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Method for the microbiological isomerisation of alpha-hydroxy carboxylic acids

A technology of hydroxycarboxylic acid and microorganism, which is applied in the field of microbial isomerization of α-hydroxycarboxylic acid, which can solve the problems of high substrate specificity and other problems

Inactive Publication Date: 2006-07-26
BASF SE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, currently known racemases are characterized by high substrate specificity
Therefore, mandelate racemase is only suitable for the biocatalytic racemization of β, γ-unsaturated α-hydroxy acids

Method used

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  • Method for the microbiological isomerisation of alpha-hydroxy carboxylic acids
  • Method for the microbiological isomerisation of alpha-hydroxy carboxylic acids

Examples

Experimental program
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example

[0136] a) Racemization of the [alpha]-hydroxycarboxylic acid after withdrawal of the desired product in order to recycle the stereoisomer which is not desired in conventional racemate separations. The advantage is a purely enzymatic racemization reaction; in contrast, chemically catalyzed racemization reactions usually react under severe reaction conditions leading to decomposition, formation of secondary products and side reactions (elimination, etc.). (cf. also: Kontrollierte Racemisierung von organischen Verbindungen [Controlled racemization of organic compounds]: E.J. Ebbers, G.J.A. Arians, J.P.M. Houbiers, A. Bruggink, B. Zwanenburg, Tetra-hedron, 1997, 53, 9417-9476).

[0137] b) Stepwise deracemization of α-hydroxycarboxylic acids. If the racemase cannot be used directly in the (chemical and enzymatic) enantioselective step in a "one-pot approach" (e.g. due to the required reaction conditions), then enzymatic racemization can be performed after the enantioselective step...

Embodiment 1

[0186] Embodiment 1: the synthesis of substrate and standard substance

[0187] a) Synthesis of Enantiomerically Pure 2-Hydroxy-3-Phenylalanine

[0188] Reaction mixture: 1 g of enantiomerically pure phenylalanine (6.05 mmol) was dissolved in 12 ml of H 2 SO 4 (1M). Add 1.66 g (24 mmol) of ice-cold NaNO in portions 2 . After the addition, the cooling system was removed and the reaction mixture was stirred overnight at room temperature.

[0189] Procedure (Work-up): In each case the aqueous solution was extracted 3 times with 4 ml of diethyl ether, the organic phase was washed with saturated sodium chloride solution and the sodium chloride solution was extracted again with diethyl ether. Combine the organic phases and wash with Na 2 SO 4 Dry and evaporate the solvent on a Rotavapor. The yellow oily residue was treated with 2 mL of hexane and crystallized by scraping with a glass rod. A white crystalline precipitate formed. The crystals were washed three times with abo...

Embodiment 2

[0211] Example 2: Screening for racemase activity

[0212] To get a first indication of the racemic activity of microorganisms, observe their reaction to the (natural) substrate (S)-lactic acid 1 and / or the (unnatural) substrates (S)-phenyllactic acid 2 and (S)- Racemization of 4-fluorophenyllactate 3.

[0213] For screening, approximately 10 mg of freeze-dried cells were rehydrated in 900 μl of Bis-Tris buffer (50 mM Bis-Tris, pH=6) in Eppendorf flasks for 1 hour at 42° C. and 150 rpm. Then, 10 mg of substrate were added in each case, which had been previously dissolved in 100 μl of buffer and adjusted to pH 6-7 with NaOH. Incubate the reaction mixture at 42 °C and 150 rpm in a shaker incubator. A blank was measured in parallel with each reaction mixture, in which the same reaction conditions were maintained but without the addition of cells. Since all control experiments were negative, spontaneous racemization under these given reaction conditions could be ruled out.

[...

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Abstract

The invention relates to a method for the microbiological isomerisation of alpha-hydroxy carboxylic acids using an alpha-hydroxy carboxylic acid racemase, to the enzymes and micro-organisms that express a suitable racemase activity, to a screening method for micro-organisms with alpha-hydroxy carboxylic acid racemase activity, to nucleic acid sequences, expression vectors and recombinant micro-organisms that code for said enzyme and express said racemase and to a method for the production or isolation of a protein with alpha-hydroxy carboxylic acid racemase activity.

Description

technical field [0001] The present invention relates to a method for the isomerization of α-hydroxycarboxylic acids using α-hydroxycarboxylic acid racemase microorganisms, enzymes used in the method and microorganisms expressing appropriate racemase activity, having α-hydroxycarboxylic acid exogenous A method for screening microorganisms with racemase activity, a nucleic acid sequence encoding the enzyme, an expression vector, a recombinant microorganism expressing the racemase, and a method for producing or isolating a protein having α-hydroxycarboxylic acid racemase activity. Background technique [0002] Known in the prior art is the enzyme known as microbial mandelate racemase, which is capable of racemizing mandelic acid both in vitro and in vivo (G.L. Kenyon, J.A. Gerit, G.A. Petsko and J.W. Kozarich, "Mandelate Racemase: Structure-Function Studies of aPseudosymmetric Enzyme", Accts.Chem.Res., 28, 178-186 (1995); R.Li, V.M.Powers, J.W.Kozarich and G.L.Kenyon, "Racemiza...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/42C12P41/00C12N1/20C12N15/01C12N9/90C12N15/52C12N15/11C12N15/74C12R1/225C12Q1/04C12Q1/533
CPCC12N1/20C12R1/225C12Q1/04C12N9/90C12Q1/533G01N2500/00C12P7/42C12R2001/225C12N1/205
Inventor S·格卢克B·施内尔M·皮克尔K·费伯
Owner BASF SE
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