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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
  • C12N9/90—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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