DE20023437U1 - Hydantoin racemase - Google Patents

Abstract

Rec-hydantoin racemase, characterized in that it comes from Arthrobacter aurescens DSM 3747.

Description

The present invention relates to a hydantoin racemase from Arthrobacter aurescens (DSM 3747, hyuA).

Within the agriculture industries, There is a growing interest in the food and pharmaceutical industries Production of optically pure aminocarboxylic acids. Especially the enzymatic one Hydrolysis of hydantoins is an attractive method for synthesis of D and L amino acids with regard to inexpensive raw materials and a full one Substrate turnover.

There have been various hydantoin breakdowns Microorganisms were isolated and the enzymatic conversion of 5'-monosubstituted hydantoins was carried out in detail studied (Syldatk and Pietzsch, "Hydrolysis and formation of hydantoins "(1995), VCH Verlag, Weinheim, pp. 409-434; Ogawa et al., J. Mol. Catal. B: enzyme. 2: 163-176 (1997); Syldatk, C., May, O., Altenbuchner, J., Mattes, R. and Siemann, M. (1999) Microbiol. hydantoinases - industrial enzymes from the origin of life? Appl. Microbiol. Biotechnol. 51 293-309). Asymmetric bioconversion into L or D amino acids includes the following 3 steps:

• (i) chemical and / or enzymatic racemization of 5 'substituted hydantoins
• (ii) Ring opening hydrolysis achieved by a hydantoinase and
• (iii) Carbamoylase-catalyzed hydrolysis of the second Step generated N-carbamoyl amino acid.

The chemical racemization of hydantoins passes over enolization. The speed is electronic Condition of the rest depending on the 5 'position (Ware, Chem. Rev. (1950), 46, 403-470), usually however, racemization is a very slow process. At room temperature and a pH of 8.5, for example, within 20 hours only about 10% L-IMH racemized to D-IMH (Syldatk et al., "Biocatalytic production of amino acids and derivatives "(1992), Hanser publishers, New York, pp. 75-176). The racemization rate is increased by a very basic pH value (> 10) and a high temperature (> 80 ° C).

Under physiological conditions is a high racemization rate due to hydantoin-specific Racemases reached. So far, hydantoin racemases have been purified and characterized by 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 characterized in terms of nucleotide sequence and genetic makeup.

It was therefore a goal of the present Invention to provide another Rec-hydantoin racemase the hydantoins under physiological conditions with an acceptable Speed for their implementation in an entantiomeric production process enriched aminocarboxylic acids on an industrial scale to racemize.

By providing the recombinant derived hydantoin racemase from Arthrobacter aurescens DSM 3747 (Seq. 4) can refer to the above Task to be waived. In particular, the racemase according to the invention advantageously in an industrial scale Process for the preparation of enantiomerically enriched aminocarboxylic acids included become. The possibility the provision of the racemase in a recombinant manner the key to accept this method in terms of economic Efficiency.

Furthermore, a gene (Seq. 3), the the racemase according to the invention encoded, protected. In the context of the present invention, the gene is considered a group viewed by genes that all sorts Genes includes the protein in question according to the degeneration of the genetic Encode codes.

In another embodiment, the present one closes Invention Plasmids, Vectors and Microorganisms Containing the Gene of the present invention. As part of the present Invention are all plasmids, vectors and microorganisms that can advantageously be used to implement the invention could and are known to those skilled in the art. In particular described in Studier et al., Methods Enzymol. 1990, 185, 61-69 mentioned or those in brochures from Novagen, Promega, New England Biolabs, Clontech or Gibco BRL presented are found to be suitable. More suitable plasmids, vectors can be found in:

• DNA cloning: a practical approach. Volumes I-III by D. M. Glover, IRL Press Ltd., Oxford, Washington DC, 1985, 1387;
• Denhardt, D.T. and Colasanti, J .: A surey of vectors for regulating expression of cloned DNA in E. coli. In: Rodriguez, R.L. and Denhardt, D. T (ed.), Vectors, Butterworth, Stoneham, MA, 1987, pp. 179-204;
• Gene expression technology. In: Goeddel, D.V. (ed.), Methods in Enzymology, volume 185, Academic Press, Inc., San Diego, 1990;
• Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular cloning: a laboratory manual, 2nd edition. Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.

In addition, primers that helpful for amplifying the gene according to the invention in a PCR are protected as well. Possible Examples of primers are:

Furthermore, all other primers, which could be used to implement the present invention and that are known to the skilled person are considered useful in this sense. A suitable primer is found by comparison known DNA sequences or the translation of amino acid sequences into the codon of the organism in question (e.g. for streptomycetes: Wright et al., Gene 1992, 113, 55-65). similarities in amino acid sequences of so-called superfamily proteins are also helpful in this regard (Firestine et al., Chemistry & Biology 1996, 3, 779-783). For more information, see Oligonucleotide synthesis: a practical approach, edited by M.J. Gait, IRL Press Ltd, Oxford Washington DC, 1984; PCR Protocols: A guide to methods and applications by M.A. Innis, D.H. Gelfound, J.J. Sninsky and T.J. White. Academic Press, Inc., San Diego, 1990. These strategies are hereby included by reference.

Another embodiment of the present invention is the use of the racemase according to the invention in a process for the preparation of aminocarboxylic acids or derivatives thereof. It is preferably used according to the invention in a process for the preparation of enantiomerically enriched derivatives. It is most preferably used in a covalent enzyme membrane reactor (DE19910691.6) or after a noncovalent or covalent immobilization on solid supports ( DE 197 033 14 ).

For detection of the enzyme function, the gene was amplified by PCR from plasmid pAW16 with primers 51137 and 51138 and placed under the control of a rhamnose promoter provided by the expression system pJOE2702. The resulting plasmid was named pAW210 ( 1 ). The E. coli cells harboring the pAW210 had specific hydantoin racemase activities up to a maximum of 60 U / mg in raw cell extracts ( 2 ). Racemase activity was determined in crude extracts by polarimetry using 3 mM L-BH as substrate (Teves et al., Fresenius' J. Anal. Chem. 1999, 363, 738-743). A 31 kDa abundant protein, which represented approximately 10% of the total cellular protein, was detected by SDS-PAGE analysis in rhamnose-induced cells and was mainly present in the soluble fraction of the raw cell extracts.

The plasmid pAW210 in E. coli JM109 was used to purify the racemase. A two-step process consisting of ammonium sulfate fractionation and MonoQ anion exchange chromatography as described below. The racemase became 10-fold on homogeneity cleaned with a total yield of 35% (Tab. 1).

Table 1: Purification of Racemase HyuA from E. Coli JM109 pAW210

The specific activity of the purified enzyme was determined by a standard enzyme approach with D-Ben cylhydantoin determined as a substrate with 313 U / mg. In potassium phosphate buffer, pH 7.0, with 25% glycerol, the purified enzyme could be stored at -20 ° C for at least 6 months without any apparent loss of activity.

Matrix-assisted laser desorption / ionization spectrometry (MALDI) the purified racemase produced a peak with a molecular mass of 25078.7. In contrast to SDS-PAGE electrophoresis, this is true a relative molecular mass of 31 kDa for the racemase monomer performed quite well with the calculated value out of 25085 there. The relative molecular mass of the. Was measured on a calibrated Superose 12 HR column native enzyme estimated to be approximately 170 kDa ± 25. Because of the small subunit of 25 kDa and an inaccuracy of the gel filtration process in this area it is suggested that the native enzyme either is a hexamer, heptamer or octamer.

The effect of pH and temperature on enzyme activity and stability is in the 3 - 5 shown. The optimal pH was between 8.0 and 9.0. As a result, all standard tests were carried out at a pH of 8.5. The optimal temperature for racemization of L-BH was around 55 ° C, but the stability of the enzyme could only be maintained up to 45 ° C under test conditions (Tris, pH 8.5).

It was the racemization of the 5-substituted Hydantoine BH, IMH and MTEH examined by HyuA (Tab. 2).

Table 2: Substrate specificity of HyuA

L and D-BH had the highest activity rates, whereas the L and D isomer of MTEH is rather poorly racemized which was an indication that aromatic hydantoins were preferred as substrates.

The K _M values of IMH and BH could not be determined due to the limited solubility of the substrates. Instead, the initial rates were measured at various concentrations of L-MTEH. The kinetic diagram ( 6 ) showed that the racemase is inhibited by the substrate L-MTEH. Inhibition is observed even at low substrate concentrations (> 5 mM).

The one used for the invention Microorganism Arthrobacter aurescens was found in the German Collection for microorganisms deposited with the accession number DSM 3747.

Examples:

Bacterial strains, plasmids and growth conditions. E. coli JM109 (Yanisch-Perron et al., Gene (1985), 33, 103-109) for cloning, sequencing and expressing the hyuA gene of Arthrobacter aurescens DSM 3747 used (Groß et al., Biotech. Tech. (1987), 2, 85-90). E. coli strains were in liquid 2xYT nutrient solution or cultivated on 2xYT agar (Sambrook et al., Molecular Cloning: A Laboratory Manual (1989), Cold Spring Harbor Laboratory Press, New York). The media were enriched with 100 μg / ml ampicillin, around plasmid-bearing strains to select. The cultures were grown at 37 ° C; for hyuA expression the temperature was raised to 30 ° C reduced.

General protocols. All recombinant DNA techniques used were standard procedures (Sambrook et al., Molecular Cloning: A Laboratory Manual (1989), Cold Spring Harbor Laboratory Press, New York). PCR reactions were performed with Taq DNA polymerase according to Roche Molecular Biochemicals recommendations. DNA sequencing was performed from pUC subclones using an automated laser fluorescence DNA sequencer (Pharmacia LKB, Freiburg) using the AutoRead ^™ sequencing kit and M13 forward and backward primers.

Expression of hyuA in E. coli. The racema gene was determined by PCR with the primers S1137 (5'-AG AACATATGAGAATCCTCGTGATCAA-3 ') and 51138 (5'-AAAACTGCAGCTAGAGGTACTGCTTCTCTG-3') and pAW16 amplified as a template (Wilms et al., J. Biotechnol. (1999), 68, 101-113). The fragment was inserted between the NdeI and PstI sites of the expression vector pJOE2702 (Volff et al., Mol. Microbiol. (1996), 21, 1037-1047) to create the plasmid pAW210. Expression was induced by adding 0.2% rhamnose to the cultures at an optical density of 0.3 at 600 nm. After 6 hours, cells corresponding to an OD ₆₀₀ of 10 were harvested, washed and resuspended in 1 ml of disintegration buffer (0.07 M potassium phosphate, pH 7.0) and lysed by sonication (Ultrasonics Sonicator, microtip, 2 × 30 s, Duty cycle 50% pulsed). Clarified extracts were obtained by centrifugation at 14,000 rpm for 10 minutes.

Enzyme approaches. The racemization of L-BH was measured by ORD polarimetry (model 341, Perkin Elmer Bodenseewerk, Überlingen, Germany) at a wavelength of 295 nm in the standard approach for racemase enzyme activity. 3 mM L-BH was dissolved in 0.1 M Tris, pH 3, at 45 ° C in an ultrasonic water bath, cooled to room temperature, and the pH was adjusted to 8.5 with 3 M NaOH. 0.1 ml of enzyme, which was diluted in 0.1 M Tris, pH 8.5, was added to 1 ml of substrate solution and the change in the optical rotation at 37 ° C. was determined by polarimetry (Teves et al., Fresenius 'J. Anal. Chem. 1999, 363, 738-743). The racemization of MTEH and IMH by HyuA was determined at substrate concentrations of 0.9 mM and recorded by ORD at 253 nm and 334 nm. The specific activities were calculated from the initial reaction rates, which according to Teves et al. (1999). To determine the enzyme activity by HPLC, 1 mM L-IMH was dissolved as described above. The mixture with 900 ul enzyme solution was incubated at 37 ° C for 5 minutes. The reaction was interrupted by adding 400 μl of 14% trichloroacetic acid and centrifuging in an Eppendorf centrifuge at full speed. 100 ul of the sample was diluted with 0.9 ml of 0.1 M TrisHCl, pH 8.5, and D-IMH and L-IMH in the supernatant were determined by HPLC (Thermoseparation Products, Darmstadt, Germany) by injection of a 20- μl sample separated into a Chiralpak WH column (0.46 × 25 cm; Daicel Chemicals Industris LTD, Griesheim, Germany). The column was equilibrated with 0.25 mM CuSO ₄ , pH 5.5. The flow rate was 1 ml / min at 50 ° C and IMH was detected at 254 nm. The chemical racemization of the substrate was taken into account. The racemization of 1 μM substrate per minute was defined as a unit enzyme.

Purification of recombinant hyuA. To produce crude extract, cells from a 300 ml culture of rhamnose-induced E. coli JM109 pAW210 were resuspended in 3 ml disintegration buffer and cleaved three times by French press (Amico, SLM Instruments Inc, Illinois, USA) at a pressure of 600 bar. Solid (NH ₄ ) ₂ SO ₄ was gradually added to the cell-free extract to a concentration of 1.5 M and stirred at 4 ° C for 2 hours. The precipitate which formed was removed by centrifugation (Sorvall) and discarded. Another 0.7 M (NH ₄ ) ₂ SO _{4 was} added to the supernatant. The second precipitate which was collected by centrifugation, was dissolved in buffer A (10 mM potassium phosphate, pH 6.5), resuspended and applied to a MonoQ 5/5 column ^® HR equilibrated in buffer A, and eluted with a linear gradient from 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 with decay buffer, glycerol was added to a final concentration of 25% and stored at -20 ° C.

Protein characterization. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the method of Laemmli (Laemmli, Nature (1970), 227, 680-685). Protein concentrations were determined using the Bradford method (Bradford, Anal. Biochem. (1976), 72, 248-254) using the Biorad Protein Assay dye reagent concentrate. Standard curves were generated with bovine serum albumin. M _r of native protein was determined by gel filtration using a Superose12HR column as previously described (Wilms et al., J. Biotechnol. (1999), 68, 101-113), the column was equilibrated and eluted with a buffer from 0, 1 M potassium phosphate and 0.1 M NaCl, pH 7. The pH profile of the purified racemase was measured between pH 7.0 and 9.5 in Tris buffer. The substrate was dissolved in 0.1 M Tris, pH 3, at 45 ° C using an ultrasonic water bath. After cooling to room temperature, the pH was adjusted to the desired pH with sodium hydroxide and the enzyme activity was determined using the standard approach. The optimal reaction temperature of the purified racemase was determined using temperatures between 25 and 65 ° C in the standard batch. The stability of the enzyme was determined after preincubation at temperatures between 25 and 70 ° C. for 15 minutes in the presence of each decay buffer and 0.1 M Tris buffer, pH 8.5. The increased chemical racemization was considered at high pH and high temperatures. The effect of EDTA, DTT, HgCl ₂ and iodoacetamide on HyuA was tested by incubating the respective substance (10 mM) and the purified enzyme (12 μg) in decay buffer (final volume 20 μl) at 30 ° C. After 1 hour, the specific activities were determined using the standard enzyme approach.

Claims (5)

Rec-hydantoin racemase, characterized in that it comes from Arthrobacter aurescens DSM 3747. Gene encoding the racemase according to claim 1. Vector comprising the gene of claim 2. A microorganism comprising the gene of claim 2. Primer for a gene according to claim 2.