WO2007011156A1 - Novel microorganism catalyzing regioselective hydroxylation of 6 cyclic compound containing nitrogen and method for producing hydroxylated 6 cyclic compound containing nitrogen using the same - Google Patents

Novel microorganism catalyzing regioselective hydroxylation of 6 cyclic compound containing nitrogen and method for producing hydroxylated 6 cyclic compound containing nitrogen using the same Download PDF

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WO2007011156A1
WO2007011156A1 PCT/KR2006/002823 KR2006002823W WO2007011156A1 WO 2007011156 A1 WO2007011156 A1 WO 2007011156A1 KR 2006002823 W KR2006002823 W KR 2006002823W WO 2007011156 A1 WO2007011156 A1 WO 2007011156A1
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microorganism
formula
compound represented
acid
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Junhyeong Cho
Sanghyun Lee
Joowon Rhee
Si Jae Park
Ohjin Park
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Lg Chem, Ltd.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
    • C12P17/165—Heterorings having nitrogen atoms as the only ring heteroatoms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/05—Alcaligenes

Definitions

  • the present invention relates to a novel microorganism of the genus Alcaligenes, which catalyzes the regioselective hydroxylation of nitrogen-containing six- memhered cyclic compounds, including nicotinic acid, pyrazine carboxylic acid and picolinic acid, as well as a method for producing a nitrogen-containing hydroxylated six-membered cyclic compound using said microorganism.
  • Imidacloprid and related compounds which are used as insecticides, have a significant difference from existing insecticides such as organophorphorus compounds, carbamates and pyrethroids with respect to the structure and function thereof.
  • Substances which are commonly present in nicotinoid compounds, including Imidacloprid include 6-hydroxynicotinic acid.
  • the Kolbe-Schmitt reaction is used, but has shortcomings in that it has a yield of only about 45% and requires a high-pressure (130 arm) carbon dioxide atmosphere and a high-temperature (250 ° C) reaction condition. For this reason, there has been a need to develop a process of producing 6-hydroxynicotinic acid with a higher yield in a milder condition compared to the Kolbe-Schmitt reaction.
  • Processes of producing 6-hydroxynicotinic acid from nicotinic acid using a microbial biocatalyst have been reported, but these processes mainly employed a method of accumulating 6-hydroxynicotinic acid in the metabolic process of nicotinic acid degradation products which are used as a nitrogen source and carbon source for microorganisms.
  • microorganisms accumulating 6- hydroxynicotinic acid, the first product of the nicotinic acid degradation process were isolated mainly by an enrichment process.
  • Examples of the isolated microorganisms include Achromobacter xylosoxidans LKl) (Kulla, H., Chimia, 45:81, 1991), Pseudomomas fluorescens TN5) (Nagasawa et ah, Biosci. Biotechnol. Biochem., 58:665, 1994) and Serratia marcescens IFO 12648) (Hurh et al, J. Ferment. Technol, 77:382, 1994).
  • JP Publication No.1998-262691 discloses a microorganism of the genus Serratia, which can produce 3-hydroxynicotinic acid by catalyzing a hydroxylation reaction involving the decarboxylation of the carboxyl group of nicotinic acid.
  • JP Registration No. 3220210 discloses a microorganism of the genus Agrobacterium, which can convert 3-cyanopyridine into 6- hydroxynicotinic acid.
  • the present inventors have made extensive efforts to isolate a microorganism catalyzing the regioselective hydroxylation of nitrogen-containing six-membered cyclic compounds.
  • the present inventors have isolated a novel microorganism Alcaligenes faecalis LGNA2 capable of effectively producing 6- hydroxynicotinic acid by examining the conversion of nicotinic acid in microorganisms isolated during the degradation process of nitrile compounds (e.g., 3-hydroxypropionitrile, 3-cyanopyridine, etc.), and found that this microorganism can convert nicotinic acid into 6-hydroxynicotinic acid, thereby completing the present invention.
  • nitrile compounds e.g., 3-hydroxypropionitrile, 3-cyanopyridine, etc.
  • a main object of the present invention is to provide a novel microorganism which not only catalyzes the regioselective reaction of nicotinic acid to produce 6- hydroxylicotinic acid with high efficiency, but also catalyzes the regioselective hydroxylation of nitrogen-containing six-membered cyclic compounds.
  • Another object of the present invention is to provide a method for producing a nitrogen-containing hydroxylated six-membered cyclic compound using said microorganism.
  • the present invention provides a microorganism of the genus Alcaligenes, which catalyzes the regioselective hydroxylation of a compound represented by Formula 1 : [Formula 1]
  • R is a carbon atom or a nitrogen atom
  • Rl and R2 are each independently hydrogen, a carboxyl group, a carbamoyl group, a cyano group, a formyl group, a C 1 -Cs hydroxyalkyl group, a C 2 -C 6 alkoxycarbonyl group, a carboxylvinyl group, a carboxymethyl group or an oxime group, with the proviso that one of Rl and R2 must be a carboxyl group.
  • the present invention provides a method for producing a compound represented by Formula 2, the method comprising carrying out the selective hydroxylation of a compound represented by Formula 1 using said microorganism of the genus Alcaligenes: [Formula 2]
  • R is a carbon atom or a nitrogen atom
  • Rl and R2 are each independently hydrogen, a carboxyl group, a carbamoyl group, a cyano group, a formyl group, a C 1 -C 5 hydroxyalkyl group, a C 2 -C 6 alkoxycarbonyl group, a carboxylvinyl group, a carboxymethyl group or an oxime group, with the proviso that one of Rl and R2 must be a carboxyl group.
  • the present invention provides a method for preparing an enzyme catalyzing the regioselective hydroxylation of the compound represented by Formula 1, the method comprising culturing said microorganism of the genus Alcaligenes; and recovering the enzyme catalyzing the regioselective hydroxylation of the compound represented by Formula 1 from the cultured microorganism. Also, the present invention provides a method for producing the compound of Formula 2, the method comprising carrying out the selective hydroxylation of the compound of Formula 1 using the enzyme prepared by said method, or a crushed substance or fraction of the microorganism containing said enzyme.
  • said microorganism of the genus Alcaligenes is preferably Alcaligenes faecalis, and has 16S rRNA having a homology of more than 99% to a base sequence of SEQ ID NO: 1. More preferably, said microorganism of the genus Alcaligenes is Alcaligenes faecalis LGNA2 (KCTC 10779BP).
  • the compound represented by Formula 1 is preferably selected from the group consisting of nicotinic acid, pyrazine carboxylic acid and picolinic acid.
  • FIG. 1 shows identification results obtained by microbial fatty acid analysis (MIDI) of Alcaligenes faecalis LGNA2, a novel microorganism isolated according to the present invention.
  • MIDI microbial fatty acid analysis
  • FIG. 2A and 2B show HPLC chromatograms of a product at 0 hour and 63 hours after performing the conversion of nicotinic acid using Alcaligenes faecalis LGNA2, and
  • FIG 2C shows HPLC chromatogram of standard 6-hydroxynicotinic acid.
  • FIG. 3 A shows the 1 H-NMR spectrum of standard 6-hydroxynicotinic acid
  • FIG. 3B shows the 1 H-NMR spectrum of a product resulting from the conversion of nicotinic acid, caused by Alcaligenes faecalis LGNA2.
  • FIG. 4A shows the mass spectrometry (MS) spectrum of standard 6- hydroxynicotinic acid
  • FIG. 4B shows the MS spectrum of a product resulting from the conversion of nicotinic acid, caused by Alcaligenes faecalis LGNA2.
  • FIG. 5A, 5B and 5C show an HPLC chromatogram of a product resulting from the conversion of pyrazine carboxylic acid, caused by Alcaligenes faecalis LGNA2 at 0 hour, 4 hours and 20 hours after the conversion reaction, respectively.
  • FIG. 6 shows 1 H-NMR spectra illustrating the conversion of pyrazine carboxylic acid, caused by Alcaligenes faecalis LGNA2.
  • FIG. 6A shows the 1 H-NMR spectrum of reactant pyrazine carboxylic acid
  • FIG. 6B shows the H-NMR spectrum of a reaction product.
  • FIG. 7 illustrates the mass spectrometry (MS) spectra of a product resulting from the conversion of pyrazine carboxylic acid, caused by Alcaligenes faecalis LGNA2, and shows an LC-MS spectrum, the MS spectrum of peak 1 and the MS spectrum of peak 2, starting with the top of the figure, respectively.
  • MS mass spectrometry
  • Alcaligenes faecalis LGNA2 a novel microorganism of the genus Alcaligenes, was obtained using a method of isolating the microorganism during the degradation process of nitrile compounds, instead of using a method of isolating a microorganism producing 6-hydroxynicotinic acid through the degradation of nicotinic acid, and it was found that the obtained microorganism converts nicotinic acid into 6-hydroxynicotinic acid with high efficiency.
  • the present invention in one aspect, relates to a microorganism of the genus Alcaligenes which catalyzes the regioselective hydroxylation of a compound represented by Formula 1 and a method for producing a compound represented by Formula 2, the method comprising carrying out the selective hydroxylation of a compound represented by Formula 1 using said microorganism of the genus Alcaligenes: [Formula 1]
  • R is a carbon atom or a nitrogen atom
  • Rl and R2 are each independently hydrogen, a carboxyl group, a carbamoyl group, a cyano group, a formyl group, a C 1 -Cs hydroxyalkyl group, a C 2 -C 6 alkoxycarbonyl group, a carboxylvinyl group, a carboxymethyl group or an oxime group, with the proviso that one of Rl and R2 must be a carboxyl group.
  • a hydroxide represented by Formula 2 could be produced directly by catalyzing the regioselective hydroxylation of a nitrogen-containing 6- membered cyclic compound represented by Formula 1.
  • the microorganism according to the present invention not only can catalyze the regioselective hydroxylation of nicotinic acid to produce 6- hydroxynicotinic acid, but also can convert 2-pyrazinecarboxylic acid and picolinic acid into 5-hydroxy pyrazine-2-carboxylic acid and 6-hydroxypicolinic acid, respectively.
  • an enzyme catalyzing the regioselective hydroxylation of the compound represented by Formula 1 recovered from the microorganism of the genus Alcaligenes can be used.
  • the present invention in another aspect, relates to a method for preparing an enzyme catalyzing the regioselective hydroxylation of the compound represented by Formula 1 , the method comprising culturing said microorganism of the genus
  • Alcaligenes and recovering the enzyme catalyzing the regioselective hydroxylation of the compound represented by Formula 1 from the cultured microorganism, and a method for producing the compound of Formula 2, the method comprising carrying out the selective hydroxylation of the compound of Formula 1 using the enzyme prepared by said method or a crushed substance or fraction of the microorganism containing said enzyme.
  • the enzyme catalyzing the regioselective hydroxylation of the compound represented by Formula 1 can be retrieved from the cultured microorganism of the genus Alcaligenes using the conventional method.
  • a method for recovering the enzyme comprising the steps of: collecting the cells from the culture broth by centrifugation, etc; crushing the collected cells; and applying to chromatography, etc.
  • crushed substance refers to a substance which is obtained by crushing cultured microorganism with, for example, a grinder, and contains the inventive enzyme capable of catalyzing the hydroxylation of nitrogen- containing six-membered cyclic compounds.
  • fraction refers to a substance which is obtained by partially purifying an enzyme from said crushed substance and contains the inventive enzyme capable of catalyzing the hydroxylation of nitrogen-containing six-membered cyclic compounds.
  • Example 1 Isolation of microorganism converting nicotinic acid into 6- hydroxynicotinic acid
  • a microorganism that degrades 3-hydroxypropionitrile and uses the degraded substance as a single carbon source was isolated from a soil source.
  • GHPN medium and HPN medium were prepared and used.
  • the GHPN medium was prepared by adding 5 g/L of glucose and 20 g/L of 3-hydroxypropionitrile as a nitrogen source to a minimal medium containing the components shown in Tables 1 and 2.
  • the HPN medium a medium of using 3-hydroxypropionitrile as a single carbon source and nitrogen source, was prepared by adding 20 g/L of 3-hydroxypropionitrile as a nitrogen source to a minimal medium containing the components shown in Tables 1 and 2.
  • solid media HPN agar prepared by adding glucose (10 g/L) as a carbon source and 3-hydroxypropionitrile as a nitrogen source (5 g/L) to LB medium plate and minimal media were used.
  • a soil microbial source was added into 5 ml of liquid medium (GHPN medium and HPN medium) and subjected to enrichment culture in a shaking incubator under conditions of 200 rpm and 30 ° C for 3 days. Then, 1 ml of the culture broth as a microbial source was seeded again into the same liquid medium as described above, and then subjected to enrichment culture in the same conditions as described above. The culture process was repeated three times, and then the culture broth was plated onto an HPN agar solid medium, and cultured to produce a single colony. Microorganisms from the single colony were cultured in an HPN minimal medium to validate the degradability of nitrogen thereof. The culture broth showing the growth of the microorganisms was plated onto a solid medium and cultured at 30°C , and the microorganisms were purely isolated.
  • Example 1 To identify the microorganism isolated in Example 1 , an MIDI method, a cell wall fatty acid analysis method, was carried out, and for a more accurate analysis, the base sequencing of 16S rRNA was conducted. The results of the MIDI analysis showed that said microorganism had similarity index of 0.735 to Alcaligenes faecalis (FIG. 1).
  • 16S rRNA was amplified using the purified chromosome as a template and primers of SEQ ID NOs: 2 and 3 (Wilson et al., Appl. Environ. Microbiol, 71 :290, 2005).
  • SEQ ID NO: 2 forward primer AF16S rRNAF: 5-TTG GAT CCA GAG TTT GAT CMT GGC TCA G-3
  • SEQ ID NO: 3 reverse primer AF16S rRNAR: 5-GTT GGA TCC ACG GYT ACC TTG TTA CGA YT-3
  • Each sequence of 500 bases in the 16S rRNA base sequence (SEQ ID NO: 1) of said microorganism was determined. On the basis of the determined base sequence, it was found from the Clustal X program that said microorganism has a similarity more than 99% to the Alcaligenes faecalis KCTC 2678 strain.
  • Example 1 the microorganism isolated in Example 1 was named "Alcaligenes faecalis LGNA2" and deposited with the Korean Collection for Type Cultures (KCTC), the Korea Research Institute of Bioscience and Biotechnology, on February 15, 2005 under accession No. KCTC 10779BP).
  • KCTC Korean Collection for Type Cultures
  • the regioselective hydroxylation of nitrogen-containing six-membered cyclic compounds was carried out using a novel microbial strain Alcaligenes faecalis LGNA2, and then the structures of the resulting products were determined in order to validate the hydroxylation of the compounds.
  • a microbial strain was collected from the culture medium by centrifugation, washed with 50 mM Tris-HCl buffer (pH 7.0), and then suspended in 2.8 mL of the same buffer.
  • 50 mM Tris-HCl buffer (pH 7.0) and nicotinic acid (final concentration: 20 g/L) were added, the mixture was allowed to react in a shaking incubator under conditions of 200 rpm and 30 °C , and then nicotinic acid and the product were detected by HPLC.
  • a solvent for HPLC a mixed solution of methanol and water (29:60 v/v) was used.
  • FIG. 2A and 2B show HPLC chromatograms of a product at 0 hour and 63 hours after performing the conversion of nicotinic acid using Alcaligenes faecalis LGNA2, respectively and
  • FIG 2C shows HPLC chromatogram of standard 6- hydroxynicotinic acid. From the results in FIG. 2, it could be observed that 10.5 g/L of 6-hydroxynicotinic acid was produced at 63 hours after the reaction.
  • FIG. 3 shows the 1 H-NMR spectrum of the product resulting from the conversion of nicotinic acid, caused by Alcaligenes faecalis LGNA2. More specifically, FIG. 3A shows the 1 H-NMR spectrum of standard 6-hydroxynicotinic acid, and FIG.
  • FIG. 3B shows the 1 H-NMR spectrum of the product resulting from the conversion of nicotinic acid, caused by Alcaligenes faecalis LGNA2.
  • FIG. 4 shows the mass spectrometry spectrum of the product resulting from the conversion of nicotinic acid, caused by Alcaligenes faecalis LGNA2. More specifically, FIG. 4A shows the MS spectrum of standard 6-hydroxynicotinic acid, and FIG. 4B shows the MS spectrum of the product by converting nicotinic acid using Alcaligenes faecalis LGNA2.
  • Alcaligenes faecalis LGNA2 was seeded into 2 ml of YEPD (10g/L yeast extract, 20g/L Bacto peptone, 20g/L glucose), and cultured in a shaking incubator at 200 rpm and 30 ° C for 16 hours.
  • YEPD yeast extract, 20g/L Bacto peptone, 20g/L glucose
  • nicotinic acid final concentration of 5 g/L
  • the cultured Alcaliganes faecalis LGNA 2 was cultured for 20 hours (50% of nicotinic acid was converted into 6-hydroxynicotinic acid at 20 hours).
  • the culture medium was centrifuged to obtain a microbial fraction, which was then washed with 50 mM potassium phosphate buffer and suspended in 8 mL of the same buffer.
  • 50 mM potassium phosphate buffer 50 mM potassium phosphate buffer and suspended in 8 mL of the same buffer.
  • 2 mL of pyrazine carboxylic solution 6.21 g/L
  • was added to a final concentration of 4.14 g/L followed by reaction.
  • FIG. 5A, 5B and 5C are HPLC chromatograms of a product resulting from the conversion of pyrazime carboxylic acid using Alcaligenes paecalis LGNA2, which show HPLC results measured at 0 hour, 4 hours and 20 hours after the conversion reaction, respectively. From the results in FIG. 5, it could be observed that 100% of pyrazine carboxylic acid was converted within 20 hours after the reaction.
  • Microbial cells were removed from the reaction solution by centrifugation, and the remaining solution was adjusted to pH 1.2 with 6N HCl. The formed precipitate was collected and washed with water (pH 1.2), followed by drying at 30 ° C (52% yield). The dried substance was dissolved in DMSO-d6 solvent and then subjected to 1 H NMR analysis and MS analysis (FIGs. 6 and 7).
  • FIG. 6 shows the 1 H-NMR spectrum of the product resulting from the conversion of pyrazine carboxylic acid, caused by Alcaligenes faecalis LGNA2. More specifically, FIG. 6A shows the 1 H-NMR spectrum of reactant pyrazine carboxylic acid, and FIG. 6B shows the 1 H-NMR spectrum of the reaction product. Also, FIG. 7 illustrates the mass spectrometry (MS) spectra of the product resulting from the conversion of pyrazine carboxylic acid, caused by Alcaligenes faecalis LGNA2.
  • MS mass spectrometry
  • Peak 1 coincides with the molecular weight of hydroxypyrazine carboxylic acid
  • peak 2 is the MS spectrum of a trace amount of a dimmer.
  • the present invention provides the microorganism
  • the microorganism according to the present invention catalyzes the direct conversion of said compounds, the use of the inventive microorganism leaves little or no byproducts after the final reaction, thus making it easy to isolate and purify the product, and also results in high conversion efficiency. Accordingly, the inventive microorganism can be widely used in synthesizing nicotine and pyrazine derivatives in large amounts.

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Abstract

The present invention relates to a novel microorganism of the genus Alcaligenes, which catalyzes the regioselective hydroxylation of nitrogen-containing six-membered cyclic compounds, including nicotinic acid, pyrazine carboxylic acid and picolinic acid, as well as a method for producing a nitrogen-containing hydroxylated six-membered cyclic compound using said microorganism. The novel microorganism Alcaligenes faecalis LGNA2 according to the present invention is effective in catalyzing the regioselective hydroxylation of nitrogen-containing six-membered cyclic compounds such as nicotinic acid, pyrazine carboxylic acid and picolinic acid and has a high efficiency for converting the six-membered cyclic compounds into other compounds. Accordingly, it can be widely used in synthesizing nicotine and pyrazine derivatives in large amounts.

Description

Novel Microorganism Catalyzing Regioselective Hydroxylation of 6
Cyclic Compound Containing Nitrogen and Method for Producing
Hydroxylated 6 Cyclic Compound Containing Nitrogen Using the Same
TECHNICAL FIELD
The present invention relates to a novel microorganism of the genus Alcaligenes, which catalyzes the regioselective hydroxylation of nitrogen-containing six- memhered cyclic compounds, including nicotinic acid, pyrazine carboxylic acid and picolinic acid, as well as a method for producing a nitrogen-containing hydroxylated six-membered cyclic compound using said microorganism.
BACKGROUND ART
Imidacloprid and related compounds, which are used as insecticides, have a significant difference from existing insecticides such as organophorphorus compounds, carbamates and pyrethroids with respect to the structure and function thereof. Substances which are commonly present in nicotinoid compounds, including Imidacloprid, include 6-hydroxynicotinic acid. For the synthesis of 6- hydroxynicotinic acid, the Kolbe-Schmitt reaction is used, but has shortcomings in that it has a yield of only about 45% and requires a high-pressure (130 arm) carbon dioxide atmosphere and a high-temperature (250 °C) reaction condition. For this reason, there has been a need to develop a process of producing 6-hydroxynicotinic acid with a higher yield in a milder condition compared to the Kolbe-Schmitt reaction.
Processes of producing 6-hydroxynicotinic acid from nicotinic acid using a microbial biocatalyst have been reported, but these processes mainly employed a method of accumulating 6-hydroxynicotinic acid in the metabolic process of nicotinic acid degradation products which are used as a nitrogen source and carbon source for microorganisms. For example, microorganisms accumulating 6- hydroxynicotinic acid, the first product of the nicotinic acid degradation process, were isolated mainly by an enrichment process. Examples of the isolated microorganisms include Achromobacter xylosoxidans LKl) (Kulla, H., Chimia, 45:81, 1991), Pseudomomas fluorescens TN5) (Nagasawa et ah, Biosci. Biotechnol. Biochem., 58:665, 1994) and Serratia marcescens IFO 12648) (Hurh et al, J. Ferment. Technol, 77:382, 1994).
Meanwhile, JP Publication No.1998-262691 discloses a microorganism of the genus Serratia, which can produce 3-hydroxynicotinic acid by catalyzing a hydroxylation reaction involving the decarboxylation of the carboxyl group of nicotinic acid. Also, JP Registration No. 3220210 discloses a microorganism of the genus Agrobacterium, which can convert 3-cyanopyridine into 6- hydroxynicotinic acid.
However, there is still no report on a microorganism which not only catalyzes the direct regioselective hydroxylation of nicotinic acid to produce 6-hydroxynicotinic acid, but also catalyzes the regioselective hydroxylation of other kinds of nitrogen- containing six-membered cyclic compounds such as pyrazine carboxylic acid and picolinic acid.
Thus, the present inventors have made extensive efforts to isolate a microorganism catalyzing the regioselective hydroxylation of nitrogen-containing six-membered cyclic compounds. As a result, the present inventors have isolated a novel microorganism Alcaligenes faecalis LGNA2 capable of effectively producing 6- hydroxynicotinic acid by examining the conversion of nicotinic acid in microorganisms isolated during the degradation process of nitrile compounds (e.g., 3-hydroxypropionitrile, 3-cyanopyridine, etc.), and found that this microorganism can convert nicotinic acid into 6-hydroxynicotinic acid, thereby completing the present invention.
SUMMARY OF THE INVENTION
A main object of the present invention is to provide a novel microorganism which not only catalyzes the regioselective reaction of nicotinic acid to produce 6- hydroxylicotinic acid with high efficiency, but also catalyzes the regioselective hydroxylation of nitrogen-containing six-membered cyclic compounds.
Another object of the present invention is to provide a method for producing a nitrogen-containing hydroxylated six-membered cyclic compound using said microorganism.
To achieve the above objects, the present invention provides a microorganism of the genus Alcaligenes, which catalyzes the regioselective hydroxylation of a compound represented by Formula 1 : [Formula 1]
Figure imgf000005_0001
wherein R is a carbon atom or a nitrogen atom, and Rl and R2 are each independently hydrogen, a carboxyl group, a carbamoyl group, a cyano group, a formyl group, a C1-Cs hydroxyalkyl group, a C2-C6 alkoxycarbonyl group, a carboxylvinyl group, a carboxymethyl group or an oxime group, with the proviso that one of Rl and R2 must be a carboxyl group.
In another aspect, the present invention provides a method for producing a compound represented by Formula 2, the method comprising carrying out the selective hydroxylation of a compound represented by Formula 1 using said microorganism of the genus Alcaligenes: [Formula 2]
Figure imgf000006_0001
wherein R is a carbon atom or a nitrogen atom, and Rl and R2 are each independently hydrogen, a carboxyl group, a carbamoyl group, a cyano group, a formyl group, a C1-C5 hydroxyalkyl group, a C2-C6 alkoxycarbonyl group, a carboxylvinyl group, a carboxymethyl group or an oxime group, with the proviso that one of Rl and R2 must be a carboxyl group.
In still another aspect, the present invention provides a method for preparing an enzyme catalyzing the regioselective hydroxylation of the compound represented by Formula 1, the method comprising culturing said microorganism of the genus Alcaligenes; and recovering the enzyme catalyzing the regioselective hydroxylation of the compound represented by Formula 1 from the cultured microorganism. Also, the present invention provides a method for producing the compound of Formula 2, the method comprising carrying out the selective hydroxylation of the compound of Formula 1 using the enzyme prepared by said method, or a crushed substance or fraction of the microorganism containing said enzyme.
In the present invention, said microorganism of the genus Alcaligenes is preferably Alcaligenes faecalis, and has 16S rRNA having a homology of more than 99% to a base sequence of SEQ ID NO: 1. More preferably, said microorganism of the genus Alcaligenes is Alcaligenes faecalis LGNA2 (KCTC 10779BP).
In the present invention, the compound represented by Formula 1 is preferably selected from the group consisting of nicotinic acid, pyrazine carboxylic acid and picolinic acid.
The above and other objects, features and embodiments of the present invention will be more clearly understood from the following detailed description and accompanying claims.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows identification results obtained by microbial fatty acid analysis (MIDI) of Alcaligenes faecalis LGNA2, a novel microorganism isolated according to the present invention.
FIG. 2A and 2B show HPLC chromatograms of a product at 0 hour and 63 hours after performing the conversion of nicotinic acid using Alcaligenes faecalis LGNA2, and FIG 2C shows HPLC chromatogram of standard 6-hydroxynicotinic acid.
FIG. 3 A shows the 1H-NMR spectrum of standard 6-hydroxynicotinic acid, and FIG. 3B shows the 1H-NMR spectrum of a product resulting from the conversion of nicotinic acid, caused by Alcaligenes faecalis LGNA2.
FIG. 4A shows the mass spectrometry (MS) spectrum of standard 6- hydroxynicotinic acid, and FIG. 4B shows the MS spectrum of a product resulting from the conversion of nicotinic acid, caused by Alcaligenes faecalis LGNA2.
FIG. 5A, 5B and 5C show an HPLC chromatogram of a product resulting from the conversion of pyrazine carboxylic acid, caused by Alcaligenes faecalis LGNA2 at 0 hour, 4 hours and 20 hours after the conversion reaction, respectively. FIG. 6 shows 1H-NMR spectra illustrating the conversion of pyrazine carboxylic acid, caused by Alcaligenes faecalis LGNA2. FIG. 6A shows the 1H-NMR spectrum of reactant pyrazine carboxylic acid, and FIG. 6B shows the H-NMR spectrum of a reaction product.
FIG. 7 illustrates the mass spectrometry (MS) spectra of a product resulting from the conversion of pyrazine carboxylic acid, caused by Alcaligenes faecalis LGNA2, and shows an LC-MS spectrum, the MS spectrum of peak 1 and the MS spectrum of peak 2, starting with the top of the figure, respectively.
DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS
In the present invention, Alcaligenes faecalis LGNA2, a novel microorganism of the genus Alcaligenes, was obtained using a method of isolating the microorganism during the degradation process of nitrile compounds, instead of using a method of isolating a microorganism producing 6-hydroxynicotinic acid through the degradation of nicotinic acid, and it was found that the obtained microorganism converts nicotinic acid into 6-hydroxynicotinic acid with high efficiency.
The present invention, in one aspect, relates to a microorganism of the genus Alcaligenes which catalyzes the regioselective hydroxylation of a compound represented by Formula 1 and a method for producing a compound represented by Formula 2, the method comprising carrying out the selective hydroxylation of a compound represented by Formula 1 using said microorganism of the genus Alcaligenes: [Formula 1]
Figure imgf000009_0001
[Formula 2]
Figure imgf000009_0002
wherein R is a carbon atom or a nitrogen atom, and Rl and R2 are each independently hydrogen, a carboxyl group, a carbamoyl group, a cyano group, a formyl group, a C1-Cs hydroxyalkyl group, a C2-C6 alkoxycarbonyl group, a carboxylvinyl group, a carboxymethyl group or an oxime group, with the proviso that one of Rl and R2 must be a carboxyl group.
In the case of using the microorganism of the genus Alcaligenes according to the present invention, a hydroxide represented by Formula 2 could be produced directly by catalyzing the regioselective hydroxylation of a nitrogen-containing 6- membered cyclic compound represented by Formula 1.
For example, the microorganism according to the present invention not only can catalyze the regioselective hydroxylation of nicotinic acid to produce 6- hydroxynicotinic acid, but also can convert 2-pyrazinecarboxylic acid and picolinic acid into 5-hydroxy pyrazine-2-carboxylic acid and 6-hydroxypicolinic acid, respectively.
Meanwhile, in the preparation of the hydroxide represented by Formula 2, instead of the microorganism of the genus Alcaligenes, an enzyme catalyzing the regioselective hydroxylation of the compound represented by Formula 1 recovered from the microorganism of the genus Alcaligenes can be used.
The present invention, in another aspect, relates to a method for preparing an enzyme catalyzing the regioselective hydroxylation of the compound represented by Formula 1 , the method comprising culturing said microorganism of the genus
Alcaligenes; and recovering the enzyme catalyzing the regioselective hydroxylation of the compound represented by Formula 1 from the cultured microorganism, and a method for producing the compound of Formula 2, the method comprising carrying out the selective hydroxylation of the compound of Formula 1 using the enzyme prepared by said method or a crushed substance or fraction of the microorganism containing said enzyme.
The enzyme catalyzing the regioselective hydroxylation of the compound represented by Formula 1 can be retrieved from the cultured microorganism of the genus Alcaligenes using the conventional method. For example, a method for recovering the enzyme comprising the steps of: collecting the cells from the culture broth by centrifugation, etc; crushing the collected cells; and applying to chromatography, etc.
As used herein, the term "crushed substance" refers to a substance which is obtained by crushing cultured microorganism with, for example, a grinder, and contains the inventive enzyme capable of catalyzing the hydroxylation of nitrogen- containing six-membered cyclic compounds. Also, the term "fraction" refers to a substance which is obtained by partially purifying an enzyme from said crushed substance and contains the inventive enzyme capable of catalyzing the hydroxylation of nitrogen-containing six-membered cyclic compounds.
Examples Hereinafter, the present invention will be described in more detail by specific examples. However, the present invention is not limited to these examples, and it is obvious to those skilled in the field of the present invention that numerous variations or modifications could be made within the spirit and scope of the present invention.
Example 1; Isolation of microorganism converting nicotinic acid into 6- hydroxynicotinic acid
First, a microorganism that degrades 3-hydroxypropionitrile and uses the degraded substance as a single carbon source was isolated from a soil source.
As liquid media for the isolation of the microorganism, GHPN medium and HPN medium were prepared and used. The GHPN medium was prepared by adding 5 g/L of glucose and 20 g/L of 3-hydroxypropionitrile as a nitrogen source to a minimal medium containing the components shown in Tables 1 and 2. Also, the HPN medium, a medium of using 3-hydroxypropionitrile as a single carbon source and nitrogen source, was prepared by adding 20 g/L of 3-hydroxypropionitrile as a nitrogen source to a minimal medium containing the components shown in Tables 1 and 2. In solid culture, solid media (HPN agar) prepared by adding glucose (10 g/L) as a carbon source and 3-hydroxypropionitrile as a nitrogen source (5 g/L) to LB medium plate and minimal media were used.
To screen microorganisms degrading nitrile, 1 g of a soil microbial source was added into 5 ml of liquid medium (GHPN medium and HPN medium) and subjected to enrichment culture in a shaking incubator under conditions of 200 rpm and 30°C for 3 days. Then, 1 ml of the culture broth as a microbial source was seeded again into the same liquid medium as described above, and then subjected to enrichment culture in the same conditions as described above. The culture process was repeated three times, and then the culture broth was plated onto an HPN agar solid medium, and cultured to produce a single colony. Microorganisms from the single colony were cultured in an HPN minimal medium to validate the degradability of nitrogen thereof. The culture broth showing the growth of the microorganisms was plated onto a solid medium and cultured at 30°C , and the microorganisms were purely isolated.
Table 1 : Components of minimal medium
Figure imgf000012_0001
Table 2: Components of metal solution and vitamin solution
Figure imgf000012_0002
Example 2; Identification of isolated LGN A2 microorganism
To identify the microorganism isolated in Example 1 , an MIDI method, a cell wall fatty acid analysis method, was carried out, and for a more accurate analysis, the base sequencing of 16S rRNA was conducted. The results of the MIDI analysis showed that said microorganism had similarity index of 0.735 to Alcaligenes faecalis (FIG. 1).
To determine the base sequence of 16S rRNA, the chromosome of each of said microorganism and an Alcaligenes faecalis KCTC 2678 strain known as the standard bacterial strain of Alcaligenes faecalis was isolated and purified using reagents for isolating and purifying chromosomes. Then, 16S rRNA was amplified using the purified chromosome as a template and primers of SEQ ID NOs: 2 and 3 (Wilson et al., Appl. Environ. Microbiol, 71 :290, 2005).
SEQ ID NO: 2 (forward primer AF16S rRNAF): 5-TTG GAT CCA GAG TTT GAT CMT GGC TCA G-3
SEQ ID NO: 3 (reverse primer AF16S rRNAR): 5-GTT GGA TCC ACG GYT ACC TTG TTA CGA YT-3
Each sequence of 500 bases in the 16S rRNA base sequence (SEQ ID NO: 1) of said microorganism was determined. On the basis of the determined base sequence, it was found from the Clustal X program that said microorganism has a similarity more than 99% to the Alcaligenes faecalis KCTC 2678 strain.
On the basis of said analysis results, the microorganism isolated in Example 1 was named "Alcaligenes faecalis LGNA2" and deposited with the Korean Collection for Type Cultures (KCTC), the Korea Research Institute of Bioscience and Biotechnology, on February 15, 2005 under accession No. KCTC 10779BP).
Example 3: Regioselective hydroxylation of nitrogen-containing six-membered cyclic compounds
The regioselective hydroxylation of nitrogen-containing six-membered cyclic compounds, including nicotinic acid and pyrazine carboxylic acid, was carried out using a novel microbial strain Alcaligenes faecalis LGNA2, and then the structures of the resulting products were determined in order to validate the hydroxylation of the compounds.
3-1. Hydroxylation of nicotinic acid
Alcaligenes faecalis LGNA2 was cultured in 3 mL of YEPD (10 g/L yeast extract, 20 g/L Bacto peptone, and 20 g/L glucose) for 16 hours. To induce an enzyme catalyzing hydroxylation, nicotinic acid was then added to 50 mL of the same YEPD medium to a final concentration of 20 g/L, and the medium was incubated for 39 hours (OD600= 1.73).
A microbial strain was collected from the culture medium by centrifugation, washed with 50 mM Tris-HCl buffer (pH 7.0), and then suspended in 2.8 mL of the same buffer. To 0.5 mL of the microbial suspension, 3.5 mL of 50 mM Tris-HCl buffer (pH 7.0) and nicotinic acid (final concentration: 20 g/L) were added, the mixture was allowed to react in a shaking incubator under conditions of 200 rpm and 30 °C , and then nicotinic acid and the product were detected by HPLC. As a solvent for HPLC, a mixed solution of methanol and water (29:60 v/v) was used. The flow rate of the solvent in a Capcellpak Cl 8 column was maintained at 1 ml/min, and HPLC detection was performed at UV 230 nm (FIG. 2). FIG. 2A and 2B show HPLC chromatograms of a product at 0 hour and 63 hours after performing the conversion of nicotinic acid using Alcaligenes faecalis LGNA2, respectively and FIG 2C shows HPLC chromatogram of standard 6- hydroxynicotinic acid. From the results in FIG. 2, it could be observed that 10.5 g/L of 6-hydroxynicotinic acid was produced at 63 hours after the reaction.
After 5 days of the reaction, the reaction solution was centrifuged to remove microbial cells, and adjusted to pH 1.8 with 6N HCl. Then, the precipitate was collected, washed with water (pH 1.8), followed by drying at 30 °C (52% yield). The dried precipitate was dissolved in DMSO-d6 solvent and then subjected to 1H NMR and MS analysis (FIGs. 3 and 4). FIG. 3 shows the 1H-NMR spectrum of the product resulting from the conversion of nicotinic acid, caused by Alcaligenes faecalis LGNA2. More specifically, FIG. 3A shows the 1H-NMR spectrum of standard 6-hydroxynicotinic acid, and FIG. 3B shows the 1H-NMR spectrum of the product resulting from the conversion of nicotinic acid, caused by Alcaligenes faecalis LGNA2. Also, FIG. 4 shows the mass spectrometry spectrum of the product resulting from the conversion of nicotinic acid, caused by Alcaligenes faecalis LGNA2. More specifically, FIG. 4A shows the MS spectrum of standard 6-hydroxynicotinic acid, and FIG. 4B shows the MS spectrum of the product by converting nicotinic acid using Alcaligenes faecalis LGNA2.
As shown in FIGs. 3 and 4, it could be observed that the 1H-NMR spectrum and the MS spectrum of the reaction product coincided with those of the standard 6- hydroxynicotinic acid.
3-2. Hydroxylation of pyrazine carboxylic acid
Alcaligenes faecalis LGNA2 was seeded into 2 ml of YEPD (10g/L yeast extract, 20g/L Bacto peptone, 20g/L glucose), and cultured in a shaking incubator at 200 rpm and 30°C for 16 hours. To the 100 mL of the same culture medium, nicotinic acid (final concentration of 5 g/L) was added, and then the cultured Alcaliganes faecalis LGNA 2 was cultured for 20 hours (50% of nicotinic acid was converted into 6-hydroxynicotinic acid at 20 hours). The culture medium was centrifuged to obtain a microbial fraction, which was then washed with 50 mM potassium phosphate buffer and suspended in 8 mL of the same buffer. To 1 mL of the microbial suspension, 2 mL of pyrazine carboxylic solution (6.21 g/L), was added to a final concentration of 4.14 g/L, followed by reaction.
To analyze the reaction solution, a mixed solution of acetonitrile and water (4:96 v/v; adjusted to pH 2.5 with H3PO4) was used as an HPLC solvent, the flow rate of the solvent in a Capcellpak Cl 8 column was maintained at 1 ml/min, and HPLC detection was performed at UV 230 nm. FIG. 5A, 5B and 5C are HPLC chromatograms of a product resulting from the conversion of pyrazime carboxylic acid using Alcaligenes paecalis LGNA2, which show HPLC results measured at 0 hour, 4 hours and 20 hours after the conversion reaction, respectively. From the results in FIG. 5, it could be observed that 100% of pyrazine carboxylic acid was converted within 20 hours after the reaction.
Microbial cells were removed from the reaction solution by centrifugation, and the remaining solution was adjusted to pH 1.2 with 6N HCl. The formed precipitate was collected and washed with water (pH 1.2), followed by drying at 30 °C (52% yield). The dried substance was dissolved in DMSO-d6 solvent and then subjected to 1H NMR analysis and MS analysis (FIGs. 6 and 7).
FIG. 6 shows the 1H-NMR spectrum of the product resulting from the conversion of pyrazine carboxylic acid, caused by Alcaligenes faecalis LGNA2. More specifically, FIG. 6A shows the 1H-NMR spectrum of reactant pyrazine carboxylic acid, and FIG. 6B shows the 1H-NMR spectrum of the reaction product. Also, FIG. 7 illustrates the mass spectrometry (MS) spectra of the product resulting from the conversion of pyrazine carboxylic acid, caused by Alcaligenes faecalis LGNA2. More specifically, and shows an LC-MS spectrum, the MS spectrum of peak 1 (hydroxypyrazine carboxylic acid) and the MS spectrum of peak 2 (hydroxypyrazine carboxylic acid dimmer), starting with the top of the figure, respectively. Peak 1 coincides with the molecular weight of hydroxypyrazine carboxylic acid, and peak 2 is the MS spectrum of a trace amount of a dimmer.
As shown in FIGs. 6 and 7, it could be observed that the reaction product was 5- hydroxypyrazine-2-carboxylic acid (mass analysis: 139=[M-H]-, 253=[M+CF3COO-]). However, a trace amount of a dimmer was produced as a byproduct.
INDUSTRIAL APPLICABILITY
As described in detail above, the present invention provides the microorganism
(Alcaligenes faecalis LGNA2) effective in catalyzing the regioselective hydroxylation of nitrogen-containing six-membered cyclic compounds such as nicotinic acid, pyrazine carboxylic acid and picolinic acid, as well as the method for producing a nitrogen-containing hydroxylated six-membered cyclic compound using said microorganism. Because the microorganism according to the present invention catalyzes the direct conversion of said compounds, the use of the inventive microorganism leaves little or no byproducts after the final reaction, thus making it easy to isolate and purify the product, and also results in high conversion efficiency. Accordingly, the inventive microorganism can be widely used in synthesizing nicotine and pyrazine derivatives in large amounts.
Although a specific embodiment of the present invention has been described in detail, those skilled in the art will appreciate that this description is merely a preferred embodiment and is not construed to limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims

THE CLAIMS
What is claimed is:
L A microorganism of the genus Alcaligenes, which catalyzes the regioselective hydroxylation of a compound represented by Formula 1 below: [Formula 1]
Figure imgf000018_0001
wherein R is a carbon atom or a nitrogen atom, and Rl and R2 are each independently hydrogen, a carboxyl group, a carbamoyl group, a cyano group, a formyl group, a C1-C5 hydroxyalkyl group, a C2-C6 alkoxycarbonyl group, a carboxylvinyl group, a carboxymethyl group or an oxime group, with the proviso that one of Rl and R2 must be a carboxyl group.
2. The microorganism according to claim 1, wherein said microorganism of the genus Alcaligenes is Alcaligenes faecalis.
3. The microorganism according to claim 1, wherein said microorganism of the genus Alcaligenes has 16S rRNA having a homology of more than 99% to a base sequence of SEQ ID NO: 1.
4. The microorganism according to claim 1, wherein said microorganism of the genus Alcaligenes is Alcaligenes faecalis LGNA2 (KCTC 10779BP).
5. A method for producing a compound represented by Formula 2, the method comprising carrying out the selective hydroxylation of a compound represented by Formula 1 using the microorganism of the genus Alcaligenes: [Formula 1]
Figure imgf000019_0001
[Formula 2]
Figure imgf000019_0002
wherein R is a carbon atom or a nitrogen atom, and Rl and R2 are each independently hydrogen, a carboxyl group, a carbamoyl group, a cyano group, a formyl group, a C1-C5 hydroxyalkyl group, a C2-C6 alkoxycarbonyl group, a carboxylvinyl group, a carboxymethyl group or an oxime group, with the proviso that one of Rl and R2 must be a carboxyl group.
6. The method according to claim 5, wherein said microorganism of the genus Alcaligenes is Alcaligenes faecalis.
7. The method according to claim 5, wherein said microorganism of the genus Alcaligenes has 16S rRNA having a homology of more than 99% to a base sequence of SEQ ID NO: 1.
8. The method according to claim 5, wherein said microorganism of the genus Alcaligenes is Alcaligenes faecalis LGNA2 (KCTC 10779BP).
9. The method according to claim 5, wherein the compound represented by Formula 1 is selected from the group consisting of nicotinic acid, pyrazine carboxylic acid and picolinic acid.
10. A method for preparing an enzyme catalyzing the regioselective hydroxylation of the compound represented by Formula 1 , the method comprising culturing the microorganism of any one claim among claims 1-4; and recovering the enzyme catalyzing the regioselective hydroxylation of the compound represented by Formula 1 from the cultured microorganism: [Formula 1]
Figure imgf000020_0001
wherein R is a carbon atom or a nitrogen atom, and Rl and R2 are each independently hydrogen, a carboxyl group, a carbamoyl group, a cyano group, a formyl group, a C1-C5 hydroxyalkyl group, a C2-C6 alkoxycarbonyl group, a carboxylvinyl group, a carboxymethyl group or an oxime group, with the proviso that one of Rl and R2 must be a carboxyl group.
11. A method for producing the compound represented by Formula 2, the method comprising carrying out the selective hydroxylation of the compound represented by Formula 1 using the enzyme prepared by the method of claim 10, or a crushed substance or fraction of the microorganism containing said enzyme: [Formula 1]
Figure imgf000020_0002
[Formula 2]
Figure imgf000020_0003
wherein R is a carbon atom or a nitrogen atom, and Rl and R2 are each independently hydrogen, a carboxyl group, a carbamoyl group, a cyano group, a formyl group, a C1-C5 hydroxyalkyl group, a C2-C6 alkoxycarbonyl group, a carboxylvinyl group, a carboxymethyl group or an oxime group, with the proviso that one of Rl and R2 must be a carboxyl group.
12. The method according to claim 11, wherein the compound represented by Formula 1 is selected from the group consisting of nicotinic acid, pyrazine carboxylic acid and picolinic acid.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5082777A (en) * 1984-02-21 1992-01-21 Lonza Ltd. Process for the production of 6-hydroxynicotinic acid
US5182197A (en) * 1991-02-04 1993-01-26 Lonza Ltd. Microbiological process for the production of 6-hydroxypicolinic acid
US5273893A (en) * 1991-06-21 1993-12-28 Lonza Ltd. Microbiological process for the production of hydroxypyrazinecarboxylic acid
US5516661A (en) * 1991-12-05 1996-05-14 Lonza Ltd. Microbiological process for the production of aromatic hydroxy-heterocyclic carboxylic acids

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5082777A (en) * 1984-02-21 1992-01-21 Lonza Ltd. Process for the production of 6-hydroxynicotinic acid
US5182197A (en) * 1991-02-04 1993-01-26 Lonza Ltd. Microbiological process for the production of 6-hydroxypicolinic acid
US5273893A (en) * 1991-06-21 1993-12-28 Lonza Ltd. Microbiological process for the production of hydroxypyrazinecarboxylic acid
US5516661A (en) * 1991-12-05 1996-05-14 Lonza Ltd. Microbiological process for the production of aromatic hydroxy-heterocyclic carboxylic acids

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