Method of producing polymer containing monomeric units o. ch(ch sub three) ch sub two co

专利摘要:
The invention allows for the preparation of a polymer containing —O units. CH (CHD) CH "CO - with reduced crystallinity and brittleness. The process of fermentation of producing poly-oxobutyric acid microorganisms in an aqueous nutrient medium is carried out until the nitrogen and / or phosphorus source is completely assimilated, and then 4-100 wt.% Of the assimilated carbon source is introduced into the fermentation medium, and the cultivation is continued until 20–70 mash accumulation .% polymer produced. Acid is used as an assimilable carbon source: propionic or isobutyric, or acrylic, or 3-oxpropionic, or 3-chloropropionic, or 2-hydroxybutyric or their water-soluble alkali metal salts. Analysis of the obtained polymers isolated from bacterial cells shows a decrease in the melting endotherm zone, which indicates a decrease in the crystallinity and brittleness of the polymer. 2 Cpf. 3 tab. (L od sl 4: oo
公开号:SU1375143A3
申请号:SU813359451
申请日:1981-11-17
公开日:1988-02-15
发明作者:Артур Холмс Пол；Хью Коллинз Стефен；Фредерик Райт Леонард
申请人:Империал Кемикал Индастриз Плс (Фирма)；
IPC主号:

专利说明:


and
. This invention relates to biotechnology and relates to the production of a thermoplastic polyether of a repeating O. structure. CH (CH, g) CH 2 CO - (1), which quickly crystallizes to a relatively high level (on the order of 70% or more) and is a poly-hydroxy methyl acid (PHB) The purpose of the invention is to increase the thermoplasticity of a polymer by lowering its crystal strength and brittle
Tio
The method is as follows.
As microorganisms that produce poly - p-hydroxybutyric acid, are used kulrzture, capable of A. srmilirovani acid or its salt. The preferred strains or mutants of Alcaligenes eutrophus nos. KC IB 11600, NCI B 11599s NCIB 11598, NClB 11597, ATCC 17699 and Nocardia salmonicolor bacterium n. ATCC 19149 and ATCC 21243.
I
Fermentation is carried out in such a way that the dry weight of polyether-containing cells is at least 5 g / l of aqueous medium. To obtain, for example, 10 g / l of PHB-containing cells with a PHB content of 40% by weight,%, the amount of nutrients supplied to the fermenter to limit cell growth is set at the rate of growth of b g / l of cells, that do not contain PHB. For example, if you use nutrient growth as a limiting factor, the nitrogen content in RHB-free bacterial cells should be 8–15 wt.%, And the required amount of assorted p-nitrogen should be About ,, 5-0.9 g / l, for example 0.6 1.2 g of ammonium ions per 1 liter.
Suitable acid substrates should be soluble in water and therefore can be added as such or in the form of a water-soluble alkali metal salt. Preferred are propionic and isobutyric acids hydroxy-substituted derivatives of these acids and butyric acid, such as 3-chloropropionic, 3-hydroxypropionic 5 and also unsaturated acids or their gado-substituted ones, such as acrylic, methacrylic, 2-chloropropionic acid. The acids used should give rise not only to the repeating units (I), when
The growth is carried out in a state of limiting growth.
The polymer is formed as granules inside the cells of the microorganism. Cells containing a polymer can be used as a molding polymer. It is desirable to separate the polymer from the cells by splitting them, followed by extraction with a suitable solvent.
The polymer introduced has a molecular weight of between 100,000 and above 200,000.
With a normal metabolism of the substrate, for example propionate, the latter is converted to succinate, which yes. It is the beginning of acetyl CoA by oxidizing the element of tricarboxylic acid, TCA, the Krebs cycle, into oxaloacetic acid, followed by decarboxylation. When decarboxylated with oxaloacetic acid, both terminal acid groups are removed as carbon monoxide. Consequently, if the propionate having a carbon atom in

a carboxyl group that is radioactively labeled; 5 i.e., 1-C-propnonTd is supplied to the cells, then its conversion to acetyl Co A will take place with a simultaneous loss of radioactivity
1-1
WITH,
Any nonregregtivities in the form of ov., C, C should be the solution of converting propionyl CoA into p. Shchroksivaleryl CoA and subsequent half-sterilization, 1
EXAMPLE 1 Mutant Alcaligenes eutrophus NCIB 11599 is grown under aerobic conditions in a 5 liter periodic fermenter containing the following medium per liter of deionized water;
Glucose
(NH) 2SO
MgSO, -7H
HjPO (, lM)
Feso. 7Н ,, 0 Traces of elements
 g 2.0 g 0.8 g 0.45 g 1 2 ml 1 5 mg 24 ml
The solution of trace elements:
Cuso

0.02 0.1 OJ 2.6
 ZnSO,
MnSO
SAS1.2N ,, 0
When the concentration of cells reaches 4.5 g / l 5 t, e, after the assimilation of the assimilated nitrogen source is exhausted, 1 g / l of sodium propionate containing 1-С-propionate is added to the medium, along with glucose and fermentation.
within 5 min. The cells are separated by filtration and the polymer is extracted with chloroform. Labeled carbon is found exclusively in chloroform solution. This indicates that the labeled carbon end atoms are not lost in the form of carbon dioxide. Consequently, a certain amount of propionate was incorporated into the polymer, different from acetyl CoA,
The content of the polymer in the cells 60% So pl. 122 ° C, Melting Zone 51 JHP,
Example 2. The mutant Alcaligenes eutrophus NCI B 11599 is grown under aeration conditions at pH and 34 ° C in a 5-liter batch fermenter containing 4000 g-w of aqueous medium containing 1 l of deionized water:.
(Shr ,, g
MgSO 7H O 0.8 g
0.45 g
12 ml 1 5 ml
The solution of trace elements by
example 1) 24 ml
Glucose is added at a rate of 8 g / h. The amount of azo assimilated in the medium is sufficient to form 26 g / l of polyhydroxybutyric acid (PHB) -free cells,
After 40 hours, the cells are centrifuged by freezing, dried by freezing, and extracted with chloroform.
The content of the polymer in the cells 70% So pl. 91 ° C. The melting endotherm zone is 127 J-h.
Example 3 The process is carried out as in Example 2, except that when the cell concentration reaches 34 g / l, propionic acid is added to the fermentation medium instead of glucose at a rate of 2 g / h. The amount of polymer in the cells is 70%.
KjSOj, HjPO FeSO .7H O
Example 4: The process is carried out as in Example 3, except that at the time the cells reach a concentration of 28 g / l, iso-oil is added to the fermentation medium instead of glucose at a rate of 2 g / h in the form of a solution containing 150 g Per liter of acid. Fermentation is carried out until the cell concentration reaches 26 g / l of PHB with the addition of glucose.
Polymer content in cells 50%,

-
about

0
five
0
five
PRI me R 5. The process is carried out as in example 4, except that when the cell concentration reaches 30 g / l, 4 g of 3-chloropropionic acid at a concentration of 50 g / l is fed into the fermentation medium and fermentation is continued for 7 hours with a constant addition of glucose at a rate of 6.8 g / h.
The amount of polymer in the cells is 35%. EXAMPLE 6 The method is carried out according to Example 2, except that when reaching a cell concentration of 31 g, acrylic acid is added to the medium at 5 g / h with a concentration of 100 g / l for 5 hours.
Polymer content in cells is 25%. In the polymers obtained, the amount of comonomer units is determined by the hydrolysis and gas chromatography method.
Polymer molecular weights are determined by gel permeation chromatography.
The results are presented in Table L
Example 7. A 5-liter fergmenter ze, forms 3 liters of medium of the following composition per 1 liter of deionized water: (NH), SO, 6.2 g
H, (1, W) 1.5 ml MgSO g. THgO 15 mg A solution of trace elements (as in
example 1). 36 ml of the fermenter is inoculated with the culture of mutant Alcaligenes eutrophus NCIB 11599 kept in a shaking flask for 48 hours and then 5 g / l of glucose is added. Fermentation is carried out aerobically at a controlled pH of 6.8 automatically with the addition of 9; 1 volume / volume of a mixture of 4m KOH and 4M NaOH. The amount of phosphorus is sufficient to maintain 8 g / l RNB cells free from polymer o
When all glucose has been utilized (at this stage, the cell concentration is about 2.5 g / l), propionic acid is added as a solution containing 300 g / l propionic acid at a rate of 0, -8 g / h for 54 h. J
The cells are then harvested, the polymer is extracted with chloroform, and analyzed.
ziruyut. The following results are obtained: final cell concentration of 20 g / l; polymer content 60% by weight.
The polymer contains 60 mol% of beta-hydroxy-butyrate units and 40 mol% of beta-oxyalkylene units (i.e., where R is ethyl).
Example The mutant Alcaligenes eutrophus NC1B 11599 is grown under aerobic cultivation at pH 6.8 and 34 ° C in a 5-liter fermenter containing 4000 ml of aqueous medium having the following composition per one liter of deionized water:
Glucose 17 g
(NH) 80. 4 g
MgSO 7H O 0.8 g
HjPO (1, W) 12 ml
FeSO 15 mg
The solution of trace elements (as
in example 1) 36 ml
The pH is maintained at a level of 6.8 with the automatic addition of 9: 1 v / v mixtures of 4 M potassium hydroxide and 4 M sodium hydroxide. This addition of a KOH / NaOH mixture to adjust the pH also serves to supply the culture medium with potassium and sodium.
The amount of assimilated nitrogen (provided by the use of 4 g / l ammonium sulfate) was sufficient to maintain about 6.5 g / l PHB; polymer free cells.
Since 16 g / l of glucose is required to produce 6.5 g / l of polymer cells, the amount of glucose present is sufficient to provide a slight excess of carbon .. By adjusting the concentration of residual glucose and also traces of dissolved oxygen pressure, an indication of when the system becomes insufficiently assimilated nitrogen. At this stage, the delivery of 3-hydroxypropionic acid in two experiments and 2-hydroxy-oil
acid in the third experiment.
I
After the addition of the acid is complete, the cells are harvested by centrifugation. The sample of the centrifuged cells is freeze dried and the polymer is extracted with chloroform. The polymer is analyzed by means of

0
5 o
,
Q 5
five
five
gas chromatography / mass spectroscopy (GHMS) techniques.
The results are presented in table 2. Examples 9-13. In each example, a 250 ml shake flask was charged with 50 ml of aqueous medium containing 1 liter of deionized water: Glucose 10 g
1.9 g
NaH., 56 g
(NH), 8 g
MgSO 7H O0.2 g
FeCl bNT) 0.001 g
The solution of trace elements (as in Example 1) is 1 ml. The pH of the aqueous medium is 7.0. The flask is inoculated with Nocardia salmonicolor and carried out by spinning under rotating (gyroscopic) shaking for 24 hours. The resulting suspension is then centrifuged. The cell mass obtained by centrifugation is then resuspended in 50 ml of aqueous medium identical (medium above, except that 10 g / l of glucose is replaced with 1 g / l of acid, and 1.8 g / l of sulfate ammonium is not applied. The resuspended cells are shaken for 24 hours at 30 ° C. and then the cell suspension is centrifuged. The centrifuged pellet or cell tablet is washed twice with methanol and analyzed for polymer content.The polymer is analyzed by gas chromatography / mass spectroscopy. Rezult The data are presented in Table 3. Higher polymer contents could be obtained by adding an additional amount of acid after culturing the resuspended cells for 24 hours and continuing the cultivation.
PRI and MER 14. Mutant A. eutrophus ClB 1 1599 is grown by aerobic | cultivation at pH 6.8 and 34 ° C in a 5-liter batch ferment
ture containing from 3500 to 4000 ml of aqueous medium containing per liter of deionized water: Glucose 18 g
(NH) g
MgSO 7H, 0 0.8 g (1.1M) 12 ml FeSO 7H, 0 15 mg
713
Trace solution
elements of the same
that in example 1 36 ml
The pH is adjusted to 6.8 by automatically adding 9: 1 v / v mixtures of 4 M potassium chloride and 4 M sodium hydroxide. This addition of a KOH / NaOH mixture of pH control is used to supply potassium and sodium to the growth medium.
The amount of assimilated nitrogen provides 4 g / l of acmokk sulfate sufficient to maintain a total of about 6.5 g / l of poly-free HB cells.
Since approximately 16 g / l of glucose is required to produce 6.5 g / l of polymer-free cells, the amount of glucose present is sufficient to provide a slight excess of carbon.
When the cell concentration reaches 8.0 g / l for 4.5 hours, 1 g / l of 2-hydroxybutyric acid is added, and then the cells are harvested by centrifugation. A sample of these cells is freeze dried and the polymer is extracted with chloroform. Polymer content in cells 24%. Melting point is 174 ° C, and the zone of melting endo-POJ-g.
Example 15. In this example, the aqueous medium and fermentation conditions, i.e. The pH, temperature are the same as in Example 14, with the exception that the glucose concentration is 16 g / L and a laboratory continuous fermenter is used with a working volume of 2 L (approximately). Continuous cultivation conditions are established by continuously feeding an aqueous medium into the fermenter and continuously removing an appropriate amount of medium from the enzyme, ensuring an average pre-aging time of 12.5 hours (i.e., dilution rate 0.08 h M 4.4 g of hydroxypropionic acid was added in an amount of 4.4 g per liter of aqueous medium.
After reaching a stable state, the content of residual glucose is 0.6 g / l, the residual concentration of assimilated nitrogen is less than I mg / l, and the concentration of cells is about 10 g / l.
The polymer is collected from the medium removed from the fermenter by centrifugation, freeze drying and extrusion.
five
0
5 o
O 5 q
five
five
438
by chloroform. The polymer content in the cells is 33%. M.p. . Zone of melting endotherm 106 J g
EXAMPLE 15 Example 15 was repeated, except that the feed rates were adjusted so that the average residence time was 11.8 hours (i.e., the dilution rate was 0.095 hours) and 3-hydroxypropionic acid was added. acids in the amount of 2.8 g / l.
After reaching a stable state, the residual glucose content is 0.3 g / l, the residual concentration of assimilated nitrogen is less than 1 mg / l, and the concentration of cells is about 9 g / l.
The content of polymer in cells is 28%. Mp 166 ° C, zone of melting i 00 j g
The analysis determines the glass transition temperature of the amorphous phase. Zones of the melting endotherm indicate the relative degree of crystallinity.
The polymers from Examples A, B, and C were analyzed by nuclear magnetic resonance specgroscopy C. The melting behavior of the polymers was determined by differential scanning calorimetry (DSC) on annealed samples obtained by compression molding at 190 ° C.
After annealing, the polymers obtained had a significantly lower degree of crystallinity than the control copolymer from Example 2.

权利要求:
Claims (3)
[1]
1. A method of producing a polymer containing units — 0, CH (CHJCH, CO—, by cultivating a microorganism capable of producing poly-p-hydroxy-butyric acid in an aqueous nutrient medium containing organic acid throughout its entire cultivation period , or its derivative as an assimilable carbon source and assimilable sources of nitrogen and phosphorus, characterized in that, in order to increase thermoplasticity by lowering its crystallinity and fragility, cultivation is carried out until complete assimilation source of nitrogen and / or phosphorus, and then the process of pro- using from 4 to
913751
100 wt.% Of an assimilable source, carbon in the form of glucose or propionic, or isobutyric, —OR acrylic or 3-hydroxypropionic 4 or 3-chloropropionic or 2-hydroxy acid, or their water-soluble alkali metal salts, to accumulation by the producer from 20 to 70 wt.% of the polymer obtained.
[2]
2. Method POP.1, which is different from the fact that the cultivation process after the assimilation of nitrogen
310
and / or phosphorus is carried out when using as a carbon source a mixture of glucose and one of the above-mentioned organic acids or its salt when the content of this acid or salt in the mixture is 4-75% by weight,
[3]
3. The method of pop. 1, which differs from the fact that the cultivation process to complete assimilation of nitrogen and / or phosphorus is carried out using glucose as a carbon source.
h
Table 1
Not
Propionov
Isomasl on
3 Chloropropionic
Acrylic
ABOUT
27
thirty
2 0.6
.6,5
Acid
Z-Oksipro-pionova
2-Oxymasl - on
table 2
ethyl and hydrogen in both cases
ethyl
eleven
19149 19149 21243 21243 21243
Isomasl on
2-Chlorpropionov
Propionov
Isomasl on
2-Chlorpropionov
Note. - beta-c-units;
Z-NU - beta-oxivalerate units.
137514312 Table 3

类似技术:

---

公开号 | 公开日 | 专利标题

---

EP0069497B1|1987-04-15|Copolyesters and process for their production

---

Byrom1987|Polymer synthesis by microorganisms: technology and economics

---

US4477654A|1984-10-16|3-Hydroxybutyrate polymers

---

EP0052459B1|1985-12-04|Beta-hydroxybutyrate polymers

---

US5200332A|1993-04-06|Process for preparation of copolymer

---

JP3684150B2|2005-08-17|Polyhydroxyalkanoate

---

EP0046344B1|1985-06-19|Fermentation process

---

JPH0615604B2|1994-03-02|β-hydroxybutyrate copolymer

---

US4336334A|1982-06-22|Microbiological process for the production of poly|

---

US5036009A|1991-07-30|Biochemical process

---

JP2004535754A|2004-12-02|Preparation of 3-hydroxycarboxylic acids and use in branched polymers

---

CA1313635C|1993-02-16|Copolymer production

---

EP0396289B1|1995-10-25|Copolymer production

---

JPH057492A|1993-01-19|Production of copolymer

---

Sugimoto et al.1999|Control of acetic acid concentration by pH‐stat continuous substrate feeding in heterotrophic culture phase of two‐stage cultivation of Alcaligenes eutrophus for production of P | from CO2, H2, and O2 under non‐explosive conditions

---

EP0015669B1|1983-05-25|Microbiological process for the production of poly | and micro-organisms for use therein

---

Linko et al.1993|Production of poly-β-hydroxybutyrate on lactic acid by Alcaligenes eutrophus H16 in a 3-l bioreactor

---

SU1375143A3|1988-02-15|Method of producing polymer containing monomeric units o. ch| ch sub two co

---

KR100223341B1|1999-10-15|Process for producing copolymer

---

Takeda et al.1995|Biosynthesis of poly-3-hydroxybutyrate by Sphaerotilus natans

---

JP2000072865A|2000-03-07|Polyester and its production

---

US5266470A|1993-11-30|Copolymer production

---

EP0643138B1|2003-03-12|Process for production of bacterial cells containing poly-3-hydroxybutyric acid

---

JP3114148B2|2000-12-04|Method for producing biopolyester

---

RU2051968C1|1996-01-10|METHOD OF PREPARING HETEROPOLYMER OF β-HYDROXYBUTYRIC AND b-HYDROXYVALERIC ACIDS

同族专利:

---

公开号 | 公开日

---

JPH0438763B2|1992-06-25|

---

JPS5869224A|1983-04-25|

---

UA6302A1|1994-12-29|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

DE2948023A1|1979-11-29|1981-06-04|Bayer Ag, 5090 Leverkusen| HYDROXIBUTTER ACID POLYESTER, METHOD FOR THE PRODUCTION THEREOF AND THEIR USE AS LACQUER RAW MATERIALS|

---

DE3168826D1|1980-11-18|1985-03-21|Ici Plc|Polymer blends|JPH0412712B2|1987-04-28|1992-03-05|Yoshiharu Doi|

---

US4876331A|1987-08-18|1989-10-24|Mitsubishi Kasei Corporation|Copolyester and process for producing the same|

---

JPH07113055B2|1987-08-18|1995-12-06|三菱化学株式会社|Polyester copolymer and method for producing the same|

---

JPH0412713B2|1987-09-09|1992-03-05|Mitsubishi Gas Chemical Co|

---

JPH0465425A|1990-07-06|1992-03-02|Showa Denko Kk|Copolymer and its production|

---

GB9314577D0|1993-07-14|1993-08-25|Zeneca Ltd|Adhesion process|

法律状态:

优先权:

---

申请号 | 申请日 | 专利标题

---

GB8120991|1981-07-07|

---