Method of producing amide

专利摘要:
A process for the production of amides utilizing microorganisms is described, which comprises subjecting nitriles having from 2 to 6 carbon atoms to the action of a microorganism belonging to the genus Rhodococcus, the genus Arthrobacter or the genus Microbacterium having an ability to hydrate the nitriles to form the corresponding amides in an aqueous medium.
公开号:SU1512488A3
申请号:SU864011809
申请日:1986-01-07
公开日:1989-09-30
发明作者:Ватанабе Итиро；Окумура Масами
申请人:Нитто Кемикал Индастриз Ко., Лтд (Фирма)；
IPC主号:

专利说明:

one
(21) 4011809 / 30-13
(22) 07.01.86
(31) 452/85
(32) 01/08/83
(33) JP
(46) 30.09.89.BSN. (36
(73) Nitto Chemical Industries Co.,
Co., Ltd. (JP)
75) Ichiro Watanabe and Pazami Okumura
(JP)
(53) 631.841 (088.8)
(56) French Application No. 2421212,
cl. C 12 D 13/06, 1979,
(54) METHOD FOR OBTAINING ANIDL (57) The invention relates to methods for producing amides with the aid of microorganisms, more specifically to methods for hydrating nitriles under the action of microorganisms, resulting in the corresponding amides. The purpose of the invention is to increase the yield of the target product. This nitrile containing 2-6 carbon atoms is exposed to the microorganism RhodocQCcus sp. S-6 FERM-BP No. 687 - with the ability to hydrate nitriles with the formation of the corresponding amides in an aqueous medium, 1 tab.
G
about
The invention relates to methods for producing amides using microorphisms, in particular to methods of hydrating nitriles under the action of microorganisms, with the result that the corresponding amides are obtained,
The purpose of the invention is to increase the yield of the target product.
Rhodococcus sp. S-6 has a particularly high nitrilase activity, it is deposited at the Fermentation Research Institute of the Industrial Science and Technology Agency of the Ministry of International Trade and Industry, Japan, under the number FERM-BP No. 687.
Morphologists, Small sticks 0.5–0.8 microns in diameter and 1–5 microns in length. At the initial stage of cultivation, the cells have an elongated rod-shaped form, irregular branching is observed. Then they break up and split into spherical or short rod-shaped forms (pleomorphism). .- Mobility is not observed.
No spore formation was observed.
Gram staining is positive (+).
Acid resistance is negative (-).
Growth pattern on various culture media (30 ° С).
Culture on broth plates on agar: colonies are circular, irregular, the surface is smooth with a slight pink tinge.
Culture on oblique agar: good growth, trapezoidal cross-section, without gloss slightly pinkish.
sl hd
4i
00 CX)

O1
Liquid broth culture: intensive growth during pellicle formation, transparent liquid, sediment as it grows.
Physiological characteristics.
Nitrate reduction: positive (+).
Urea decomposition: positive (+).
Indole production: negative (Starch hydrolysis: negative (-).
Decomposition of gelatin: negative (-).
Decomposition of cellulose: negative (-). Oxidase: negative (-).
Catalase: positive (+).
The need for free oxygen is positive (+).
Growth under anaerobic conditions: negative (-).
0 / F test: 0.
Growth at 37 ° C
The need for (-).
Gas production from glucose: negative (-).
positive (+). vitamins: Negative Production of acid from glucose: 30 0.01 to 10 wt.% of cells and from about
positive (+).
The chemical composition of cells.
Contains the mesa of o-diaminopimelic acid, arabinose and galactose.
0.1 to 10% by weight of the nitrile compound, at a temperature from its freezing point to 30 ° C, preferably 7-9, for 0.5 to 10 hours.
Contains fatty acids (n, 1), 35, Nitrile compounds used
C, (F,)
18
AND
C ,, (10-СНз) in
As a substrate, they are biologically very toxic and have a detrimental effect on the enzyme reaction. Therefore, the nitrile compound is added gradually, controlling that the concentration of nitriles in the system preferably does not exceed 2 wt.%.
40
45
new fatty acids.
Contains C ,, -C g-microfoil acids as a type of mycolic acids.
Strain S-6 is defined as Bacillus, which is Gram-positive, negative in sporulation, aerobic, polymorpholysis, and negative acid resistance.
This strain includes mesodiaminopimelic acid, arabinose and galactose, (n, F), C, g (F) and) (0-CU-) in the form of fatty acids and С „, - С ,,, - mycolic acid in the form of myco3Z Po
lovoy acid.
Based on the bacteriol; the genetic characteristics of the present strain; identifies as a bacterium belonging to the genus Rhodococcus.
When cultivating 1 microorganisms using the proposed method, the usual culture medium containing a carbon source is used (for example,.
55
glucose y, glycerin and maltose), a source of nitrogen (for example, ammonium sulfate and ammonium chloride) and sources of organic
nutrients (e.g. yeast extract, peptone, meat extract, soy protein hydrolyzate and liquid after soaking maize (CSZ), source of inorganic nutrient
substances (for example, phosphate, magnesium,
potassium, zinc, iron and manganese), etc. Such cultivation is carried out under aerobic conditions with stirring at a pH of 6-8 and 20-35 ° C, preferably 25-30 ° C, for 1-3 days.
In practice, during the implementation of the method, one of the strains selected from these microorganisms is cultured for 2-3 days by this method, and the resulting cultures or cells are isolated from the culture or treated cells (crude enzymes, immobilized cells, etc.) are suspended in water, buffer or
physiological saline, and then a nitrile compound is added. The nitrile compound acts on the cells when interacting with an aqueous medium, usually containing from about
0.1 to 10% by weight of the nitrile compound, at a temperature from its freezing point to 30 ° C, preferably 7-9, for 0.5 to 10 hours.
Nitrile compounds used
Nitrile compounds used

as a substrate, are biologically very toxic and have a deleterious effect on the enzyme reaction. Therefore, the nitrile compound is added gradually, controlling that the concentration of nitriles in the system preferably does not exceed 2 wt.%.
If the rP is outside the specified range of values, the resulting amusement is further hydrolyzed and the stability of the cells decreases. Therefore, it is preferable to control the pH value in the range of 7-8 by gradually adding a caustic (for example, NaOH and KOH) or by preferentially adding a buffer to the system.
If the reaction conditions are controlled appropriately, the target amide can be obtained and isolated from the nitrile compound with a conversion of approximately 100% and with almost no formation of by-products.
515
The resulting amide can be isolated from the reaction mixture by conventional methods, for example, cells are isolated from the reaction mixture using a centrifuge.

g, treatment with activated carbon or ion exchange resin, etc., and then concentrated under reduced pressure to obtain the desired amide, such as acrylamide.
Various nitriles and their corresponding amides were quantitatively analyzed by gas chromatography, and the corresponding organic acids by means of high-performance liquid chromatography.
Example 1. The strain Rhodococcus sp. S-6 is cultivated under aerobic conditions on a medium (pH 7.2) containing weight,%: glucose 1, peptone 0.5J yeast extract- 0.3-, meat extract 0.3, at 30 ° С within 48 hours. The cells thus formed are isolated using a centrifuge and washed with 0.05 M phosphate buffer (pI 7.7). This procedure is repeated until the washed cells of strain S-6 are obtained (water content is 80%).
Prepare a mixture of 0.5 weight.h. washed cells and 84.5 weight.h. 0.05 M phosphate buffer (pH 8.5), and then
15 weight.h. acrylonitrile is added by
about
while stirring at 0–3 ° C, control the conditions so that the concentration of acrylonitrile in the reaction system does not exceed 2%, i.e. not subjecting acrylonitrile to hydration reaction. The addition of acrylonitrile is completed in about 3 hours. To ensure completion of the reaction, stirring is continued for several more hours. The cells are then isolated using a centrifuge until a clear solution is obtained. This
the solution contains 20% acrylamide., the yield of acrylamide exceeds 99.9%. Unreacted acrylonitrile is not at all
detected, and the content of acrylic acid by-products does not exceed 0.1% (based on the weight of acrylamide).
Water is distilled from a clear solution at a temperature of not more than 50 CJ. The clear solution is concentrated to precipitate crystals. These crystals are recrystallized from methanol to give colorless plate-like crystals. This compound is defined as acrylamide based on
35
45
50
55
25 30 40
elemental
melting point, analysis and IR data.
Example 2. Washed cells of strain S-6 are prepared in the same way as in Example 1, and their reactivity with respect to different nitriles is determined in the following conditions.
Reaction conditions:
Nitrile compound5
0
five
five
0
five
performance
2.5 0.05
Potassium phosphate buffer (pH 7.7), M Cells (as uhrh cells), mg 5 Temperature, with 10 Reaction time, min 10 Number of reaction solution, ml 10 The results of the reaction are shown in the table.
PRI and MER 3. 100 ml of culture medium containing wt.%: Glycerin 1; . 0.1i 0.05, 5 FeSO -7H20 0.001j soy protein protein hydrolyzate 0.5 yeast extract (pH 7.5) 0.1, which is sterilized and to which 0.5% sterile isobutyronitrile is added, is prepared in a Erlenmeyer bottle capacity of 500 ml. Then, 1 ml culture of the culture strain type is added, which is cultured for 48 hours in the same culture medium as in Example 1, and the culture is carried out with vibration for 48 hours. After completion of the culture, the cells are isolated using a centrifuge then washed with 0.05 And 0 phosphate buffer (pH 7.7). This procedure is repeated until the washed cells are obtained. .The activity of these cells is determined by the formation of acrylamide from acry o-nitrile in the same way as in Example 2. The results obtained are:
Strain type
Rhodococcus crythro-polis IFM 155 Rhodococcus rhodoch- rous IFM 153 Arthrobacter oxydans
IFO 12138
Arthrobacter aures- cens lAl I 12340 Microbacteriura-. flavum lAM 1642
Activity in the formation of acrylamide, μI / mg h
2.5 5.0 2., 0 2.0
7 15124888

权利要求:
Claims (1)
[1]
Formula invented and. due to the fact that, in order to increase
Biotrans yield of the target product. A method of obtaining an amide by bioforming is subjected to nitrile with 2-6 transformations of nitrile with a bacterial strain with carbon atoms, and Rhodococcine pH and temperature are used as bacteria in a buffer solution otlichayu- sp. S-6 FERM-BP No. 687.
Relative value in relation to acrylonitrile as 100

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法律状态:

优先权:

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申请号 | 申请日 | 专利标题

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JP60000452A|JPH044873B2|1985-01-08|1985-01-08|

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