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WO2003031634A1 - Method for selective oxidation of substituted toluenes by microbial coprinus peroxydases - Google Patents

Method for selective oxidation of substituted toluenes by microbial coprinus peroxydases Download PDF

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WO2003031634A1
WO2003031634A1 PCT/EP2002/010867 EP0210867W WO03031634A1 WO 2003031634 A1 WO2003031634 A1 WO 2003031634A1 EP 0210867 W EP0210867 W EP 0210867W WO 03031634 A1 WO03031634 A1 WO 03031634A1
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coprinus
microbial
peroxidase
oxidation
peroxydases
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German (de)
French (fr)
Inventor
Bernhard Hauer
Thomas Zelinski
Tilo Habicher
Michael Breuer
Timm Anke
Rainer Russ
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BASF SE
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Priority to JP2003534604A priority patent/JP2005504553A/en
Priority to US10/491,536 priority patent/US20040254083A1/en
Publication of WO2003031634A1 publication Critical patent/WO2003031634A1/en
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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group

Definitions

  • the present invention relates to a process for the selective oxidation of toluene derivatives by microbial peroxidases from microorganisms of the genus Coprinus.
  • Substituted benzaldehydes are valuable intermediates in chemical synthesis.
  • the chemical production of these benzaldehydes by oxidation of the corresponding substituted toluenes is often difficult to achieve, since the methyl side chain is often oxidized to the carboxyl function by the oxidation reaction and does not remain at the stage of the aldehyde function.
  • the present invention is therefore based on the object of providing a process for the selective oxidation of substituted toluenes by microbial peroxidases which both ensures a high conversion of the substrates and achieves the lowest possible proportion of substituted benzoic acids.
  • the peroxidases used can be used in different degrees of purity. A partially purified peroxidase solution that no longer has laccase activity is preferred.
  • the peroxidase from Coprinus spec DSM 14525 (deposited on September 25, 2001 at DSM) is particularly preferably used.
  • the peroxidase can be used in a variety of solvents. Buffered solutions are preferred which consist of at least 30%, preferably at least 50%, water.
  • the hydrogen peroxide causing the oxidation can be added in stoichiometric or superstoichiometric amounts. A continuous feed of the hydrogen peroxide into the reaction medium is preferred.
  • the reaction temperature can be freely selected between 0 and 50 ° C. At low temperatures, the enzymatic conversion is slowed down considerably, while high temperatures can lead to inactivation of the enzyme.
  • the optimal reaction temperature is therefore between 10 and 30 ° C and can easily be determined by a person skilled in the art on the basis of series tests.
  • the oxidation of the methyl side chain in the process according to the invention depends on the further substituents on the aromatic nucleus and their position in relation to the methyl chain.
  • Preferred further substituents R are methyl, isopropyl, t-butyl, halogen, methyl and nitro groups.
  • the position of the radical R relative to the methyl chain is obviously more important than the nature of the radical R.
  • An exception to this are nitrotoluenes.
  • o-Nitrobenzaldehyde was obtained in a relatively low yield, while the m-Nitrobenzaldehyde yield was comparable to p-Nitrobenzaldehyde.
  • o-Nitrotoluene was also the only substrate on which the corresponding alcohol derivative was found. No oxidation of the methyl chain to alcohol was observed for any other substrate.
  • Another object of the invention is a microbial peroxidase from Coprinus, which is capable of oxidizing toluene so selectively to benzaldehyde by H 2 0 2 that less than 0.4 mol of benzoic acid is formed per mole of oxidized toluene.
  • Coprinus spec. DSM 14525 was fermented in 20 liter medium (soy meal). Mycelium and soy meal were removed by centrifugation. The resulting supernatant was clarified by ultrafiltration (pore size 0.16 ⁇ m). The filtrates were then concentrated by ultrafiltration with a membrane with 10kDa cutoff. The peroxidase was purified from the filtrate using FPLC. Two chromatography steps on Q-Sepharose with pH 5 and pH 7.3 gave a 12-fold activity enrichment (specific activity was 7.6 U / mg protein with ABTS, 2.2 azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) Enzyme preparation showed no laccase activity.
  • This peroxidase preparation was used for the following reactions.
  • reaction batches were incubated at room temperature. With a syringe, 4.5 ⁇ l hydrogen peroxide (50 mM) was added through a membrane every 10 minutes (a total of nine times). At the halfway point of the biotransformation, an additional 25 ⁇ l peroxidase solution was added. The concentration of the compounds was determined by comparison with authentic standards by HPLC. The structure of the compounds obtained was confirmed by GC-MS.

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  • Bioinformatics & Cheminformatics (AREA)
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  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

The invention relates to a method for selective oxidation of toluene derivatives by microbial peroxydases from the Coprinus species of microorganisms.

Description

Verfahren zur selektiven Oxidation von substituierten Toluolen durch mikrobielle Peroxidasen aus CoprinusProcess for the selective oxidation of substituted toluenes by microbial peroxidases from Coprinus

Beschreibungdescription

Die vorliegende Erfindung betrifft ein Verfahren zur selektiven Oxidation von Toluolderivaten durch mikrobielle Peroxidasen aus Mikroorganismen der Gattung Coprinus .The present invention relates to a process for the selective oxidation of toluene derivatives by microbial peroxidases from microorganisms of the genus Coprinus.

Substituierte Benzaldehyde sind wertvolle Zwischenprodukte in der chemischen Synthese. Die chemische Herstellung dieser Benzaldehyde durch Oxidation der entsprechenden substituierten Toluole ist oft schwierig zu erreichen, da die Methylseitenkette durch die Oxidationsreaktion häufig bis zur Carboxylfunktion oxi- diert wird und nicht auf der Stufe der Aldehydfunktion bleibt .Substituted benzaldehydes are valuable intermediates in chemical synthesis. The chemical production of these benzaldehydes by oxidation of the corresponding substituted toluenes is often difficult to achieve, since the methyl side chain is often oxidized to the carboxyl function by the oxidation reaction and does not remain at the stage of the aldehyde function.

Auch mikrobielle Verfahren zur Oxidation sind häufig technisch nicht brauchbar, da sie entweder eine zu geringe Umsetzung der substituierten Toluole bewirken oder die Oxidation bis zur Benzoesäure abläuft.Even microbial processes for oxidation are often technically unusable, since they either cause the substituted toluenes to be converted too little or the oxidation takes place up to the benzoic acid.

Der vorliegenden Erfindung liegt daher die Aufgabe zugrunde, ein Verfahren zur selektiven Oxidation von substituierten Toluolen durch mikrobielle Peroxidasen bereitzustellen, das sowohl eine hohe Umsetzung der Substrate gewährleistet und einen möglichst geringen Anteil an substituierten Benzoesäuren erzielt.The present invention is therefore based on the object of providing a process for the selective oxidation of substituted toluenes by microbial peroxidases which both ensures a high conversion of the substrates and achieves the lowest possible proportion of substituted benzoic acids.

Gefunden wurde ein Verfahren zur Herstellung von Aldehyden der Formel (I) durch Oxidation von Toluolen der Formel (II) mit Wasserstoffperoxid in Gegenwart einer mikrobiellen Peroxidase isolierbar aus einem Mikroorganismus der Gattung CoprinusA process has been found for the preparation of aldehydes of the formula (I) by oxidation of toluenes of the formula (II) with hydrogen peroxide in the presence of a microbial peroxidase which can be isolated from a microorganism of the genus Coprinus

Figure imgf000002_0001
Figure imgf000002_0001

II I wobeiII I being

R = Cι-C4-Alkyl, Cι-C4-Alkoxyl, F, Cl, Br, J, N02, OH und n = 0, 1, 2 bedeutet.R = -C 4 alkyl, -C 4 alkoxyl, F, Cl, Br, J, N0 2 , OH and n = 0, 1, 2 means.

Die verwendeten Peroxidasen können in verschiedenen Reinheitsgraden eingesetzt werden. Bevorzugt wird eine teilgereinigte Peroxidaselösung, die keine Laccaseaktivität mehr besitzt. Besonders bevorzugt wird die Peroxidase aus Coprinus spec DSM 14525 (hinterlegt am 25.09.01 bei DSM) verwendet.The peroxidases used can be used in different degrees of purity. A partially purified peroxidase solution that no longer has laccase activity is preferred. The peroxidase from Coprinus spec DSM 14525 (deposited on September 25, 2001 at DSM) is particularly preferably used.

Die Peroxidase kann in einer Vielzahl von Lösungsmitteln ein- gesetzt werden. Bevorzugt werden gepufferte Lösungen, die zu mindestens 30 % bevorzugt mindestens 50 % aus Wasser bestehen.The peroxidase can be used in a variety of solvents. Buffered solutions are preferred which consist of at least 30%, preferably at least 50%, water.

Das die Oxidation bewirkende Wasserstoffperoxid kann in stöchiometrischen oder überstöchiometrischen Mengen zugegeben werden. Bevorzugt ist eine kontinuierliche Zuführung des Wasserstoffperoxids in das Reaktionsmedium.The hydrogen peroxide causing the oxidation can be added in stoichiometric or superstoichiometric amounts. A continuous feed of the hydrogen peroxide into the reaction medium is preferred.

Die Reaktionstemperatur kann in breiten Bereichen zwischen 0 und 50°C frei gewählt werden. Bei niedrigen Temperaturen ist die enzy- matische Umsetzung stark verlangsamt, während hohe Temperaturen zu einer Inaktivierung des Enzyms führen können. Die optimale Reaktionstemperatur liegt demzufolge zwischen 10 und 30°C und kann vom Fachmann leicht anhand von Reihenversuchen ermittelt werden.The reaction temperature can be freely selected between 0 and 50 ° C. At low temperatures, the enzymatic conversion is slowed down considerably, while high temperatures can lead to inactivation of the enzyme. The optimal reaction temperature is therefore between 10 and 30 ° C and can easily be determined by a person skilled in the art on the basis of series tests.

Die Oxidation der Methylseitenkette im erfindungsgemäßen Verfahren hängt von den weiteren Substituenten am aromatischen Kern und deren Position zur Methylkette ab. Bevorzugte weitere Substituenten R sind Methyl, Isopropyl, t-Butyl, Halogen, Methoyy und Nitrogruppen.The oxidation of the methyl side chain in the process according to the invention depends on the further substituents on the aromatic nucleus and their position in relation to the methyl chain. Preferred further substituents R are methyl, isopropyl, t-butyl, halogen, methyl and nitro groups.

Die Position des Restes R zur Methylkette ist offenbar entscheidender als die Natur des Restes R. Im erfindungsgemäßen Verfahren sind solche Substituenten R bevorzugt, die ortho und para zur Methylkette stehen gegenüber solchen in meta-Position. Eine Ausnahme hiervon sind Nitrotoluole. o-Nitrobenzaldehyd wurde in einer relativ niedrigen Ausbeute erhalten, während die m-Nitro- benzaldehydausbeute vergleichbar zu p-Nitrobenzaldehyd war. o-Nitrotoluol war auch das einzige Substrat, bei dem auch das entsprechende Alkoholderivat gefunden wurde. Bei allen anderen Substraten wurde keine Oxidation der Methylkette zum Alkohol beobachtet .The position of the radical R relative to the methyl chain is obviously more important than the nature of the radical R. In the process according to the invention, preference is given to those substituents R which are ortho and para to the methyl chain compared to those in the meta position. An exception to this are nitrotoluenes. o-Nitrobenzaldehyde was obtained in a relatively low yield, while the m-Nitrobenzaldehyde yield was comparable to p-Nitrobenzaldehyde. o-Nitrotoluene was also the only substrate on which the corresponding alcohol derivative was found. No oxidation of the methyl chain to alcohol was observed for any other substrate.

Ein weiterer Gegenstand der Erfindung ist eine mikrobielle Peroxidase aus Coprinus, die in der Lage ist Toluol, durch H202 so selektiv zu Benzaldehyd zu oxidieren, daß pro mol oxidiertem Toluol weniger als 0,4 mol Benzoesäure entstehen. Beispiele 1Another object of the invention is a microbial peroxidase from Coprinus, which is capable of oxidizing toluene so selectively to benzaldehyde by H 2 0 2 that less than 0.4 mol of benzoic acid is formed per mole of oxidized toluene. Examples 1

Herstellung der PeroxidaseProduction of the peroxidase

Coprinus spec. DSM 14525 wurde in 20 1 Medium (soy meal) fermen- tiert. Mycel und soy meal wurden durch Zentrifugation entfernt. Der entstandene Überstand wurde durch Ultrafiltration (Porengröße 0,16 μm) geklärt. Die Filtrate wurden anschließend durch Ultrafiltration mit einer Membran mit lOkDa cutoff konzentriert. Die Partiaireinigung der Peroxidase erfolgte aus dem Filtrat mit FPLC. Zwei Chromatographieschritte an Q-Sepharose mit pH 5 und pH 7,3 ergaben eine 12 fache Aktivitätsanreicherung (spezifische Aktivität betrug 7,6 U/mg Protein mit ABTS, 2,2 Azino- bis (3-ethylbenzthiazolin-6-sulfonsäure) . Die Enzympräparation wies keine Laccase Aktivität auf.Coprinus spec. DSM 14525 was fermented in 20 liter medium (soy meal). Mycelium and soy meal were removed by centrifugation. The resulting supernatant was clarified by ultrafiltration (pore size 0.16 μm). The filtrates were then concentrated by ultrafiltration with a membrane with 10kDa cutoff. The peroxidase was purified from the filtrate using FPLC. Two chromatography steps on Q-Sepharose with pH 5 and pH 7.3 gave a 12-fold activity enrichment (specific activity was 7.6 U / mg protein with ABTS, 2.2 azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) Enzyme preparation showed no laccase activity.

Diese Peroxidasepräparation wurde für die folgenden Umsetzungen verwendet .This peroxidase preparation was used for the following reactions.

Beispiel 2 Allgemeine Vorschrift zur Oxidation mit PeroxidaseExample 2 General instructions for oxidation with peroxidase

10 μl der entsprechenden Ausgangsverbindung (substituiertes Toluol) gelöst in Aceton (100 mM) wurde verdünnt in 350 μl Tartrat-Puffer (200 mM pH 5). Es wurden 4,5 Ml Wasserstoffperoxid (50 mM) , 45 μl Wasser und 25 μl einer Peroxidaselösung, die nach Beispiel 1 hergestellt worden war, und eine Aktivität von 30 U/ml aufwies; d.h. 1 ml dieser Peroxidaselösung oxidiert 30 μmol ABTS pro Minute.10 μl of the corresponding starting compound (substituted toluene) dissolved in acetone (100 mM) was diluted in 350 μl tartrate buffer (200 mM pH 5). 4.5 ml of hydrogen peroxide (50 mM), 45 μl of water and 25 μl of a peroxidase solution, which had been prepared according to Example 1, and had an activity of 30 U / ml; i.e. 1 ml of this peroxidase solution oxidizes 30 μmol ABTS per minute.

Die Reaktionsansätze wurden bei Raumtemperatur inkubiert. Mit einer Spritze wurden durch eine Membran alle 10 Minuten 4,5 μl Wasserstoffperoxid (50 mM) zugegeben (insgesamt neun mal) . Zur Halbzeit der Biotransformation wurde zusätzlich 25 μl Peroxidaselösung zugegeben. Die Konzentration der Verbindungen wurde durch Vergleich mit authentischen Standards durch HPLC ermittelt. Die Struktur der erhaltenen Verbindungen wurde durch GC-MS bestätigt.The reaction batches were incubated at room temperature. With a syringe, 4.5 μl hydrogen peroxide (50 mM) was added through a membrane every 10 minutes (a total of nine times). At the halfway point of the biotransformation, an additional 25 μl peroxidase solution was added. The concentration of the compounds was determined by comparison with authentic standards by HPLC. The structure of the compounds obtained was confirmed by GC-MS.

Gemäß Beispiel 2 wurden die in der Tabelle 1 gezeigten Oxidationen durchgeführt . Tabelle 1According to Example 2, the oxidations shown in Table 1 were carried out. Table 1

Oxidation verschiedener Toluole mit Peroxidase und WasserstoffperoxidOxidation of various toluenes with peroxidase and hydrogen peroxide

Figure imgf000005_0003
Figure imgf000005_0003

Figure imgf000005_0001
Figure imgf000005_0002
Figure imgf000005_0001
Figure imgf000005_0002

Figure imgf000006_0001
Figure imgf000006_0001

Figure imgf000006_0002
Figure imgf000006_0002

Figure imgf000007_0001
Figure imgf000007_0001

Figure imgf000007_0002
Figure imgf000007_0002

Figure imgf000008_0001
Figure imgf000008_0001

00

Figure imgf000009_0001
Figure imgf000009_0001

Figure imgf000010_0001
Figure imgf000010_0001

Claims

Patentansprüche claims 1. Verfahren zur Herstellung von Aldehyden der Formel (I) durch Oxidation von Toluolen der Formel (II) mit Wasserstoffperoxid in Gegenwart einer mikrobiellen Peroxidase isolierbar aus einem Mikroorganismus der Gattung Coprinus1. Process for the preparation of aldehydes of the formula (I) by oxidation of toluenes of the formula (II) with hydrogen peroxide in the presence of a microbial peroxidase isolatable from a microorganism of the genus Coprinus
Figure imgf000011_0001
Figure imgf000011_0001
 II I wobeiII I being R = Cι-C4-Alkyl, Cι-C4-Alkoxyl, F, Cl, Br, J, N02, OH und n = 0, 1, 2 bedeutet.R = -C 4 alkyl, -C 4 alkoxyl, F, Cl, Br, J, N0 2 , OH and n = 0, 1, 2 means. 2. Verfahren nach Anspruch 1, dadurch gekennzeichnet, dass die mikrobielle Peroxidase aus Coprinus DSM 14525 verwendet wird.2. The method according to claim 1, characterized in that the microbial peroxidase from Coprinus DSM 14525 is used. 3. Verfahren nach Anspruch 1 oder 2, dadurch gekennzeichnet, daß R = CH3 bedeutet.3. The method according to claim 1 or 2, characterized in that R = CH 3 . 4. Mikrobielle Peroxidase aus Coprinus, die in der Lage ist4. Coprinus microbial peroxidase that is capable Toluol, durch H202 so selektiv zu Benzaldehyd zu oxidieren, daß pro mol oxidiertem Toluol weniger als 0,4 mol Benzoesäure entstehen. Toluene, so selectively oxidized to benzaldehyde by H 2 0 2 that less than 0.4 mol of benzoic acid are formed per mole of toluene oxidized.
PCT/EP2002/010867 2001-10-05 2002-09-27 Method for selective oxidation of substituted toluenes by microbial coprinus peroxydases Ceased WO2003031634A1 (en)

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EP02774660A EP1438414A1 (en) 2001-10-05 2002-09-27 Method for selective oxidation of substituted toluenes by microbial coprinus peroxydases
JP2003534604A JP2005504553A (en) 2001-10-05 2002-09-27 Selective oxidation of substituted toluene with microbial peroxidase derived from microorganisms of the genus Coprinus
US10/491,536 US20040254083A1 (en) 2001-10-05 2002-09-27 Method for selective oxidation of substituted toluenes by microbial coprinus peroxidases

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DE10149266.9 2001-10-05
DE10149266A DE10149266A1 (en) 2001-10-05 2001-10-05 Production of benzaldehydes useful as intermediates comprises oxidizing toluenes with hydrogen peroxide in the presence of a peroxidase from Coprinus microorganism

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022171606A2 (en) 2021-02-09 2022-08-18 F. Hoffmann-La Roche Ag Methods for base-level detection of methylation in nucleic acids

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4503153A (en) * 1982-03-29 1985-03-05 Cetus Corporation Method for producing aldehydes from primary alcohols
US5968883A (en) * 1996-09-03 1999-10-19 Novo Nordisk A/S Peroxidase variants

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4503153A (en) * 1982-03-29 1985-03-05 Cetus Corporation Method for producing aldehydes from primary alcohols
US5968883A (en) * 1996-09-03 1999-10-19 Novo Nordisk A/S Peroxidase variants

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LOOLA AL-KASSIM ET AL: "ENZYMATIC REMOVAL OF SELECTED AROMATIC CONTAMINANTS FROM WASTEWATERBY A FUNGAL PEROXIDASE FROM COPRINUS MACRORHIZUS IN BATCH REACTORS", JOURNAL OF CHEMICAL TECHNOLOGY AND BIOTECHNOLOGY. (INTERNATIONAL JOURNAL OF BIOTECHNICAL AND CHEMICAL PROCESSES), ELSEVIER APPLIED SCIENCE PUBLISHERS. BARKING, GB, vol. 61, no. 2, 1 October 1994 (1994-10-01), pages 179 - 182, XP000469825, ISSN: 0268-2575 *
PETERSEN J F W ET AL: "CRYSTALLIZATION AND X-RAY DIFFRACTION ANALYSIS OF RECOMBINANT COPRINUS CINEREUS PEROXIDASE", JOURNAL OF MOLECULAR BIOLOGY, LONDON, GB, vol. 232, no. 3, 1993, pages 989 - 991, XP000985610, ISSN: 0022-2836 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022171606A2 (en) 2021-02-09 2022-08-18 F. Hoffmann-La Roche Ag Methods for base-level detection of methylation in nucleic acids

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