L-Amidase from Bhxzobivan hua tlense
The present invention relates to a polypeptide with L- amidase activity from the organism of the genus Rhizobium, and to the nucleic acids which code this enzyme and vehicles containing these nucleic acids.
The invention relates in particular to an amidase from Rhizobium huautlense 38-2 DSM 14983, and to the nucleic acids which code this enzyme and the strain per se. Enzymes which cleave carboxylic acid amide bonds by hydrolysis are called amidohydrolases and are classified according to the E.C. system into peptidases (E.C. 3.4.) and into amidases, which convert linear (E.C. 3.5.1.) or cyclic amides (E.C. 3.5.2.). Amidohydrolases are widespread within microorganisms and occur inter alia in the genera of Corynebacterium, Pseudomonas, Bacillus, Brevibacterium, Rhodococcus and Alcaligenes. They are usually inducible enzymes, of which the specificity varies greatly from organism to organism (Martinkova, L.; Kren, V. (2002); Nitrile- and Amide- converting Microbial Enzymes: Stereo-, Regio- and Che oselectivity; Biocatal. Biotrans. 20, 73-93; aestracci, M. ; Bui, K. ; Thiery, A.; Arnaud, A.; Galzy, P. (1988), The Amidases from a Brevibacterium Strain: Study and Applications, Adv. Biochem. Eng. 36, 69-115) .
The enzymes from Mycobacterium neoaurum ATCC 25795 {Hermes, H. F. M.; Tandler, R. F. ; Sonke, T.; Dijkhuizen, L.; Meijer, E. M. (1994), Purification and Characterization of an L-Amino Amidase from Mycobacterium neoaurum ATCC 25795, Appl. Environ. Microbiol. 60, 153-159), Ochrobactrum anthropi NCIMB 40321 (Vandentweel, . J. J. ; Vandooren, T.; Dejonge, P. H.; Kaptein, B.; Duchateau, A. L. L.; Kamphuis, J. (1993), Ochrobactrum anthropi NCIMB 40321: a new
biocatalyst with broad-spectrum L-specific amidase activity, Appl. Microbiol. Biotechnol. 39, 296-300) and Pseudomonas putida ATCC 12633 (Hermes, H. F. M. ; Sonke, T • / Peters, P. J. H.; van Balken, J, A. M. ; Kamphuis, J. ; Dijkhuizen, L.; Meijer, E. M. (1993), Purification and Characterization of an L-Aminopeptidase from Pseudomonas putida ATCC 12633, Appl. Environ. Microbiol. 59, 4330-4334) are of industrial importance in particular in respect of the hydrolysis of L-amino acid amides. Both enzymes show a relatively high affinity for N-branched amino acid amides and dipeptides and are therefore classed as aminopeptidases (E.C. 3.4.). The L-amino-peptidase from Pseudomonas putida ATCC 12633 shows a relatively high affinity for N-branched amino acid amides or dipeptides and is thus classed with the aminopeptidases (E.C. 3.4.). It is employed for stereospecific cleavage of a D,L-phenylglycinamide mixture into D-phenylglycinamide and L-phenylglycine. A process developed by the DSM uses whole cells of Pseudomonas putida for the preparation of pure D- and L-amino acids from D,L- amino acid amides (Sonke, T.; Kaptein, B.; Boesten, W. H. J. ; Broxterman, Q. B.; Schoemaker, H. E.; Kamphuis, J. ; Formaggio, F. ; Toniolo, C; Rutjes, F. P. J. T. (2000); Aminoamidase-Catalyzed Preparation and Further Transformations of Enantiopure α-Hydrogen- and α,α- Disubstituted α-Amino Acids; In: Stereoselective
Biocatalysis. Patel, R. N. (eds.), Marcel Dekker, Inc., New York, 23-58).
Schering AG has furthermore patented a process for the preparation of L-amino acids and amino acid amides from D,L-α-aminonitriles (Klages, U.; Weber, A. (1988), Verfahren zur Herstellung von L-Aminosauren und Aminosaureamiden, DE 3 816 063 Al; WO 8 910 969) . In this biotransformation with whole cells, the D,L-aminonitriles are first hydrolysed to D, L-amino acid amides with AcinetoJacter calcoaceticus DSM 3875. In principle, complete conversion to the L-amino acid is possible with an L-amino acid amidase and an amino acid racemase in
Arthrobacter sp. ATCC 31652 or Corynebacterium sp. ATCC 31662.
In principle, however, there is still a need for further, possibly improved L-amidases in order to be able to employ these in an advantageous manner in an industrial process for the preparation of chiral organic compounds, especially since their substrate spectra are not covered 100% and previously poorly convertible substrates could now be prepared on an industrial scale under economically advantageous aspects by the discovery of new enzymes.
The object of the present invention was therefore to provide new polypeptides with L-amidase activity. In particular these polypeptides should be very suitable for use in an industrial process, i.e. should be as superior as possible to the amidases of the prior art in an economic and ecological aspect.
The object is achieved according to the claims. Claim 1 relates to nucleic acid sequences which code for a polypeptide with L-amidase activity. Claim 2 relates to a polypeptide with L-amidase activity. Claim 3 in turn relates to vehicles which contain the nucleic acids according to the invention, and claim 4 protects preferred primers. Claim 5 relates to a process for improving the polypeptides with L-amidase activity starting from the nucleic acids according to the invention, while claim 6 relates to particular improved rec-polypeptides and the nucleic acids which code them. Claim 7 and 8 relate to particular uses of the polypeptides and nucleic acid sequences according to the invention. Claims 9 and 10 relate to whole cell catalysts. Claim 11 protects the new organism Rhizobium huautlense 38-2 DSM 14983.
By providing isolated nucleic acid sequences which code for a polypeptide with L-amidase activity, containing a nucleic acid sequence chosen from the group consisting of: a) a nucleic acid sequence which codes for a polypeptide
with L-amidase activity from Rhizobium huautlense 38-2 DSM 14983, b) a nucleic acid sequence which hybridizes under stringent conditions with the nucleic acid sequence which codes for a polypeptide with L-amidase activity from Rhizobium huautlense 38-2 DSM 14983, or the sequence complementary thereto, c) a nucleic acid sequence which has a homology of at least 70% to the nucleic acid sequence which codes for a polypeptide with L-amidase activity from Rhizobium huautlense 38-2 DSM 14983, d) a nucleic acid sequence which codes for a polypeptide with L-amidase activity which has at least 80% homology at the amino acid level with the polypeptide with L-amidase activity from Rhizobium huautlense 38-2 DSM 14983, without the activity and/or the selectivity and/or the stability of the polypeptide being substantially reduced compared with the polypeptide with L-amidase activity from Rhizobium huautlense 38-2 DSM 14983, e) a nucleic acid sequence which codes for a polypeptide with L-amidase activity with improved activity and/or selectivity and/or stability compared with the polypeptide with L-amidase activity from Rhizobium huautlense 38-2 DSM 14983, prepared by i) mutagenesis of the nucleic acid sequence which codes for a polypeptide with L-amidase activity from Rhizobium huautlense 38-2 DSM 14983, ii) cloning of the nucleic acid sequence obtainable from i) into a suitable vector with subsequent transformation into a suitable expression system and iii) detection of the decisive polypeptide with improved activity and/or selectivity and/or stability, the possibility is arrived at, in a preferred manner, of being able to prepare the enzymes needed for an enzymatic industrial process for the production of enantiomerically
enriched compounds in a sufficient amount via recombinant techniques. With the nucleic acid sequences it is possible to obtain the polypeptides in high yields from fast-growing host organisms. In addition to having an unusually broad substrate spectrum with exceptionally high spec. activities, the polypeptides according to the invention, against expectations, are also heat-stable. Sequence data of the nucleic acid sequences can in principle be obtained by methods familiar to the expert (lit. see below) .
In the present invention, in addition to the original nucleic acid sequences, those which hybridize under stringent conditions with the nucleic acid sequence or the complement thereto and further sequences which have been improved by suitable mutagenesis processes are thus also claimed.
The procedure for improving the nucleic acid sequences according to the invention or the polypeptides coded by them by mutagenesis methods is adequately known to the expert. Possible mutagenesis methods are all the methods available to the expert for this purpose. These are, in particular, saturation mutagenesis, random mutagenesis, in vitro recombination methods and site-directed mutagenesis (Eigen, M. and Gardiner, W. (1984), Evolutionary molecular engineering based on RNA replication, Pure Appl. Chem. 56, 967-978; Chen, K. and Arnold, F. (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 Loeb, L. (1986) , Promoters Selected From Random DNA-Sequences, Proc Natl Acad Sci USA 83, 7405-7409; Dube, D. and L. Loeb (1989), Mutants Generated By The Insertion Of Random Oligonucleotides Into The Active-Site Of The Beta-Lactamase Gene, Biochemistry 28, 5703-5707; Stemmer, P.C. (1994), Rapid evolution of a protein in vi tro by DNA shuffling,
Nature 370, 389-391 and Stemmer, P.C. (1994), DNA shuffling
by random fragmentation and reassembly: In vi tro recombination for molecular evolution. Proc Natl Acad Sci USA 91, 10747-10751) .
The new nucleic acid sequences obtained are cloned into a host organism by the methods described below (lit. see below) and the polypeptides expressed in this way are detected with suitable screening methods and then isolated. All possible detection reactions for the molecules formed are suitable in principle for the detection. In particular, methods which are in principle suitable are all the possible detection reactions for ammonia and ammonium ions, such as Nessler's reagent (Vogel, A. I., (1989), Vogel's textbook of quantitative chemical analysis, John Wiley & Sons, Inc., 5th ed., 679-698, New York), the indophenol reaction, also called Berthelot's reaction (Wagner, R., (1969) , Neue Aspekte zur Stickstoffanalytik in der Wasserchemie, Vom Wasser, VCH-Verlag, vol. 36, 263-318, Weinheim) , in particular enzymatic determination by means of glutamate dehydrogenase (Bergmeyer, H. U., and Beutler, H.-O. (1985), Ammonia, in: Methods of Enzymatic Analysis, VCH-Verlag, 3rd edition, vol. 8: 454-461, Weinheim) and also detection with ammonium-sensitive electrodes. HPLC methods are furthermore used for detection of amino acids, such as e.g. a derivative method based on o- phthaldialdehyde and N-isobutyryl-cysteine for enantiomer separation of amino acids (Bruckner, H., Wittner R., and Godel H., (1991) Fully automated high-performance liquid chromatographic separation of DL-amino acids derivatized with o-Phthaldialdehyde together with N-isopropyl-cysteine. Application to food samples, Anal. Biochem. 144, 204-206) .
As stated, the invention also includes nucleic acid sequences which hybridize under stringent conditions with the single-stranded nucleic acid sequences according to the invention or single-stranded nucleic acid sequences complementary thereto. Specific gene probes or the primers necessary for a PCR are to be regarded e.g. as such.
The term "under stringent conditions" is understood herein as described by Sambrook et al. (Sambrook, J. ; Fritsch, E. F. and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York) . A stringent hybridization preferably exists according to the invention if, after washing for one hour with 1 x SSC (150 mM sodium chloride, 15 mM sodium citrate, pH 7.0) and 0.1% SDS (sodium dodecyl sulfate) at 50°C, preferably at 55°C, more preferably at 62°C and most preferably at 68°C and more preferably for 1 hour with 0.2 x SSC and 0.1% SDS at 50°C, more preferably at 55°C, even more preferably at 62°C and most preferably at 68°C, a positive hybridization signal is still observed.
The invention furthermore also relates to polypeptides with L-amidase activity chosen from the group consisting of a) the polypeptides coded by a nucleic acid sequence according to the invention, b) the polypeptides with a homology of min. 80% to the polypeptides with L-amidase activity from Rhizobium huautlense 38-2 DSM 14983, without the activity and/or the selectivity and/or the stability of the polypeptide being substantially reduced compared with the polypeptide with L- amidase activity from Rhizobium huautlense 38-2 DSM 14983, c) the polypeptides having L-amidase activity from Rhizobium, in particular Rhizobium huautlense .
The polypeptides according to the invention are very suitable for use in industrial processes because of the stability already indicated and the broad substrate spectrum.
In a next embodiment the invention relates to plasmids or vectors containing one or more of the nucleic acid sequences according to the invention.
Possible plasmids or vectors are in principle all the embodiments available to the expert for this purpose. Such plasmids and vectors can be seen e.g. from Studier and colleagues (Studier, W. F.; Rosenberg A. H.; Dunn J. J.;
Dubendroff J. W.; (1990), Use of the T7 RNA polymerase to direct expression of cloned genes, Methods Enzymol. 185, 61-89) or the brochures of Novagen, Promega, New England Biolabs, Clontech or Gibco BRL. Further preferred plasmids and vectors can be found in: Glover, D. M. (1985), DNA cloning: a practical approach, vol. I-III, IRL Press Ltd., Oxford; Rodriguez, R.L. and Denhardt, D. T (eds) (1988), Vectors: a survey of molecular cloning vectors and their uses, 179-204, Butterworth, Stoneham; Goeddel, D. V. (1990), Systems for heterologous gene expression, Methods Enzymol. 185, 3-7; Sambrook, J. ; Fritsch, E. F. and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York. Plasmids with which the gene construct containing the nucleic acid according to the invention can be cloned in a very preferred manner into the host organism are: pUC18 (Roche Biochemicals) , pKK-177-3H (Roche Biochemicals) , pBTac2 (Roche Biochemicals) , pKK223-3 (Amersham Pharmacia Biotech), pKK-233-3 (Stratagene) or pET (Novagen).
The invention likewise relates to microorganisms containing one or more nucleic acid sequences according to the invention.
The microorganism into which the plasmids containing the nucleic acid sequences according to the invention are cloned are used for multiplying and obtaining a sufficient amount of the recombinant enzyme. The processes for this are well-known to the expert (Sambrook, J. ; Fritsch, E. F. and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York) . Microorganisms which can be used are in principle all the organisms possible to the expert for this purpose, such as e.g. yeasts, such as Hansenula polymorpha, Pichia sp . and Saccharomyces cerevisiae, prokaryotes, such as E. coli and Bacillus subtilis or eukaryotes, such as mammalian cells and insect cells. E. coli strains are pref-erably to
be used for this purpose. The following are very particularly preferred: E. coli XL1 Blue, NM 522, JM101, JM109, JM105, RR1, DH5α, TOP 10" or HB101. Plasmids with which the gene construct containing the nucleic acid according to the invention is preferably cloned into the host organism are mentioned above.
A following aspect of the invention relates to primers for the preparation of the gene sequences according to the invention by means of all types of PCR. These also include the sense and antisense primers which code for the corresponding amino acid sequences, or complementary DNA sequences. Suitable primers can in principle be obtained by processes known to the expert. The discovery of the primers according to the invention is undertaken by comparison with known DNA sequences or by translating the amino acid sequences under consideration into the preferred codon of the organism in question (e.g. for Streptomyces: Wright F. and Bibb M. J. (1992), Codon usage in the G+C-rich Streptomyces genome, Gene 113, 55-65) . Common features in the amino acid sequence of proteins of so-called super- families are also of benefit for this (Firestine, S. M.; Nixon, A. E.; Benkovic, S. J. (1996), Threading your way to protein function, Chem. Biol. 3, 779-783) . Further information in this respect can be found in Gait, M. J. (1984), Oligonucleotide synthesis: a practical approach, IRL Press Ltd., Oxford; Innis, M. A.; Gelfound, D. H.; Sninsky, J. J. and White, T.J. (1990), PCR Protocols: A guide to methods and applications, Academic Press Inc., San Diego.
In a further embodiment, the present invention relates to a process for the preparation of improved rec-polypeptides with L-amidase activity starting from the nucleic acid sequences according to the invention, wherein a) the nucleic acid sequences are subjected to a mutagenesis, b) the nucleic acid sequences obtainable from a) are cloned
into a suitable vector and this is transferred into a suitable expression system, and c) the polypeptides with improved activity and/or selectivity and/or stability formed are detected and isolated.
The present invention also provides rec-polypeptides or nucleic acid sequences which code these, which are obtainable by a process as has just been described. The preparation of the nucleic acid sequences required for generation of the improved rec-polypeptides and expression thereof in hosts is referred to above and applies here accordingly.
The polypeptides and rec-polypeptides according to the invention are preferably used for the preparation of chiral enantiomerically enriched organic compounds, such as e.g. chiral carboxylic acids, such as α- or β-amino acids, with one or more stereogenic centres.
The nucleic acid sequences according to the invention and moreover those which are further improved, which code for the polypeptides in question, are furthermore preferably suitable for the preparation of whole cell catalysts. The preparation in principle of such biocatalysts is dealt with below and is adequately familiar to the expert.
The invention also provides whole cell catalysts containing a cloned gene for a polypeptide with amidase activity, preferably L-amidase activity, and a cloned gene for a polypeptide with nitrile hydratase activity and optionally a cloned gene for a . polypeptide chosen from the group consisting of polypeptides with α-aminonitrile racemase activity, with cyanohydrin racemase activity, with α- hydroxycarboxylic acid racemase activity or with (α- or β-) -amino acid amide racemase activity. The whole cell catalyst according to the invention preferably has a polypeptide with L-amidase activity from
Rhizobium, preferably R. huautlense DSM 14983. Nitrile hydratases (E.C.4.2.1.84) are enzymes which are capable of converting nitriles into acid amides. Such polypeptides are adequately familiar to the expert (Enzyme Catalysis in Organic Synthesis, Ed. : K. Drauz, H. Waldmann, vol. I, VCH, 1995, p. 365 et seq. ) .
Corresponding racemases are known e.g. from Pseudomonas putida and Rhodococcus sp. (Godtfredsen, S. E.; Clausen, K. ; Ingvorsen, K.; Hermes, H. F. ; Van Balken, J. A. ; Meijer, E. M. (1989), EP 0 307 023; WO 8 901 525). Further amino acid racemases in Klebsiella oκytoca are described by Hermes and colleagues (Hermes, H. F. M. ; Peeters, W. P.; Peters, P. J. (1990), EP 0 383 403).
An organism as mentioned in DE10155928 is preferably employed as the host organism.
The advantage of such an organism is the simultaneous expression of both polypeptide systems, which means that only one rec-organism has to be grown for the reaction of a nitrile or cyanohydrin or aminonitrile, which is easy to prepare, to give the corresponding enantiomerically enriched acid.
To coordinate the expression of the polypeptides in respect of their rates of conversion, the corresponding coding nucleic acid sequences can be accommodated on different plasmids with different numbers of copies and/or promoters of different potency can be used for an expression of the nucleic acid sequences of different intensity. In such coordinated enzyme systems, advantageously no accumulation of an intermediate compound which may have an inhibiting effect occurs, and the reaction in question can proceed at an optimum overall rate. However, this is adequately known to the expert (Gellissen, G.; Piontek, M.; Dahlems, U.; Jenzelewski, V.; Gavagan, J. W.; DiCosimo, R. ; Anton, D. L.; Janowicz, Z. A. (1996), Recombinant Hansenula polymorpha as a biocatalyst. Coexpression of the spinach glycollate oxidase (GO) and the S. cerevisiae catalase T
(CTT1) gene, Appl. Microbiol. Biotechnol. 46, 46-54; Farwick, M.; London, M. ; Dohmen, J. ; Dahlems, U.; Gellissen, G.; Strasser, A. W. ; DE19920712).
The nucleic acid sequences according to the invention can thus preferably be employed for the preparation of rec- polypeptides. By recombinant techniques which are adequately known to the expert, organisms which are capable of providing the polypeptide in question in an amount sufficient for an industrial process are arrived at. The preparation of the rec-polypeptides according to the invention is carried out by genetic engineering processes which are known to the expert (Sambrook, J. ; Fritsch, E. F. and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nd ed. , Cold Spring Harbor Laboratory Press, New York; Balbas, P. and Bolivar, F. (1990), Design and construction of expression plasmid vectors in E. coli, Methods Enzymol. 185, 14-37; Rodriguez, R.L. and Denhardt, D. T (eds) (1988), Vectors: a survey of molecular cloning vectors and their uses, 205-225, Butterworth, Stoneham) . In respect of the general procedure (PCR, cloning, expression etc.) reference may also be made to the following literature and that cited there: Universal GenomeWalker™ Kit User Manual, Clontech, 3/2000 and literature cited there; Triglia T.; Peterson, M. G. and Kemp, D.J. (1988), A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences, Nucleic Acids Res. 16, 8186; Sambrook, J. ; Fritsch, E. F. and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York; Rodriguez, R.L. and Denhardt, D. T (eds) (1988),
Vectors : a survey of molecular cloning vectors and their uses, Butterworth, Stoneham.
For the use, the polypeptide in question can be used in the free form as homogeneously purified compounds or as an enzyme prepared by a recombinant method. The polypeptide
can furthermore also be employed as a constituent of an intact guest organism or in combination with the broken- down cell mass of the host organism, which has been purified to any desired extent. The use of the enzymes in immobilized form is also possible (Sharma B. P.; Bailey L. F. and Messing R. A. (1982), Immobilisierte Biomaterialiern - Techniken und Anwendungen , Angew. Chem. 94, 836-852) . The immobilization is advantageously carried out by lyophilization (Paradkar, V. M.; Dordick, J. S. (1994), Aqueous-Like Activity of α- Chymotrypsin Dissolved in Nearly Anhydrous Organic Solvents, J. Am. Chem. Soc. 116, 5009-5010; Mori, T.; Okahata, Y. (1997), A variety of lipi-coated glycoside hydrolases as effective glycosyl transfer catalysts in homogeneous organic solvents, Tetrahedron Lett. 38, 1971-
1974; Otamiri, M. ; Adlercreutz, P.; Matthiasson, B. (1992), Complex formation between chymotrypsin and ethyl cellulose as a means to solubilize the enzyme in active form in toluene, Biocatalysis 6, 291-305) . Lyophilization in the presence of surface-active substances, such as Aerosol OT or polyvinylpyrrolidone or polyethylene glycol (PEG) or Brij 52 (diethylene glycol monocetyl ether) (Kamiya, N.; Okazaki, S.-Y.; Goto, M. (1997), Surfactant-horseradish peroxidase complex catalytically active in anhydrous benzene, Biotechnol. Tech. 11, 375-378), is very particularly preferred.
Immobilization on Eupergit®, in particular Eupergit C® and Eupergit 250L® (Rohm) (Eupergit . RTM. C, a carrier for immobilization of enzymes of industrial potential. Katchalski-Katzir, E.; Kraemer, D. M. Journal of Molecular Catalysis B: Enzymatic (2000), 10(1-3), 157-176) is extremely preferred.
Immobilization on Ni-NTA in combination with His-Tag (hexa- histidine) supplemented polypeptide (Purification of proteins using polyhistidine affinity tags. Bornhorst,
Joshua A.; Falke, Joseph J. Methods in Enzymology (2000), 326, 245-254) is likewise preferred.
The use as CLECs is also conceivable (St. Clair, N.; Wang, Y.-F.; Margolin, A. L. (2000), Cofactor-bound cross-linked enzyme crystals (CLEC) of alcohol dehydrogenase, Angew. Chem. Int. Ed. 39, 380-383). By these measures it can be possible to generate from polypeptides which become unstable due to organic solvents those which can operate in mixtures of aqueous and organic solvent or entirely in an organic medium.
The invention also provides the strain Rhizobium huautlense DSM14983. The strain Rhizobium huautlense DSM14983 was deposited on 06.05.02 at the Deutsche Sammlung fur Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg lb, D-38124 Braunschweig in accordance with the terms of the Budapest Treaty.
Investigation of soil samples by means of a screening process in which tert-leucinamide in minimal medium M-2 served as the sole source of nitrogen resulted in the most diverse isolates with L-amidase activity. The best strain in respect of the conversion of tert-leucinamide was identified as a Rhizobium huautlense . It had a surprisingly high activity of 550 mU/ml for the reaction in question. A zinc ion dependency is characteristic of the strain DSM14983. When 5 mM Zn2+ was present in the PD-10 filtered crude extract, a rel. increase in activity by about a factor of 28 in relation to the above conversion resulted. The following concentrations were found to be the most suitable M2+ combination:
Table 1:
This dependency on metal ions is unusual for amidases, since no co-factor dependency has been described for the typical representatives of this class. It may therefore be possible that the present amidohydrolase is a so-called aminopeptidase, and it is known of these that they have metal ion dependencies and often also hydrolyse acid amides as a secondary activity.
With the PD-10 filtered crude extract, for DL-tert- leucinamide a high L-enantioselectivity with ee values for L-tert-leucine of greater than 99% resulted, at a conversion of 48%.
The temperature dependency of the amidase according to the invention showed a further peculiarity. As can be seen from fig. 2, the amidase from Rhizobium huautlense has an optimum temperature of >90°C (determined with partly purified enzyme after IEC) . Incubation of the amidase at 70°C for 2 h led to no significant loss in activity.
To determine the linearity range for the enzyme test, the KM value of the L-amidase was estimated using the standard substrate DL-Tle-NH2. For this, using partly purified enzyme (after IEC) the activity in the range from 0.5 to 0 mM DL-Tle-NH2 was determined after incubation for 20 and 30 min. With the aid of the course of the activity in relation to the substrate concentration, a KM value of less than 0.5 mM for DL-Tle-NH2 can be estimated for the L- amidase (fig. 1) . No substrate excess inhibition was to be found in the range up to 40 mM.
The temperature stability of the L-amidase at 70°, 80° and 90°C was investigated. For this, the enzyme (after IEC) was incubated in MES buffer, 100 mM pH 7.5 + 0.1 mM Zn2+ at the particular temperature and the residual activity was determined at intervals of time.
The residual activities measured could be fitted to a plot under the simplified assumption of an enzyme activation according to a time law of the first order (fig. 3) , according to which:
A = activity at time t
A = A •e~k°*t A° = activity at time t = 0 k = inactivation constant
= time
With the inactivation constant k obtained, it was possible to calculate the half-life τ for the particular temperatures from the following relationship:
Table 2
Similarly to the optimum temperature, the L-amidase showed an exceptionally high temperature stability in the range up to 80°C. The inactivation half-lives here were 52 h and
150 min at 70 and 80°C respectively. In the region of the optimum temperature of 90°C, a half-life of 2.7 min was determined. Since a high temperature stability consequently also results in a good operational stability (Suzuki, Y., K.
Oishi, H. Nakano and T. Nagayama. Appl . Microbiol .
Biotechnol . 26: 546), it is to be assumed that the amidase
according to the invention is very suitable for use in an industrial process.
Further amino acid amides were investigated as possible substrates of the L-amidase, these being predominantly present as a racemate and employed at 40 mM in the enzyme test. The activity was determined with partly purified enzyme (after IEC) in comparison with DL-Tle-NH2. The following relative activities resulted in relation to the spec. act. of 4.3 U/mg.
Table 3:
All the substrates employed so far were converted by the L- amidase to a significantly better degree compared with DL-
Tle-NH2. The increases were between 3-fold for L-His-NH2 and 11-fold for racemic tryptophanamide with 50 U/mg. In the series of aliphatic branched amino acid amides valin-, isoleucin- and leucinamide, the activity increased from 17 to 29 U/mg. For the substrate with the smallest side-chain, Ala-NH2, a virtually 8-fold increase was determined.
For all the racemic substrates listed, the L-amidase according to the invention showed a virtually exclusive hydrolysis of the particular L-enantiomer . With conversions of less than 10%, ee values of greater than 98% resulted. For the substrates DL-Tle-NH2, DL-Val-NH2 and DL-Leu-NH2 a detailed investigation of the enantioselectivity as a function of the conversion was carried out for a possible use of the L-amidase for cleavage of the racemates.
Using partly purified enzyme after IEC, it was possible for the following ee values to be determined for the particular L-amino acids as a function of the conversion.
Table 4
For racemic Tle-NH
2 and also for Val-NH
2, high enantioselectivities with ee values of greater than 99% existed at a conversion of up to 50%. Compared with this, the ee values measured for L-Leu at a conversion of up to 44% were somewhat lower at about 98.5%. The E values calculated from this (enantiomeric ratio E; Chen, C.-S.; Fujimoto, Y.; Girdaukas, G. ; Sih, C. J. (1982); Quantitative analysis of biochemical kinetic resolutions of enantiomers; J. Am. Chem. Soc. 104, 7294-7299) were in the region of greater than 200 for DL-Tle-NH
2 and DL-Val-NH
2, while an E value of greater than 100 was calculated for DL- Leu-NH
2.
The amide cleavages were carried out in the pH range typical for these enzymatic reactions, pH values in the range of 5 to 10, preferably 6 to 9, very preferably 6.5 to 8 and extremely preferably of approx. 7.5 having proved to be suitable.
The reaction temperatures for the amidases according to the invention are preferably in the range between 30 and 85°C, in particular between 60 and 80°C, extremely preferably 70°C.
To prepare the native polypeptides according to the invention, harvested cells of Rhizobium huautlense DSM14983 can be broken down by grinding in a glass bead mill and the solid constituents can be separated off by centrifugation. After precipitation under heat and purification of the cell-free supernatant of the centrifugation via various evident chromatography processes in which the activity of the fractions is constantly tested, a polypeptide fraction which allows amino acid sequencing can be obtained. The initial sequence determined and the conservative patterns obtained by comparison with known amidohydrolases serve for construction of degenerated primers, with the aid of which a nucleic acid fragment of corresponding size can be
obtained by a PCR, this serving for the preparation of a gene probe in connection with homologous primers in a PCR.
After genomic digestion of the purified DNA from Rhizobium huautlense DSM14983, the gene library obtained can be screened with the gene probe by southern blotting and a specific hybridization signal can be identified. The total sequence of the gene for the L-amidase can subsequently be determined with the aid of further specific primers, derived from the cleavage sites of restriction enzymes, and a PCR with the aid of overlapping nucleic acid sequences and the stop codon.
In the context of the invention, optically enriched (enantiomerically enriched, enantiomer-enriched) compounds is understood as meaning the presence of one optical antipode as a mixture with the other in >50 mol%.
The term nucleic acid sequences subsumes all types of single-stranded or double-stranded DNA and also RNA or mixtures thereof.
The improvement in the activity and/or selectivity and/or stability means, according to the invention, that the polypeptides are more active and/or more selective or less selective or, under the reaction conditions used, more stable. While for industrial use the activity and the stability of the enzymes should of course be as high as possible, in respect of the selectivity an improvement is referred to if either the substrate selectivity decreases, but the enantioselectivity of the enzymes is increased. The same applies mutatis mutandis for the expression not substantially reduced (<10%, preferably <5%, particularly preferably <2%) used in this connection.
The polypeptides claimed and the nucleic acid sequences also include, according to the invention, those sequences which have a homology (exclusive of natural degeneration) of greater than 70% (in respect of the nucleic acid sequence)
or 80% (in respect of the polypeptides) , preferably greater than 90%, 91%, 92%, 93% or 94%, more preferably greater than 95% or 96%, and particularly preferably greater than 97%, 98% or 99%, to one of these sequences, as long as the mode of action or aim of such a sequence is retained. The expression "homology" (or identical) as used herein can be defined by the equation H (%) = [ 1 - V/X] x 100, wherein H denotes homology, X is the total number of nucleobases/amino acids of the comparison sequence and V is the number of different nucleobases/amino acids of the sequence in question with respect to the comparison sequence. In all cases, the term nucleic acid sequences which code for polypeptides includes all sequences which seem possible according to the degeneration of the genetic code.
The literature references cited in this specification are regarded as also included in the disclosure.
Examples :
1. Culture of the strain Rhizobium huautlense DSM 14983 in minimal medium M-2
For induction of the L-amidase, the strain Rhizobium huautlense 38-2 DSM 14983 was cultured in a minimal medium with racemic Tle-NH2 as the source of nitrogen; composition of the minimal medium:
Table 5: Minimal medium for Rhizobium huautlense
Table 7 : Composition of the vitamin solution
The sterilization was carried out by autoclaving at 121°C under 1.2 bar for 20 minutes. Since glucose, CaCl2, MgS0 x 7 H20, vitamin solution and DL-Tle-NH2 react sensitively to this type of sterilization, they were added to the nutrient solutions only after the autoclaving, by sterile filtration over 0.2 μm membranes (Sartorius) .
2. Culture of the strain Rhizobium huautlense DSM 14983 in DSMZ complete medium no . 1
The strain Rhizobium huautlense was furthermore cultured in DSMZ complete medium no. 1; composition (DSMZ, Catalogue of Strains (1998), Braunschweig): Table 8: Composition of DSMZ complete medium no. 1
Culturing of Rhizobium huautlense in DSMZ complete medium no. 1 also led to an L-amidase activity in the crude
extract. Under similar growth conditions, after cell breakdown with a protein content of 4.5 mg/ml a spec. act. of 0.11 U/mg was also to be determined. Compared with the activity in the minimal medium, in the absence of induction a residual activity of the L-amidase of about 30% was thus present.
3. Obtaining of crude extracts from Rhizobium huautlense
After harvesting by centrifugation, the cultures were adjusted to a 20% cell suspension with potassium phosphate buffer (20 mM; pH 6.5) with 0.1 mM ZnS04 or MES buffer (MES = 2- (N-morpholino) ethanesulfonic acid; 100 mM; pH 7.5) with 0.1 mM ZnS04. Depending on the volume of the cell suspension, the cell breakdown was carried out either by wet grinding by means of a vibratory mill from Retsch
(Hummel, W. ; Kula, M.-R. (1989), A Simple Method for Small- Scale Disruption of Bacteria and Yeasts, J. Microbiol. Methods 9, 201-209) , by ultrasound by means of a Pulses Sonifier from Branson, or by means of a Disintegrator S from IMA.
To lower protease activities, all further working steps were carried out while cooling to 4°C. The Bradford protein content determination (Bradford, M. M. (1976), A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein- Dye Binding, Anal. Biochem. 72, 248-254) in the crude extract enabled the quality of the breakdown to be evaluated.
Breakdown on an analytical scale by means of a vibratory mill:
1.2 g of glass beads (diameter 0.3 mm) and 0.6 ml of cell suspension were introduced into 1.5 ml Eppendorf cups and the cells were broken down by means of a vibratory mill for
ten min at maximum vibration frequency. The glass beads and cell debris were separated off from the cell homogenate by means of centrifugation for ten minutes at 10,000 rpm and
4°C and the supernatant was employed as the crude extract in the enzyme test.
Breakdown for volumes up to 20 ml by means of ultrasonic breakdown :
Rhizobium huautlense in portions of 10 ml maximum were broken down with 8 x 60 s bursts at 70% impulse, 80% intensity and in each case 60 s intermediate cooling.
Breakdown for volumes up to 200 ml by means of a disintegrator :
The breakdown for purification of the L-amidase from Rhizobium huautlense in volumes of between 20 and 200 ml was carried out in a Disintegrator S from IMA. For this, the cell suspension and glass beads (diameter 0.3 mm) were mixed in a ratio of 1:1.5 and the cells were broken down for 20 min at 3,500 rpm.
Demonstration of the L-amidase activity
In the conversion of tert-leucinamide (gen. acid amides) by the L-amidase, the formation of ammonia or ammonium ions and tert-leucine (gen. acid) in equimolar amounts occurs. The determination of the increase in ammonium ions or the tert-leucine formation by means of HPLC was thus used to demonstrate the amidase activity. The change in concentration of the components participating can be measured in an enzyme test, which comprises incubation of the following test batch at 70°C for 10 - 60 minutes.
Table 9 Composition of the enzyme test
The enzyme test was started by addition of the substrate, the reaction was stopped by addition of an enzyme test volume of 0.01 M NaOH or direct addition of 40 μl of the enzyme test in 140 μl Na borate buffer (pH 10.4) for derivatization for the subsequent HPLC analysis.
The ammonium ion reaction products were analyzed by enzymatic determination of ammonium by means of glutamate dehydrogenase (Bergmeyer, H.,U., and Beutler, H.-O. (1985) Ammonia. In: Methods of Enzymatic Analysis. VCH-Verlag, 3rd edition, vol. 8: 454-461, Weinheim) and D- and L-tert- leucine by means of HPLC (Bruckner, H., Wittner R. , and Godel H., (1991) Fully automated high-performance liquid chromatographic separation of DL-amino acids derivatized with o-phthaldialdehyde together with N-isopropyl-cysteine. Application to food samples. Anal. Biochem. 144(1): 204- 206) . Batches to which no substrate was added served as controls .
5. Enzymatic determination of the ammonium ions by means of glutamate dehydrogenase
The enzyme glutamate dehydrogenase (GluDH; E.C.1.4.1.3) converts 2-oxoglutarate into L-glutamate, ammonium ions being consumed and NADH being oxidized to NAD+ (Bergmeyer, H.,U. and Beutler, H.-O. (1985), Ammonia. In: Methods of
Enzymatic Analysis. VCH-Verlag, 3rd edition, vol. 8: 454- 461, Weinheim) .
The amount of NADH consumed during the reaction is equivalent to the amount of ammonium ions. The change in the concentration of NADH is the measurement parameter and can be determined spectrophotometrically at a wavelength of 340 nm.
Test solutions:
2-Oxoglutarate/ADP/TEA buffer: 9.3 g TEA, 95 mg ADP, 670 mg 2-oxoglutarate in aq. demin. to 100 ml, pH 8.0
NADH solution: 30 mg NADH, 60 mg NaHC03 dissolved in 6 ml aq. demin.
Glutamate dehydrogenase: From bovine liver in 50% glycerol, 120 U/mg
Table 10: Composition of the ammonium ion determination by means of glutamate dehydrogenase
The test components were pipetted into cells of plastic (1.5 ml semimicro disposable cells, Brand) and mixed and the extinction at 340 nm was determined after 5 min. 10 μl GluDH were then added, and after the reaction had gone to completion, as a rule after 30 min, the extinction was measured again.
The change in extinction ΔE was obtained by subtraction of the second value from the first. A measurement range up to 2 mM ammonium ions results for the particular samples.
A comparison between sample batches and associated controls gave information on whether the values measured were to be attributed to ammonium ions liberated from D,L-Tle-NH2 or to those already present in the crude extract.
By plotting a calibration line by means of defined amounts of ammonium chloride, it was possible to determine the ammonium ion concentration from the changes in extinction.
6. OPA/IBC derivatization for the determination of L- and D-tert-leucine by means of HPLC
The separation and quantitative determination of the enantiomers D- and L-tert-leucine were carried out by a "chiral derivatization" on the basis of o-phthaldialdehyde (OPA) /N-isobutyryl-L-cysteine or N-isobutyryl-D-cysteine (Bruckner, H.; Wittner R. and Godel H. (1991), Fully automated high-performance liquid chromatographic separation of DL-amino acids derivatized with o- phthaldialdehyde together with N-isopropyl-cysteine . Application to food samples. Anal. Biochem. 144, 204-206). The diastereomeric isoindole derivatives formed were separated on an RP-18 (reversed phase) column and detected by fluorescence. A two-buffer system of aqueous acetate buffer and an acetonitrile/water mixture was used as the mobile phase. Chromatography conditions:
Stationary phase: Kromasil™ HPLC column, 250 x 4 mm, 5 μm, 100 A (Eka Nobel)
Mobile phase:
Mobile phase A: 23 mM sodium acetate, pH 6.0 Mobile phase B: acetonitrile (HPLC grade) and aq. demin.: 10:1.5 (v:v)
Flow rate: 1 ml/min
Sample volume: 20 μl
Detection: fluorescence: ex. 340 nm/em. 440 nm
Table 11: Gradient programme of the HPLC
7. Purification of the L-amidase from -Rhizobium huautlense
The partial purification of the L-Amidase from Rhizobium huautlense DSM14983 has so far been carried out in three purification steps under the following conditions.
Table 12:
On the basis of a spec. act. of 0.40 U/mg in the crude extract, an act. of 42 U/mg resulted for the L-amidase after the HIC, and therefore a purification factor of 105 at an overall yield of about 60%.
Analysis of the protein composition of the fraction pool after IEC and HIC was possible with an SDS-polyacrylamide gel. Even after the 3rd purification step, a band pattern with at least three protein bands of relatively high intensity and further impurities was still to be seen in the HIC pool.
Fig. 4 shows the result of the SDS-PAGE analysis; marker: Premixed Standard, low range" from Roche in track 1; IEC pool in track 2 (20 μg protein) and 3 (10 μg protein); HIC pool in track 4 (10 μg protein) and 5 (5 μg protein) . A
12.5% separating gel with a 4% collecting gel, which was prepared by the method of Lae mli (Laemmli, U. K. (1970), Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685) was used.
8. Determination of the optimum temperature and the temperature stability of the L-amidase
Partly purified enzyme with a spec. act. of 4 U/mg was used for determination of the optimum temperature and the temperature stability. The activity was determined, as described under 4., with DL-Tle-NH2 as the substrate from 202 to 95eC in 5SC steps, the range from 702 to 95SC being investigated in more detail with further measurements. The incubation time chosen was relatively short at 15 min. The course of the activity as a function of the temperature is shown in fig. 2. The temperature stability of the L-amidase at 70, 80 and 90aC was investigated. For this, the enzyme was incubated in MES buffer (MES = 2-(N- morpholino) ethanesulphonic acid; 100 mM; pH 7.5) with 0.1 mM ZnS04 at the particular temperature and the residual activity was determined at intervals of time. The residual activities measured and the fit to a time law of the first order for the enzyme activity (simplified assumption) is shown in fig. 3.
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FOR THE PURPOSES OF PATENT PROCEDURE
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L IDENTIFICATION OF THE MICROORGANISM
Identification reference given by the DEPOSITOR: Accession number given by the INTERNATIONAL DEPOSITARY AUTHORITY:
38-2
DSM 14983
H. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION
The microorganism identified under I. above was accompanied by:
( ) a scientific description
( x) a proposed taxonomic designation
(Maifc with a cross where applicable).
ffl. RECEIPT AND ACCEPTANCE
This Internationa] Depositary Authority accepts the microorganism identified under L above, which was received by it on 2002-05-06 (Date of the original deposit)1.
IV. RECEIPT OF REQUEST FOR. CONVERSION
The microorganism identified under I above was received by this International Depositary Authority on (date of original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request fcrcoovσsico).
V. INTERNATIONAL DEPOSITARY AUTHORTΓY
Name: DSMZ-DEUTSCHE SAMMLUNG VON Signature^) of person(s) havin the power to represent the MKROORGANISMEN UND ZELLKULTUREN GmbH International Depository Authority or of authorized oft-ά&Ks):
Address: Mascheroder Weg lb D-38124 Braunschweig
Date: 2002-0S-15
Where Rule 6.4 d) applies, such date is the date on which the status of international depositary authority was acquired.
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RECOGNITION OF THE DEPOSIT OF MICROORGANISMS
FOR THE PURPOSES OF PATENT PROCEDURE
INTERNATIONAL FORM
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VIABHJTY STATEMENT 63457 Hanau-Wolfgang issued pursuant to Rule 10.2 by the INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page
L DEPOSITOR π. IDENTIFICATION OF THE MICROORGANISM
Name: Degussa AG Accession number given by the
Projekthaus Biotechnologie INTERNATIONAL DEPOSITARY AUTHORITY: Address: Rodenbacher Chaussee 4
DSM 14983 63457 Hanau-Wolfgang Date of the deposit or the transfer1:
2002-05-06
ΠL VIABILITY STATEMENT
The viability of the microorganism identified under II above was tested on 2002-05-08 On that date, the said microorganism was
lχ? viable
( Y no longer viable
IV. CONDITIONS UNDER WHICH THE VIABILITY TEST HAS BEEN PERFORMED4
V. INTERNATIONAL DEPOSITARY AUTHORITY
Name: DSMZ-DEUTSCHE SAMMLUNG VON Signatures) of person(s) having the power to represent the
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Date: 2002-05-15
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Form DSMZ-BP/ (sole page) 12/2001