EP1220898A2 - Hydanto ne-rac mase - Google Patents

Hydanto ne-rac mase

Info

Publication number
EP1220898A2
EP1220898A2 EP00964094A EP00964094A EP1220898A2 EP 1220898 A2 EP1220898 A2 EP 1220898A2 EP 00964094 A EP00964094 A EP 00964094A EP 00964094 A EP00964094 A EP 00964094A EP 1220898 A2 EP1220898 A2 EP 1220898A2
Authority
EP
European Patent Office
Prior art keywords
racemase
hydantoin
gene
enzyme
racemization
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP00964094A
Other languages
German (de)
English (en)
Inventor
Josef Altenbuchner
Ralf Mattes
Markus Pietzsch
Christoph Syldatk
Anja Wiese
Andreas Bommarius
Wilhelm Tischer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universitaet Stuttgart
Evonik Operations GmbH
Original Assignee
Degussa GmbH
Roche Diagnostics GmbH
Universitaet Stuttgart
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.)
Filing date
Publication date
Application filed by Degussa GmbH, Roche Diagnostics GmbH, Universitaet Stuttgart filed Critical Degussa GmbH
Priority to EP00964094A priority Critical patent/EP1220898A2/fr
Priority to DE20023437U priority patent/DE20023437U1/de
Priority claimed from PCT/EP2000/008580 external-priority patent/WO2001023535A2/fr
Publication of EP1220898A2 publication Critical patent/EP1220898A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C12N9/90—Isomerases (5.)
    • 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
    • C12P41/009—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 by reactions involving hydantoins or carbamoylamino compounds

Definitions

  • the instant invention is directed to a hydantoin-racemase from Arthrobacter aurescens (DSM 3747, hyuA) .
  • the chemical racemization of hydantoins proceeds via enolisation.
  • the velocity depends on the electronic nature of the residue at the 5 '-position (Ware, Chem. Rev. (1950), 46, 403-470) but usually, the racemization is a very slow process.
  • the racemization is a very slow process.
  • at room temperature and pH 8.5 only about 10 % of -IMH is racemized to D-IMH in 20 hour (Syldatk et al . , "Biocatalytic production of amino acids and derivatives" (1992), Hanser publishers, New York, pp. 75-176) .
  • the rate of racemization is increased by a very basic pH (>10) and high temperature (>80 °C) .
  • Arthrobacter (Syldatk et al . , "Biocatalytic production of amino acids and derivatives” (1992), Hanser publishers, New York, pp. 75-176; Syldatk et al . , "Hydrolysis and formation of hydantoins” (1995), VCH Verlag, Weinheim, pp. 409-434) and a Pseudomonas species (Watabe et al . , J. Bacteriol . (1992a), 174, 3461-3466; Watabe et al . , J. Bacteriol. (1992b), 174, 7989-7995). Only the latter is also characterised in terms of nucleotide sequence and genetic organisation .
  • an object of this invention to provide another rec-hydantoin-racemase, which is able to racemize hydantoins under physiological conditions with an acceptable rate for their implementation in a process for the production of enantiomerically enriched amino carboxylic acids on industrial scale.
  • the racemase according to the invention can advantageously be incorporated in a large scale process for the production of enantiomerically enriched amino carboxylic acids.
  • the feasibility of providing the racemase in a recombinant manner is the clue for acceptance of this process in view of economic efficiency.
  • a gene (Seq. 3) encoding for the racemase according to the invention is protected.
  • the gene with relation to the framework of this invention is seen as a group of genes comprising all possible genes encoding for the protein in question according to the degeneration of the genetic code.
  • this invention encompasses plasmids, vectors and micro-organisms, which comprise the gene of instant invention.
  • plasmids, vectors and micro-organisms which could advantageously be used to carry out the invention and are known to the skilled worker are incorporated herewith.
  • those mentioned in Studier et al . , Methods Enzymol . 1990, 185, 61-69 or those presented in brochures of Novagen, Promega, New England Biolabs, Clontech or Gibco BRL are deemed to be suitable. More applicable plasmids, vectors can be found in:
  • Denhardt, D. T. and Colasanti, J. A surey of vectors for regulating expression of cloned DNA in E. coli .
  • Rodriguez, R.L. and Denhardt, D. T (eds) Vectors, Butterworth, Stoneham, MA, 1987, ppl79-204;
  • primers useful for the amplification of the gene of the invention in a PCR are protected similarly.
  • Primers which are feasible are for example: 51137 5' -AGAACATATGAGAATCCTCGTGATCAA-3 ' (Seq. 1)
  • racemase of the invention is used in a process for the production of amino carboxylic acids or derivatives thereof.
  • it is used according to the invention in a process for the production of enantiomerically enriched derivatives.
  • the use is conducted in a covalent enzyme-membrane-reactor (DE19910691.6 ) or after non-covalent or covalent immobilisation to solid carriers (DE 197 033 14) .
  • the gene was amplified by PCR from plasmid pAWl ⁇ using the primers S1137 and S1138 and placed under the control of a rhamnose promoter provided by the expression system pJOE2702.
  • the resulting plasmid was designated pAW210 (Fig. 1) .
  • the E. coli cells harbouring pAW210 exhibited specific hydantoin racemase activities up to a maximum of 60 U/mg in crude cell extracts (Fig. 2) .
  • the racemase activity was determined in crude extracts by polarimetry using 3 M L-BH as substrate (Teves et al . , Fresenius ' J. Anal. Chem. 1999, 363, 738-743) .
  • the plasmid pAW210 in E. coli JM109 was used for purification of the racemase.
  • a two step procedure consisting of ammonium sulfate fractionation and MonoQ - anion exchange chromatography was accomplished as described down under.
  • the racemase was purified 10-fold to homogeneity, with 35 % overall recovery (Tab. 1) .
  • Protein was purified on MonoQ in 4 separate runs using 4 mg for each run.
  • the specific activity of the purified enzyme was determined by standard enzyme assay with D-Benzylhydantoin as substrate at 313 U/mg. In potassium phosphate buffer, pH 7.0 with 25 % glycerol, the purified enzyme could be stored for at least 6 months at -20 °C without noticeable loss of activity.
  • the matrix assisted laser desorption ionisation spectrum (MALDI) of the purified racemase gave a peak at a molecular mass of 25078.7. This is in good agreement with the calculated value of 25085 Da in contrast to the SDS-PAGE electrophoresis which gave a relative molecular mass of 31 kDa for the racemase monomer.
  • MALDI matrix assisted laser desorption ionisation spectrum
  • the relative molecular mass of the native enzyme was estimated to be approximately 170 kDa ⁇ 25. Due to the small subunit of 25 kDa and inaccuracy of the gel filtration method within this range the native enzyme is suggested to be either a hexamer, heptamer or octamer.
  • Fig. 3-5 The effect of pH and temperature on the enzyme activity and stability are illustrated in Fig. 3-5.
  • the pH optimum was determined between pH 8.0 and 9.0. Consequently, all standard assays were performed at pH 8.5.
  • the optimum temperature for racemization of L-BH was around 55 °C, however the stability of the enzyme under assay conditions (Tris, pH 8.5) was only maintained up to 45 °C.
  • the K M values of IMH and BH could not be determined due to the limited solubility of the substrates. Instead the initial velocities at different concentrations of L-MTEH were measured. The kinetic plot (Fig.6) showed that the racemase is inhibited by the substrate L-MTEH. Even at low substrate concentrations (> 5 mM) inhibition is observed.
  • microorganism Arthrobacter aurescens used for the invention was desposited at Deutsche Sammlung fur
  • E. coli JM109 (Yanisch-Perron et al . , Gene (1985), 33, 103-109) was used for cloning, sequencing and expression the hyuA gene from Arthrobacter aurescens DSM 3747 (Gro ⁇ et al . , Biotech. Tech. (1987), 2, 85-90) .
  • E. coli strains were cultivated in 2xYT liquid broth or on 2xYT agar (Sambrook et al . , Molecular Cloning: A Laboratory Manual (1989), Cold Spring Harbour Laboratory Press, New York) . The media were supplemented with 100 ⁇ g/ml ampicillin to select plasmid carrying strains. The cultures were grown at 37°C, for hyuA expression the growth temperature was reduced to 30°C.
  • the racemase gene was amplified by PCR using the primers S1137 (5'- AGAACATATGAGAATCCTCGTGATCAA-3 ' ) and S1138 (5'- AAAACTGCAGCTAGAGGTACTGCTTCTCTG-3 ' ) and pAW16 as template (Wilms et al . , J. Biotechnol . (1999), 68, 101-113).
  • the fragment was inserted between the Ndel and Pstl sites of the expression vector pJOE2702 (Volff et al . , Mol . Microbiol. (1996), 21, 1037-1047) to create plasmid pAW210.
  • Expression was induced by addition of 0.2 % rhamnose to cultures at an optical density of 0.3 at 600 nm. After 6 h, cells corresponding to OD 60 o of 10 were harvested, washed and resuspended in 1 ml desintegration buffer (0.07 M potassium phosphate, pH 7.0) and lysed by sonification (Ultrasonics sonicator, microtip, 2 x 30 s, duty cycle 50 % pulsed) . Clarified extracts were obtained by centrifugation at 14000 rpm for 10 min .
  • the second precipitate obtained by centrifugation was resuspended in buffer A (10 mM potassium phosphate, pH 6.5) and applied to a MonoQ ® HR 5/5 column equilibrated in buffer A and eluted with a linear gradient of 0 to 1.0 M NaCl in buffer A.
  • HyuA was eluted at a concentration of 0.37 M NaCl .
  • Peak fractions were pooled and dialyzed against desintegration buffer, glycerol was added to a final concentration of 25 % and stored at -20 °C.
  • Protein characterisation Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was done according to the method of Laemmli (Laemmli, Nature (1970), 227, 680-685) . Protein concentrations were determined by the method of Bradford (Bradford, Anal. Biochem. (1976), 72, 248-254) using the Biorad protein assay dye reagent concentrate. Standard curves were generated with bovine serum albumin. The M r of native protein was determined by gel filtration using superosel2HR column as described previously (Wil s et al . , J. Biotechnol .
  • the column was equilibrated and eluted with buffer consisting of 0.1 M potassium phosphate and 0.1 M NaCl, pH 7.
  • buffer consisting of 0.1 M potassium phosphate and 0.1 M NaCl, pH 7.
  • the pH profile of the purified racemase was measured between the pH range 7.0 to 9.5 in Tris buffer.
  • the substrate was dissolved in 0.1 M Tris, pH 3 at 45 °C using an ultrasonic waterbath. After cooling to room temperature, the pH was adjusted to the desired pH with sodium hydroxide and enzyme activity was determined using the standard assay.
  • the reaction temperature optimum of purified racemase was determined using temperatures between 25 and 65 °C in the standard assay.
  • the stability of the enzyme was measured after preincubation at temperatures between 25 and 70 °C for 15 minutes in the presence of desintegration buffer and 0.1 M Tris buffer, pH 8.5, respectively. The increased chemical racemization at high pH and temperatures, respectively, was considered.
  • the effect of EDTA, DTT, HgCl 2 and iodoacetamid on HyuA was tested by incubation of respective substance (10 mM) and purified enzyme (12 ⁇ g) in desintegration buffer (final volume 20 ⁇ l) at 30°C. After 1 h specific activities were determined by the standard enzyme assay.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Analytical Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Cette invention a trait à une rec-hydantoïne-racémase issue de <i>Arthrobacter aurescens</i> DSM 3747. De plus, il faut protéger le gène codant la racémase et les plasmides ainsi que les vecteurs et les micro-organismes renfermant ce gène. Il est également question de l'usage qui en fait, s'agissant de produire des acides amino carboxyliques ou leurs dérivés.
EP00964094A 1999-09-27 2000-09-02 Hydanto ne-rac mase Withdrawn EP1220898A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP00964094A EP1220898A2 (fr) 1999-09-27 2000-09-02 Hydanto ne-rac mase
DE20023437U DE20023437U1 (de) 1999-09-27 2000-09-02 Hydantoin-Racemase

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP99118956 1999-09-27
EP99118956 1999-09-27
EP00964094A EP1220898A2 (fr) 1999-09-27 2000-09-02 Hydanto ne-rac mase
PCT/EP2000/008580 WO2001023535A2 (fr) 1999-09-27 2000-09-02 Hydantoïne-racémase

Publications (1)

Publication Number Publication Date
EP1220898A2 true EP1220898A2 (fr) 2002-07-10

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EP00964094A Withdrawn EP1220898A2 (fr) 1999-09-27 2000-09-02 Hydanto ne-rac mase

Country Status (2)

Country Link
EP (1) EP1220898A2 (fr)
DE (1) DE20023437U1 (fr)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0123535A2 *

Also Published As

Publication number Publication date
DE20023437U1 (de) 2004-07-29

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