WO2005017171A2 - Procédé de production d'acides aminés d - Google Patents

Procédé de production d'acides aminés d Download PDF

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Publication number
WO2005017171A2
WO2005017171A2 PCT/EP2004/009000 EP2004009000W WO2005017171A2 WO 2005017171 A2 WO2005017171 A2 WO 2005017171A2 EP 2004009000 W EP2004009000 W EP 2004009000W WO 2005017171 A2 WO2005017171 A2 WO 2005017171A2
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WO
WIPO (PCT)
Prior art keywords
process according
amino acids
nadh oxidase
leucine
leudh
Prior art date
Application number
PCT/EP2004/009000
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English (en)
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WO2005017171A3 (fr
Inventor
Werner Hummel
Birgit Geueke
Mutlu Kuzu
Harald GRÖGER
Original Assignee
Degussa Ag
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Degussa Ag filed Critical Degussa Ag
Publication of WO2005017171A2 publication Critical patent/WO2005017171A2/fr
Publication of WO2005017171A3 publication Critical patent/WO2005017171A3/fr

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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
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/006—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures
    • 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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids

Definitions

  • the present invention focuses on a process for the production of enantiopure D-amino acids.
  • a mixture of D- and L-amino acids is treated with an enzymatic reaction system in such a way that the L-form of the amino acid is removed from the system, leaving only the D-form of the amino acid.
  • the present invention also provides a reaction system that operates in such a way.
  • Enantiopure D-amino acids and in particular sterically demanding representatives thereof are still rewarding targets for organic synthesis, but they can be useful intermediates for the production of bioactive molecules or catalysts .
  • the blood values of branched-chain L-amino acids are measured, and the H 2 0 2 that is formed is analysed by luminol fluorescence (Kiba et al . Analyt. Chim. Acta 1998, 375, 65-70; Kiba et al . J. Chrom. 1996, 724, 354-7) .
  • the object of the present invention was therefore to provide another process for the production of enantiomer- concentrated, in particular sterically demanding D-amino acids, which helps to overcome the disadvantages of the prior art processes.
  • the process according to the invention should offer advantages from an economic and ecological perspective, particularly also with regard to the number of synthesis steps.
  • Claims 2 to 8 relate to preferred embodiments of the process according to the invention.
  • Claim 9 focuses on a reaction system.
  • Claim 10 protects a whole-cell catalyst operating according to the invention.
  • the stated object is advantageously achieved in that, in a process for the production of D-amino acids, a mixture of D- and L-amino acids is reacted with an enzymatic reaction system displaying a leucine dehydrogenase (LeuDH), NAD(H) or NADP(H) and an agent for oxidising NADH or NADPH selected from the group containing an NADH oxidase or an electrochemical arrangement.
  • LeuDH leucine dehydrogenase
  • NAD(H) or NADP(H) an agent for oxidising NADH or NADPH selected from the group containing an NADH oxidase or an electrochemical arrangement.
  • the abbreviation NOX stands for NADH oxidase.
  • an electrochemical oxidation of the cofactor NADH can be performed, with formation of NAD + .
  • the agents that can be used for the oxidation of NADH or NADPH are known to the person skilled in the art. They are in particular the NADH oxidases. Particularly suitable representatives are described in EP1285962 and in the literature cited therein (see also below) . Electrochemical processes can also be used here. The suitability in principle of electrochemical oxidation to regenerate NAD + from NADH is described for example - for oxidation reactions with alcohols - in Kelly, R. M. ; Kirwan, D. J., Biotechnol. Bioeng. 19 (1977) 1215-1218 and Schroder, I . ; Steckhan, E.; Liese, A. Journal of Electronanalytical Chemistry 541 (2003) 109-115. An overview of electrochemical studies in this regard can be found in Gorton, L. Journal of the Chemical Society, Faraday Transactions 1: Physical Chemistry in Condensed Phases 82 (1986) 1245-1258.
  • Sterically demanding in the present case denotes amino acids having a branched radical in the ⁇ position to the carboxyl group.
  • the compounds mentioned in EP692538 in this respect can be regarded as being sterically demanding, for example. They are in particular amino acids selected from the group comprising tert.- leucine, neopentyl glycine, adamantyl glycine.
  • LeuDH that is familiar to the person skilled in the art and can be used for the purpose under consideration can be used in principle as leucine dehydrogenase.
  • LeuDHs can be found for example in A. Bommarius in: Enzyme Catalysis in Organic Synthesis (Editors: K. Drauz, H. Waldmann) , Volume III, Wiley-VCH, Weinheim, 2002, chapter 15.3.
  • the LeuDH in accordance with that from Bacillus species can preferably be used. Most particularly preferred is the LeuDH in accordance with that from Bacillus cereus, B . stearothermophilus or B. sphaericus .
  • Mutagenesis processes which can bring about improved stability, for example heat stability, storage stability and/or selectivity of the LeuDH, are known to the person skilled in the art. They are in particular saturation mutagenesis, random mutagenesis, shuffling methods and site-directed mutagenesis (Eigen M. and Gardinger W. (1984) Evolutionary molecular engineering based on RNA replication. Pure & Appl . Chem. 56(8), 967- 978; Chen & Arnold (1991) Enzyme engineering for nonaqueous solvents: random mutagenesis to enhance activity of subtilisin E in polar organic media. Bio/Technology 9, 1073-1077; Horwitz, M. and L. Loeb (1986) "Promoters
  • NADH oxidases that can be used in the process according to the invention are familiar to the person skilled in the art (Nishiyama, Y. et al . J. Bacteriol. Vol. 183 (2001), 2431- 2438; Lopez de Felipe, F. et al . , J. Bacteriol. Vol. 180 (1998) , 3804-08) .
  • Examples which yield irreversibly inert water as the reduction product are preferably used, since a formation of secondary products can be avoided in this way, in contrast to hydrogen peroxide-forming NADH oxidases. These can come from the Lactobacillus organism, for example.
  • NADH oxidase in accordance with that from Lactobacillus kefir or Lactobacillus brevis is particularly advantageous.
  • An NADH oxidase that can most particularly preferably be used can be taken from DE1014088.
  • the enzymatic reaction system is preferably used in the form of a whole-cell catalyst displaying cloned genes for both enzymes .
  • the whole-cell catalyst according to the invention should preferably display an NADH oxidase or leucine dehydrogenases .
  • the representatives claimed above in particular are especially suitable.
  • the process is preferably performed at pH values in the range from 4 to 9 , particularly preferably 5 to 8.
  • the temperature range in the preferred embodiment is 15 to 100°C, preferably 20 to 50°C and in particular 25 to 40°C. It can also be advantageous to perform the process in a two-phase mixture consisting of water and an organic phase, in which the keto acid dissolves very readily. The enzymatic reaction takes place in the aqueous phase, wherein the keto acid that is formed concentrates in the organic phase and can therefore be readily separated from the remaining mixture of substances.
  • the invention also provides an enzymatic reaction system displaying a leucine dehydrogenase (LeuDH), NAD(H) or NADP(H) and an NADH oxidase, together with a mixture of the D- and L-form of an amino acid.
  • LeuDH leucine dehydrogenase
  • NAD(H) or NADP(H) an NADH oxidase
  • the enzyme under consideration can be used for the application in free form as a homogeneously isolated compound.
  • the enzyme can also be used as a component of an intact guest organism or in conjunction with the digested cell mass, isolated to the desired level, of the host organism.
  • the enzymes can also be used in immobilised form (Bhavender P. Sharma, Lorraine F. Bailey and Ralph A. Messing, "Immobilinstrumente Biomaterialien -techniken und füren” , Angew. Chem. 1982, 94, 836- 852) .
  • the immobilisation is advantageously performed by lyophilisation (Dordick et al . J. Am. Chem. Soc. 194, 116, 5009-5010; Okahata et al .
  • a racemic mixture of a preferably aliphatic amino acid is dissolved in water at a pH of around 8.5 and brought into contact with an appropriately buffered medium displaying NAD + , LeuDH and NADH oxidase. This initiates the reaction of the L-amino acid to the corresponding keto acid.
  • the enantiopure D-amino acid remains, which can easily be separated from the reaction mixture and isolated by common methods .
  • keto acid produced in the reaction undergoes reductive amination again.
  • This process can either be non-selective or as strongly D-selective as required. In this way up to 100% of the D-amino acid can be obtained from a batch of racemate if the cycle described is executed several times in succession (diagram below) .
  • optically concentrated (enantiomer-concentrated, enantiopure) compounds is understood to refer to the presence of an optical antipode combined with the other in >50 mol%.
  • Lactobacillus brevis DSM 20054 or Lactobacillus kefir DSM 20587 is stored in the Deutsche Sammlung fur Mikroorganismen und Zellkulturen under the corresponding number and is available to the public.
  • the volume of the reaction mixture for enzymatic oxidation is 1 ml.
  • This reaction mixture containing 290 ⁇ l tris buffer (50 mM, pH 8.0), 100 ⁇ l D, L- tert-leucine (100 mM) , 10 ⁇ l NAD+ (20 mM) , lOO ⁇ l NADH oxidase (42 U-ml "1 ) , und 500 ⁇ l leucine dehydrogenase (0.5 U-ml "1 ), is incubated at 30°C.
  • leucine dehydrogenase corresponds here to the amount of leucine dehydrogenase that catalyses the oxidation of 1 ⁇ mol of L- ert-leucine per minute in the presence of 20 mM D,L- tert-leucine at a pH of 8.0 (0.1 M potassium phosphate buffer) .

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Genetics & Genomics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Abstract

La présente invention concerne un procédé de production d'acides aminés D énantiopurs. En particulier, un mélange d'acides aminés D et L est traité à l'aide d'un système de réaction enzymatique de telle sorte que la forme L de l'acide aminé est éliminée du système, laissant uniquement la forme D de l'acide aminé. La présente invention concerne également un système de réaction qui fonctionne de ladite façon et un catalyseur de cellule entière qui peut être utilisé pour le procédé selon l'invention.
PCT/EP2004/009000 2003-08-16 2004-08-12 Procédé de production d'acides aminés d WO2005017171A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE2003137614 DE10337614A1 (de) 2003-08-16 2003-08-16 Verfahren zur Herstellung von D-Aminosäuren
DE10337614.3 2003-08-16

Publications (2)

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WO2005017171A2 true WO2005017171A2 (fr) 2005-02-24
WO2005017171A3 WO2005017171A3 (fr) 2005-06-23

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WO (1) WO2005017171A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006113085A3 (fr) * 2005-03-28 2007-05-10 Biocatalytics Inc Deshydrogenase d'acides amines d et procede de production d'acides amines d

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995011296A1 (fr) * 1993-10-18 1995-04-27 Degussa Aktiengesellschaft Procede de modification controlee d'enzymes, enzymes modifiees et utilisation
US5416019A (en) * 1984-12-19 1995-05-16 Degussa Aktiengesellschaft Rhodococcus microorganisms that produce phenylalanine dehydroganase
EP0692538A2 (fr) * 1994-07-15 1996-01-17 Degussa Aktiengesellschaft Procédé pour la préparation des acides L-aminés optiquement actifs, nouveaux acides L-aminés optiquement actifs avec des groupements latéraux à encombrement spatial et leur utilisation
EP0919630A1 (fr) * 1997-04-22 1999-06-02 Kaneka Corporation Procede de preparation d'acetals de l-allysine

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Publication number Priority date Publication date Assignee Title
JPS5365781A (en) * 1976-11-25 1978-06-12 Mitsui Toatsu Chemicals Reagent for quantitative analysis of llamino acid and alphaaketo acid
EP0735143B1 (fr) * 1995-03-28 1998-11-18 Tanabe Seiyaku Co., Ltd. Procédé pour la préparation des acides D-aminés
JP4125809B2 (ja) * 1997-10-01 2008-07-30 天野エンザイム株式会社 Ｄ−アミノ酸の製造法
DE10055512A1 (de) * 2000-11-09 2002-05-23 Degussa L-Aminosäure-Oxidase aus Rhodococcus-Arten
DE10218689A1 (de) * 2002-04-26 2003-11-20 Degussa ADH aus Rhodococcus erythropolis

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5416019A (en) * 1984-12-19 1995-05-16 Degussa Aktiengesellschaft Rhodococcus microorganisms that produce phenylalanine dehydroganase
WO1995011296A1 (fr) * 1993-10-18 1995-04-27 Degussa Aktiengesellschaft Procede de modification controlee d'enzymes, enzymes modifiees et utilisation
EP0692538A2 (fr) * 1994-07-15 1996-01-17 Degussa Aktiengesellschaft Procédé pour la préparation des acides L-aminés optiquement actifs, nouveaux acides L-aminés optiquement actifs avec des groupements latéraux à encombrement spatial et leur utilisation
EP0919630A1 (fr) * 1997-04-22 1999-06-02 Kaneka Corporation Procede de preparation d'acetals de l-allysine

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
BOMMARIUS A. S. ET AL: "Comparison of different chemoenzymatic process routes to enantiomerically pure amino acids" CHIMIA, vol. 55, no. 1-2, 2001, pages 50-59, XP001204656 *
BOMMARIUS A.S. ET AL: "Some new developments in reductive amination with cofactor regeneration" BIOCATALYSIS, vol. 10, no. 1-4, 1994, pages 37-47, XP008045061 *
DATABASE WPI Section Ch, Week 197829 Derwent Publications Ltd., London, GB; Class B05, AN 1978-52387A XP002323148 & JP 53 065781 A (MITSUI TOATSU CHEM INC) 12 June 1978 (1978-06-12) *
DRAUZ K. H.: "Chiral amino acids. A versatile tool in the synthesis of pharmaceuticals and fine chemicals" CHIMIA, vol. 51, no. 6, 1997, pages 310-314, XP001204657 *
HUMMEL W. ET AL: "An efficient and selective enzymatic oxidation system for the synthesis of enantiomerically pure D-tert-leucine." ORGANIC LETTERS, vol. 5, no. 20, 2 October 2003 (2003-10-02), pages 3649-3650, XP002323147 ISSN: 1523-7060 *
KIBA N. ET AL: "Chemiluminometric branched chain amino acids determination with immobilized enzymes by flow-injection analysis" ANALYTICA CHIMICA ACTA, vol. 375, no. 1-2, 27 November 1998 (1998-11-27), pages 65-70, XP002323146 ISSN: 0003-2670 cited in the application *
KIBA N. ET AL: "Postcolumn co-immobilized leucine dehydrogenase-NADH oxidase reactor for the determination of branched-chain amino acids by high-performance liquid chromatography with chemiluminescence detection" JOURNAL OF CHROMATOGRAPHY A, vol. 724, no. 1, 16 February 1996 (1996-02-16), pages 354-357, XP004039579 ISSN: 0021-9673 cited in the application *
KRIX G ET AL: "Enzymatic reduction of alpha-keto acids leading to l-amino acids, d- or l-hydroxy acids" JOURNAL OF BIOTECHNOLOGY, vol. 53, no. 1, 28 February 1997 (1997-02-28), pages 29-39, XP004075445 ISSN: 0168-1656 *
LAUMEN K. ET AL: "Enzyme-assisted preparation of D-tert.-leucine" BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, vol. 65, no. 9, September 2001 (2001-09), pages 1977-1980, XP002323144 ISSN: 0916-8451 cited in the application *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006113085A3 (fr) * 2005-03-28 2007-05-10 Biocatalytics Inc Deshydrogenase d'acides amines d et procede de production d'acides amines d
US7550277B2 (en) 2005-03-28 2009-06-23 Codexis, Inc. D-amino acid dehydrogenase and method of making D-amino acids

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WO2005017171A3 (fr) 2005-06-23
DE10337614A1 (de) 2005-03-17

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