The way to obtain 6-oxycodonebuy acid

 

(57) Abstract:

Usage: microbiological industry. The inventive method of obtaining 6-oxycodonebuy acid of nicotinic acid by enzymatic hydroxylation in the presence of microorganisms of the genera Pseudomonas or Achromobacter, in particular Pscedomonas acidovorans DS M 4746 or Achromobacter xylosoxydans DS M 2783. By complying with certain limits of concentration (from 0 to 10 g/l) for adding nicotinic acid is able to carry out the multiplication of the microorganism and to obtain the target product at one stage of the process without loss of product.

The invention relates to a method for producing 6-oxycodonebuy acid enzymatic hydroxylation of nicotinic acid.

Known methods for producing 6-oxycodonebuy acid using living microorganisms of the genera Pseudomonas, Bacillus or Achromobacter (patent Switzerland N 658866). According to the method of using a suspension of biomass relevant microorganisms that receive separately by multiplication of the starting culture. The hydroxylation process carried out periodically by a single addition of nicotinic acid as sodium salt. Due to the fact that in this way because p is enact a separate preliminary stage to obtain the total number of effective biomass.

The purpose of the invention is the development of a more simple way, which receive a high concentration and output 6-oxycodonebuy acid.

Discovered that it is possible to dispense with separate biomass production, if on the basis of obtained in a known manner starter cultures nicotinic acid be added so that its concentration in the suspension of microorganisms was mainly supported below the concentration above which retards the growth of microorganisms. The expression "mainly" means that the concentration of short-term and/or topically, in particular, in place of adding acid to its mixing with the suspension may exceed the maximum permissible value. In these conditions it was possible to expect formation of biomass, but suddenly it turned out that during this phase of growth is produced 6-oxycotine acid, which does not undergo further metabolisation, so after adding nicotinic acid and after complete consumption of 6-oxycodoneao acid it is possible to select virtually quantitative yield. Increasing with the addition of nicotinic acid concentration of 6-oxycodonebuy acid inhibits the growth of cells. However, the latter olisaemeka strains of microorganisms, these concentrations are about 50 g/l or more. Suddenly, what then 6-oxycotine acid is no longer subject to decay, but the inhibition of hydroxylase, i.e., enzyme, hydroxyperoxide nicotinic acid, and therefore, the limit of attainable concentration of the target product at 100% output only at higher concentrations. Significantly for the proposed method to the concentration of nicotinic acid for growth of the cells was never reduced to zero, because otherwise the decomposition of the formed 6-oxycodonebuy acid. Only when the concentration of 6-oxycodonebuy acid reaches the limit value, inhibited its degradation, so that after the addition of nicotinic acid can wait for its full flow without losses in output. As microorganisms, hydroxyperoxy nicotinic acid, it is advisable to use such genera as Pseudomonas, Bacillus, Achromobacter. Preference is given to the species Pseudomonas pituda and Achromobacter xylosoxydans, in particular the strain Achromobacter xylosoxydans DSM2783. Preferred microorganisms are described in the patent Switzerland N 658866. The concentration of nicotinic acid in the suspension of microorganisms for the entire period of its addition is less than 10 g/is ptx2">

Nicotinic acid can be introduced in solid or dissolved form, in each case in the form of the free acid or water soluble salt. Preference is given to adding the aqueous solution, in particular an aqueous solution of sodium or potassium salt.

It is advisable to aerate and stir the suspension of microorganisms, but can be limited to a mixing action of the blown air. The partial pressure of dissolved oxygen (po2should range 1-200 mbar, preferably 40 to 80 mbar. Preferably using a mechanical stirrer. The process of reproduction of microorganisms and getting 6-oxycodonebuy acid it is advisable to carry out at 20-40aboutC and pH 5.5-9.

Together with nicotinic acid to promote the growth of biomass, it is recommended to add other nutrients. These include carbon sources such as glycerol or glucose, nitrogen sources such as ammonium salts or glutamic acid, and mineral salts, trace elements and vitamins. These substances should be added in such quantities that their concentration in the suspensions of the microorganisms were neither limiting nor in the acts of the process pretalk such or in the form of complex natural or obtained by synthetic mixtures. Particularly preferred complex nutrient is a yeast extract.

The proposed method can be carried out continuously so that upon reaching the concentration of 6-oxycodonebuy acid, high enough to prevent its further decomposition to other products, begin to separate the target product with simultaneous recirculation cells. This can be accomplished, for example, continuous or periodic centrifugation or ultrafiltration.

In order to maintain a constant concentration of the target product feed rate nicotinic acid should be equal to the speed of separation of the product.

P R I m e R 1. a). Obtain starter cultures

From 5,19 g dihydrate sodium hydrogen phosphate, 2.0 g of potassium dihydrophosphate, 0.25 g yeast extract, and 1.00 g of nicotinic acid and 500 ml of water prepared liquid nutrient medium, which for 20 min sterilized at 120aboutC. After cooling medium 30aboutSince it was introduced a sterile concentrated solution of trace elements in number, ensuring the achievement in the medium specified concentration, mg/l: Dihydrate calcium chloride 20 Sulfate Mar is 11) 0.1 Heptahydrate zinc sulfate 0.1 molybdate Dihydrate sodium 0,1

Nutrient medium were seeded strain Achromobacter xylosoxydans DSM 2783. The culture was grown for 24 h at 30aboutC and pH 7.

b). Received 6-oxycodonebuy acid.

In the fermenter with a capacity of 20 l equipped with a stirrer, aeration system and device for regulating the pH value, in 12 l of water was dissolved in 90 g of nicotinic acid, 19,44 g of sodium hydroxide, 90 g of yeast extract, 12 g of potassium sulfate, 9.6 g of hexanitrate magnesium chloride, 1.92 g of calcium chloride, 2.4 ml of polypropylenglycol 2000, 180 g of L-glutamic acid and 300 g of glucose.

The solution was sterilized for 30 min at 121aboutC. After cooling the solution to 30aboutSince it was introduced starter culture and with the air supply and mixing were cultured for 10 h at pH 7. After this period of time the concentration of nicotinic acid decreased from 7.5 to 2 g/l (according to a high-speed liquid chromatography), and the biomass concentration increased to 10 g/l (dry matter). At this point, began to add sterilized for 20 min at 121aboutTo a solution of 1.13 g of nicotinic acid and 0,365 kg of sodium hydroxide in 3 liters of water. The rate of addition was set so that the concentration of nicotinic acid in the farm, the new acid and glucose had been completely consumed and the concentration of biomass increased to 15 g/l (dry matter). After additional 4 h, i.e., through a total of 25 h after seeding, stopped fermentation. The final concentration of 6-oxycodonebuy acid was 74 g/l, which corresponds to a virtually quantitative conversion of nicotinic acid 6-oxycodonebuy. The reaction suspension was subjected to centrifugation to distinguish cells. Transparent centrifugal brought to a pH of 1.5 by addition of conc.hydrochloric acid, and 6-oxycotine acid was precipitated as a white solid. The product is filtered, washed with water and dried at 60aboutC and pressure of 20 mbar.

P R I m m e R 2. a). Obtain starter cultures.

Analogously to example 1A) made of a liquid nutrient medium, were seeded strain of Pseudomonas acidovorans DSM 4746. The culture was grown for 24 h at 30aboutC and pH 7.

b). Getting 6-oxycodonebuy acid.

The fermenter with a capacity of 20 l described in example 1 b), were loaded with liquid nutrient medium and sterilized as described in this example. The composition of the liquid nutrient medium was identical to example 1B) except that did not contain glucose.

After cooling the solution to 30aboutSince it was introduced starter culture and with the air supply and peremeschennoe acid decreased from 7.5 to 2.5 g/l (according to a high-speed liquid chromatography), as the biomass concentration increased to 9.5 g/l (dry matter). At this point he started to continuously add a solution of nitronicotinic [receive and composition are consistent with those in example 1B)]. The rate of addition was set so that the concentration of nicotinic acid in the fermenter during the adding of the solution was between 1 and 9 g/l After 12 h the process of adding was completed, glutamic acid was completely consumed and the concentration of biomass increased to 14 g/l (dry matter).

After additional 4 h, i.e. over the entire 24 h after the veil was stopped fermentation. The final concentration of 6-oxycodonebuy acid was 73 g/l, which corresponds to a virtually quantitative conversion of nicotinic acid 6-oxycodonebuy. Processing of the product was carried out as in example 1.

The WAY to OBTAIN 6-OXYCODONEBUY ACID by microbiological hydroxylation of nicotinic acid by a microorganism of the genus Pseudomonas or Achromobacter under aerobic conditions at 20 - 40oC and at pH values of 5.5 to 9, characterized in that the microorganism used one of the strains of Pseudomonas acidovorans DSM 4746 or Achromobacter xylosoxydans DSM 2783, nicotinic acid or its soluble salt or its solution to what you are culture in the process of adding support in the interval 0 - 10 g/l, at least until the braking formed 6-oxycodonebuy acid on the growth of the microorganism and the process is conducted with simultaneous multiplication of the microorganism and education 6-oxycodonebuy acid.

 

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