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Патент USA US3044951

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3,044,942
1
United States Patent 0 ‘ICC
1
Patented'July 17, 1962v ,
2
much too low for this process to be of any value.
3,044,942
"
PROCESS FOR PREPARING POLY-,B-HYDROX‘I
.
BUTYRKC ACID
In ,
order to increase product yields, a second method was
devised whereby the dried bacterial cells are ?rst digested
in sodium hypochlorite and'the resulting insoluble residue,
James Noel Baptist, Laurel, Md., assignor to‘W. R. Grace 5
crude poly-,G-hydroxybutyric acid, is extracted with chloro
& (30., New York, N.Y., a corporation of ‘Connecticut
form. The product obtained by this second method is so
No Drawing. Filed Sept. 27, 1960, Ser. No. 58,614
21 Claims.
(Cl. 195-47)
degraded that, it is useless as a plastic.
.
‘
It is, therefore, an,_object of this invention to provide
Thisinvention relates to the synthesis of poly-[3:hydrox
an improved process for producing greater product yields
ybutyrlc acid, a polymer consisting of repeating units of 10 of poly-,B-hydroxybutyric acid without signi?cantly de
the formula [—CH(CH3)CH2C(O)O—]n. In one par
»ticular aspect it is a method for obtaining greater yields
of poly-?-‘hydroxybutyric acid without signi?cantly degrad
ing the polymer. In another particular aspect it is a
grading the polymer than methods heretofore employed. '
It is a further object of this invention to produce a
polymer with valuable properties as a plastic.
It has been found that yields of poly-?-hydroxybutyric
method for obtaining a polymer with valuable properties 15 acid are greatly increased by extracting the polyester with
7 as a plastic.
a solution of a solvent and a mild hydrolyzing agent. -
In summary, the invention in its broadest aspect is di
rected to extracting poly-B-hydroxybutyric acid from bac-‘
vteria containing the same, by means of a solvent contain
?ciently broken up by dispersing them in acetone.
To carry out this improved process, large quantities .of
It has also been foundthat the bacteria cells are suf
20 appropriate bacteria are grown in a suitable nutrient me
ing a hydrolyzing agent.
In my copending application, Serial No. 58,154 ?led
dium. .The bacteria are collected by known means, e.g., .
Sept. 26, 1960, of which this application is a continuation
centrifugation, and the mass of wet cells is dispersed in'
in-part, there is described a method for isolating poly-,8
acetone. A cellszacetone weight ratio of 1:1 to 10 is
hydroxybutyric acid by extraction with pyridine. I have
now found that a mixture of methylene chloride and 25
In addition to breaking up the cell walls, the acetone
suitable.‘
ethanol will not only extract about as much polyester as
pyridine, but it will also give a product with considerably
higher viscosity.
.
The poly-B-hydroxybutyric acid produced by this novel
'
"
’
'
'
i
removes water and dissolves lipids and other materials '
which ‘would otherwise contaminate the product. The use
of acetone at this stage makes the polyester readily ex»
tractable.
'
method is a translucent, plastic-like material which can 30 The acetone is removed and the bacterial residue is
be utilized in several ways. It is easily cast into a ?lm
easily dried to a powder. The powder is treated with a
or molded into articles by conventional methods. It is
poly-li-hydroxybutyric solvent containing a hydrolyzing
also useful for surface coatings and as a ?ber.
agent. Methylene chloride asa solvent with ethanol as
It is especially useful in the ?eld of medicine. Medical
a hydrolyzing agent is preferred. \ Suitably, a methylene
sutures made of poly-?-hydroxybutyric acid need not be 35 jchloridezethanol weight ratio of about 5:1 and a bacteria
removed since they eventually decompose to naturally
residuenn'ethylene chloride/ ethanol solution weight ratio
occurring substances without harm to the patient. Films
of about 1:10 to 100 is used. The bacterial residue pow‘.
of poly-p-hydroxybutyric acid can be used to support in
der can be treated with the methylene chloride/ethanol
jured arteries and blood vessels‘until the tissues heal.
solution at re?ux temperature or below.
40
Poly-?-hydroxybutyric acid can be synthesized by vari
The polyester solution is ?ltered and the polyester can
ous bacteria under suitable conditions. The choice of
be recovered from the ?ltrate by various methods. The
bacteria depends not only on one capable of forming this
polyester can be recovered by evaporating the solution.
polyester, but'also on one which can effect the desired
Suitable precipitants such as petroleum ether and petro
biochemical change within a relatively short time and can
leum hydrocarbons in general can be used. A means
produce the highest yield with a minimum of attention.
that is particularly suitable for precipitating the polyester,
The families where this polyester-is known to be a
and which is a preferred embodiment of this invention, is major constituent include Athiorhodaceae, Pseudomona
to add ether to the'methylene chloride/ ethanol solution.
daceae, Spirillaceae, Rhizobiaceae, Bacillaceae and Azo
tobacteriaceae. Rhodospirillum rubrum of the Athio 50 An etherzmethylene chloride/ethanol ratio of about 3:1
is suitable.
'
rhodaceae family and Bacillus megaterium of the Bacil
Other methods of recovering the polyester include add
laceae family are notable examples. Cultures of bacteria
ing water, alcohol and nonpolar solvents such as benzene
used in established processes are usually available from
to the solvent/hydrolyzing agent solution. Generally,
scienti?c culture collections such as the >A.T.C.C. (Amer
ican Type Culture Collection, Georgetown University,
School of Medicine, Washington, D.C.), the collections of
various universities and the U.S. Department of Agricul
ture.
liquids miscible in the polyester solvent will precipitate
the polyester.
This invention can be varied without departing from
its scope.‘ For example, means other than acetone can
The individual bacteria cells are physiologically inde
pendent. A thin membrane determines the outside bound
be used to dry and to break up the bacterial cell mass
sufficiently for extraction of the polyester. This can be
ary of the cell and constitutes the cell wall. Within the
cell is a colloidal solution known as cytoplasm. Some of
done by placing the bacterial cell mass under a vacuum
or in an oven at about 105° C. in the absence of oxygen.
Mixing the cell mass with an antioxidant and placing it .
the granules suspended in the cytoplasm have been found
in an oven at about 105° C. is another method.
to be composed largely of poly-,B-hydroxybutyric acid.
Under appropriate conditions unusually large amounts of 65. Obviously, other solvents suchv as chloroform, 1,4
this polyester can be built up in the cells.
'
There are two processes presented in the literature for
isolating’ poly-?-hydroxybutyric acid from bacteria. In
one process the polyester is extracted from the dried bac
terial cells with chloroform. Although this method gives
a polymer with useful properties as a plastic, the yield'is
dioxane or pyridine can be used as the polymer solvent
7 in place of methylene chloride.
I
Other hydrolyzing agents which can be used include
vother alcohols, carboxylic acids and amines. Any of
‘these compounds when present in small amounts will
break a few of the'polyester bonds and thus reduce the
3,044,942
3
4.
This data illustrates the effect of a hydrolyzing agent.
average molecular weight of the polyester enough to make
it dissolve.
Methylene chloride, alone, extracted only a negligible
The following examples will serve to illustrate the in~
vention:
the methylene chloride/ ethanol solution. The methylene
EXAMPLE I
.
.
.
-
amount of polyester as compared to that extracted by
5 chloride and triethanolamine mixture extracted an un
- -
usually large amount of polyester even though a large
A nutnenémedmm havmg the followmg composition
amount of quaternary ammonium salt was also produced
was Prepare '
under these conditions.
Glucose
—
g
240-0
Mineral solutions1 ------------------ --I111--
500-0
Yeast extract ________________________ __g__
9.0
Water
ml
12000.0
1Mineral solution: 10.0 g. (NH4)2SO1, 10.0
..
~HP-7
iritiilfiolddfo mgimldio
.2.
0 g
. KHgPOl,
.Ms0,o.2.ch12,o.o6.
g
4
g
a
g
EXAMPLE 1v
10
_
,
.
.
- -
A nutrlent medium havlng the followlng composmon
was prepared:
phos hate buffet-1
_P_
1 ——————————————————— --
In}
--
3200
-
Mixed mineral SOllltlOnz _______________ _..ml__ 320.0
c001,
(A,T,c_c_
The A
nutrient
500
#3245)
m1.
medium
from
inoculum
awas
24-hour
autoclaved
of old
Bacillus
culture
and megaterium
allowed
was added
to
Yeast
Water
Sodmmextract
---------------------------------acetate
-----------------------""""""""""""""""""""" "g"
"g1"
"
15'
to the nutrient medium. The medium was continuously
d t
t
ature for about sixt hours with
1 Phosphate buffer: 46-0 g- KH2P04, 86-4 g- NMHPOPTHQO,
2000 {111' H2O‘
'
?rmed
a T001111!
empelf'
_ te. 20 S0 l?ltgxed?lglgueraé
solutlliiii:dl0itgIi
tere air
att e rate
0 ab out 200 to 500 cc.Y per mlnu
ve in
all Ben
m 29 W
=1nltl‘ilt?l‘l?cegii'?
so \1 0n 0
- gacldKgllsi
g
Aeration was vigorous enough to mix the nutrient medi-
l§%.§‘f?3§<)§,?411$?d9$4?§ghg?igh?g? gjoc?i?gdidgrgd
um continuously. At the end of the incubation period,
the cells were harvested by cent?fuga?on and mixed With
tlifltcg rmrtletgl solution. It Add H20 to give 1 1- and adjust to
p » Sthhdal'd trace metal solution: 0.106 g. COCOa, 1.14 g.
200 ml. of acetone. The acetone was ?ltered off and the 25 laiilfllom 5%1Bgé Zngij 5-0 gérl‘s‘gggtgl'ggforsgégalg- (?gsggfézo bacterial residue was added to another 200 ml. of acetone
and allowed to stand for about 15 hours.
The acetone
was then removed and the bacterial residue was air dried
until the odor of acetone disappeared.
The dried residue
Weighed 11.4 g_
EXAMPLE H
_
lhouoiu‘ E120?’
Th
‘
.
g"
1
di
1
P
d
d
n
4’
d
1 e Ring’? H116 . um 1Was an“; aveb _an a 0g‘: to
00.0 '
.
m ' mocu um of
' r” ' um (C'
‘ van
30 Niel strain 1.1.1.) from a seven day old culture was added
to the nutrient medium. Two 150~watt ?oodlights were
.
_
focused on the culture and the temperature was man
one gram 9f dry B- megalerlllm c6113 pl‘odllced 111 the
tained at about 30° C. The medium was continuously
manrler described In EXamPIe I Was added dlfeclly to '3
aerated with a stream composed of 5% N2 and 95% CO2.
solution consisting of 50 ml.‘ of methylene chlorlde and 3_ After Six days the Stream of N2 and CO2 was stopped
10 ml. of ethanol. The solution was re?uxed for 10 mmutes and ?ltered. 2Q‘). ml. of ether was added i0 1116
clear ?ltrate t0 preelpltate the polyester- After three
hours the solution was chilled, ?ltered and the polyester
precipitate was dried in a vacuum dessicator.
The dried
product weighed 0.173 g.
D and the culture was agrated with H2 for six hours. The
PH rose from 7 to 9_ The Hz was stgpped and aeration
with N2 and CO2 was resumed for 18 hours and then
Stopped. Aeration with Hz was resumed and after 6
hours the H2 was stopped_
The cells were harvested
by centrifugation and weighed 37.6 g.
EXAMPLE HI
EXAMPLE V
Samples. of dry B. megaterium produced in the manner
The elfectiveness of various solvents in extracting poly
descnbed 1:1 Example I were placed 1n solvents as listed
B-hydroxybutyric acid is illustrated in the table below.
below. Each sample was ?ltered and 200 m1. ether Was 45 The degree of polymerization of the ?nal product is indi
added to each ?ltrate to precipitate the poly-?-hydroxycated by the intrinsic viscosity. R. rubrum cells obtained
butyric acid. After about ?fteen hours each sample was
in Example IV were used.
Sample
No.
'
R. rubrum cells
Solvent
Time
Temp.
Yield
“alga/tog.
Cells, g.
1 _____ __
Dried (1.7g.1)._.
50ml. Pyridine _________ __
15
R n
__ - .22
._
2 __________ .-<1o ......... -. 50111101101. ___________ __ ZOEiEHHUElOILIiN 3.135%“
1.
3.3%
..
88%
3 __________ __do ......... .- 5oetirlli?olCHzclz-l-l0 ml.
41/2 days___ 30° 0..-- 0.23011“
2.20
0.401
4 ..... -- Wet(7-50g.)---- 50etggio1CHzCh+10 ml.
5% days... 30°C---. 0.115 g--
1.90
0.152
5 .......... -_do _________ _.
50 ml. 0121 or ______________ __d _____ __
s ..... -_ Wet (5.6 g.).._.. 50 1111. 01120132. _____________ "<13 _____ __
e
,
_
Z3€i3nlilgé IIIIIILIII
1 From 5.0 g. of wet cells.
?ltered. Samples A and B were air dried while samples
C, D and B were washed with water several times be
fore being air dried.
Sample
Sample
Weight,
and allowed to stand at room temperature for three hours.
Solvent
Temp,
0.
Time, Yield,
days
mg.
g.
A.-.---.
1
50 ml. C3201: _______ ._
25
2%
2.7
B
1
40 ml. CHzCh-l-IO ml.
25
2%
1-5
l
50 ml. CHrClz-l-‘l ml.
25
2%
1
50 ml. CHzClz-HO ml.
30
2
86. 4
1
50 m1. CHrCl2+5 ml.
30
2
14
.
EXAMPLE VI
3 g. of wet R. rubrum cells were dispersed in acetone
pyridine.
78
triethanolamine.
ethanol.
triethylamine.
65 The acetone was ?ltered oil and the bacterial mass was
air dried. The dried bacterial mass was dissolved in a
solution of 100 ml. of methylene chloride and 20 ml. of
ethanol. The cells were ?ltered off and the clear ?ltrate
was placed in a ?at dish to dry slowly in the air. After
70 all the solution had evaporated, a thin translucent ?lm
remained. The ?lm was easily peeled from the dish.
To determine the type of product obtained when the
bacterial mass is initially digested in sodium hypochlorite
solution, 3 g. of wet R. rubrum cells were dispersed in
75 sodium hypochlorite solution and allowed to stand at
3,044,942
5
e
room temperature for three hours. vThe solution was ?l~
tered off and the bacterial mass was air dried. The same
procedure was then followed as described above. After
is added to the re?uxed methylene chloride/ethanol solu
tion in-an ether:methylene chloride/ethanol weight ratio
between about 1:1 and 10:1, whereby poly-?-hydroxy-i
all the solution had evaporated, a greyish-white material
remained which crumbled easily.
butyric acid is precipitated, followed by’ recovering and
'
J ~
drying the said precipitate.
'
12. The - process of recovering poly-?-hydroxybutyric
acid from a bacterial cell mass containing this polyester w
EXAMPLE VII
5 g. of R. rubrum‘ cells produced‘in the manner de
which comprises adding the bacterial cell mass to meth
scribed in Example IV, were mixed with 4 mg. of 2,6
ylene ' chloride/ethanol solution to extract ‘the poly-18
di'tertiary-‘butyl-4-methyl~phenol and dried in an oven at 10 hydroxybutyric acid, separating the methylene chloride/
105 ° C. The dried sample, weighing 1.7 g., was dis
ethanol/polyester solution from the cell residue, and re
persed in a solution of 50 ml. of methylene chloride and
covering the polyester product from solution.
10 ml. of ethanol and allowed to stand at 30° C. After
’ :13. The process of preparing poly-p-hydroxybutyric
four days the solution was ?ltered and 200 ml. of ether
acid which comprises growing appropriate bacteria in a
was added to the clear ?ltrate‘to precipitate the polyester.
culture medium under conditions conductive to the for
After three hours the solution was chilled, ?ltered and
mation of poly-,B-hydroxybutyric acid, adding the bacterial
the polyester precipitate was dried in a vacuum dessicator.
cell mass to methylene chloride/ethanol solution to ex-‘
The dried product weighed 0.21 g.
'
tract the poly-B-hydroxybutyric acid, separating the meth
ylene chloride/ethanol/polyester from the cell residue,
EXAMPLE VIII
20 and recovering the polyester product from solution.
The procedure of Example VII was followed except that
14. The processlof recovering poly-B-hydroxybutyric
the bacterial cell mass was dried in an oven at 105 ° C.
acid from a ‘bacterial cell mass containing this polyester
‘for six hours in the absence of oxygen before dispersing
in methylene chloride/ethanol solution.
which comprises drying the bacterial cell mass, dispersing
‘
it in methylene chloride/ethanol solution to extract the
25
EXAMPLE IX
The procedure of Example VII was followed except‘
that the bacterial cell mass was dried in a vacuum (650
poly-,S-hydroxybutyric acid, separating the methylene
chloride/ethanol/polyester solution from the .cell residue,
and recovering the polyester product from solution.
15. The method according to claim 14 in which the
mm. Hg) for 16 hours before dispersing in methylene
bacterial cell mass is dried by extracting it with acetone.
chloride/ ethanol solution.
30
16. The method according to claim 14 in which the
I claim:
bacterial cell mass is dried in an oven at 105° C. in the
1. In the process of preparing poly-?-hydroxybutyric
absence of oxygen.
acid by the growth of bacteria in a culture medium
17. The method according to claim 14 in which'the
whereby the bacteria acquire deposits of said polyester
bacterial cell mass is mixed with an antioxidant and dried
within their cell walls followed by recovery of the poly 35 in an oven at about 105° C.
ester-bearing bacteria and the solvent extraction of the
‘18. The method according to claim 14 in which the.
polyester from the bacteria, the improvement comprising
bacterial cell mass is dried under a vacuum.
the steps of dispersing the polyester-bearing bacteria in
19. The process of preparing poly-?-hydroxybutyric acid
acetone to break up the bacteria cell walls and to remove
from Rhodospirillum rubrum by inoculating Rhodospiril
lipids and water therefrom, separating the thus treated 40 lum rubrum into a culture medium comprised of 320 1111.,
bacterial residue and dispersing it in a methylene chlo
phosphate buffer composed of 46.0 g. KH2PO4, 86.4 g. .
ride/ethanol solution to cause mild partial hydrolysis of
poly-B-hydroxybutyric acid and to extract it from the
bacterial residue, separating the insoluble bacterial resi
due from the methylene chloride/ethanol solution, and
recovering the poly-B-hydroxybutyric acid from the meth
ylene chloride/ ethanol solution.
2. The method according to claim 1 in which the poly
B-hydroxybutyric acid is‘ recovered from the methylene
chloride/ethanol solution by adding liquids miscible with
methylene chloride thereto to precipitate the polyester.
3. The method according to claim 1 in which the meth
ylene chloridezethanol weight ratio is substantially 1:1
to 20:1.
Na2HPO4-7H2O, 2000 ml. H20; 320- ml. mixed mineral
solution composed of 10 g. nitrilotriacetic >acid7dissolved
in H20 and neutralized with a solution of 7.3 g. KOH
and adding 14.45 g. MgSO4, 2.5 g. CaC2, 9.2 mg.
standard trace metal solution, enough H2O to make 1 l.
' adjusting pH 6.5 to 7.0, the standard trace metal solu
tion being composed 0.106 g. CoCO3, 1.14 g. MnCO3, '
5.21 g. ZnCl2, 5.0 g._FeSO4-7H2O, 0.39 g. CuSO4-5H2O;
0.117 g. H3BO3, 2.50 g. versene acid, several drops
H2SO4, 1000.0 ml. H2O; aerating the inoculated culture
medium with a nitrogenzcarbon dioxide mixture under
illumination; stopping the nitrogenz-carbon dioxide ?ow
4. The method according to claim 1 in which the poly 55 and aerating with hydrogen under illumination; harvesting B-hydroxybutyric acid is recovered from the methylene
the bacterial cell mass; dispersing the bacterial cell mass
chloride/ethanol solution by adding ether thereto to pre
in acetone to break up the bacterial cell walls and to
cipitate the polyester.
remove lipids and water therefrom; separating'the thus
5. The method according to claim 1 in which the bac
treated ‘bacterial cell mass and dispersing it in methylene
teria is selected from the group of families consisting of 60 chloride/ ethanol solution to extract poly-,B-hydroxybutyric
Anthiorhodaceae, Psuedornonadaceae, Spirillaceae, Rhizo
acid; separating the polyester solution from the cell resi
biaceae, Bacillaceae and Azotobacteraceae. ,
6. The method according to claim 5 in which the bac
terium is Bacillus megaterium.
7. The method according to claim 5 in which the bac
terium is Rhodospirillum rubrum.
'
due; adding ether to the polyester solution to precipitate
the polyester and recovering the polyester product. ,
20. The process of preparing poly - B - hydroxybutyric
65 acid from Bacillus megaterium. by inoculating Bacillus
megaterium into a culture medium comprised of 240 g.
8. The method according to claim 1 in which the weight
glucose, 9 g. yeast extract, 600 ml. mineral solution and
ratio of bacteriazacetone is substantially 1:1 to 10.
12,000 ml. water, the mineral solution being composed
9. The method according to claim 8 in whichthe weight
of 10 g. (NH4)2SO4, 10 g. KH2PO4, 18.9 g.
ratio of bacterial residuezmethylene chloride/ethanol is 70 Na2HPO4-7H2O, 2 g. MgSO4, 0.2 g. CaCl2, 0.06 g. FeCl3
substantially 1:10 to 100.
and 1000 ml. H2O; continuously aerating the inoculated
10. The method according to claim 9 in which the ex
culture medium during the incubation period with ?ltered
traction with methylene chloride/ ethanol solution is car~
air at about room temperature during the incubation
ried out under re?ux for about 5-30 minutes.
period; harvesting the bacterial cell mass; dispersing the
11. The method according to claim 10 in which ether 75 ‘bacterial cell mass in acetone to break up the bacterial
3,044,942
7
cell walls and to remove lipids and water therefrom; sepa
rating the thus treated bacterial mass and dispersing it in
methylene chloride/ethanol solution to extract poly-1S
hydroxybutyric acid; separating the polyester solution from
the cell residue; adding ether to the polyester solution
5
by drying the bacterial cell mass under nonoxidative con
ditions, dispersing it in a poly-B-hydroxybutyric acid sol
vent/hydrolyzing agent solution to cause mild partial hy
drolysis of the polyester and to dissolve the polyester,
to precipitate the polyester and recovering the polyester
separating the solution of polyester from the cell residue,
and recovering the polyester product from the solvent/
product.
hydrolyzing agent solution.
21. The process of recovering poly-B-hydroxybutyric
acid from a bacterial cell mass containing this polyester
No references cited.
i
UNITED STATES PATENT OFFICE
CERTIFICATF. OF CORRECTION
Patent No; 3,044,942
July 17, 1962
James Noel Baptist
It is hereby certified that error appears in the above numbered pat
ent requiring correction and that the said Letters Patent should read as
corrected below.
Column 6, line 45, for "CaC2"- read —- CaC12 --.>
I
Signed and sealed this 8th day of January 1963,
(. SEAL)
Attest:
‘ERNEST w. SWIDER
Attesting Officer
DAVID L- LADD
‘
Commissioner of Patents
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