures corl- ¦
21 tained in 1.0 ml: 150 mM potassium phosphate buffer, pH 7.0 22 containing 5 mM MgC12, 0.6 ml; 10~ moles NADH7 and cellular 23 ~raction.
24 Reaction mixtures were contained ln 10 ml vials a~
4C. Vials were sealed with rubber caps. The gase~u~ phase 26 in the vials was removed using vacuum and then was replaced 27 with a gas ~ixture o~ methane or propylene and oxygen at a `:
~``

- 31 -1 1:1, v/v ratio. Oxidation of other gaseous n-alkanes and 2 n-alken~s was examined as described above. For liquid sub-3 strates, 10~1 of substrate was used directly. Vials were 4 then incubated at 30C. on a rotary shaker at 200 RPM.
The products of epoxidation of n-alkenes and 6 hydroxylation of n-alkanes were assayed by fla~e ioniz~ion ;. 7 gas chromatography using a stainless steel column (12' x 1/8") 8 packed with 10% earbowax 20M on 80/100 Chromosorb W and Porapak 9 Q column. The column ~emperature was maintained isothermally at 120C. The carrier gas flow rate was 30 ml/min. o~ helium, 11 The various produc~s were identified by retention time compar-12 isons and co-chromatography with authentic s~andards.
13 Specific activi~ies were expressed as ~moles of 14 products formed per houx per mg. protein. Concentrations of ` 15 protein in vaxious fractions were determined by the method 16 of Lowry et al.~ 193: 265-275 (1951).
17 Distribution of n-Alkanes- and n-Alkenes-18 Oxidizing Activities in Cell-Frac~ions 19 . Three distinct groups of methane-utilizing organisms were selected to examine oxidation of n-alkanes (Cl-S4) and 21 n-alkenes (C2-C4) in ~ell-free sys~em~. C~llular fractions 22 were prepared from Type I obl~gate methane-utilizing organisms, 23 MethYlomonas sp. (C~L-17, NRRL B-11,208) and 24 caDsulatus (Texas, ATCC 19,069); ~ype II obligate methane-utilizing organisms, ~ (OB3b, NRRL
26 B-11,196) and Methylosinus sp. (CRL-15, NRRL B-ll, 202); and 27 a facultative methane-utilizing bacteri~m, Methylbacterium sp.
28 (CRL~26, NRRL B-11,222).
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- 32 -1 Table III shows ~he distribution of the methane-2 and propylene-oxidizing activity in various fractions derived : 3 from these organisms. About 85~90% of the total activity was 4 detected in the P(40) ~raction and lOa/o was detected in the P(803 fraction. The soluble fraction S(80) did not contain 6 any activity. The specific activities or the methane and 7 the propylene oxidation in fractions P(40) and P(80~ did not 8 vary significantly in the various organisms examined (Table IV).
9 Epoxidation of propylene and hydroxylation o~ methane were both 10 dependent upon the presence of oxygen and NADH. NADP~ or as-11 corbat~ and other electron carriers could also be utilizedO
12 Both reaction~ were linear during the first 15 min. as meas-13 ured by detection of product by gas ch~omatography.

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. 1The particulate rrac~ions [P(40) and P(80~ ~rom 2 various organisms also catalyzed the epoxidation of 3 other n-al~enes (ethylene, l-butene, and 1~3-butadiene) 4 to the corresponding 1,2-epo~,sides and the hydroxylation of methane and ethane to the correspondlng alcohols.
6 Table V shows th~ rate of oxida~ion of various n-alkanes 7 and n-alkenes by the P(40) particulate fraction of Methy~-8 5~L~Y~ . (CRL-15, MRRL B-11,202). The product of ; 9 oxidation was identified by gas chromatography after in-cubating P(40) fraction with various substrates a~ 30C.
11 for 10 min.

13 OXIDATION OF n-ALKENES AND n-ALKANES BY P(40) 14PARTICU1ATE FRACTION OF ~ V Q~ I~n ~-~
: 15(CRL-15? NRRL B~ 202)_ 16Rate of Product Formation ) 17 Substrate ~ e _ 18 Ethylene Ethylene Oxide 1.27 19 Propylene Propylene Oxide 4.1 l-Butene Epoxy butane 2.18 21 Butadiene Epoxy butene 0.63 22 l-Pentene -- 0 23 Methane Methanol 4.8 24 Ethane Ethanol 3.2 25 a) Reactions were carried out as described in Example 1.
- 26 The product of the reaction was estimated by gas 27 chromatography after 5, l0~ and 15 min. of incubation ~ 28 of reaction mixture at 30C. on a rotary shaker.

: ' '`~' .; . .
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t~

Methylosinu_ sp. (CRL-15, MRRL B-11,202) was selected for further studies on the influence of various environmental factors on the methane- and propylene- oxidizing activities in cell-free systems.
Effect of Particulate Fraction Concentration The effect of the P(40) particulate fraction concentra--tion on the hydroxylation of methane and epoxidation of propylene was examined. The production of methanol and propylene oxide was directly dependent upon the concentration of particulate fraction ranging from 1-6 mg. of protein per ml. The rate of reaction was decreased upon further increasing the particulate ; protein concentration to 8 mg./ml.
Time Course of Reactions The rate of formation of methanol and propylene oxide by hydroxylation of methane and epoxidation of propylene respec-tively, by the P(40) particulate fraction of Methylosinus sp.
(CRL-15, MRRL B-11,202) was linear with time up to 15 minutes.
Effect pH
The effect of pH on the hydroxylation of methane and epoxidation of propylene by the P(40) particulate fraction of Methylosinus sp. (CRL-15, MRRL B-11,202) was examined by esti-mating the amount of methanol and propylene oxide formed after ~; 10 min. incubation of reaction mixtures. The optimum pH for both hydroxylation of methane ancl epoxidation of propylene ' was found to be 7Ø In carrying out these tests.

.

;~
''~ ~ , ' .
'" ` ` , .

~ the reactions were carried out as described in Example 1. The - product of reaction was estimated by gas chromatography a~ter 5, 10 and 15 minutes of incubation of reaction mixture at 30 C on a rotary shaker. 100% activity equals 4.0 and 4.1 moles of ~
methanol or propylene oxide formed respectively, per hour, per mg protein.

Effect of Temperature The effect of temperature on the production of methanol and propylene oxide by the Pt40) particulate fraction of Methylo-sinus sp. (CRL-15, MRRL B-11,202) was examined after incubation of reaction mixtures for 10 min. at various temperatures. The optimum temperature for epoxidation of propylene and hydroxyl-ation of methane was found to be 35C. In carrying out these tests the reactions were carried out as described in Example 1.
The product of reaction was estimated by gas chromatography after 5, 10 and 15 minutes of incubation of reaction mixture at 30 C
on a rotary shaker. 100% activity equals 5.0 and 4.2f~noles of methanol and propylene oxide formed respectively, per hour per mg of protein.

Effect of Storage It was noted that both the activity for hydroxylation of methane and the epoxidation of propylene by the P(40) particulate fraction of Methylosinus sp. (CRL-15, MRRL B-11,202) decreased simultaneously when stored at refrigerator (0-4C) temperature.
In carrying out these tests the reactions were carried out as described in Example 1. The product of reaction was estimated ~; by gas chromatography after 5, 10 and 15 minutes incubation of the reaction mixture at 30C on a rotary ```.
.
.
.`.` ' ' ' ' .
.

:, ' .

1 shaker. 100% activity equals 4.8 and 4.1 ~.moles of methanol and 2 propylene oxid~ formed respectively per hr, per mg. of pro~ein.
3 Effect of Inhibitors 4 It has been reported that the oxidation of methane by cell suspensions of methane-utilizing b~c~eria was in-6 hibited by ~arious metal-binding or metal-chela~ing agents 7 (Patel et al., J. Bacteriol. 126: 1017-1019 (1976)). Hence, 8 the effect of inhibitors on methane- a~d propylene-oxidizing 9 activities by the P(40) particulate fraction of ~
sp. (CRL-15, NRRL B^11,202) was examined. The production of 11 methanol and propylene oxide was inhibited by various me al~
12 binding compounds with different ligand combinat ions, i. e~, 13 nitrogen-nitrogen (~ ,.~ -bipyridyl), oxygen-nitrogen (8-14 hydroxyquinoline) and sulfur-nitrogen (thiourea, thiosemi-carbazide) as shown in Table VI. This suggests the in~olve~
16 ment of metal ion(s) in the oxidation of both hydroxylation 17 of methane, and epoxidation of propylene. Similarly, as 18 shown in Table VIa these compounds also inhibit the hydroxyl-19 ation of methane and epoxidation of propylene when using 20 cell-containing enzyme preparations.

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. .

2 EFFECT OF INHIBITO~ ON THE ACTIVITY FOR EPOXIDATION
3 OF PROPYLENE AND HYDROXYLATION OF METHANE BY ME~YLOSINUS SP.
4(CRL-15 NRRL B-ll 202) ., ~_~

6Concentra- Epoxidizing Hydroxylating 7 Inhibitor ~ y _Acti~itY
. .
8 Control -- 0 0 9 ,~ ,~ -Bipyridyl 10-3 98 99 1,10-Phenanth~oline 10-3 93 90 11 Potassium cyanide 10-3 98 100 12 Thiosemicarbazide 1~-3 97 100 13 Thiourea 10-3 98 98 14 8-Hydroxyquinoline 10W3 75 80 16 a) Reactions were carried out as described in Example 1. The 17 product of the reaction was estimated by gas chro~atography 18 after 5, 10, and 15 min. incubation of the reaction mixture 19 of 30~C on a rotary shaker. The uninhibited rates of methane and proPylene oxidation were 4.5 and 4.1 ,~moles 21 of methanol and propylene oxide formed, respectively, per 22 hr. per mg. of protein in P(40) fraction of ~ y~
23 sp. (CRL-15, NRRL B-11,202).
24 Effect of Metals Since the methane mono-oxygenase from ~ethane-26 utilizing bacteria is a copper or i~onQcontaining protein 27 (Tonge et al., J. Biochem.,161: 333-344 (1977)~ we have ex-28 amined the effect o~ copper and iron sal~s on the oxidation 29 of methane and propylene by the P(40) particulate fraction of ethvlosinus ~Q. (CRL-15, NRRL B-11,202). The rate of hydroxyl-31 ation of methane to methanol and e~oxidation of propylene to 32 propylene oxide was increased 2 fold in the presence o~ added

33 copper salts (Table VII).

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1 ub ~ ~r~ ~ Coo~e tion ExPeriments ; 2 The hydroxylation of methane and the eDoxidation of 3 propylene by particulate frac~ion~ of methane-utilizing : 4 bacteria required oxygen and ~ADH. The question of whether 5 the same or a similar enzyme was in~olved in the oxidation 6 of both substrates was examined by substrate competition 7 experiments. The experiment consisted of determining the 8 effect o ~ethane on the oxidation of propylene to 3ropylene 9 oxide by the P(40) particulate frac~ion of Methylosinus sp.
. 10 (CRL-15, NRRL B-11,202). As shown in Table VIII, there ~as 11 a reduction in the amount of propylene oxide formed in the 1~ presence of methane. Hence, metha~e inhibited the conversion 13 of propylene to propylene oxide, presumably by competing for 14 the available enzymatic site.

16E~ECT O~ ME~IAN~: ON PROPYLENE EPOXIDIZING ACTIVITY BY
~` 17P (40) PA~TICULATE FRACTION OF _~IYLOSINUS S P
18 (CRL-15? ~R~L B~11.202) 19 Propylene Oxide Produceda) _ sub~
. 21 Propy lene 4 . 3 :: 22 Propylene ~ Methane 1.8 . 23 (1.:1, v/Y) . 24 Methane O

.
: 26 a) Reactions were carried out as described in Example lo 27 The product of the reaction was estimated by gas 28 chrom~tography after 5, 10, and lS min. of incubation 29 of the reaction mixture at 30C on a rotary shaker.

.
. .

- ~ 42 -1 Similarly, methane effects the epaxidat ion o~
2 propylene ~rom cell-suspensions o methane-grown Me~
3osinus trichosDorium OB3b (NRRL B-11,196) as shown in 4 Table VIIIa.
5TABL:E VIIIa 6 ~ ~=~) 7 CCT~pOSitiOn of Propylene Oxide %
8 Gaseous Phase F~rm:d ~ ~ Inhibition . _ _ 10 (2o~yl5n.5e0,vH/e)iu~ 1 2 1.~ 0 11 Propylene~Me~hane~02 0.8 50 12 (25:25:50 v/~) 14 a) The reactions were conducted as ~escribed in Example 1 except tha~.various gaseous compositions ~ere used to 16 m~intain a constant propylene partial pressure. Cell-17 suspansions of methane-grown MethYlosinus trichosPorium 18 OB3b (NRRL B-11,196) (3.6 mg.
19 oxide was estimated by gas chrom~tography after 15 minutes of the incubation.
21 ~y ~
22 ~ par~iculate P(4û) fraction of Methylosinus ~.
23 (CRL-15, NRRL B-11,202) was used to determine the stoichi-24 ometry o hydroxylation and epo~idation reactions. The 25 stoichiometry o~ methan~or propylene-dependen~ NAD~ oxi-26 dation, ox~7gen cons~lmp~ion and product formation was ap-27 proximately 1~ Table I~]. This is consistent with 28 methane or propyl2~e oxygenation being catalyzed by a 29 mono-oxygenase.

2 S~OICHIOMETRY OF PROPYLENE EPOXIDATION A~D ~ETHANE
3 HYDRO~YLATION BY P(40) PARTICTJLATE FRACTION QF METHYLOSINUS
4 ~
Substrate Product For~ed NADH Oxidized 2 Consumed 6 ~ (,u=cles~ moles) (~umoles) 7 Propylene Propylene Oxide 8 5.0 4~5 5,0 4.8 9 Methane Methanol 5.0 ~.2 4.8 4.5 11 , 12 a) Under identical condition o~ reaction, the estimation of 13 NADH oxidized was carried out spectropho~ometrically, the 14 estimation o~ oxygen consum~d was measured polarographic-ally, and the estima~ion of produ~t formed was carried 16 out by gas chromatography.
17 As a comparison the s~oichiome~ry of the epoxida-18 tion of propylene by a cell-suspension of Methylosinus 19 trichosDorium OB3b (NRRL B-11,196) was determined as follows.
The reaction mixture (3.0 ml.) contained O.05~ sodium 21 phosphate buffer, pH 7.0 and 3.6 ~ moles of propylene. The ., 22 raac~ion w~s initiated by the injection of 0.1 ml, of cell-23 suspension (3.1 mg. protein3. A correction was m2de ~or the 24 endogenous consumption of oxygen. The amount of oxygen con-sumed during the reaction (3 man.) was determined polaro-26 graphlcally with a Clar~ oxygen electrode. The propylene 27 consumed and the propylene oxide form~d was estimated by 28 gas chromatography. The propylene consumed was 0.29 ,~moles, 29 the oxygen consu~ed was 0.30 v~moles and the propylene oxide formed was 0.28~mo1es.

.
:.~

- ~4 -1 To ~urther demonstrate that the enzyme activity 2 is in the par~iculate fraction (not in the supernaten~) the 3 following e~eriments were carried out. Cells of methane-4 grown M thylococcus caDsulatus (CRL ML3 N~RL B-11,219) were obtained by the method of Example 1. The crude extrac~
6 after 10,000X g.centrifugation of sonically disrupted (3 x 7 50 S2C., Wave Energy Ultrasonic Oscillator, Model W 201) was 8 found to have no activity for either epoxidation or h~droxyl-9 ation. However, when the cells were disrup~ed by passing twice ehrough a French pressure cell (1000 ~g. pressure)9 11 bo~h acti~7ities were found in the crude extract after 10,000 x 12 g. centrifugation. All of ~he activi~y in the crude extract 13 was collected as a particulate fraction by fur~her centri~u-14 gation of the crude extract at 40,000 x g. for 90 min. at 4C.
NADH stimNlated both the epoxidation and the hydroxylation 16 reactions as shown in Table X.

19 ~ ~ a) Oxidation Rate tr.moles/30 min/assa ) 21 ~ ~ u~r ~
22 Cell-Frea Fraction~ o~ylene Methane 23 (1) Particulate fraction 750 500 24 (10,000 g.-40,000 ~.) (1) + NA~H 900 650 26 (2) Supernatant fraction O O
27 of 40,000 g.
28 (2) + NADH O

30 ~ rI~ were disrupted by French Press as described above.
31 NADH (2.5,~moles) was added into the reaction mixture where 32 indicated. The amount o~ p~otein in the particulate frac-33 ~ion and the 40,000x g. supernatant fraction used was 1 mg.

34 and 2.5 mg., respectively. Each assay contained 0~5 ml.
reaction mixture.

. .
: ' .
. .

SU~ARY
. . .
2 Both the system of _~c~dc~ aer~_no~ demon-3 strated ~y van der Linden, ~ C _~ ggb~_ c~a.~
4 77: 1S7-159 (1963) and the system of Pseudomonas oleovorans , Abbott and Hou~ ADD1. Microbiol., 26: 86-91 (1973) epoxidiz4d liquid l~alkenes rom C6 to C12, but not gaseous alkenes.
` 7 ~he present in~ention provides for the epoxidation ; 8 o ethylene, propylene, l-butene and butadiene by C211 :~ 9 suspensions of all three distinct groups of methane utilizing bacteria. The epoxidation of alkenes and the . ~; 11 hydroxylation o~ methane were not found under anaerobic 12 co~ditions or iQ methanol-grown cells, suggesti~g that the 13 enzyme system is inducible . The produc 1,2-epoxides accumu-14 lated extracellularly. The non-enzymic degradation of pro-' 15 pylene oxide in ~he assay system disclosed was not signi~icanc . ~
16 even after a prolonged incubation time. Van der Linden9 17 suora, demonstrated the production of 1,2-epoxyoctane from 18 l-octene by heptane-grown cells of Pseudomonas s~. and also 19 stated that the epoxide was not further oxidized enzymatically.
20 However, May and Abbo~t, B~-hem. Blo~ ~. Res. Commun., 48:
21 1230-1234 (1972) and ~ æ ~ 248: 1725-1730 (1973~
22 repor~ed that ~hen l-octene was supplied as a subs~rate to 23 the ~-hydroxylation enzyme system of P. oleovorans, both 24 8 hydroxy-l-octene and 1,2~epoxyoctane were formed. In addition, Abbo~t and Hou, ~ , found that the methyl group 26 of ~he latter compound was also susceptible to hydroxylation.
27 The present results obtained from the 3tudies of viable cell 1 suspensions of the me~hane-utilizing bacteria, however, 2 indicated that propylene ox~:de was not further metabolized . 3 enzy~atically.
4 Van der Linden, su~r~ showed that the epoxide accumu-lation from l-octene by Pseudomonas ~ a was accompanied 6 by the metabolism of a large quantity of l-octene via m~thyl 7 group epoxidation. In the epoxidation o~ propylene by cell 8 suspensions o~ methane-utilizing bacteria, however, no forma-9 cion of 3-hydroxy propene 1 was detected.
Both the epoxidatian of the C2-C4 l-alkenes and ~he 11 hydroxylation of methane with the cell suspensions r"ere in-12 hibited by various metal-binding and metal-chela~ing agents, 13 indicating the involvement of metal(s)-containing enzyme 14 system(s). The similar extent of inhibition ~or both pro-pylene and methane oxidation (Table VIa) indicated that the 16 ep3xidation and hydroxylation reaction may be catalyzed by 17 the same or a similar enzyme system. The epoxidation of 18 propylene to propylene oxide by a cell suspension of methane-19 grown strain MethYlococcus caDsulatus NRRl B-11,219 was in-hibitPd (50qO) in the presence of the hydroxylation substrater 21 methane (Table X). This clearly suggests a competition 22 between the hydroxylation subs~rate and the epoxidation 23 substrate for a single enzyme system. It is likely that 24 the methane mono-oxygenase enzyme system catalyzes both the epoxidation of alkene and the hydroxylation of methane.
26 May and Abbott publications, supraJhave reported that the 27 ~ -hydroxylation system from Pse~domonas oleovoxa~ catalyzed 28 both the epoxidation of l-octene and the hydroxylation of 29 n-octane.

.

: ~ .
.

1 The optimum conditions for th~ in vivo epoxidation of . 2 propylene by cell suspensions o~ the three dis~inct groups of 3 methane-utilizing bacteria are quite similar. The pH oDtima 4 were around 6 ~ 7 and the temperature optimum was around 35C.
The apparent decrease in epoxidation above 40C may be due to 6 both the instability of the mono-oxygenase s~stem and the 7 volati~ity of the product propylene oxide (b.p, 35C).
8 Both the hydroxylation and epoxidation activities 9 are located in the cell-free particulate fraction precipitated between 10,00~x g.and 80,000x ~ centrifugation. Tonge et al., 11 Biochem. J., 161: 333-344 (1977~ and FEBS Lett., 58: 293-299 12 (1975) have reported the purification of a membrane-bound ` 13 methane mono-oxygenase from the particulate fraction (sed-14 imented between 10,000x g.and lS0,000x g. centrifugation) ~ Y~5~Y~ D'iJa~ e~ OB3b- Recently, but subse-1~ quent to our discoveries Colby et al., Biochem. 3., 165:
17 395-402 (1977) demonstrated a unique soluble methane mono-18 oxygenase from MethYlococcus capsulatus (Bath strain) which 19 catalyzes the oxidation of n-alkanes, n-alkenes, ethers and ` 20 alicyclic, aromatic and heterocyclic compounds. The strains 21 from the three distinct group~ of methane-u~ilizing bacteria 22 th~t we have examined all catalyze the epoxidation of gaseous ~3 alkenes (C2-C4) and the hydroxylation of gaseous aLkanes :
24 (Cl-C4). Also, we unexpectedly found the e~zyme activity ~ in the particulate fraction (i.e., the mate~ial whlch sediments 26 when the superna~ant af~er centrifuging broKen cells at 10, C00 27 x g. for 30 minu~es is cen~rifuged for 1 hour at 10, oao x g.
28 or greater), not the soluble ~raction (i.e., the supernatan~
29 after centrifuging broke~ cells a~ 80,G00 x g. or greater for 1 hr.

.
, ~ ~
` . , ~. -48 ~

1 Differen~ial centrifugation of broken-cell 2 suspensions of Methylomonas sp. (CRL-17, NRRL B~ 208 3 and ~ (Texas ATCC 19,069), (Type I
4 obligate methylotrophs); _e~, a ~y~_~9. (CRL-15, NRRL B-11,202) -` S and Methylosinus triehos~orium (OB3b~ NRRL B-11,196) (Type II
6 obligate methyloerophs), and Methylobacterium s~. CRL-26, 7 NRRL B-11,222) (a facultative methylotroph) has yielded : 8 cell-free particulate fractions that catalyzed the hydroxyla-9 tion of n-alkanes and the epoxidation of n-alkenes. Both aetivities mainly resided in the P(40) fraction and were 11 dependent upon the presence of oxygen, as well an electron 12 2arrier, e.g., ~ADH.
13 The hydroxylation of methane to me~hanol and the 14 e~oxidation of propylene to propylene oxide catalyzed by . 15 the P(40) partic~late fraction of ~ ~a~ (C~L-15, ; 16 NRRL B-11,202) have similar pH and temperature oDtima 17 (Figures3&4). Both activities were lost simultaneously 18 during storage of the P(40) particulate fraction at re-19 frigera~or temperature.
The hydroxylation of methane and the epoxidation 21 of propylene with the cell-free extracts were strongly in~
22 inhibited by various metal-binting or metal-ehelating agents 23 (Table VI). The rate of both reactions were increased 2 fold 24 in the presence of copper or iron salts (Table VII). This suggests the involvement of a metal-containing enzyme system 26 in the oxidation of both substrate, These results, an~ the 27 stoichiometry of the hydroxylation and the epoxidation re-1 4~
. .

..
actions indicat~ that both reactions ~ be catalyzed by 2 the same metal-containing mono-oxygenase system. The act 3 that conversion of propylene to propylene oxide was in-4 hibited by methane support this proposition.
S It has been reported that the cell-free particu-6 late fractions derived from Methylococcus capsulatus (Texas) . 7 (Ribbons e~ al., J.BacterioL., 122: 1351-1363 (1975)), 8 ~ca (Ferenci et al., J. Gen. Microbiol.
9 91: 79-91 ~97~)) and Meth~losinus trichos wrium (OB3b) (Tonge et al., Biochem. J., 161: 333-344 (1977)) catalyzed 11 oxygen- and NAD~-dependent oxidation of methane, ethane, 12 proPane~ butane, and carbon monoxide. The oxidation o 13 methane by particulate fractions of these organisms was 14 inhibited by various metal-binding or metal-chelating agents. ~owever, epoxidation of n-al~enes was not re-16 ported for these organisms.
17 Th~ methane mono-~xygenase from Methylosinus 18 trichos~orium (OB3b, NRRL B-ll, 196~ has been purified and 19 shown to be consisting of three components: a soluble CO-binding cytochrome c, a copper-containing Drotein 21 ~ethane mono-oxygenase), and a small molecular weight 22 protein (Tonge et al., 1977, su~ra).
23 In contras~ to the above organi~ms, Colby et al., 24 supra have reported the unique soluble methane monowoxygenase activity from 2~13h .L ~-~ L L:a~91~5~ (Bath). The oxida-26 tion of methane by the solubLe fraction o~ this organism was 27 not inhibited by variou meta~-binding agents. Recently, .:

. .
, -. ~ 50 1 Colby and Dalton (Biochem. J., 171: 461-468 (1978)) resolYed 2 the methane mono-oxygenase of Methylococcus caPsulatus (Bath) 3 into three components and identified one of the components as 4 an iron-containing flavoprotein~
The methane-oxidizing activities from ~he methylo~
6 trophic bacteria described above i~ in the par~iculate frac-7 tion and different from the sQ~uble activity o~ y~
8 ca~sulatus (Bath) disclosed by Colby et al.
9 Van der Linden (1963, supra) demonstrated the produc-tion o~ 1,2-epoxides from l-octene by heptane-grown resting 11 cells o Pseudomonas sp. Epoxides were not detected as __ 12 products of alkane metabolism a~d were not oxidized by 13 Pseudomonas sp. l'hus, the roLe of epoxides in alkane 14 metabolism is uncertain. Van der Linden postulated that the enzyme system that forms epoxides may be the same as 16 the system that catalyzes the initial oxidation of alkanes.
17 Cardini and Jurtshuk (J.Biol. Chem., 245: 2789-2796 (1970)) 18 found that a cell~free extract of a C~r~oe~ c~riom sp.
19 carried out the oxidation of l~octene to epoxyoctane in addition to hydro~ylation of octane to octanol. McKenna ~l and Coon (J. Biol. Che~ , 245: 3883-3889 (1970)) isolated 22 an enzyme system from Pse~ O-c~ L -v-e-r~ that catalyzed 23 the hydroxylation of n-alkanes (C6-C12) and fa~ty Qcids.
24 Subsequently, Abbott and Hou7 ~Q~ and May and Abbot~, 25 supra reported that the enzyme system from Pseudomonas 26 oleo~orans also catalyæed the epoxidation of l-alkenes in 27 addition to the hydroxylation reac~ions, Th~ enzyme systems ` 1 from Pse~do~onas and Corynebacterium 5~. catalyzed ; 2 epoxidation of C6-C12 n-alkenes. Epoxidation of C2-Cs 3 n-alkenes was not catalyzed by the Pseudomonas enzyme 4 systems.
We ha~e unexpectedly demonstrated that the thre~
6 distinct groups o methane-oxidizing bacteria catalyze 7 the hydroxylation of n-alkanes (Cl-C4) as well as the 8 epoxidation of n-alkenes ~C~-C4). Furthermore, the hy-9 droxylation and the epoxidation reactions are catalyzed by the same or a similar NADH-dependent mono-oxygenase.
In additi~n ~o methylotrophic bacteria, o~er ~2 microorga~isms can be ~sed to carry out the epox~datlon of 13 C~-C4 alkenes. These ~clude bacteria~ f~gi and yeast 14 which grow on short chairl alkaDes. The methylotrophic bac-15 teria (obligat~ or facultative) or the other microo~ganisn~s 16 are grown either on methane as a sole source of car~on, or 17 OT~ another car~o~ compo~d (in the presence o~ methane or 18 a~other i~ducer), and the celLs, or enzymes derived ~here-19 from, may be used i~ the procass of the prese~ invention.
While the i~vention has been described in c~n~ec-21 tion with specific ~nbodi~Dents ~here~f, it will be u~der-~` 22 stood that it is capable of further modificatiorl, aad this 23 application is interlded to cover a~y variations, uses, or 24 adaptations of the ~nvention foLlow:ing, isl general, the 25 principles of ~he invesltiorl and including such depar~res 26 ~rom the pres`ent disclosure as come within t~own or custoin-27 ary practice i~ the art to which the invention pertains and 28 as may be applied to the essential fe~tures hereinbefore set forth, and as fa:Ll wiehin the scope of the :Lnvention.

. ~ .
,

Claims (21)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for the epoxidation of a C2-C4 n-alkene or diene selected from the group consisting of ethylene, propylene, butene-1 and butadiene which comprises contacting said alkene or diene under aerobic conditions With resting or washed cells of microorganisms or enzyme preparations derived therefrom/ wherein said microorganisms are obligative or facultative methylotrophs and have been cultivated in a nutrient medium containing methane under aerobic conditions.

2. The process of claim 1 wherein the enzyme preparation is derived from a cell-free extract of the microorganisms.

3. The process of claim 2 wherein an electron carrier is added.

4. The process of claim 1 wherein NADH is the electron carrier.

5. The process of claim 4 wherein a metal is added.

6. A process for the epoxidation of propylene which comprises: contacting propylene under aerobic conditions with resting or washed cells of microorganisms or enzyme preparations derived therefrom, wherein said microorganisms are obligative or facultative methylotrophs and have been cultivated in a nutrient medium containing methane under aerobic conditions.

7. The process of claim 6 wherein said microorganisms belong to the genera selected from the group consisting of Methylosinus, Methylocystis, Methylomonas, Methylobacter, Methyl-ococcus and Methylobacterium.

8. The process of claim 6 wherein said microorganisms are species selected from the group consisting of: Methylosinus trichosporium, Methylosinus sporium, Methylocystis parvus, Methyl-omonus methanica, Methylomonas albus, Methylomonas streptobacter-ium, Methylomonas agile, Methylomonas rubrum, Methylomonas rosa-ceus, Methylobacter chroococcum, Methylobacter bovis, Methylo-bacter capsulatus, Methylobacter vinelandii, Methylococcus capsu-latus, Methylococcus minimus and Methylobacterium organophilum.

9. The process of claim 6 wherein said microorganisms are strains having the designations selected from the group con-sisting of: Methylosinus trichosporium OB3b (NRRL B-11,196);
Methylosinus sporium 5 (NRRL B-11,197); Methylocystis OBBP
(NRRL B-11,198); Methylomonas methanica S1 (NRRL B-11,199); Methyl-omonas albus BG 8 (NRRL B-11,200); Methylobacter capsulatus Y
(NRRL B-11,201); Methylobacterium organophilum sp nov. (ATCC
27,886); Methylomonas sp AJ-3670 (FERM P-2400); Methylococcus 999 (NCIB Accession No. 11,083); and Methylomonas SM3 (NCIB Accession No. 11,084).

10. The process of claim 6 wherein the epoxidation is carried out at a temperature in the range from about 5 to about 55°C. at a pH in the range from about 4 to about 9.

11. The process of claim 6 wherein the epoxidation is carried out at a temperature in the range from about 25 to about 50°C. and at a pH in the range from 5.5 to 7.5.

12. The process of claim 6 wherein the oxygen is used in the form of air.

13. The process of claim 6 wherein the epoxidation is carried out batchwise.

14. The process of claim 6 wherein the epoxidation is carried out in a batchwise manner and the enzyme preparation is immobilized.

15. The process of claim 6 wherein the epoxidation is carried out in a continuous manner and the enzyme preparation is immobilized.

16. A process for the epoxidation of propylene, which comprises the sequential steps:
(a) incubating an obligative or facultative methylotro-phic microorganism in a nutrient medium under aerobic conditions and recovering therefrom an alkene epoxidase enzyme system, and (b) contacting propylene under aerobic conditions with said alkene epoxidase enzyme system in the absence of a nutrient medium until at least a portion of said propylene is converted to propylene oxide.

17. The process of claim 16 wherein said microorganisms belong to the genera selected from the group consisting of Methyl-osinus, Methylocystis, Methylomonas, Methylobacter, Methylococcus, and Methylobacterium.

18. The process of claim 16 wherein said microorganisms are species selected from the group consisting of: Methylosinus trichosporium, Methylosinus sporium, Methylocystis parvus, Methyl-omonas methanica, Methylomonas albus, Methylomonas streptobac-terium, Methylomonas agile, Methylomonas rubrum, Methylomonas rosaceus, Methylobacter chroococcum, Methylobacter bovis, Methylo-bacter capsulatus, Methylobacter vinelandii, Methylococcus capsu-latus, Methylococcus minimus and Methylobacterium organophilum.

19. The process of claim 16 wherein said microorganisms are strains having the designations selected from the group con-sisting of: Methylosinus trichosporium OB3b (NRRL B-11,196);
Methylosinus sporium 5 (NRRL B-11,197); Methylocystis parvus OBBP
(NRRL B-11,198); Methylomonas methanica S (NRRL B-11,199);
Methylomonas -albus BG 8 (NRRL B-11,200); Methylobacter capsulatus Y (NRRL B-11,201); Methylobacterium organophilum sp nov (ATCC
27,886); Methylomonas sp AJ-3670 (FERM P-2400); Methylococcus 999 (NCIB Accession No. 11083); and Methylomonas SM3 (NCIB Accession No. 11084).

20. The process of claim 16 wherein the cultivation in step (a) and the epoxidation in step (b) are carried out at a temperature in the range from about 25 to about 50°C. and at a pH in the range from about 5.5 to 7.5.

21. The process of claim 16 wherein steps (a) and (b) are carried out in a continuous manner.