JPS5948088A - Microbiological oxidation of alkane, vinyl compound and secondary alcohol - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to microbial cells of microorganisms or -'F: i that utilize C3-C6 alkanes as main carbon and energy sources.
This invention relates to a method for oxidizing alkanes and other saturated hydrocarbons, alkenes, dienes, vinyl aromatic compounds, and secondary alcohols by the action of an enzyme derived from t and oxygen.

The present invention also provides C3-C6 alkyl group-donating compounds such as C3
- A method for oxidizing secondary alcohol using a C6-chain primary alcohol and a C2-C6 alkylamine by the action of a microbial cell or an enzyme agent derived therefrom and a printing plate.

Methane is not the only hydrocarbon substrate on which microorganisms can grow. It is known that gaseous and liquid hydrocarbons are also useful as growth substrates, depending on the particular strain of bacteria used. Microbial genera that are known to utilize hydrocarbons other than methane as carbon and energy supplies include, for example, Achromobacter.
), Ac1netobacter
), Pseudomonas
, Nocardia, Bacillus (Ba
cillus), Mycobacterium (Mycoba
cterlum), Corynebactorium (Coryn
ebacterum), Brevibacterium (Br
evlbacterlum), Candida (Candl)
da ), Arthrobacterium
r), Streptomycetes
es ), Flavobacterium (Flavobacterium
rlum) and various filamentous fungi. However, for a given combination of strain and hydrocarbon substrate for growth, it is often difficult to predict the details of the nature of the strain in the medium.

U.S. Pat. No. 3,311,11θ37 uses a microorganism that grows on a hydrocarbon (substrate) that corresponds commercially to an oxidizable hydrocarbon in terms of the number of carbon atoms to produce an oxidized hydrocarbon, such as an alcohol or an aldehyde, with the same carbon atoms. Discloses a method for enzymatically targeting several oxidation products. The microorganism is preferably selected from the genera Achromobacterium, Nonidomonas, Nocardia, Bacillus and Mycobacterium, preferably from the Achromobacterium species growing on decane.

7λ Stell [J, W, Foster, Ant,
v. Leeu.

J,Mlcroblol,and Ser,,2 g,
2'l/(/qA, 2)) provides a comprehensive review of microorganisms growing on hydrocarbons and has recently been published by Perry [J, J
, Perry, Mlcroblol, Rev.

q3.59 (/ri79)] is a co-edition of substrates using microorganisms that grow on hydrocarbons (coox I dat l).
We are scrutinizing the ri research base on ). In co-oxidation, non-growing ginseng substances are present as co-substrates in a growth medium containing the growth substrate, and the non-growing substrates are oxidized. Co-oxidation therefore does not apply to downstream oxidation of resting cells.

Case f le [A, S,” ester, PhD D
lss,, Univ.

of Texas (/9b/)] are Norcalcia, Brevibacterium, Pseudomonas, Alcaligenes or Achromobacterium, Corynebacterium, Mycobacterium, Streptomyces and Fusarium (F
belongs to the ``usarlum''. About 23 types of microorganismsC
□～C engineering. Although the ability to grow on hydrocarbons is tested, the ability to grow varies with the sex of the particular microorganism being tested.

In this specification, alkanes, saturated alicyclic or aromatic hydrocarbons, alkenes, dienes, vinyl aromatic compounds or rl'
Disclosed is a method for oxidizing a secondary alcohol or a selected substrate to the corresponding oxidation product, i.e., product, which is carried out in a reaction medium under aerated conditions with microorganisms (provided that these strains are Microbial cells derived from microorganisms (selected from the strains listed in the following text), cells derived from genetically engineered strains of microorganisms, or cells derived from natural microorganisms, or enzyme preparations prepared from the cells. contacting the microorganism, derivative strain, mutant strain or enzyme agent until a separable amount of the corresponding product is produced, the microorganism acts as an oxidase or dehydrogenase, and the microorganism It has been previously grown under aerobic conditions in a Kato culture medium containing a lactone compound, or in a normal medium containing a C2-C6 alkyl group donating compound when the substrate is an alcohol.

In a preferred embodiment, the substrate is 02-C6 alkanes, cyclohexane, benzene, 02 for microorganisms growing on alkanes.
~C7 alkene, butadiene, carbon or C5~C
6 Secondary alcohol, alkyl group donor 4&+
03-C5 secondary alcohol for top-growing microorganisms. Most preferably the substrate is propane, n-butane, cyclohexane, benzene, ethylene, propylene, euglopanol or u2-butanol. The 02-C6 alkane on which the microorganism is grown is preferably a C2-C4 alkane, such as ethane, propane or butane, and the 02-C6 alkyl group donating compound is preferably a 02-C4 compound, such as ethanol, Gropanol, butanol, ethylamine, propylamine or butylamine.

In another preferred embodiment, the microbial cells are resting cells, more preferably in the form of a resting cell suspension.

In addition, in other preferred embodiments, pJ! A, The enzyme agent shown in a is a cell-free soluble fraction.

The term "microorganism" herein is used in its broadest sense and includes all strains of microorganisms hereinafter generally bacteria.

The term "genetically engineered derivatives of microorganisms" is used in the sense recognized by those in the art and includes artificial mutants and recombinant DNA-producing microorganisms.

The term ``enzyme agent'' is used to mean any composition that produces the desired dehydrogenase action. The term includes, for example, living whole cells, dried cells, cell-free powders and soluble fractions, cell extracts. It refers to purified and &km preparations spun from substances and cells. Enzyme preparations can be in dry or liquid form.

The term also refers to immobilized enzymes such as whole cells of microorganisms grown on 02-C6 alkanes or alkyl group-donating compounds, or chemical covalent bonds, absorption and optical spectroscopy that allows free passage of one substrate molecule and one product molecule. The term "enzyme agent" includes enzyme extracts that are bound to an insoluble matrix by entrapment of the enzyme within a cell lattice with large openings but small enough to retain the enzyme. Rony, Biotechnology
and Bioengineering C/97/)]
includes an enzyme retained within a perforated fibrous membrane as disclosed in .

The term ``02-C6 alkyl group donating compound'' is ethyl\
02-C6 alkyl compounds that donate propyl, butyl, pentyl or hexyl groups, such as ethanol, glopanol, butanol, pentanol, hexanol, ethylamine, propylamine, butylamine, ethyl chloride, butyl carbonate, etc. are meant. These compounds can also be characterized as growth substrates capable of supporting the dehydrogenase action of the microorganisms described below.

The term ``resting #J cell'' refers to cells that are not cantilevered in a growth medium or a normal medium (i.e., containing a carbon source and a chlorine source) and therefore cannot actively grow or proliferate, but perform oxidative functions. Resting cells typically refer to cells in culture that are harvested and washed with a buffer and suspended in a buffer, thus obtaining a resting cell suspension for use in oxidation. Especially!
ll prepared.

That is, no growth medium can normally be present during enzymatic conversion. However, various substances may be present during the enzymatic conversion, such as cofactors, salts, water, promoters, etc.

As one aspect of the present invention, examples of 02-C6 aluminum growth substrates,
t Microorganisms shown in the text that have been grown in advance under aerobic conditions in a normal medium containing flyethane, propane, butane, pentane, co-methylbutane, hexane, etc., preferably 02-C4 alkanes, most preferably propane. Microbial cells derived from bacterial strains (or equivalent cells of, for example, genetically engineered 'fc microorganisms) and corresponding alkanes or saturated alicyclic or aromatic hydrocarbons. It has been discovered that dimethyl ketones and alcohols can be produced by contacting them under aerobic conditions. Preferred microorganisms for this embodiment include Gutuidomonas aeruginosa strains, especially those that grow on 02-C, alkanes.

The oxidizing alkane substrates used in the present invention include the following alkanes, such as propane, n-butane, isobutane, n-pentane, impentane, neopentane, n-hexane, co, 2-dimethylbutane, and co.

It can be selected from 3-dimethylbutane and the like. The most preferred alkanes are propane or n-butane.

In the present invention, saturated alicyclic (i.e., cycloaliphatic) or aromatic hydrocarbons include cycloalkanes such as cyclopropane, cycloaliphatic,
Includes cyclobutane, cyclopentane and cyclohexane, and aromatic and arylalkyl compounds such as benzene and toluene.

Preferred compounds according to the invention are cyclohexane and benzene.

Other embodiments of the invention include C2-C6 alkanes such as ethane, propane, butane, pentane, comethylbutane,
Resting microbial cells derived from the strains shown below (or equivalent resting cells of genetically engineered microorganisms) grown in advance under aerobic conditions in a normal medium containing hexane, etc. It has been found that 2-epoxides can be prepared by contacting enzymatic agents prepared from them with the corresponding alkenes, dienes or vinyl aromatic compounds under aerobic conditions. . Preferably the growth substrate is 02
~C, an alkane, most preferably propane. Preferred microorganisms I''i according to the invention include, in particular in the case of the use of 02-C4 alkanes, Nonydomonas aeruginosa strains.

The olefin Jlt in the invention is 511, ethylene, nolopyrene, /-butene, cis or trans-butene, 2
-ene, isobutyne, pentene, copene, butadiene, isoprene, styrene, etc. Preferably the olefin substrate is a C3-C7 alkene (more preferably a 02-C6 alkene), butadiene or styrene. The most preferred alkenes are ethylene or propylene.

In a third aspect of the present invention, microbial cells derived from the microorganisms shown below (or genetically engineered by contacting the corresponding secondary alcohol, preferably a 03 to C6 secondary alcohol, under aerobic conditions. It has been found that dimethyl ketones are produced. Examples of C8-C6 alkanes are ethane, propane, butane, imbutane, pentane, comethyl-butane, hexane, etc. The most preferred flucan in this method is propane. Preferred orchid organisms for this method are Pseudomonas aeruginosa strains, especially when using 02-C4 alkanes.

In a fourth aspect of the present invention, from a microorganism grown in advance under aerobic conditions in a normal medium containing an 02-C6 alkyl group-donating compound, preferably a 02-C6 linear primary alcohol or an 02-C4 alkylamine. Guided microorganisms 11i1
11 cells (or equivalent cells) and corresponding secondary alcohols,
It has been found that dimethyl ketones are produced by contacting preferably with a 03-C5 secondary alcohol under aerobic conditions.

The secondary alcohol to be oxidized can be chosen, for example, from coprobanol, coptanol, coprobanol, 3-pentanol, coprobanol and 3-hexanol. Preferably the alcohol is a linear secondary alcohol, most preferably coprobanol or coptanol.

The present invention includes the following types: iaj The microbial cell suspensions shown below oxidize 03-C6 alkanes to the corresponding secondary alcohols and methyl ketones. Among the n-alkanes, propane and butane are oxidized at the highest rate.

(bl f Cell-free soluble extract derived from Nonidomonas fluorescens NRRL B-/2 daq grown on lopane. Fi02-C6 alkanes, cyclohexane and toluene are converted to their respective corresponding alcohols.

(C) The convenient strain has an optimal oxidation temperature of 3, t'O and an optimal oxidation pH of 7, for propane and butane oxidation.
Indicates t.

(d) Cell suspensions of at least three strains of the microorganisms listed below generally oxidize cyclohexane and benzene to their respective corresponding alcohols. Brevibacterium growing on butane Fuscum ATCC/!
;993 has been found to oxidize toluene to benzyl alcohol.

(e) Suspensions of resting cells of the microbial strains indicated below oxidize (epoxidize) alkenes, dienes and vinyl aromatics to their respective corresponding 2-epoxides. The product, co-epoxide, is enzymatically produced and accumulates outside the cell. Among the alkenes, ethylene and propylene are oxidized to the highest extent.

(The cell strain according to the invention grown on fJ propatool does not convert olefins to /,2-epoxides.

(gJ The optimum growth temperature of the strain used is 35"O, and the optimum p11 is O, θ~7.0.

(h) Epoxidation can be carried out in the presence of electron-carrying NADH or other biochemicals that promote epoxidation. The nyreclone-supported material or other biochemical material can be regenerated, for example, by adding ethanol to the reaction medium.

(1) E-biological 9-cell suspensions grown on 02-C6 alkanes shown in the text oxidize 03-06 secondary alcohols to the corresponding methyl ketones. C8-C6
Cell suspensions of microorganisms grown on alkyl group-donating compounds oxidize C5-C6 secondary alcohols to the corresponding methyl ketones. Among the C3-C6 secondary alcohols, Cope-co-butanol is oxidized at the highest rate.

fjl The optimum oxidation temperature of the strain used is 3jt'O and the optimum ptl for oxidation is about g, θ~9. θ.

The microorganisms used in the present invention are all known microorganisms and are described in Bergey's Manual of Dete.
rminative Bacteriology, Ro
bert S, Breed at al, eds
,.

gth ed, (Ba1t Imore: Will
l Iams and Wl 1klnsCo,,
/q7+)] is classified as classification method K (IE). New cultures of each strain are classified as ATCC [American
Type to culture Co11ection
(ATC:C:) in Rockville, Mar
yland 20gk2] or NRRLPeorla,
l1llnols b/1sOL). ATC from the depository of each new culture
:C or l'J The RRL deposit number is the unwritten serial number. The microorganism consisting of λ has an accession number of AT
CC Catalog (ATCC Catalog of Stra
Ins I, /&th ed, 19g, 2). Microorganisms of lower dC are useful in the oxidation reactions described above: Microorganisms ATCC or Deposit No./
, odococcus rhodoch 8T to C/7ノ4tθ (glaze of the genus Arthrobacour) [Rhodococcus rhodoch, rous
(8 rthrobacter sp,)) Yu, Pseudomonas aeruginosa ATCC/S!
;M (alkaline earth metal species) [Pseudomonas aeruglnosa (A
icaligenes sp, )] 3, Arthrobacter petroleophagus ATCC2/lIq'1
Arthrobacter simplexus ATC
C2IO3,2 [Arthrobacter slmp
lex ] Left, Gutuidomonas aeruginosa
AT to C/uncle-g [Pseudomonas ae
ruglnosa6, Plevibacterium spp.
ATCCl'16'lde(Brevlba
cterium sp, ) 7, Brevibacterium fuscum ATCC/! 99.3[Brev
lbacteriun'+ fuscum) microorganism
Article 8 TCC or deposit number g,
Alcaligenes eutrophus ATCC
/7697 (Hydrokesomonas eutrophus) [Alca order genes eutrophus (Hyd
rogenomonas eutropha))? ,
Mycobacterium alpum ATC
Cxqbqb[Mycobacterlum aIbU
m ] π, a dokokusu rhodochrous
ATCC291>70 (Mycobacterium rhodochrous) [Rhodococcus rhodochrous]
(Mycobacterium rhodochrou
sl]〃, Rhodococus Rhodoclaus
ATCC2qb72 (Mycobacterium rhodochrous) [Rhodococcus rhodochrous]
(Mycobacterum rhodochrou
s))/2. Rhodococcus yuzu
C to A'T: l/1I99 (Nocardia neopaca) [Hhodococcus sp.

(Nocardia neopaca))
Article ATCC or deposit number/3
．． Pseudomonas kurufle NRRL
B-102/LPseudomonas crucu
rae] thick, pseudomonas fluorescens
NRRL E3-/2'l'1[Pseudomon
as f Iuorescens] 15, 7' Soitomonas se/zosia ATCC/77.
11゜ (Pseudomonas multivirans) [Pseudomonas cepacia (Pseudomonas
domonas mu iti vor ans
))/6. Puso (tomo t, x, ffl”Am
ATCC/74'! ;3 [Pseudom
onas putlda Type A)/7. Pseudomonas aeruginosa ATCC/translation 42
(Pseudomonas ligustri) [Pseudomonas aeruglnosa (
Pseudomonas llgustrl)] If the substrate is an alkene, diene, or vinyl aromatic compound, the microorganisms used are the following microorganisms: Mycobacterium paraffinlc.
um) ATCC/267θ and Mycobacterium rhodochrous
(Mycobacterium rhodochrous
)] to 8 CC/[further selected from 4.

If the basic substance is a secondary alcohol, the reusable microorganisms are the following microorganism groups: Mycobacterium paraffinicum
um ] A TCC/2 Otsu 70 and Rhodococcus rhodochrous (Nocardia and Roughinica) [Rhodococcus rhodochrous
(Nocardlaparafflnlca))
Historically selected from ATCC2//7g.

If the substrate is an alkane or a saturated alicyclic or aromatic hydrocarbon, the microorganism used is also Rhodococcus rhodochrous (Nocardia parafinica).
Rhodococcus rhodochrous (N
ocardla paraffinlca ) ) A
TCC2//9g Oh! Get 7.

The morphological and taxonomic characteristics of the above-mentioned strains as well as references or patent specifications describing the strains are given below: Rhodococcus rhodochrous (species in the genus Arthrobacterium) ATCC/9/lIOCM; Goldbe
rget at,, Can, J, Mlcrobl
o'1. , 3.3.29 (/937)] This bacterium is a Durham-negative bacillus, and during the logarithmic growth phase, it usually has a short culm shape and bulges out, and during the stationary phase, it is nearly spherical, and is mainly unpaired and short chain shaped. Legs, no movement, catalase activity positive. It grows aerobically on normal broth, succinate, glucose, aliphatic hydrocarbons and n-A' rough-in. Will not grow on methane.

Pseudomonas aeruginosa (species sensitive to alkaline earth metals) ATCC/! ;! r25CLJ-5-Pat,
No, Jl, 30g I 03g, Re, ,
zg, ,tθ, l andU, S, Pat,
No, 3. ．． 30/, 764 to Ess.

Research and Engineering
Co, 3 Ho is a small Gram-negative bacillus that is immobile and ferments glucose. 02-C3o Aliphatic hydrocarbons such as C3-C30 n-paraffins and C6-C
Grows aerobically on 3o olefins.

Arthrobactyl Vetroleophagus ATC 2
/lI9'l (strain mu-/, t) [U, s.

pat, No, 3, 762.997 to N1
ppon Oil Co, Ltd.

Ho is a gram-negative or immotile bacillus. C2-C5 and Cyu,~Cza jl-/rifin, grown aerobically on glucose, citrate and glycerol. Will not grow on methane.

Arthrobacter simgrex ATCC21θ32
(FA-/; 19 stocks) [U, S, Pat, No
.

3, /,,,22. lI Otsu5 to A11ied
hemlcal Corp,] This bacterium is a Durham cell and has both rod-shaped and spherical shapes? None, exercise,
Does not form spores. 03~C□8(7)n-z? Grows aerobically on rough-in. Will not grow on methane.

Pseudomonas aeruginosa (Plevipacterium infectiphilum) ATCC/3jr2g [u, s,
pat, No-3+ Jθg, θ3k and R
e.

21z,! r02 to Es5o Re5ear
ch and EnglneerlngCo, ) Ho is a small, nonmotile, Durham-positive bacillus. Ferment glucose. 02~C3o Aliphatic hydrocarbons such as C2~
Grows aerobically on C3o 0-4 roughheins and C6-C3o olefins.

Species belonging to the genus Brevibacterium ATCC/+Otsudawa CU, S, Pat, No, 3.239.39
'lt.

Merck and Co, Inc.) Ho is a Durham-positive short bacillus. 02-C0°Grows on alkanes and alcohols and on normal medium. Will not grow on methane.

S7.7 C/91. ! ;) Eho is a Durham-negative short bacillus that does not branch, reproduces by simple cell division, belongs to the Brevibacteriaceae family, and is motile. Produces brown, yellow or orange pigments. Grows aerobically on normal medium, glucose, succinate, aliphatic hydrocarbons and /J rough-in. Will not grow on methane.

Alcaligenes eutrophus (Hydrogenomonas eutrophus) 7) ATCC/7 Otsuwo 7 [Intern.

J, 5yst, Bacterol, /9.
3g! t(/deotwa)(type 5train
), B., F., Johnson et.
al., J.

Bacterol, / 07, +4g (/97/
) and D, H, Davls et al., Ar.
ch, Mlkroblol,, 76%2 (/970
) Eho is a Durham-negative unicellular bacillus that is motile and has peritrichous flagella. Does not form spores. Coconi is opaque, white or cream colored. Normal medium, glucose,
Grows aerobically on succinate, aliphatic hydrocarbons and paraffin. Will not grow on methane.

Mycobacterium album ATCC, 29671° [J, J, Perry et al, J, 8
acterlo1. , ／ ／ ,2,! ;/3(/9
72), J, Gen, MIcroblol,, S2
,/Otsu 3 (/97gu)] This strain is not easily stained by the Gram method, but is usually considered to be Durham positive.It is non-motile and has a slightly curved or straight rod shape, sometimes with branching lines. Growth occurs in a hyphal-like or hyphal-like manner.Growth faster during the growing season with acid-alcohol mixtures.Grows aerobically on paraffin and aliphatic hydrocarbons.Does not grow on methane.

Rhodococcus Rhodocurus (Mycobacterium rhodoculata, x, ) ATCC29470 [J, J, Pe
rry et al., J. Bacterol.
/ / 2. ! ; / 3 (lqix>: + Ho is not easily stained by the Gram method or is usually considered to be Durham positive. It is a short rod that is round, non-motile, and has a slightly crooked shape.
It has rounded or sometimes thick ends, rarely branched, but sometimes Y-shaped cells are seen. Acid-alcohol mixtures speed up growth. Produces yellow or u tit color t=f.

]+Grows aerobically on rough-in and aliphatic hydrocarbons. Will not grow on methane.

Rhodococcus (Mycobacterium 1=l)
)'riI=lus) ATCC, 291, ri, 2 [
J, J, Perry et al, J, 8ac.
terio1. , ? 4t, / 9 / 9 (/
9 Otsu 7), J, Bacterol,, Wa Otsu, 3
/gCI9 6 g), Arc, Mlk
ro,,9/,g7(/973),J
, Gen, Mlcroblol,, g 2, /1
, 3(/97'l), and J, Bacterlo
l.

11g, 391I1. (/9741)FATCC294
He is the same sex as 70.

ar: y Kusuki Kaede (Nocalsia neoopaka) ATCC2/1199 C2-! ;3 stocks) [LI・
S, Pat, N(L, J', 7b, 2.997
to N1ppon oil Co.

Ltd.] This bacterium is a Gram-positive rod-shaped or linear bacterium and is not motile. It grows aerobically on C2-C4 and C, -c1B n-/, roughin, glucose, gluconate, citrate and succinate. Will not grow on methane.

! Sodomonas Kuruku L/NRRL F3-10
2/[Bergey's Manual of
Determnative8acterlog
y, gth ed,, /97'I, 5up
ra ] This bacterium is a Durham-negative aerobic bacillus. Motion is carried out by a minimal monomeric flagellum. Fluorescent pigments were born in 7th grade. ~C Alkane, 02~C Engineering. First H full 2
10 Grows aerobically on cole, butylamine and ordinary agar. Do not live on methane.

Pseudomonas fluorescens N RRLB −
/ 24t'l [Bergey's Manual
of Determinative Bacteria
logic, gth ed,, / rough 4t, 5
[upra] This bacterium is a Durham-negative aerobic bacillus and is motile.

Produces fluorescent pigment. Thaw the gelatin. Does not form spores. 02~C engineering. Yellow colonies are formed on a flat medium containing inorganic salts in the presence of alkanes and primary alcohols. It also grows on regular culture medium. Will not grow on methane.

Pseudomonas sepasi (Pseudomonas multiporans) ATCC/7b/Otsu [R, Y, 5tanler
et al., J. Gen., Mlcrobiol
,, II 3,is'y(/9 otis') and
H.W., Ballard et al., J.

Gen, Microbiol,, Otsu0, /9qC19
, 70)] This bacterium is a Durham-negative unicellular bacillus with a microscopic, multi-flagellated, motile body. Liquefy gelatin. Produces phenazine pigment. Does not form spores. Does not hydrolyze starch. It grows aerobically on phosphate, glucose, pro-4, paraffin & aliphatic hydrocarbons including C2-C4 alcohols and butylamine. Will not grow on methane.

Fu0 Soitomonas guchida type A A TCC/7'1
33 [R,Y, 5tanler et at,...
J. Gen.

Mlcroblol,, ,113,/! ;9 (/9
1,1. ) E-main is a Durham-negative unicellular bacillus that is motile and has microscopic polytrichous hairs. Gelatin does not thaw or denitrify. Produces a diffusible fluorescent pigment. Does not form spores. Grows aerobically on aliphatic hydrocarbons, including co/phosphate, common broth, glucose, 4-loughin, and C, alcohols, butylamine. It does not grow on methane.

Pseudomonas aeruginosa (f. Pseudomonas aeruginosa) ATCC/kk22 CLl,
S, Pat, N(C3,,30g, 03!
;, Re, 24.302 and U, S.

Pat, Na 3, 30/, 71,6 to
Es5o Research and Engineering
ring Go, ) is motile in Durham-negative small q bacilli. Ferment starch and glucose.

It produces a fluorescent phenazine dye. Does not form spores. It grows aerobically on C2-C3o aliphatic hydrocarbons such as C2-C30 n-pR paraffins and 06-C3° olefins.

Mycobacterium parafinicum AT C/247
0 [J, B. Davis et al., Ap
p.

Mlcroblol,, II, 3 /θ(/[54
)t.

rv'lagnol la Petroleum Co
), Durham-positive long rods are non-motile, and at a certain stage of growth, acid-alcohol mixtures accelerate their growth. Contains lipids (Ir1sh 1ipid) within the cells and cell walls. Colonies are normally yellow or yellow-orange in color and can vary from time to time due to carotenoid pigments. It grows aerobically on paraffinic hydrocarbons such as 02 to C1o n-alkanes, aliphatic hydrocarbons, ethanol and acetate. Will not grow on methane.

Rhodococcus rhodochrous (Mycobacterium rhodochrous) AT C / g 'I [R,S,
Breed.

J, Bacterol, 73.1g (/937
)': Has the same properties as the JATCC2 De Otsu 70 bacterium.

Rhodococcus (Nocardia) Dried Finica) AT CC2/ / 9 g [U, S,
Pat.

Na3,7. ! ;1,337 to yowa Hak
ko to ogyo to o.

Ltd.] This bacterium is a Durham-positive bacillus or cell and is not rapidly moving. Ferment sugar. C2-C4 and C engineering, ~C engineering.

It grows aerobically on n-alkanes. Will not grow on methane.

It will be appreciated that genetically engineered strains of these microorganisms may also be used in the production of microbial cells and enzyme preparations derived therefrom.

Storage of these strain cultures should be carefully controlled. A preferred method for preserving the culture is described below.

a on an inorganic salt agar plate medium consisting of the culture medium described in 101 Obscene Existing Example/
It is advisable to perform new cultures of microorganisms every week.

A glass blocker with a seal that prevents air from entering and an external sheath that connects the pipe.
The above-mentioned plated culture medium is maintained at a constant temperature. The assicator top wall is typically filled with 02-C4 alkanes and air (/:/V/V). It should be kept warm at 30°C. The culture is kept in this desiccator for 3 months at 20°C. However, it is preferable to transplant the culture from time to time.

In order to propagate microorganisms commercially, it is generally necessary to carry out the propagation in stages. Fewer or more steps may be taken l), depending on the nature of the process and the nature of the microorganisms. Propagation is usually caused by transplanting the cells from an agar slant into a sterile medium, usually contained in a flask.

Various methods can be used to promote microbial growth in the flask, such as shaking for aeration and maintaining an appropriate temperature. This step or step is carried out in regular flasks or large flasks with the same amount of medium:
Incubate seven or more times in a container containing i'(r). These steps are referred to as the culture enhancement phase for convenience. At the final stage of the enhancement phase, the microorganism is grown into a large-scale generator with or without a culture medium. The microorganisms or microorganisms derived therefrom can be commercially produced by transplanting them into the microorganism.

The reasons for propagating microorganisms in multiple stages are diverse, but the main reason lies in the requirements for microbial growth and/or enzyme production therefrom. This includes microbial resistance, normal nutrients,
Includes various relationships of 91Js osmotic pressure, degree of aeration, temperature conditions during fermentation, and pure culture conditions. For example, to achieve the maximum yield of microbial cells, the final fermentation conditions must be somewhat different, Fi, from the fermentation conditions for achieving microbial propagation during the culture enhancement phase. Maintaining the purity of the medium is also particularly important, particularly when fermentation 7 is carried out under aerobic conditions, as in the case of the microorganisms utilizing 02-C6 alkyl compounds in the present invention. If the fermentation is started from the beginning in a relatively large fermenter, the time required to achieve an acceptable production amount of oxidase or dehydrogenase from it is considerably longer. Become. This, of course, increases the possibility of foreign material contamination of the medium and microbial mutation.

The medium used for microbial growth and induction of the oxidase system is inorganic phosphate, sulfate and nitrate as well as oxygen and C3-C.
6 alkyl compound W.

The fermentation is carried out at a temperature of S to about Sθ°C, preferably about λS to about S′
Generally, it is set to 0. The pH of the medium is about 3.5 to 6.5 g, preferably about 3.5 to 6.5 g.
0-7. , tK should be controlled. Light hF may be carried out at atmospheric pressure, but may be carried out at pressures below and above about S atmosphere.

Typically, enzyme-inducing breeding substrates and energy substrates (e.g., ethane, butane, propane, ethanol, propatool, butanol, ethylamine, propylamine, microorganisms are inoculated into a medium containing oxygen (butylamine, etc.) and oxygen.

For continuous flow culture, the microorganisms are propagated in suitable fermenters, such as stirrer baffle fermenters or sparge tower fermenters with internal cooling rings or external circulation cooling lines. The culture is removed in a short time by continuously pumping fresh medium into the culture at a rate corresponding to each volume of culture from 90.02 per hour to keep the volume of the culture constant. Enzyme spinning--common substrate--oxygen mixture and possibly carbon dioxide or other gases are brought into contact with the medium by bubbling continuously, preferably via an oil bottom sparger. The source of boron for culture is nitrogen, oxygen or boron-enriched air. The waste gas is discharged from the top of the tank. External or internal means 2 can be used to circulate the waste gas by means of a blower for guiding the gas. The gas flow and gas circulation should be arranged to achieve the highest degree of reproduction of the microorganisms and the highest degree of utilization of the oxygen induction-propagation substrate.

Harvesting microbial cells from the propagation medium may be performed using any standard technique in common use, such as flocculent sedimentation, sedimentation, and/or precivitation. ), followed by centrifugation and/or filtration. The biomass can be dried, for example by freeze-drying or r@f drying, and used in this form in the oxidative conversion step. In the case of alcohol dehydrogenase systems, the enzyme may be conveniently available in the form of a soluble extract, optionally immobilized on an inert carrier.

In the practice of the present invention, for example, microorganisms that have been propagated aerobically in a normal culture medium containing a substrate for enzyme induction and propagation are obtained. A common medium for inoculating and propagating microorganisms is Foster and Davis [F
oster and Davls, J.

Bacterol,, 9 /, /9.2 (IC/9
/, B)] is the medium described. Microbial inoculation and breeding rAa
Afterwards, the microbial cells are collected and washed, and the resulting microbial cell arrest or iI enzyme preparation is then used to produce the substrate under aerobic conditions (in the presence of oxygen) in a buffered solution. Transform into living things. The mixture of substrate raw material and produced microbial cell population or enzyme agent during buffer infusion is maintained at a constant temperature to achieve the desired degree of conversion. The rainbow product is then recovered by conventional methods such as distillation and other flat methods.

To facilitate the necessary effective contact between oxygen and the enzyme system (which system may be an enzyme system or a microbial cell population derived from a C2-C6 alkyl compound). It is preferred to deliver a strong atomizing air stream into the dispersion of the substrate in the oxidation medium with vigorous stirring to give the best results. The oxidation medium typically contains water and a buffer in which the enzyme preparation or derived microbial cell line is suspended. The enzyme preparation or the attracted biological cell line is then separated from the liquid medium, preferably by filtration or centrifugation. In this way, sorted raw materials/& products can be obtained.

The process of the invention can be carried out batchwise, semi-continuously, continuously, co-currently or counter-currently. Optionally, the suspension 7 containing the buffer solution and the enzyme preparation or the microbial cell population is passed downwards with stirring countercurrently to the rising air current in the reaction tube.

Removing the upper layer from the downwardly flowing suspension, at least partially circulating the culture component and the residual M solution component on the one hand, and adding the obsolete base 11
4 and [If necessary, add additional amount of raw material or induced microbial cell line.

The microbial cultivation and oxidation steps are performed simultaneously but separately, and the oxidation step is highly aerated (e.g., at least twice as high, preferably at least three times as high as the amount necessary for growth). It is convenient to combine microbial growth and oxidation process.Both the growth process and oxidation process are carried out in the same reactor V3, and normal aeration and strong aeration are alternated while operating 111 next or simultaneously. It can be used for

In order to further illustrate the present invention, a 1V bow 1 will be used below, but these are not intended to limit the scope of the present invention. All parts and percentages are based on weight and are not listed in Section 4.

Example 1 Foscou et al. [Foster and Davis,
J.

Bac,teriol,,de/,/9117 C/9
A normal culture medium containing water and the following Ml was prepared as described in [66], supra: Na2HP0. 0.2/9NaH,P
o, 0.09 gNaNC),
Q(H'Mg5O4・7H200.2g to C10.0dal CaCl, 0.0/5gFeS
o, 27H, O/ mfi CuSO, s5H, OO, 0/ rrryH, BO, Q
,Q:1mg Mn5O,-,tH,OO-02m? Zn5O, θ, iqyy Mobr3Q, θ, 1rnq The pH of the above-mentioned normal medium was adjusted to 7.0 by adding acid or base.
700 samples of this medium were added to 0 gt shake flasks. Shake one loopful of inoculating cells from these shake flasks with a homogeneous colony of microorganisms on agar plates (the purity of the isolate was confirmed by microscopy). The culture medium in a flask was inoculated.The isolate was maintained on an agar plate in an atmosphere consisting of ethane, propane or butane and air at a gas ratio of t'il:l / The transplanted one was torn. Gas phase in the flask after inoculation Y/:
/v/v ratio of ethane, groin or butane and air. After seeding, the flask was tightly closed, isolated from air, and kept at constant temperature at 30° C. on a rotary shaker at 23 Orpm for about 1 day until turbidity appeared in the medium. #1 cells were collected by centrifugation at 10OOOXI for 3θ minutes at 7°C.

Pellets consisting of cell groups y, -pi17. θ at θ, θ5
rvl phosphate buffer (0,0θ, S-M MgCl
□Contains)y! Washed twice using Next, when the washed cell group is suspended in 0°03M phosphate buffer at ps+ 7.0, the optical density at 60 nm is 0.
．． It was 5.

# of cell tespensions after each wash, □ of Jml Samples (groups of 2 cells) were placed in separate 10-volume vials at 3°C and sealed with rubber stoppers. . The gas phase within the vial was removed under vacuum and the interior of the vial was replaced with a gas mixture of substrate reactant (ie, alkane or hydrocarbon) and acidic reactant in a ratio of 1:1V/V. (Liquid substrates such as hexane, benzene and toluene are placed in a vial with a volume of /θ μt.) The vial was then kept at a constant temperature of 30° C. on a rotary shaker at 300 rpm. A sample (7 to 3 μt) of the product is periodically taken out using a microsyringe and subjected to gas chromatography (ionizing flame extractor column) at a carrier gas flow rate of 1θ0 or i, 2o, and 2o at a column temperature of 3θ or 3
! ? Analyzed using tnl 1 min (helium). By comparing the heat retention times of various products, and by using chromatography with a standard product for confirmation, the product was identified. A standard map constructed using a standard product for confirmation was used to generate a peak area from the highest value (peak) area.

Determination of proteins in cell suspensions using the method of Lowry et al.
l, Chem,.

/93, J&5 (/de, t/)) [! ! I measurement position.

According to the procedure described in Example 1, several microbial strains shown in Table 1 below were grown under aerobic conditions in the ground containing propane as a growth substrate. Cell clusters were washed as described in Example 1. The resulting resting imitative cell population was then treated with h-6i in a V titer solution.
6 n-alkane.

The results of these experimental systems are shown in Table 1.

1 According to the procedure of Example 1, in a normal medium containing ethane or taybutane as a base for increasing fm, under aerobic conditions,
Rice strains were grown. The plasmodia rM was harvested, washed and contacted with C to C n-alkanes in a buffered solution according to the procedure of Example 1. The results of these 6 oxidative conversions are shown in Table H.

Example 3 strains of Example 3 were grown under aerobic conditions in a normal medium containing guethane, pronoyanine or butane, harvested and washed, and then C, C n- It was contacted with alkanes and analyzed for the presence of secondary 6 alcohols and methyl ketones.

The oxidative conversion rates for the production of alcohol gradients are shown in Table 1.

Example S According to the procedure of Example 1, the bacterial strains of the species listed in Table 1 were grown under aerobic conditions in a private medium containing ethane, propane or butane as propagation material. A cell group was collected and washed, and then brought into contact with cyclohexane, benzene or badruene in a slow rinsing solution RFFL according to the procedure described in Example 1 to produce secondary alcohol. The results of these oxidative conversions are shown in Table 2.

Optimal Conditions for the Oxidation of Alkanes Various tests conducted regarding the optimal conditions for the production of methyl ketones from alkanes are summarized as follows. Those skilled in the art should understand that these are found to be the prevailing conditions and the scope of the present invention is not limited to such conditions. Even when departing from these optimum values, conversion can still be achieved, but yields and conversions are reduced.

Temporal Process The rate of production of methyl ketones from n-alkanes by suspensions of cells grown on various C2-C4 alkanes is linear for the first 7 hours of incubation. Therefore, when testing for the influence of variables, measurements were taken on methyl ketone loading within 7 hours of incubation.

, -1- Using cell suspension 7 of the fungus Rhodococcus rhodochrous (Nocardia parafinica) A1'CC2//9g grown on propane, Prono? production of seven tons from n or n
- pJ on the production of 2-butanone from butane
The influence of j was examined. In this case, the pH value is 5.5-
0.0, t M IJ phosphate buffer for adjustment of ? Use p
H value 3.0~/ To adjust Q and θ, a tris-(hydroxy-methyl)aminomethane-Hα buffer of θ, θ, and tM was used. The optimum p11 for acetone and cobutanone production was approximately 7.5.

TemperatureThe optimal temperature for the preparation of cobutanone from propane to acetone or from n-butane when using a cell suspension of Rhodococcus rhodochrous (Nocardia paraphini) ATCC2/9g grown on propane is approximately 3 &'O
Met.

Substrate properties 1. As can be seen from If and Table 1, methyl ketones and secondary alcohols were produced from the n-alkanes. Among the n-alkanes, n-propane and n-butane were oxidized at the highest rate.

As can be seen from Table 1V, alcohol was produced from cyclohexane, benzene, and hydrene for at least one strain of the bacteria tested.

Product and enzyme analysis showed that all of the tested bacteria were catalytic for the production of methyl ketone. Certain cultured microorganisms, such as Rhodococcus rhodochrous (Nocardia parafinica IATcc2//9g and Plevibacillium fuscum ATCC/!;9931"), have shown relatively large methyl ketone-producing capacities. At least three microorganisms that grow on alkanes have shown that C6 alkane Z
Oxidation L7' to the corresponding co-alcohol and methyl ketone
c.

In bacteria growing on pronozone, propane is terminally oxidized (te
It is metabolized by either terminal oxidation or subterminal oxidation.

Vestal et al. [J, R, Vestal et al.
J.

Bacteriol, , q9.2/b (/q Otsu9)] was used by Brevibacterium species JOB, t'Y, Leadbetter et al. [E, H, Leadbetter et al.
,, Arch.

Mikroblol,,3! i%? 2 (/
9 b θ )] is M6 smegmatis (M, sm
'egrnatls 4122) 'l was used to suggest that propane is subjected to quasi-terminal oxidation, J:9 generation.

In the present invention, most of the test bacteria grown on propane produced coglobanol and acetone from gushinogun, but did not produce 7-propatool.

Whether the glonon monooxyrnase system is a specific enzyme for terminal oxidation or whether a non-specific enzyme catalyzes both terminal and sub-terminal oxidation, l-propertools are produced but undetectable. In order to test whether this is the case, we conducted the following experiment.

Using a cell suspension of both live bacterial cells and heat-sterilized cells of 2//9 g of the fungus Rhodococcus rhodochrous C nocardia or Roughinica) grown on bread,
These and l-propatool, coglobanol or acetone in an amount of 0.3 to 0.4 t pmol/d/cell suspension were kept at constant temperature.

The rate of substrate disappearance was followed by gas-liquid chromatography. Heat-killed cells do not oxidize any of these substrates; however, live bacterial cells disappear within 6 minutes of iLY incubation with all additions of propatool fc, as a substrate. The reaction mixture was acidified using propatool, extracted with benzene, and then compared with methyl ester using BF-methanol. The presence of propionized methyl ester was confirmed by gas-liquid chromatography. Coglobanol is also produced by suspension of live bacterial cells9.
It was oxidized after 90 minutes of moisturizing time. Coprobanol is quickly oxidized to acetone and acetone accumulates, and /-pronozonol is oxidized even more rapidly and converted from an aldehyde to a single compound.

τ1''7 No #lll IJN soluble fraction (alkane monooxidanase): Melting microorganisms of alkanes, cyclic and aromatic compounds! Fe/I Nonidomonas Fluorescens NR
RL F3-/, 21111Y30t8 fermenter (Ne
wBrunswlck 5scientific Co.
The cells were grown at 3θ°C on propane (7.5% glopane and 93% oxidation) by batch culture on inorganic salt-containing media as described in Example 1 in Edison, N.J.). This generator was inoculated with 2 tons of culture in a flask.

The cells thus grown were incubated at pH 7.0, l 3
Wash twice with 3 mM potassium phosphate buffer, then 3 mM potassium phosphate buffer.
gCl, a and deoxyliposphrease (0,0
1V-) suspended in the same buffer solution containing Kaoru.

b OrrPa T: 71i ychi pressurization + A/ [
: French pressure cell (Am
erlcan Instruments Co,,S
The suspensions were disintegrated by passing through a silver spring, MD, ) twice at 20°C. Cell suspension after disintegration /30θOXl/
The unbroken cell group was separated by centrifugation for 30 minutes. Next, the supernatant VI1. The mixture was centrifuged for 60 minutes at Ooooooy g, and the supernatant liquid was taken.

A reaction mixture of the soluble enzyme fraction obtained as described above and a 7θμmol potassium phosphate buffer at pH 7.0 and Daμmol NADH was prepared at 0.2-vig°C using several 3d I put it in Tani's vial. The gas phase of the vial is removed under the gaseous nitrogen gaseous mixture and oxygen (/:/V/
Direct replacement with the gas mixture of V); if the substrate to be oxidized was a liquid, tm or μt of substrate was added. The vial was kept at a constant temperature of 13° C. on a reciprocating water bath shaker at Sθ for a number of vibrations.

The rate of dialkene and styrene epoxidation was determined by gas chromatographing each sample of .mu.t of the reaction mixture immediately after substrate addition (time zero) and after 5 to 70 minutes of incubation. The specific activity of the product was expressed in µmoles after 12 minutes of drying for 10 minutes. Control tests were carried out using different substrates, no addition of NADH2, no addition of oxygen, and using boiled extracts. result? Shown in Table V.

Marriage 8 F in a normal medium containing fluoropane as a growth substrate.
A plurality of bacterial strains listed in Table 1 were propagated under J-air conditions according to the procedures described. Example of collecting a group of cells/
Washed as shown in U-Pi. The resting cell group thus obtained was subjected to the following procedure to obtain 02-C6n during slow dissolution.
-Touched 4 curses with Alkene and Butadiene. The results of this experimental yarn are shown in Table ①.

1,・1.7 Add the 1-glaze orchid powder shown in Table 1 under an aerobic button in a medium containing either ethane or d-butane as a growth substrate for fishing rods.
The procedure in Example/was used to mix the 1'7 ills.

Collect the cell group, wash it, and then gently wash it by the procedure described in Example/.
! 1-Alkenes of 02-C5 and butadiene in 'H solution.Only Mycobacterglyrum or 1 Aφ strain was used for the conversion of isobutyne. Shown in the table.

1st edition I O engineering d? 7.2 from propylene where tfj ■ conditions for oxygenation preferred AI = Jtjj,
- I'i11 tests conducted on suitable conditions for the production of epoxypropane lead to the following results. However, the Ministry should understand that these Q factors have been found as optimal conditions, and that this investigation is not limited to sales in this area. Even if one deviates from these optima, conversion can still be achieved, although yields and conversions are reduced.

Propylene was used in batch experiments for all strains tested over time.
)'s propylene oxide morning comb reached its maximum value after 2 to 3 hours of constant temperature maintenance. Propylene oxide + lI'
II, constant f++X retention - did not fall even after 0 hours. The rate of propylene oxide production was linear during the first 4θ minutes. In testing for the influence of variables, the amount of propylene oxide produced was measured within 7 hours of incubation.

pH In a study of the influence of pH on the production of propylene oxide, the pH value was 5.5-, θ' and θ, θ! Using rM phosphate sodium buffer, pH value g, θ ~ 10.0
About 0.0! ;M tris-(hydroxymethyl)aminomethane-HO green agent was used. The optimum p11 for the production of C propylene oxide for all of the sample cytostatic suspensions was 6. Within the range of θ~7.01. It was discovered that

The optimum temperature for propylene oxide production by temperature cell suspension was approximately 35°C for all of the strains tested.

Product and Conversion Analysis Cell groups grown on n-no-4 or nol did not show the ability to epoxidize propylene. A group of cells growing on a cell in the form of a resting cell suspension produces propylene oxide, which accumulates outside the cell. Control experiments using heat-killed cell populations showed that niboxide was enzymatically limbated. Regarding substances other than propylene oxide,
Jy, one bundle of extremely thick amount (Heari) was not delivered. [S, Vv, May etat, J, Blo
l, Chem, , 2'l g, / 72k
(/973D reported that epoxide is semi-accumulated from /-octene by Bunidomonas aeruginosa cells, and along with this, major metabolism of l-octene occurs via narrowing of the methyl group. Epoxidation of propylene by suspensions of bacterial cells grown on propane did not result in 3-hydrocarbons.

General properties/ir properties As can be seen from Table 1, general oxides are produced both from gaseous l-alkenes and fulkenes, and also from butadiene. It was found that all of the test bacterial strains produced the most epoxide when ethylene or 70-robylene was used as the substrate.

The application properties for the epoxidation of alkenes and butadiene by Frfi##ftl cells on ethane and butane were also studied (see No. 2).

The preferred group is propylene, although both of the mosquito test boxes oxidize to the corresponding 2-epoxides/-alkenes and butadiene t'.

Example 1/ Soitomonas fluorescens TjRRL
B-/, 2Q+ and 3θ fermenter (NewBrun)
swick 5clentlflc Co,, Edi
The cells were grown at 30° C. on cypropane (7 Pronozone MU93 Nitrogen) in a batch culture method on mineral salt-containing medium as described in Example 1. The fermenter was inoculated with 2 tons of flask culture.

The cells grown in this manner were washed twice with No5r City's potassium phosphate buffer, pH 7.0, and then treated with 3 mM MgCl.
, and deoxylyphosphrease (0,0!; 1ny/
into the same buffer solution containing ml of turbidate.

French pressurized cell [French p
Ressurecell (American Ins
trume, nts Co,, SilverSprf
The cells were transduced by passing the cells through the suspension for -1 times. The cells after resolution are begging for help/! A group of unbroken cells was separated by centrifugation at 000XI for 1S minutes. Next, Kasumi Attenuation 11.0
The supernatant was centrifuged at 0000 x 17 for 60 minutes to produce a soluble fraction.

The soluble enzyme fraction purified as above and pt17. 70 μmol of phosphorus at θ is 1 and 4
0.2 ml of reaction mixture with 4 mol of NADH,
At C, the sample was placed in a 3 ml vial. In the gas phase of the vial, remove the gaseous branched oxidized substrate and the TFL (/:/V/V) gas mixture.

Tit, 2 when the substrate to be converted is a dispersion;
A group of μt groups was added. Number of vibrations per minute 5θ
The vial was held at a constant ηiA of 3S on a reciprocating water bath shaker.

After 1 mouse addition of 1M Kyo (zero time) and constant temperature maintenance 3-70
The epoxidation rate of the VC dialkenes and styrene was determined by chromatographing each sample of the reaction mixture after 10 minutes. Protein/■ Our/
The specific activity was expressed by expressing the product produced in θ minutes in μmol. Control tests were carried out using various substrates, no addition of NADH, no addition of oxygen, and the use of boiled extracts. The results are shown in Table 1.

Q Here, El is the monooxygenase enzyme, E2 is the protein oxygenase, and S is the enzyme for cofactor regeneration.
lengthen.

Cofactor Pj- mediated by ethanolic enzyme
The λ7 stone strain shown in color Mj, IXi was grown on Pro-Sen and IU-1. / record ↓ uni no 0 robiren vh C) 1 change was used for epoxidation to norobi 1/nogiside, but based on 6 simultaneous experiments, cofactor 7++
The six basic i; of ethanol as η were added to the reaction mixture. Part 4i) Alcohol, small 90 genease and jetanol? Converts to acetaldehyde (in biological reactions, it is also called acetaldehyde A) and produces 1 mol of NAD.
H7 < produced, followed by middle 1 = J chemical formation acetyl co-A
was oxidized to Nitorifuda charcoal 9, and 3 mol of 80H was added to it.

The results are shown in Table 1 and demonstrate that the addition of S μmol of ethanol improved the epoxidation rate. When the ethanol strength is high (2 S μmol), the initial 2
An inhibition of 8 degrees was observed within an hour.

τ Li / 3 Raw W phase system η includes ethane, butane or li propane,! In a normal medium containing glonocene, the bacterial strain with the number of rice grains shown in the table is grown under aerobic conditions.
, y distribution school. Cell groups were collected and washed using the same route as described in Example 1. Thus obtained dormant i collected organisms = 0 cells group
Contact with C7 straight lead secondary alcohol. The results of these experiments are shown in Table X.

Example 1/ According to the procedure of Example/7 λ bacterial strains shown in Table j I filtered out the colonization. Collect the female spores, wash them, and gently wash them according to the instructions in Example/. In contact with the C3 to C7 secondary alcohol in the il + i melt! I let it happen. . The result of these oxidative conversions is ya1″6B ) [1
Shown in pine.

619- 4N, + / 3 Ai□*f1 for alcohol hrg conversion and methyl ketone forgery from alcohol.
j) Regarding the condition, if we stop wave 4 in f+j trial with r7, we get r ゛ (no) 111 and 2). However, these iI town jf;'
3jZj 'jr- sweat, and the present invention is not limited to these Article 1 Hayabusa.
Bribery! You should be angry. Even if these are shifted by 1 shift, the conversion t'i is still achieved and 1 (
However, the yield and conversion rate are as below.

time) t:'r:! '1' Each of 11 (shi, ~ C4 alkane growing country strain H; g
The speed of methyl ketone production from shogi secondary alcohol () is (jj 7j" 77 for 1 h,
-First 31i; tl) lki about Sej, 1tjJ
It was a target. He tested the influence of variables on C''r, methyl ketone formation within 3 hours of holding 14)) 1 to 71! li>Koshita.

■) 11 Gropan epigenetic bronze Rhodococcus rhodochrous (Nocardia parafinica) 8T (1: 2//9g (')
4. Effect of pH on the production of acetone from lovanol or λ-butanol from α-butanol under N:d cell suspension properties. , r, lr, t and 1311 fifi-5, 3~g, θ is 0.0
! ; Tamilin soil (Iij agent Kaoru was used to adjust the pH value and θ~
10.0 t' θ, θ, S-M) 'Jss-
hydroxy-methyl)-aminomethane-Hα setona kr
1] was also used. Acetone and Cobutanoy 1.
'4Ji Noah'c me(1) *'t 1f4e p
Htj, 'about g, o ~9.00 l:< fill
There was VJ Ic.

Temperature □□--1阿訃1-□--■1-1 Tote house-1■1 Pronoyan top % Iku Rq Rhodococcus Rhodochrous (Meso1 Rugia parafinica) 8T with C: l//9δ' under use Chisel sigma vortex for the production of coptanone from acetone or -butanol:
, tel J at about 3S°C.

It is understood from Table Alcohol is king, rainbow is used for the 71'llJ cells grown on alkylamine, methyl ketone from r↓ secondary alcohol is a dream! The formula is written from Chapter XI = 4th century). Among the secondary alcohols, &,j, 2-70 lobanol and 1-1-butanol are oxidized at the highest rate. For example, Rhodococcus rhodochrous (Nocarnoa nocerafinica) ArCC2//9g&U Brevivac-7rium fuscum AT /, 5-993 (I
The methyl-ketone raw material exhibited relatively high oiliness.

fermentation purple multi l i11 mechi/l/ 4 'I% go搐U'A and thin iM (m
ethylotrophlcbacteria) and methanol grown r! If, if you use the mother, the secondary alcohol line is L-like bu'hydroquinase (υ' to S)
It is possible to mediate the conversion of l'L, 2-alcohol to the corresponding methyl ketone.

1OLI et al,, App,
Environ, Microbiol,...
3g, /3shio (/def9)]. Honko blJ) Here, fσ (4 ni Mono Aro 4
A young boy with 5ADH activity was placed in the J4 group and the /-7'' o i9 Nord group.

1"About Urutokizu6" is the number of C2~06 alkanes.
・If 1 or the hydrocarbon is a secondary alcohol-
202-C6 Alkyl group-donating compounds such as alcohols or alkyl amines are added to selected enemy biological cells or their enzyme preparations, which have been grown aerobically, under aerobic conditions. -C provides a method for converting hydrocarbons by grafting.

1/385 6760-4
B priority claim June 28, 1982 ■United States (US)
■392610 @ June 1982 2880 United States (US) [Yes] 3926
12! ￥ Author: Ramesh N. Patel 15 Motsuking Bird Road, Edison, New Chassis, United States of America 0 Inventor: Allen I. Ruskin 3 Box 392, R.D., Somerset, New Chassis, United States of America

Claims (1)

[Scope of Claims] (1) Cells derived from a microorganism that exhibits #transferase action or dehydrogenase action, cells derived from genetically engineered strains or natural mutant strains of microorganisms, or cells prepared from such cells enzymes and alkanes, saturated alicyclic or aromatic hydrocarbons,
a substrate selected from alkenes, dienes, vinyl aromatic compounds or secondary alcohols under aerobic conditions in a reaction medium until at least an isolable amount of the corresponding product or products has been exposed. In the method of oxidizing G4 to a corresponding oxidation product or product by contacting, the microorganism is
It has been previously grown under aerobic conditions in a normal medium containing a C6 alkylene compound, or if the substrate is an alcohol, further containing a 02-C6 alkyl group donating compound; and the strain of the microorganism is as follows: Rhodococcus rhodochrous ATCC/9/110 (
Species of the genus Arthrobacterium) Pseudomonas aeruginosa ATCC/! Sf;2j
t (alkaline earth metal ridge) Arthrobactyl petroleum ATCC2/lI9'1
Offagepseudomonas aeruginosa ATCC/3! ; 2g Brevibacterium & ATCC/Daotda 7 Psida 7 Previbacterium ATCC/! ;9? ,? Alcaligenes eutrophus ATCC/'N, 97 Mycobacterium album ATC, 291.71 > Rhodococcus rhodochrous ATCC 291, 7θ Rhodococcus rhodochrous ATCC 29 Otsu 720 Species of the genus Dococcus ATCC Yu/1lqq (Nocardia neopaca) Pseudomonas kulfure NRR L F3-102
/ Punidomonas fluores NRRL B-/2'
/-11 Sensepseudomonas Senososia ATCC/71. #Pseudomonas guchida type A ATCC/7'l! ;3 Punidomonas aeruginosa ATCC/3! ;22, and if the substrate is an alkene, diene or vinyl aromatic compound, the microorganism is further selected from the following strains: Mycopafuglyrum paraphyi ATC (,: /Sike0nicum and Rhodococcus rhodochrous AT, C/ glIt, and if the group η is a secondary alcohol, the microorganism is further selected from the group consisting of the following strains: is an alkene or a saturated alicyclic or aromatic hydrocarbon. %#f The method according to claim 181, characterized in that the strain is an alkane. (3) The method according to claim 1 or 2, characterized in that the bacterial strain is Sinidomonas aeruginosa. ( 2) The cape of the patent claim, boxes 1 to 3, characterized in that the substrate is a 02 to C6 alkane, cyclohequinane, benzene, C11 to C, alkene, butadiene, styrene, or a 03 to C6 secondary alcohol. The method according to any one of the above. f!sI The method according to any one of claims 1 to 7, characterized in that the microorganism cells are resting cells. (B) The microorganism cells are resting cell suspensions. The method according to any one of claims 1 to 7, characterized in that the enzyme agent is a cell-free soluble fraction. The method described in any of the above paragraphs. The method according to any one of claims 1 to 3 characterized in that The method according to item 3. (10) Patent 8h, characterized in that the 02-C6 alkyl group donating compound is ethanol, glopanol, butanol, ethylamine, propylamine, or butylamine, and the substrate is a 03-C5M secondary alcohol. The method described in item 7 of the scope of the request.