HU220465B1 - Microbiological process for production of 6-hydroxinicotinic acid - Google Patents

Abstract

The present invention relates to a process for the preparation of 6-hydroxynicotinic acid from a nicotinic acid microbiologically, such that the Pseudomonasacidovorans DSM 7205 and / or Pseudomonas acidovorans DSM 7203 and / or Alcaligenes faecalis DSM 7204 and / or DSM 7202 are utilized with the use of Pseudomonasacidovorans acid or soluble salt thereof. ŕ

Description

The present invention relates to a novel microbiological process for the preparation of 6-hydroxynicotinic acid, starting from nicotinic acid or its water soluble salt, using aerobic biomass utilizing nicotinic acid or its water soluble salt or isolated novel strains.

Hereinafter, the salt, especially the water soluble salt of nicotinic acid, is understood to mean an alkali metal salt of nicotinic acid, for example sodium nicotinate.

6-Hydroxy-nicotinic acid is an important intermediate for the preparation of 5,6-dichloro-nicotinic acid (CH 664 754), which is itself a starting compound for pharmaceuticals (Setcliff et al., J. of Chem. And Eng. 21, 2, 246 (1976)].

Microbiological hydroxylation of nicotinic acid to 6-hydroxynicotinic acid is described, for example, in EP 152 948. Microorganisms are described as being multiplied with nicotinic acid in the presence of yeast extract, followed by the actual bio-conversion, whereby the concentration of the starting nicotinic acid is chosen so as to prevent the degradation of 6-hydroxynicotinic acid.

The disadvantage of this process is that the microorganism utilizing the nicotinic acid is propagated in the presence of a yeast extract which contaminates the cell-free fermentation solution after propagation or bio-conversion and leads to contamination of the isolated 6-hydroxynicotinic acid.

A further disadvantage is that this new process is carried out with a uniform (biologically pure) culture of microorganisms, which can be contaminated especially with large-scale fermentation.

It is an object of the present invention to overcome these drawbacks and to develop simple, ecological microbiological methods for the preparation of 6-hydroxynicotinic acid.

This object is solved by the new process according to the invention.

According to the invention, the process is carried out by:

a) The aerobic biomass utilizing nicotinic acid is propagated by a mixture of nicotinic acid and a mineral acid (1-8): 1 in a molar ratio while maintaining this molar ratio throughout the propagation period, and

b) converting the nicotinic acid or its soluble salt into the desired hydroxylated end product with this biomass.

The term "growth of biomass utilizing nicotinic acid or a salt thereof" means that if, for example, a given molar ratio of nicotinic acid and mineral acid is aerobically grown from a sewage sludge inoculum, a single aerobic biomass utilizing nicotinic acid, e.g. , using nitrogen and energy spinning in the presence of oxygen.

As inoculum, soil samples from different countries can be used, such as the soil from Santander (Spain) city park or the vineyard soil in Visper (Switzerland) in Vispertermin.

Unlike the methods mentioned above, the process of the present invention can be carried out not only with a single (biologically pure) microorganism culture but also with a mixture culture biomass.

The molar ratio of nicotinic acid to mineral acid (1-8): 1, i.e., the mixture of nicotinic acid and mineral acid is added to the cell suspension so that the molar ratio of nicotinic acid to mineral acid (1-8) is maintained throughout the growth period. between.

Suitable mineral acids are, for example, sulfuric acid, hydrochloric acid, nitric acid or phosphoric acid, preferably sulfuric acid.

Preferably, the mixture is added during the biomass propagation (step a) so that the molar ratio of nicotinic acid to mineral acid is (3-5): 1. That is, for propagation, 3-5 moles of nicotinic acid per mole of sulfuric acid are preferably used. The propagation is preferably carried out with 4-5 moles of nicotinic acid per mole of sulfuric acid.

The aerobic biomass utilizing the nicotinic acid is routinely grown in a medium containing mineral salts, preferably in the medium described in Table 1.

The biomass is propagated at pH 5-9, preferably at pH 6-8.

The temperature for biomass propagation is preferably 15-50 ° C, preferably 25-40 ° C.

Biomass is usually propagated over a period of 0.5 to 3 days.

Under these conditions, the microorganisms Pseudomonas acidovorans DSM 7205 or Pseudomonas acidovorans DSM 7203 or Alcaligenes faecalis DSM 7204 or the microorganism DSM 7202 or mixtures thereof are preferably enriched.

DSM 7205, DSM 7203, DSM 7204 and DSM

No. 7202 was deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (German Collection of Microorganisms and Cell Cultures) on 13.08.1992, Mascheroderweg lb, D-3300 Braunschweig.

These microorganisms are not yet known in the literature. The microorganism DSM 7202 was not yet taxonomically identifiable and genus-specific.

A Pseudomonas acidovorans DSM 7205, DSM

The taxonomy of microorganisms 7203 and DSM 7204 of Alcaligenes faecalis is described below.

Taxonomic description of Pseudomonas acidovorans DSM 7205 Strain characteristics Cell shape rod width pm 0.8-0.9 long pm 1.5-9.0

Mobility +

Shape polar shape 1

Gram Reactions Lysis with 3% KOH +

Aminopeptidase (Cemy) +

Spores Oxidase +

Catalase +

HU 220 465 Bl

reproduction citrate + anaerobic - glycolate + 37/41 ° C + levulinate + pH 5.7 - malate + Mac - Conkey - Agar + 5 graces + SS Agar + phenylacetate + Cetrimide-agar + L-arabinose - testosterone - fructose + pigments glucose - non-diffusing - 10 mannose - diffusing - maltose fluorescent - D-xylose - pyocyanin - mannitol + Acid (OF test) gluconate + glucose is aerobic ? 15 2-keto-gluconate + glucose is anaerobic - N-acetyl-glucosamine - glucose is alkaline + L-serine - Gas from glucose QUINN + acids D, L-Tryptophan + glucose - 20 L-tartrate + fructose + acetamide + xylose - a-amino butyrate + adonitból - ethanol w L-arabinose - A Pseudomonas acidovorans DSM cellobiose - 25 miai description dulcitol - Characteristics of the strain glycerol + Cell shape stick m-inositol - width gm 0.8-1, lactose - length gm 2.6 to 6, maltose - 30 Mobility + raffinose - Form polar al L-rhamnose - Gram reactions salicin - Lysis with 3% KOH + D-sorbitol - Aminopeptidase (Cemy) + sucrose - 35 Spores - trehalose - oxidase + ethanol + catalase + dulcitol - reproduction ONPG / PNPG - anaerobic - ADH - 40 37/41 ° C +/- VP - pH 5.7 - indole - Mac-Conkey Agar + NO _{2 from} NO ₃ + SS Agar + Phenylalanine deaminase w Cetrimide-agar + Levan from sucrose - 45 Testosterone lecithinase - pigments Uráz - non-diffusing - hydrolysis diffusing - starch - fluorescent - gelatin - 50 piocianin - casein w Acid (OF test) DNA from - glucose is aerobic ? Tween 80 + glucose is anaerobic - eszkulinból - glucose is alkaline + PHB out - 55 Gas from glucose - Tirozinlebomlás + acids Szubsztráthasznosítás glucose - acetate + fructose + adipate + xylose - caprate + Of 60 adonites -

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L-arabinose, cellobiose, dulcitol, glycerol + m-inositol + lactose, maltose, raffinose, L-rhamnose, salicin, D-sorbitol, sucrose, trehalose, ethanol -?

dulcite ONPG / PNPG

ADH

VP

indole

NO _{2 from} NO ₃ + Phenylalanine deaminase w Levan from sucrose - lecithinase - Uráz - hydrolysis starch - gelatin - casein + DNA from - Tween 80 + eszkulinból - PHB out - Tirozinlebomlás + Szubsztráthasznosítás acetate + adipate + caprate + citrate + glycolate + levulinate + malate + malonate + phenylacetate + L-arabinose - fructose + glucose - mannose - maltose - D-xylose - mannitol + gluconate + 2-keto-gluconate + N-acetyl-glucosamine - L-serine - QUINN + D, L-Tryptophan + L-tartrate + acetamide + a-amino butyrate w ethanol +

Taxonomic description of Alcaligenes faecalis DSM 7204

Characteristics of the strain

The cell shape of the rod is width pm 0.6-0.8 long pm 1.0-2.0

Mobility +

Form peritrich

Gram Reactions Lysis with 3% KOH +

Aminopeptidase (Cemy) +

Oxidase +

Catalase +

Reproduction anaerobic

37/41 ° C + / + pH 5.7

Ma-Conkey-Agar +

SS-Agar +

Cetrimid-Agar +

Pigments non-diffusing diffusing fluorescent piocyanic acid (OF-test) glucose aerobic?

from glucose anaerobic glucose alkaline +

Gas from glucose Acids

D-glucose D-fructose +

D-xylose

ONPG / PNPG

ADH

VP

indole

NO _{2 from} NOj +

Denitration Phenylalanine deaminase

Levan from sucrose Lecithinase Urase Hydrolysis from starch from gelatin to casein from DNA

Tween of 80 esculin

Tyrosine degradation +

Substrate utilization acetate + adipate caprate + citrate + glycolate + levulinate D-malate +

EN 220 465 B1 malonate + phenylacetate +

L-arabinose D-fructose

D-glucose

D-mannose maltose D-xylose mannitol gluconate 2-keto-gluconate +

N-acetylglucosamine L-serine

Hereinafter, the nicotinic acid to be hydroxylated and its salts, such as water-soluble alkali metal salts, are the substrate of the process.

After propagation, for biomass conversion (hydroxylation), the biomass is separated in a manner known to those skilled in the art, or the nicotinic acid to be hydroxylated is added directly to the enriched biomass.

The actual hydroxylation of nicotinic acid (substrate) is carried out in a manner known to those skilled in the art by non-proliferating cells.

The actual hydroxylation of the nicotinic acid is carried out with the microorganism Pseudomonas acidovorans DSM 7205, Pseudomonas acidovorans DSM 7203, Alcaligenes faecalis DSM 7204 or DSM 7202 enriched in the growth phase or a mixture thereof. In this process, the nicotinic acid used as a substrate is converted to 6-hydroxynicotinic acid.

During the bio-conversion, the substrate may be added continuously or intermittently. The szubsztrátadagolás preferably takes place in that the amount of the fermenter is not more than 20, preferably ⁰ to 15 wt / ®.

Hydroxylation can be carried out using media known to those skilled in the art, preferably hydroxylated in the medium containing the mineral salts described in Table 1 or in the A-N media described in Table 3.

Biodegradation is generally performed with cells having an optical density at 550 nm (OD ₅₅₀ ) or 650 nm (OD ₆₅₀ ) of 5-100.

The biotransformation is conveniently carried out at pH 5-9, preferably 6.5-7.5, and preferably 15-50 ° C, preferably 25-35 ° C.

After a typical reaction time of 5-24 hours, the hydroxylated nicotinic acid can be isolated by methods known to those skilled in the art, for example by acidifying the cell-free fermentation solution or precipitating it in the form of sparingly soluble salts.

Example 1 (a) Growth of biomass

Fermentation was performed in a non-sterile mineral salt medium (Table 1) with 1 g / L nicotinic acid in a 15 L fermentor, pH 7, at 30 ° C and 5-20 L / min air. An aqueous suspension containing only 307 g (2.5 moles) of nicotinic acid, 49 g (0.5 moles) of sulfuric acid and 1 liter of water was used to control the pH. The pH was adjusted by adding an aqueous suspension of the above composition from a vessel fitted with a stirrer to the fermenter lid via a pneumatically controlled ball valve. The fermentation broth was inoculated with 500 ml of sewage sludge from a wastewater treatment plant in Visp, Switzerland (Table 2). After 36 hours, the fermentor was emptied to 1 liter and filled with fresh medium. After a further 24.48 hours and 72 hours, the fermentation described herein was repeated.

(b) Hydroxylation (Preparation of 6-hydroxynicotinic acid)

The biomass was used when the optical density at 550 nm reached 5-20 to measure the rate of formation of 6-hydroxynicotinic acid. Therefore, the biomass was first washed once with 0.9% (w / v) NaCl. Subsequently, 10 μΐ of cell suspension was added to a quartz cuvette (1 cm light path) preheated to 30 ° C containing 2990 μΐ pH 7.0, 6.5 g / l nicotinic acid, 10.1 g / 1 K ₂ HPO ₄ and 4, It contained a solution containing 0 g / l KH ₂ PO ₄ . The absorbance of the cuvette was then measured at 550 nm and the linear increase in absorbance at 295 nm / min was calculated. Specific activity (U) was determined by the following formula:

A295 nm-60 U = OD ₅₅₀ nm per minute

Fermentation was repeated with soil samples from Zermatt, Switzerland, sewage sludge from Vispertermin, Lac de Joux, Switzerland and Santander, Spain (Table 2).

2-5. example

The following microorganisms were enriched from the biomass grown according to Example 1 (a):

- Pseudomonas acidovorans DSM 7205

- Pseudomonas acidovorans DSM 7203

- Alcaligenes faecalis DSM 7204

- DSM 7202 Microorganisms

These microorganisms were grown under the conditions described below and used to hydroxylate nicotinic acid to 6-hydroxynicotinic acid.

The results are summarized in Table 2.

The microorganisms were grown in a 7 liter fermenter. The fermenter contained 5 liters of A-N medium (Table 4) and 2 g of sodium nicotinate per liter. The propagation was carried out at 30 ° C at pH 7.0. 5N sodium hydroxide and 8.5% (v / v) phosphoric acid were used to control the pH. After 18 hours, the fermentation solution was supplemented with a solution containing 2 g of sodium nicotinate per liter. When the cells reached the exponential stage of growth, the fermentation was stopped and the microorganisms were separated from the medium by centrifugation. Cells were then resuspended in 500 ml of 0.27 mol (40 g) sodium nicotinate at pH 7.0. At this time, the optical density was 20. The hydroxylation of nicotinic acid to 6-hydroxynicotinic acid was followed spectrophotometrically (Table 2).

HU 220 465 BI

Table 2

Example number Time (hours) for hydroxylation of 0.27 moles of nicotinic acid (in 500 ml) The amount of 6-hydroxynicotinic acid isolated after acidification of the cell-free solution Yield in nicotinic acid (%) Second DSM 7202 • i 22 17.5 g (0.11 mol) 41 DSM 7203 4th 9 30.1 g (0.22 mol) 80 DSM 7204 5th DSM 7205 10 28.1 g (0.2 mol) 73 5 32.0 g (0.23 mol) 85

Table 3

composition Concentration (Mg / 1) Well ₂ HPO ₄ 2000 KH ₂ PO ₄ 1000 NaCl 3000 MgCl ₂ .6H ₂ O 400 CaCl ₂ .2H ₂ O 14.5 FeCl ₃ .6H ₂ O 0.8 Pyridoxal hydrochloride 10-10-3 riboflavin 5-10-3 nicotinamide 5 IO- ³ Thiamine hydrochloride 2-10-3 biotin 2-10-3 pantothenic acid 5-10-3

composition Concentration (Mg / 1) p-aminobenzoate 5-10-3 folic Acid 2 10-3 Vitamin B12 5-10-3 ZnSO ₄ .7H ₂ O 100-10-3 MnCl ₂ .4H ₂ O 90-10-3 H ₃ BO ₃ 300-10-3 CoC1 ₂ .6H ₂ O 200-10-3 CuCl ₂ .2H ₂ O ío-ίο- ³ NiCl ₂ .6H ₂ O 20-10-3 Na ₂ MoO ₄ .2H ₂ O 30-10-3 EDTANa ₂ .2H ₂ O 5-10-3 FeSO ₄ .7H ₂ O 2-10-3 (The pH of the solution was adjusted to 7.0.)

Patent Claim Point

Claims (1)

Process for the preparation of 6-hydroxynicotinic acid by hydroxylation of nicotinic acid, characterized in that the Pseudomonas acidovorans DSM 7205 and / or Pseudomonas acidovorans DSM 7203 and / or Alcaligenes faecalis DSM 7204 and / or Pseudomonas acidovorans DSM 7204 and / or soluble salts thereof are prepared. number of microorganisms.