Note: Descriptions are shown in the official language in which they were submitted.
- ~3Z92Z
Broadly, this invcn~ion rela~es to a mcthod of producing
epoxides using microorganisms. More particularly, immobilized
cells of epoxide-producing microorganisms are allowed to react
5 upon hydrocarbons having terminal double bonds in an aqueous
medium in order to produce oxygen-containing hydrocarbons by
converting the double bonds to epoxide bonds.
Oxygen-containing hydrocarbons having epoxide bonds are
useful materials for synthesis or intermediate materials of
10 polymers such as textiles and plastics as well as surfactants,
paints, agricultural medicines, and adhesives.
Heretofore, only a few kinds of such oxygen-containing
hydrocarbons, for example, ethylene oxide, propylene oxide,
and the like are most commonly produced by direct oxidation of
15 hydrocarbons having double bonds. Other epoxides, particularly
those containing long hydrocarbon chains as mentioned above
have been produced via halogenide or indirect oxidation by
peracetic acid.
The use of microorganisms to manufacture epaxides from
20 alkylenes has been disclosed in U.S. Patent 4,106,986,
Suzuki, et al, and U.S. Patent 4,102,744, McCoy, et al.
Since hydrocarbons having epoxide bonds are generally
explosive, it is desirable to produce them under moderate
conditions, for example, at mild temperatures and
atmospheric pressure.
.
V
`~
In view o the situat ~ ~ as dcscribcd above, it is of
critical importance to establish a method to pro~uce hydro-
carbons havin~ epoxide bonds under mild conditions as for
example, using the immobilized microorganisms of this
5 invention.
As the microorganisms that convert double bonds of hydro-
carbons to epoxide bonds, several microorganisms belonging to
genera of Nocardia, Brevibacterium and Corynebacterium
have already been disclosed in the above patents.
According to the conventional method, a microorganism
from the appropriate genera is cultured with any of the hydro-
carbons by the ordinary method, or resting cells of the
microorganism are allowed to react on said hydrocarbon. Since
the hydrocarbon as a substrate has minimal solubility in water,
15 a two-phase system results and conversion of the double bond of
the hydrocarbon to an epoxide bond does not proceed smoothly.
Also, according to the conventional method, separation of the
reaction products from the residue of the substrate and the
cells requires multiple and complex separative and purification
20 treatments. In many cases, complexity of the treatments
results in destruction of the microorganism itself or its'
activity preventing its'reuse or preventing adaptation of the
system to continuous production techniques.
Moreover, as products increase during the reaction,
25 product inhibition occurs and the reaction seemingly ends
despite the fact that the activity of the microorganism still
remains. To prevent this phenomenon, the products should be
--3--
~i~292Z
harvcstcd pcriodically or continuou~ly 50 that the conccn-
tration of the products can be kept low. Furthcrmore,
contamination of the medium with chemical agents to effect
separation causes degradation o epoxide-producing activity of
S the microorganism. Contamination also disrupts temperature and
pH control which cannot be avoided when chemical agents are
added to the medium. Thus, the utility of the microorganism
is hampered.
The purpose of this invention is to solve scme of the problems
existing in the conventional method by offering a novel means
of producing epoxides advantageously using microorganisms.
By the method of this invention a microorganism is used in
an immobilized condition and conversion of the double bond of
15 the hydrocarbon substrate to an epoxide bond goes smoothly and
concurrently, the produced epoxide can be separated easily
from the microorganism and reaction medium.
Thus according to the present invention, there is
provided in the process for the production of epoxides from
unsaturated hydrocarbons comprising carrying out the reaction
in an aerobic aqueous medium in the presence of an appropriate
epoxide-forming microorganism, the improvement which comprises:
(a) growing the microorganism in a nutrient medium;
(b) harvesting the microorganism;
(c) polymerizing a monomer capable of polymerization in
the presence of the organism to form immobilized cells of the
microorganism;
(d) contacting the unsaturated hydrocarbon with the
immobilized cells in an aqueous medium; and
(e) isolating the resulting epoxide.
~'13Z92Z
Hydrocarbons having double bonds which are used as sub-
strate are hydrocarbons having at least one double bond
adjacent to the terminal end and can bè of low, medium,and
higher molecular weight, including for example, ethylene,
propylene, l-butene, l-decene, and l-undecene, 1,15-
hexadecadiene and the like.
As the microorganism to be used in this invention,
those which can convert a double bond of a hydrocarbon to an
epoxide bond under the aerobic conditions will suffice.
Accordingly, microorganisms known to have such ability, for
example, Nocardia coral]ina, Corynebacterium alkanum,
Coryne~acterium hydrocarboclastus, Nocardia butanica,
Nocardia paraffinica, Brevibacterium butanicum and
Brevibacterium ketoglutamicum can be utilized.
The microbiological properties of these microorganisms
~have been reported in The Journal of General Microbiology
10(3), 107, (1967), Japanese patent publications (Kokai)
Nos. 46-41588 and;47-48673, and Japanese patent application
No. 52-75127. The following Table I summarizes those
properties.
;292Z
T~13J,~ I
Nocardia corallina
(~) Morp}~ology
Shape - Rod-shaped; cells elongate and multicellular like
5 mycelial form is found. Cells divide into daughter cells by
fragmentation.
Size - (0.6 - 0.3)x(2 - 20)~
Motility - Non-motile
Endospore forming - Non-endospore forming
Gram strain - Positive
Acid fast - Non-acid fast
(B) Cultural features
Nutrient agar plate culture - Good growth. Round shape,
smooth, wavy shape around entire circumference. Protuberances.
15 Coral color in wet light. Gradually become stronger.
Nutrient agar slant culture - Good growth. Thread shape,
smooth. Coral color in wet light. No changes in color of
culture medium are observed.
Nutrient broth liquid culture - Surface growth in form of
20 white membrane. No turbidity. Precipitates are formed.
(C) Physiological characteristics
Optimum temp. - 20 - 35C.
Optimum pH - 6.0 - 8.0
Oxygen requirement - Aerobic
Hydrogen sulfide - Trace amount produced
Indole - Negative
Starch - Not decomposed
Reduction of nitrate - Reduced
Catalase test - Positive
~1~29;22
Urease - Nec3ative
V.P. test - Negative
Utilization of sugars - Glucose, fructose, mannose,
sucrose, sorbitol, glycerol.
Norcardia butanica
(A) Morphology
Shape - Rod-shaped; cells elongate and multicellular like
mycelial form is found. Cells divide into daughter cells by
fragmentation.
Size - (0.5 - 0.75)x(2.5 - 16)~
Motility - Non-motile
Endospore forming - Non-endospore forming
Gram strain - Positive
Acid fast - Acid fast
(B) Cultural features
Nùtrient agar plate culture - Growth is rather slow.
Round shape, wrinkled shape, umbilical shape, no gloss. Color
is dull red. Become opaque.
Nutrient agar slant culture --Growth is slight. Thread
shape, no gloss, color is dull red.
Nutrient broth liquid culture - Surface growth in form of
membrane. Almost no turbidity. No precipitates are formed.
Nutrient gelatine stab culture - Growth is better on top
than on bottom. No liquefaction.
(C) Physiological characteristics
Optimum temp. - 25 - 35C.
Optimum pH - 6.0 - 8.0
--7--
-- ~i3Z92~
Oxyc~en requirc~ment - ~ro~)ic
Litmus milk - No ch~nge or alkaline
llyarogcn sulfide - ~roduced
Indole - Negative
Starch - Not decomposed
Reduction of nitrate - Reduced
Catalase test - Positive
Urease - Negative
V.P. test - Negative
Utilization of sugars - Glucose, fructose, arabinose,
mannose, sucrose, galactose, xylose, mannitol, sorbitol
Nocardia paraffinica
(A) Morphology
Shape - Rod-shaped; cells elongate and multicellular like
mycelial form is found. Cells divide into daughter cells by
fragmentation.
Size - (0.5 - 0.75)x(2.5 - 16
~otility - Non-motile
Endospore forming - Non-endospore forming
Gram strain - Positive
Acid fast - Acid fast
(B) Cultural features
Nutrient agar plate culture - Growth is rather slow.
Round shape, wrinkled shape, wavy shape, umbilical shape. No
gloss. Color is yellowish-red. Become opaque.
Nutrient agar slant culture - Growth is slight. Thread
shape. No gloss. Color is yellowish-red.
--8--
., -~ . , , - .:
-- ~132922
Nutricnt broth liquid culturc - Sur~ace growth in form of
membr~ne. Almost no turbidity. No precipitates are formed.
Nutrient gelatine stab culture - Growth is better on top
than on bottom. No liquefaction.
(C) Physiological characteristics
Optimum temp. - 25 - 35C.
Optimum pH - 6.0 - 8.0
Oxygen requirement - Aerobic
Litmus milk - No change or alkaline
Hydrogen sulfide - Produced
Indole - Negative
Starch - Not decomposed
Reduction of nitrate - Reduced
Catalase test - Positive
Urease - Negative
V.P. test - Negative
Utilization of sugars - Glucose, fructose, mannose,
sucrose, mannitol, sorbitol.
Corynebacterium a-lkanum
(A) Morphology
Shape - Generally short rods, branching is observed.
Size - (0.5 - 0.6)x(2 - 5)~
Motility - Non-motile
Endospore forming - Non-endospore forming
Gram strain - Positive
Acid fast - Non-acid fast
, . ~
~13292Z
(~) C~ltural fc.~tures
Nutrient agar plate culture - Moderate (Jrowth. Round
shape, smooth, semi-lcns sh~pe around entire circumference.
Yellow-gray under wet light. Become opaque.
Nutrient slant culture - Moderate growth. Thread shape.
Under wet light, smooth and yellowish-gray in color.
Nutrient broth liquid culture - Surface growth is weak.
Moderate turbidity occurs.
Nutrient gelatine stab culture - Growth is slight on top.
No liquefaction
(C) Physiological characteristics
Optimum temp. - 25 - 35C.
Optimum pH - 5.0 - 9.0
Oxygen requirement - Aerobic
Litmus milk - No change or alkaline
Hydrogen sulfide - Produced
Indole - Negative
Starch - Not decomposed
Reduction of nitrate - Reduced
Catalase test - Positive
Urease - Negative
V.P. test - Negative
Utilization of sugars - Glucose, fructose, mannose,
sucrose, lactose, mannitol, sorbitol
Corynebacterium hydrocarboclastus
(A) Morphology
Shape - Rod-shaped, elongate, branch; possess lipid
--10--
-- 11329:22
~ranule, ~ra~mcntation into 3~.
Size - (0.4 - 0.6)x(3 - 20)~
Motility - Non-motile
Endospore forming - Non-endospore forming
Gram strain - Positive
Acid fast - Non-acid fast
(B) Cultural features
Nutrient agar plate culture - Good growth. Round to
flower shapes. Wrinkled shapes, protuberances. Wavy shapes
around entire circumference. Under protein light color is
light yellow-orange.
Nutrient slant culture - Moderate growth. Thread shape,
smooth to wrinkled shape. Under protein light color is light
yellow-brown. No changes in color of culture medium are
observed.
Nutrient broth liquid culture - Moderate surface growth,
membrane shape. Some turbidity occurs. Precipitates are
formed.
Nutrient gelatine stab culture - Growth is better on top
than on bottom. No liquefaction.
(C) Physiological characteristics
Optimum temp. - 25 - 35C.
Oxygen requirement - None
Litmus milk - No change or alkaline
Indole - Negative
Reduction of nitrate - Reduced
Catalase test - Positive
--11--
: . `
~3Z922
Utilization of sugars - Glucose, ~ructose, lactose,
raffinose, inositol, ~lycerol
13revi.bacterium butanicum
(A) Morphology
Shape - Generally rod-shaped, snapping division is observed.
Size - (0.5)x(3.0 - 6.0)~
Motility - Non-motile
Endospore forming - Non-enaospore forming
Gram strain - Positive
Acid fast - Non-acid fast
(B) Cultural features
Nutrient agar plate culture - Good growth. Round shape,
smooth, protuberances around entire circumference. Light brown
color. Become opaque under dull light.
Nutrient slant culture - Good growth. Spiny shape. Under
a dull light, buttery consistency and light brown color. No
changes seen in culture medium.
Nutrient broth liquid culture - Surface growth in membrane
form. Almost no turbidity. Precipitates are not formed.
Nutrient gelatine stab culture - Growth is better on top
than on bottom. No liquefaction.
(C) Physiological charzcteristics
Optimum temp. - 25 - 35C.
Optimum pH - 6.0 - 9.0
Oxygen requirement - Aerobic
Litmust milk - No change or alkaline
Hydrogen sulfide - Produced
Indole - Negative
-12-
- :
--` li3Z9~2
Starch - Not dccomposed
Reduction o~ nitrate - Rcduced
Catalasc test - ~ositive
Urease - Negative
V.P. test - Negative
Utilization of sugars - Glucose, fructose, arabinose,
mannose, sucrose, xylose, mannitol, sorbitol.
-13-
-- ~132922
.~
All o~ the abovc org~nisms havc bccn dcpositcd with
Fermentation Research Institute, ~cJency of Industrial Sciencc
and Technology, Japan and with ~merican Type Culture Collection.
Corynebacterium alkanum ATCC 21194 FERM P 4598
S Corynebacterium hydrocarboclastas ATCC 15108 FERM P 4599
Nocardia butanica ATCC 21197 FERM P 4602
Nocardia corallina ATCC 31338 FERM P 4094
Nocardia paraffinica ATCC 21198 FERM P 4603
Brevibacterium butanicum ATCC 21196 FERM P 4600
Brevibacterium ketoglutamicum ATCC 15587 FERM P 4601
In this invention, microorganisms for use are
immobilized in advance so that they are useful in an
immobilized form.
For immobilization of microorganisms, any of known
immobilization techniques, for example, physical adsorption,
ionic binding, crosslinking,or entrapment can be adopted.
Materials for immobilizing cells can be anything that retains
cells by locking-up substantially in a specific space without
spoiling the abillty of the microorganism to produce epoxide.
In the preferred embodiment of this invention acrylamides
are used as monomers, as for example acrylamide,
methacrylamide, and mixtures thereof are preferred. The
microorganism is cultured in advance and is diluted with buffer
to make cell suspension. Immobilization of cells is conducted
by polymerizing the desired monomers in the presence of the
prepared suspension.
-14-
~.... . . . .
. , ~ ~ .
~ 3Z922
,,
Immobilized cells cmployed in this invention can be in any
form, ~or example, membranous, plate-like, tube-like, granular,
or rod-like.
The immobilized cells as described above are allowed to
react by contact with a hydrocarbon having a terminal double
bond in an aqueous medium containing the usual inorganic sal~s
necessary to the conversion. For example, a reaction vessel in
which the medium and the immobilized cells have been placed is
evacuated and a hydrocarbon having a terminal double bond as a
5ubstrate is added. If the hydrocarbon introduced to the
vessel is a gas, the partial pressure of the hydrocarbon is
maintained under a specific condition. The preferred
temperature for the reaction is around 30~C., the optimum one
for the growth of the microorganism. The length of the
reaction time, 24 to 72 hours, is also the optimum one for
microorganism growth. pH is also adjusted to the optimum
value for the microorganism to be used. When a column is
employed as the reaction vessel for the reaction, in order to
allow the liquid to pass through the column, either
continuous reaction or batchwise reaction is possible.
In this invention, reaction products are easily separated
by the ordinary liquid-solid separation, and the immobilized
cells can be used repeatedly.
When the immobilized cells are used repeatedly, the
epoxide-producing activity of said cells drops as the
repetition is made many times though the activity sometimes
rises at the early stage of the repetition. If the activity
-15-
:, ~ , .: .
` ~ 113Z92Z
,
of the cells becomcs deplc~cd thc rcpeatedly-uscd cells can be
cultivated in a medium containing appropriate nutrients
necessary for the growth of the microorganism to replenish the
activity of the organisms.
It is possible to maintain epoxide-producing activity in
the immobilized cells by adding to the medium the appropriate
nitrogen source necessary for the growth of the microorganisms
prior to a reaction. It is also possible to increase the
epoxide-producing activity of immobilized cells by culturing
the microorganism in advance in a medium containing nutrients
for the growth of said microorganism.
By the method of this invention the following are
illustrative of the alkenes that can be converted to the
appropriately named epoxides.
ethylene - ethylene oxide
propylene - propylene oxide
l-butene - l-epoxybutane
1,3-butadiene - 1,3-diepoxybutane
3,3-dimethyl-1-butene - 3,3-dimethyl-1-epoxybutane
l,9-decadiene - 1,9-diepoxydecane
3,4-dimethyl-4-ethyl-1-hexene - 3,4-dimethyl-4-ethyl
l-epoxyhexene
l-dodecane - l-epoxydodecane
1,15-hexadecadiene - 1,15-diepoxyhexadecane
and the like.
Thus, it can be seen that a wide range of assimilable
carbon sources can be used as a substrate for the immobilized
microorganism as provided by this invention. Preferrable
-16-
~3Z922
..,
sources, howevor, includc the -olcfins having 2 to 20
carbons and the ~ dienes having from 4 to 20 carbon atoms.
Additional carbon sources may be added with the olefins
as components of a medium as separation of the components
which is difficult to do by usual methods can be carried out
easily when a-olefins, a,~-dienes or a mixture of these are
converted to epoxides. Additionally, separation of paraffins
and a-olefins can be accomplished by reaction with the
microorganisms as shown in this invention.
In a medium containing said carbon sources, nitrogen j
sources and inorganic salts are added, and then the above
mentioned immobilized microorganism is cultivated therewith
by agitation or shaking under an aerobic condition.
The substances added into the madium as nitrogen source
can be anything that the microorganism is able to assimilate,
for example, ammonium phosphate, ammonium chloride, ammonium
sulfate, ammonium nitrate, urea, aqueous ammonia and/or
various kinds of amino acids. One kind out of these will-
suffice but a combination of more than two will also do.
As inorganic salts, potassium phosphate, sodium phosphate,
magnesium sulfate, manganese sulfate, ferrous sulfate and/or
calcium chloride are used.
~utrients such as vitamins and yeast extract may be
added to said medium in order to stimulate the growth of the
microorganism.
292~
The present invention will now be describcd specifically
with reference to the preferred cmbodiment. The following
examples, when taken together with the drawings which are ex-
plained therewithin illustrate but several modes of theinvention. They are not to be considered to demonstrate
limitations or critical parameters of the process which is the
invention.
EXAMPLE 1
(1) Preparation of cell suspension.
One hundred ml. of a medium consisting of a mixture of
10g. of beef extract powder, 10g. of bacteriological peptone,
5g. of NaCl, and 10q. of glucose in sufficient deionized water
to make the whole volume 1000ml., was placed in a 500ml.
Erlenmeyer flask and sterilized thermally. Three loopsful of
Nocardia corallina, ATCC 31338 was inoculated therein and the
whole was cultured at 30C. for 16 hours by shaking. The
produced cells were washed with 0.01m. phosphate buffer twice
and then diluted with 0.01m. phosphate buffer to make 40mg.~ml.
(ary weight) of ce~l suspension.
(2) Preparation of immobilized cells.
Ingredient Concn. Amt.
Acrylamide and N,N'-methylenebisacrylamide 20~ in 0.01m. 2ml-
phosphate
buffer soln.
25 N,N,N',N'-tetramethylethylenediamide 2% in 0.01m. 50~1.
phosphate
buffer soln.
Ammonium persulfate 8% in 0.01m.
phosphate
buffer soln. 50~1.
-18-
2g22
Ccll suspension prcpared as describcd
above 1.9ml.
The solutions and suspensions described above were mixed
in the amounts shown in a test tube after which they were
degassed under reduced pressure for 5 minutes. Polymerization
was carried out at 0C. for 30 minutes. The obtained polymer
was cut into a cube having 3mm. edges. The polymer cube was
washed twice with O.OM. phosphate buffer to prepare the
immobilized cells (19mg. dry cells/l ml of immobilized cells)
for reaction.
Production of propylene oxide
The immobilized cells prepared as mentioned above are
allowed to react on propylene by a process to be described later
using Medium A and Medium B as shown below:
Medium A
K2HP04 1.74g.
MgS04.7H20 1.50g.
FeS04.7H20 50mg.
Deionized water 1 liter
pH 8.0 pH is adjusted with
` 2N H2S4 solution
Medium B
KH2PO 13.60g.
MgS04.7H20 1.50g.
FeS04.7H20 50mg.
Deionized water 1 liter
25 ph 6.0 - 8.0 pH is adjusted with 2N KOH
solution
~i3æ922
-- Method (a)
Into a series of 500ml. Erlenmeyer flasks was placed
20ml, of Medium A, and 4ml. of immobilized cells which had been
prepared similarly to the above mentioned process using as the
monomer solutions 20% solutions varying in amounts of acryl-
amide and N,N'-methylenebisacrylamide(from 95 to 75 weight
percent of acrylamide and from S to 25 weight percent of
N,N'-methylenebisacrylamid~. Each flask was degassed under the
reduced pressure of 510mm. Hg. and filled with propylene gas to
a partial pressure of 150 mm. Hg. The medium was allowed to
shake reciprocally at 150 times per minute at 30C. After 72
hours, the medium was separated by decantation. Gas
chromatographic analysis of the concentration of propylene oxide
as compared to the concentration of N,N'-methylenebisacrylamide
is shown by the open circles O in Figure 1. The immobilized
cells used for the reaction were then washed with O.OlM.
phosphate buffer and placed in a clean series of Erlenmeyer
flasks to which 20 ml. of Medium A and propylene gas had been
added as above to make a partial pressure of propylene gas of
150 mm. Hg. The reaction was again allowed to proceed at 30C.
by reciprocal shaking at 150 times/m, and the medium was
separated by decantation. Concentration of propylene oxide as
shown by gas chromatographic analysis of the products as
plotted against the concentration of N,N'-methylenebisacryl-
amide is shown by the diamonds O in Figure 1. A review of thedata indicates there is no effect on the concentration of
propylene oxide by varying the concentration of
N,N'-methylenebisacrylamide over the range shown in manufacture
-20-
.
: .
1l3292z
of the immobilized enzymc.
Method (b)
To a serics o~ 500ml. Erlenmeyer flas~s 20ml. of
Medium B of varying pH, 4ml. of immobilized cells which had
been prepared by the method described above using 20 percent
solution of 85 weight percent of acrylamide and 15 weight
percent of N,N-methylenebisacrylamide. Propylene gas was
added to a partial pressure of 150mm. Hg. The specific pH of
~edium B was 6.0, 6.5, 7.0, 7.5 and 8.0 giving a resultant
broth of pH of 6.0, 6.5, 6.9, 7.3 and 7.5, respectively. The
reaction was allowed to occur at 30C. by reciprocal shaking
at 150 times/m. for 72 hours to produce epoxide in the
solution. The medium was separated by decantation. Gas
chromatographic analysis of the product as plotted against pH
of the Medium B is shown by the open circles O of Figure 2.
The immobilized cells were then washed with O.OlM. phosphate
buffer and again placed in a series of 500ml. Erlenmeyer
flasks to which 20ml. of the same concentration and pH of
Medium B were added. After a second reaction se~uence of 72
hours, the medium~was decanted and the concentration of
produced epoxide was determined by gas chromatography. The
concentration of propylene oxide as plotted against the pH is
shown in Fiqure B by the diamonds 0 . Again the immobilized
cells were washed with phosphate buffer and a reaction was
carried out for a third time using the same solutions,
concentrations and shaking time. The concentration of epoxide
produced from this reaction sequence was analyzed and is shown
-21-
329zz
in Fi~ure 2 by the txiangles
Method (c)
A series of 4ml. blocks of immobilized cells was prepared
from 5, 10, 15, and 20 percent aqueous phosphate buffer
solutions of a mixture of 85 weight percent acrylamide and 15
weight percent N,N'-methylenebisacrylamide by the method of
preparing immobilized cells described above (all other
ingredients being as described in the general method).
Using these immobilized cells in 20ml. of Medium A and a
partial pressure of propylene gas of 150 mm Hg. a series of
three sequential reactions was carried out. As above, the
products were analyzed after each test and the polymer block
was washed with O.OlM. phosphate buffer.
The results of the first series as plotted against
monomer concentration is shown in Figure 3 as circles O . The
results of the second series is plotted as diamonds O ; and the
results of the third series is plotted as triangles~ .
Method (d)
A series of 4ml. blocks of immobilized cells was prepared -
containing varying àmounts of cell concentrations from 2.Omg.
cells/ml. to 28.5mg. cells/ml. by the general method (all other
ingredients being as described in the general method).
Using these immobilized cells in 20ml. of Medium A and a
partial pressure of propylene gas of 150 mg. Hg. a single series
Of reactions was carried out allowing the reaction to progress
for 10 days sampling each reaction vessel once each day at the
same time. The concentration of propylene oxide was determined
-22-
" ., , . . , "
3292Z
by gas chromato(~raphy and is plottcd a~ainst timc as Figure 4.
In Figure 4 the legend is as follows:
In g.
Concentration of cells/ml. of
Symbolimmobilized cells
28.5
19
~ 14.3
X 9.5
~ 4.8
~ 2.0
EXAMPLE 2
Immobilized cells prepared by the general method of
Example 1 were contacted in 20ml. of Medium A with a partial
pressure of 150 mm. Hg. of l-butene. The mixture was shaken at
150 times/m. and at a temperature of 30C. for twenty-four
hours. The cell mass was separated, washed with O.OlM.
phosphate buffer and transferred to a new 20ml. volume of
Medium A with a partial pressure of l-butene. This process was
repeated for 7 days. Epoxide content of each broth was
determined by gas chromatography and are shown in Table 2.
Table 2
Reaction Epoxide content of broth
g./L.
1 0.23
2 0.41
3 0.30
4 0.23
0.14
6 0.10
7 0.07
-23-
~3'~92Z
,
~ xam~lc 3
(a) The procedure o~ Exam~le 2 was repeated using propylene
until the 24-hour conversion attained 0.145g./L. conversion (10
passes). The concentration of propylene oxide after each pass
is shown in Figure 2 as represented by the circles O . Upon
the eleventh pass 0,5g. of urea was added to the Medium A and
a single pass of 24 hours duration was performed. The
immobilized cells were washed with O.OlM. phosphate buffer and
again reacted on an 8-day, 24-hour sequence. The concentration
of propylene oxide after each 24-hour pass was determined and
is shown in Figure 5 by the triangles~ .
(b) The procedure of Example 2 was repeated using propylene
until the 24-hour conversion attained 0.145g./L. conversion
(10 passes). Upon the eleventh pass the growth medium of
Example 1(1) was used for a 24-hour pass. The immobilized cells
were washed with O.OlH. phosphate buffer and again reacted for
8 days on a 24-hour sequence. The concentration of propylene
oxide in the broth after each 24-hour pass was determined by
gas chromatography and is shown in Figure 5 as the squares O .
' Example 4
The samples of immobilized cells (19.0 mg./ml. of
immobilized cells) were prepared by the method of Example 1(1)
and (2).
Sample 1 was a control.
Sample 2 was cultured in Medium A containing 0.5g/L. of
urea for 24 hours and washed with O.OlM. phosphate buffer.
Sample 3 was cultured in Medium A containing 0.5g./L. of
-24-
'i3;Z92Z
urea for 48 hours and washed with O.OlM. phosphate buf~er.
~ach of the three samplcs was used to convert propylene to
propylene oxide for seven sequential 24-hour cycles as
described in Example 2. The propylene oxide concentration in
the broth after each 24-hour cycle was determined by gas
chromatography. The results are shown in Figure 6. In the
Fi~ure the circles O represent the control values, the
triangles~ represent the Sample 2 values, and the diamonds O
represent the Sample 3 values.
Example 5
Immobilized cells of Corynebacterium hydrocarboclastus
(ATCC 15108), corynebacterium alkanum (ATCC 21194),
Brevibacterium butanicum (ATCC 21196), Brevibacterium
ketoglutamicum (ATCC 15587), Nocardia butanica (ATCC 21197) and
Nocardia paraffinica (ATCC 21198) were prepared (19mg./lml.
immobilized cells) by the method described in Example 1(1) and
(2) and were cultured in Medium A at 30C. for 72 hours in the
presence of 150 mm. Hg. partial pressure of propylene. The
resultant broth was gas-chromatographically analyzed to measure
the produced propylene oxide. The results are shown in Table 3.
-Table 3
Propylene oxide in
Cells broth(mg./L.)
Corynebacterium hydrocarboclastus 3.6
Corynebacterium alkanum Trace
25 Brevibacterium butanicum 3.7
Brevibacterium ketoglutamicum 5.7
Nocardia butanica 115
~ocardia paraffinica 106
-25-
