Patent Document

This is a division of application Ser. No. 08/737,483, filed Feb. 21, 1997 now U.S. Pat. No. 5,985,632. 
     This application is based on application Ser. No. 44159714, filed in Germany on May 9, 1994, and PCT/EP95/01689, filed Apr. 29, 1995, the contents of each of which are hereby incorporated by reference. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The invention relates to a process for obtaining microorganisms containing peptide amidase, microorganisms obtained therewith, peptide amidases contained in them and the use thereof. 
     The invention relates in particular to a screening process for microorganisms exhibiting peptide amidase activity in accordance with the generic part of claim 1; microorganisms obtained according to this process and deposited in conformity with claims 2-4; peptide amidases which can be isolated from the microorganisms according to claims 5-7 and the use thereof. 
     2. Background information 
     The following publications are cited regarding the state of the art: 
     (1) DE-OS 36 29 242, 
     (2) K. Breddam, Carlsberg Res. Commun. 49 (1984) 535-554, 
     (3) DE patent 40 14 564 and 
     (4) Y. Nishida et al., Enzyme Microb. Technol., 6 (1984), 85-90. 
     A peptide amidase is an enzyme which catalyzes the selective hydrolysis of a C-terminal amide function in a peptide amidase, that is, accelerates the following conversion:                           
     Here, R′ signifies a protective group for n=0 and for n&gt;0 any amino acid, a protective group or H; n stands for zero or any whole number, R x  are the side chains of the amino acids for n&gt;0 whereas R 1  signifies the side chain of the C-terminal amino acid. 
     The selective splitting off of the C-terminal amino group of peptide amides is generally difficult to achieve by a chemical conversion since the peptide bond is also subject to a hydrolytic attack. This results in mixtures which are difficult to separate and in low yields. 
     Reference (1) teaches amidases for an enzymatic splitting off of the acid amide group which, on account of their a-amino acid amidase activity, can only be used, however, for the production of L-amino acids from a-unprotected D,L-amino acid amides. Peptide amides are not accepted. 
     Reference (4) teaches the continuous production of N-Ac-L-Met from N-Ac-D,L-methionine amide in an enzymatic process using Erwinia carotovera. 
     Erwinia carotovera does contain an amidase activity; however, it is limited exclusively to amides of methionine. Thus, the enzyme from Erwinia carotovera only “splits off amino acid amide” and is not a peptide amidase. Furthermore, the enzyme from Erwinia carotovera can obviously only convert N-acetylated amino acid amides, in which conversion it is a disadvantage that the Ac protective group can only be split off with difficulty or not at all. 
     On the other hand, peptidases are known which catalyze the hydrolytic splitting of the peptide bonds and of which it is only known that they have a certain secondary activity for splitting off the C-terminal amide protective group. An example of this is the carboxy peptidase Y, especially in chemically modified form (see reference (2)). 
     Thus, all these processes have serious disadvantages. 
     The state of the art according to reference(3) is also a peptide amidase which can be isolated from the flavedo of citrus fruits, especially of oranges. The peptide amidase described does not attack the peptide bond and catalyzes the splitting off of the free amino group from peptide amides. The peptide amidase known from (3) is characterized by the following parameters: 
     Splitting off of the C-terminal amino group from peptide amides and N-terminally protected amino acid amides; 
     No splitting of peptide bonds; 
     Optimum pH at 7.5±1.5; 
     Good stability in the pH range between pH 6.0 and pH 9.0; 
     The optimum temperature is 30° C. at a pH of 7.5; 
     Slight inhibition by inhibitors from serine proteases, especially phenylmethane sulfonyl fluoride; 
     The molecular weight is 23,000±3,000; 
     Aggregate formation is occasionally observed; 
     The isoelectric point is approximately pH 9.5; 
     The enzyme does not accept any D-amino acid groups in C-terminal position and the rate of hydrolysis thereby is distinctly less than in the case of L-amino acid groups. 
     However, the isolated enzyme can be obtained from flavedo only in slight amounts and as a function of the season. More extensive studies also did not succeed, in spite of an approximately 500-fold enrichment, in preparing the protein in homogeneous form, so that molecular and genetic studies for improving the enzyme production were not able to be included due to lack of data. 
     However, this also renders the suggestion given in reference (3) moot—that a microbial production of the enzyme can be achieved in a known manner by gene technology manipulation. The problems in the presentation of the homogeneous form do not allow manipulations of gene technology. 
     SUMMARY OF THE INVENTION 
     Therefore, in view of the problems associated with the state of the art, the invention has the object of making available a process for isolating microorganisms containing peptide amidase which makes possible a rapid selection of suitable strains. A further object is also a stable peptide amidase which is more readily available than the known peptide amidase from flavedo at an equally high selectivity of the hydrolytic splitting off of the free amino group on the C-terminal end of peptide amides. 
     These objects and others not cited in detail are achieved by a process with the features recited in claim  1 . 
     Microorganisms can be produced at practically any time of the year in any desired amount. Therefore, collection strains and isolated strains which can mobilize amide nitrogen as a source of nitrogen are analyzed for peptide amidase activity in a limited screening. Z-Gly-Tyr-NH 2  was used as test substrate and the expected hydrolysis product Z-Gly-Tyr-OH determined by HPLC. 
     It is possible, by first incubating specimens containing microorganisms in a “double screening” in a nutrient medium containing amide nitrogen as nitrogen source and inoculating colonies subsequently produced onto a nutrient medium containing N-acetyl-D,L-methionine amide, then incubating them and selecting the microorganisms which grow in both nutrient media, to find strains with an unusually good rate of success which are both relatively stable and also selective and active. 
     For the screening for microorganisms which can utilize amide nitrogen, samples of soil were suspended 4-6 hours in isotonic solution of common salt. The samples of soil were of any origin, including garden soil, forest soil, loamy or sandy soil. The solids were separated off at 2000 rpm by centrifugation. The supernatant was spread out onto agar plates and used to inoculate liquid media in Erlenmeyer flasks. The plates were incubated 3-7 days at 30° C. and the Erlenmeyer flasks agitated at the same temperature at 120 rpm. Then, individual cultures were isolated from the plates and brought into pure culture by being multiply spread out. After this time aliquots were taken from the incubated Erlenmeyer flasks and fresh medium inoculated therewith. This process was repeated up to five times before specimens of the culture liquid were spread out after a suitable dilution onto plates. The nutrient medium for the solid and also for the liquid medium had the following composition: 
     
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 K 2 HPO 4   
                 2.50 
                 g/l 
               
               
                   
                 KH 2 PO 4   
                 1.95 
                 g/l 
               
               
                   
                 NaCl 
                 1.00 
                 g/l 
               
               
                   
                 CaCl 2 *2H 2 O 
                 0.05 
                 g/l 
               
               
                   
                 MgSo 4 *7H 2 O 
                 0.3 
                 g/l 
               
               
                   
                 Yeast extract 
                 0.50 
                 g/l 
               
               
                   
                 DL-carnitine amide 
                 5.00 
                 g/l 
               
               
                   
                 Trace saline solution 
                 0.80 
                 ml/l 
               
               
                   
                 Vitamin solution 
                 2.5 
                 ml/l 
               
               
                   
                 (Agar for solid media 
                 18.0 
                 g/l) 
               
               
                   
                 pH 7.2 
               
               
                   
                   
               
             
          
         
       
     
     CaCl 2 *2H 2 O, MgSO 4 *7H 2 O as well as DL-carnitine amide and the vitamin solution (see below) were sterilized by filtration and added to the autoclaved, cooled-down medium. The trace saline solution was composed as follows: 
     
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 H 3 BO 3   
                 75.0 
                 mg 
               
               
                   
                 MnCl 2 *4H 2 O 
                 50.0 
                 mg 
               
               
                   
                 ZnCl 2   
                 187.0 
                 mg 
               
               
                   
                 CuSO 4 *5H 2 O 
                 50.0 
                 mg 
               
               
                   
                 FeCl 3 *6H 2 O 
                 625.0 
                 mg 
               
               
                   
                 (NH 4 ) 8 Mo 7 O 24 *4H 2 O 
                 25.0 
                 mg 
               
               
                   
                 CoSO 4 *7H 2 O 
                 37.5 
                 mg 
               
               
                   
                 H 2 O demin. 
                 ad. 0.2 
                 l. 
               
               
                   
                   
               
             
          
         
       
     
     Individualized microorganisms grown on this medium were used for screening for organisms with peptide amidase activity. 
     For screening for microorganisms with peptide amidase activity, a part of the organisms obtained above were spread out onto agar nutrient media and incubated 2 days at 30° C. In order to obtain higher cell masses, the cultures were enriched in 100 ml Erlenmeyer flasks with 20 ml medium. The incubation took place at 30° C. for 2 days at 120 rpm. The nutrient medium used for this had the following composition: 
     
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 K 2 HPO 4   
                 0.50 
                 g/l 
               
               
                   
                 KH 2 PO 4   
                 2.00 
                 g/l 
               
               
                   
                 NaCl 
                 1.00 
                 g/l 
               
               
                   
                 CaCl 2 *2H 2 O 
                 0.05 
                 g/l 
               
               
                   
                 MgSo 4 *7H 2 O 
                 0.10 
                 g/l 
               
               
                   
                 Glucose 
                 5.00 
                 g/l 
               
               
                   
                 DL-carnitine amide 
                 1.00 
                 g/l 
               
               
                   
                 N-Ac-DL-Met-NH 2   
                 2.00 
                 g/l 
               
               
                   
                 Yeast extract 
                 0.50 
                 g/l 
               
               
                   
                 Vitamin solution 
                 2.50 
                 ml/l 
               
               
                   
                 Trace saline solution 
                 0.80 
                 ml/l 
               
               
                   
                 (Agar for solid media 
                 18.00 
                 g/l) 
               
               
                   
                 pH 7.3. 
               
               
                   
                   
               
             
          
         
       
     
     N-Ac-D,L-Met-NH 2  was used as inductor thereby. Glucose should be avoided as a source of carbon so that the inductor is attacked from the N-terminus. Amides, glucose, CaCl 2 *2H 2 O, MgSo 4 *7H 2 O and the vitamin solution were sterilized by filtration and added to the autoclaved, cooled-down nutrient medium (see below for the composition of the trace saline solution and of the vitamin solution). 
     The cells were centrifuged, washed with 50 mM tris/HCl, pH 7.5 and taken up in this buffer (20-40% cell suspension). The cell maceration took place by wet grinding with glass beads 0.3 mm in diameter. 
     The raw extracts obtained therefrom were analyzed for the capacity to hydrolyze Z-Gly-Tyr-NH 2 . 
     Of 45 strains analyzed for deamidation, 6 exhibited the capacity to convert Z-Gly-Tyr-NH 2  to Z-Gly-Tyr-OH. Table 1 shows selected data from the screening for peptide amidases. 
     
       
         
               
               
               
               
               
             
           
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                   
                   
                 Protein 
                   
               
               
                   
                 Strain 
                   
                 content 
                 Spec. Activity 
               
               
                   
                 No. 
                 OD 660  * 
                 (mg BSAeq/ml) 
                 (mU/mg BSAeq) 
               
               
                   
                   
               
             
             
               
                   
                  4 
                 0.98 
                 1.93 
                 4.84 
               
               
                   
                 11 
                 1.22 
                 5.30 
                 9.70 
               
               
                   
                 18 
                 1.13 
                 5.96 
                 2.97 
               
               
                   
                 21 
                 0.87 
                 7.90 
                 5.22 
               
               
                   
                 22 
                 1.04 
                 9.40 
                 7.23 
               
               
                   
                 42 
                 1.58 
                 3.46 
                 2.82 
               
               
                   
                   
               
               
                   
                 * OD 660  = Optical density at a wavelength of 660 nm.  
               
             
          
         
       
     
     Isolated strains 4, 11, 18, 21, 22, 42 were deposited pursuant to the Budapest Treaty in the German Collection for Microorganisms (address Mascheroder Weqlb, D-38124 Braunschevera, Germany) DSM under the name of the applicant. 
     
       
         
               
               
               
             
           
               
                   
               
               
                 Strain No. 
                 DSM Deposit No. 
                 Deposition date 
               
               
                   
               
             
             
               
                  4 
                 9182 
                 5-2-1994 
               
               
                 11 
                 9181 
                 5-2-1994 
               
               
                 18 
                 9183 
                 5-2-1994 
               
               
                 21 
                 9184 
                 5-2-1994 
               
               
                 22 
                 9185 
                 5-2-1994 
               
               
                 42 
                 9186 
                 5-2-1994 
               
               
                   
               
             
          
         
       
     
     Strain 11 was selected from the deposited strains for further analyses. Strain 11 was determined by the DSM as Stenotrophomonas maltophilia ( Xanthomonas maltophilia ). Strains 4, 18, 21, 22 and 42 were also evaluated, with the result that strains 4, 18, 21 and 22 were likewise identified as Stenotrophomonas maltophilia ( Xanthomonas maltophilia ) and strain 42 as Ochrobactrum anthropi. 
     The invention also has as subject matter peptide amidases obtainable from the screened microorganisms. 
     The peptide amidase is formed intracellularly and can be isolated from the cells and purified according to processes familiar to the expert in the art. 
     The microbial peptide amidase is characterized by the following parameters: 
     Splitting off of the C-terminal amino group from peptide amides and N-terminally protected amino acid amides; 
     No splitting of peptide bonds; 
     Optimum pH at 6.0±0.5; 
     Good stability in the PH range between pH 7 and pH 8; 
     The optimum temperature is 35-40° C. at a pH of 7.5; 
     Inhibition of serine groups by inhibitors such as phenylmethane sulfonylfluoride as well as in particular 4-(2-aminoethylbenzylsulfonylfluoride) (Pefabloc); 
     The molecular weight is approximately 38,000 daltons (determined by gel filtration); 
     The isoelectric point is approximately pH 5.8. 
     The invention also comprises the isozymic forms of the microbial peptide amidase in accordance with the invention. The term “isozymic forms” in this connection denotes the enzymes in other microorganisms which catalyze the same reaction as the peptide amidase from  Xanthomonas maltophilia.    
     Table 2 indicates some data for the purification of the peptide amidase from  Xanthomonas maltophilia . 
     
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                 TABLE 2 
               
               
                   
                   
               
               
                   
                   
                 Spec. 
                   
                   
               
               
                   
                   
                 activity 
               
               
                   
                   
                 (mU/mg 
                 Purification 
                 Yield 
               
               
                   
                   
                 BSAeq) 
                 (−) 
                 (%) 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Raw extract 
                 4 
                 1 
                 100 
               
               
                   
                 Q-sepharose FF + 
                 224 
                 56 
                 97 
               
               
                   
                 ultrafiltration 
               
               
                   
                 Superdex G 75 + 
                 1034 
                 256 
                 75 
               
               
                   
                 ultrafiltration 
               
               
                   
                 IEF on the Mono 
                 2133 
                 533 
                 63 
               
               
                   
                 P 
               
               
                   
                   
               
             
          
         
       
     
     The purified peptide amidase from  Xanthomonas maltophilia  has in particular the following characteristic properties: 
     Purification of &gt;500 at a yield of &gt;60% 
     A molecular weight of 38000 da (gel filtration) 
     The isoelectric point is approximately pH 5.8 
     Temperature optimum between 37-45° C. 
     pH optimum between 5-6.5 
     Temperature-stable at 20, 30 and 37° C. over 3 days 
     At pH 7-8 the enzyme is stable at 30° C. over 7 days 
     Upon the addition of 20% DMF the peptide amidase exhibits after 24 h a residual activity of 32% at a pH of 7.5 
     Serine group decisive for enzyme activity. 
     The N-terminal initial sequence of the peptide amidase from  Xanthomonas maltophilia  (set forth in SEQ ID NO: 1) is: 
     
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 AS 1 
                 X 
               
               
                   
                 AS 2 
                 Arg 
               
               
                   
                 AS 3 
                 Asn 
               
               
                   
                 AS 4 
                 Val 
               
               
                   
                 AS 5 
                 Pro 
               
               
                   
                 AS 6 
                 Phe 
               
               
                   
                 AS 7 
                 Pro 
               
               
                   
                 AS 8 
                 Tyr 
               
               
                   
                 AS 9 
                 Ala 
               
               
                   
                 AS 10 
                 Glu 
               
               
                   
                 AS 11 
                 Thr 
               
               
                   
                 AS 12 
                 Asp 
               
               
                   
                 AS 13 
                 Val 
               
               
                   
                 AS 14 
                 Ala 
               
               
                   
                 AS 15 
                 Asp 
               
               
                   
                 AS 16 
                 Leu 
               
               
                   
                 AS 17 
                 Gln 
               
               
                   
                   
               
             
          
         
       
     
     The first amino acid could not be determined. No comparable sequence could be determined in the program Genepro 5.0 of the databank PIR, version 30. 
     The following table 3 compares the properties of the known, vegetable peptide amidase from flavedo of oranges (PAF) and of the microbial peptide amidase of the invention from Xanthomonas (PAX). 
     The peptide amidase of the invention shows in it neither peptidase activity nor amino acid amidase activity. 
     
       
         
               
               
               
             
               
               
               
               
             
           
               
                   
                 TABLE 3 
               
               
                   
                   
               
               
                   
                 PAF 
                 PAX 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Spec. activity 
                 100.1 
                 2.13 
               
               
                   
                 (U/mg BSAeq) 
               
               
                   
                 Amino acid amidase 
                 0 
                 0 
               
               
                   
                 activity 
               
               
                   
                 Peptidase activity 
                 0 
                 0 
               
               
                   
                 Molar mass (gel 
                 23000 
                 38000 
               
               
                   
                 filtration) 
               
               
                   
                 pH optimum 
                 6.5-8.7 
                 5-6.5 
               
               
                   
                 Temperature optimum 
                 30-35° C. 
                 37-45° C. 
               
               
                   
                 Isoelectric point 
                 9.5 
                 5.8 
               
               
                   
                 N-terminal sequence 
                 not known 
               
               
                   
                   
               
             
          
         
       
     
     The microbial peptide amidases belonging to the invention are very advantageously suited for the catalysis of quite a number of reactions. 
     Thus, the microbial peptide amidases of the invention can be used with success to produce peptides and N-terminally protected amino acids of the general formula II                           
     in which R′ is a protective group or any peptidically or isopeptidically bound amino acid- or peptide group and R 1  signifies hydrogen or any side chain with enzymatic splitting off of a C-terminal amino group from a peptide amide or from an N-terminally protected amino acid amide. 
     In a preferred variant of the process, this enzymatic reaction is carried out in a continuous manner. 
     It is also preferred that the deamidation is carried out as a process step of a coupled conversion with an enzyme system comprising proteases, peptidases, esterases and/or lipases. In this connection the selectivity of the peptide amidases of the invention is especially advantageous. 
     The microbial peptide amidases can also be used in accordance with the invention in processes for producing peptides of the above-mentioned type by the enzymatic conversion of optionally N-protected amino acid alkyl esters or optionally N-protected peptide alkyl esters with amino acid amides in aqueous phase or an aqueous-organic environment. The reaction takes place thereby in the presence of an enzyme which brings about the peptidic bonding and under enzymatic splitting off of the amide protective group; the synthesis is allowed to take place in a continuous manner, the peptide amide hydrolyzed by the peptide amidase enzymatically to the peptide and finally the peptide is separated on account of its charge from the reaction mixture and the amino acid amide returned. 
     Furthermore, the microbial peptide amidase of the invention can also be used for the splitting [separation] of racemic mixtures of N-protected amino acid amides in which a racemic mixture of N-protected amino acid amides is incubated with the peptide amidase and is reacted until the complete conversion of the N-protected L-amino acid amide, and subsequently the N-protected L-amino acid is separated from the N-protected D-amino acid amide based on the differences of charge. Furthermore, according to the invention D-amino acids can also be produced. Thus, the N α -protected L-amino acid amide can be selectively and enzymatically hydrolyzed, for example, within the scope of the invention by using the microbial peptide amidase of the invention, the N α -protected D-amino acid amide separated and converted by acid hydrolysis into the free D-amino acid. The amino acid amide racemates useful for the invention include, among others, N α -formyl-DL-methionine amide, N α -methylaminocarbonyl-DL-methionine amide, N α -methoxycarbonyl-DL-methionine amide, N α -ethoxycarbonly-DL-methionine amide, N α -benzyloxycarbonyl-DL-methionine amide, N α -acetyl-DL-neopentylglycine amide, N α -benzyloxycarbonyl-DL-neopentylglycine amide. 
     Finally, the microbial peptide amidases of the invention can also be used with great advantage to obtain non-proteinogenous D-amino acids, preferably using sterically demanding, N-protected racemic amino acid amides such as N-acetyl-neopentylglycine amide, N-acetyl-naphthylalanine amide, N-acetylphenylglycine amide or similar derivatives. The N-acetyl-L-amino acid amides are enzymatically hydrolyzed, the N-acetyl-D-amino acid amides separated from the reaction mixture by chromatography and finally converted by acid hydrolysis into the free D-amino acids. 
     The N α -protected D-amino acid amides obtainable by using the microbial peptide amidase of the invention from the preferred racemic, N α -protected amino acid amides include, for example: N α -formyl-D-methionine amide, N α -methylaminocarbonyl-D-methionine amide, N α -methoxycarbonyl-D-methionine amide, N α -ethoxycarbonyl-D-methionine amide, N α -benzyloxycarbonyl-D-methionine amide, N α -acetyl-D-neopentyiglycine amide, and N α -benzyloxycarbonyl-D-neopentylglycine amide. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The invention is explained in the following in detail using examples. Further embodiments and particularities also result in particular from the attached figures, to which reference is made in the specification. 
     FIG. 1 shows the dependency of the conversion rate (%) on the pH for peptide amidase obtained from  Xanthomonas maltophilia.    
     FIG. 2 shows a plot of relative activity vs. time for peptide amidase from  Xanthomonas maltophilia  in various buffers at 30° C. 
     FIG. 3 shows a plot of the conversion rate as a function of the temperature for peptide amidase from  Xanthomonas maltophilia.    
     FIG. 4 shows the temperature stability of the peptide amidase from  Xanthomonas maltophilia.    
     FIG. 5 shows the kinetic determination of the conversion of Z-Gly-Tyr-NH 2  with the peptide amidase from  Xanthomonas maltophilia.    
     FIG. 6 shows the splitting [separation] of racemic mixtures of Z-D,L-Ala-NH 2  with the peptide amidase from  Xanthomonas maltophilia.    
     FIG. 7 shows the enantioselective deamidation of N-Ac-D,L-Met-NH 2  with the peptide amidase from  Xanthomonas maltophilia.    
     FIG. 8 shows the influence of solvent on the enzymatic activity of the peptide amidase from  Xanthomonas maltophilia.    
     FIG. 9 shows the solvent stability of the peptide amidase from  Xanthomonas maltophilia.   
    
    
     EXAMPLE 1 
     Production and Workup of the Peptide Amidase from  Xanthomonas Maltophilia    
     1.1 Growth 
     40 g moist biomass/l and an activity of 4 U/l were obtained in the partially optimized medium with the following composition. The media optimization took place with the aid of the genetic algorithm. The nutrient medium was autoclaved, glucose, N-Ac-DL-Met-NH 2 , CaCl 2 *2H 2 O, MgSO 4 *7H 2 O and the vitamin solution were added in a sterile manner. 
     
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 N-Ac-D, L-Met-NH 2   
                 4.3 
                 g/l 
               
               
                   
                 Yeast extract 
                 4.5 
                 g/l 
               
               
                   
                 Peptone from casein 
                 19.7 
                 g/l 
               
               
                   
                 Glucose 
                 18.4 
                 g/l 
               
               
                   
                 KH 2 PO 4   
                 0.5 
                 g/l 
               
               
                   
                 K 2 HPO 4   
                 2.0 
                 g/l 
               
               
                   
                 NaCl 
                 1.0 
                 g/l 
               
               
                   
                 CaCl 2 *2 H 2 O 
                 0.05 
                 g/l 
               
               
                   
                 MgSo 4 *7 H 2 O 
                 0.1 
                 g/l 
               
               
                   
                 Vitamin solution according to Schlegel* 
                 2.5 
                 ml/l 
               
               
                   
                 Trace saline solution 
                 0.8 
                 ml/l 
               
               
                   
                 (Agar in the case of solid media 
                 18.0 
                 g/l) 
               
               
                   
                 Trace saline solution 
               
               
                   
                 H 3 BO 3   
                 75.0 
                 mg 
               
               
                   
                 MnCl 2 *4H 2 O 
                 50.0 
                 mg 
               
               
                   
                 ZnCl 2   
                 187.0 
                 mg 
               
               
                   
                 CuSO 4 *5H 2 O 
                 50.0 
                 mg 
               
               
                   
                 FeCl 3 *6H 2 O 
                 625.0 
                 mg 
               
               
                   
                 (NH 4 ) 6 Mo 7 O 24 *4H 2 O 
                 25.0 
                 mg 
               
               
                   
                 CoCl 2 *6H 2 O 
                 37.50 
                 mg 
               
               
                   
                 NiCl 2 *6H 2 O 
                 50.00 
                 mg 
               
               
                   
                 H 2 O 
                 ad 0.2 
                 l 
               
               
                   
                   
               
               
                   
                 *Schlegel, H.G. (1985): Allgemeine Mikrobiologie [German - General Microbiology], Thieme Verlag, Stuttgart  
               
             
          
         
       
     
     1.2 Cellular maceration 
     The cell maceration took place by the wet grinding of a 20-40% cellular suspension in 50 mM tris/HCl buffer, pH 7.5 with glass beads Ø 0.3 mm. The glass beads and the cellular fragments were separated by centrifugation. The glass beads were then re-suspended in 50 mM tris/HCl, pH 7.5 and separated again by centrifugation. The supernatants were purified and constituted the raw extract for the workup described in the following. The cellular maceration was evaluated by determining the amount of protein released according to Bradford (Bradford, M. M. (1976), Anal. Biochem., 72, 248-254) and by microscopic observation. 
     1.3 Purification 
     1.3.1. Ion exchange Chromatography 
     As the first step in the purification of the peptide amidase from  Xanthomonas maltophilia , an anion exchange chromatography was carried out on Q-sepharose Fast Flow (Pharmacia, Uppsala). The following conditions applied for the anion exchanger: 
     
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 Column 
                 10 cm * π * 1.3 * 1.3 * cm 2  = 53 ml 
               
               
                   
                   
                 Q sepharose FF (Pharmacia, Uppsala) 
               
               
                   
                 Travel rate 
                 10 ml/min 
               
               
                   
                 Equilibration 
                 50 mM tris, 20 mM KCl, pH 8.0 
               
               
                   
                 Specimen charging 
                 77.2 ml raw extract à 6.8 mg 
               
               
                   
                   
                 BSAeq/ml 
               
               
                   
                 Washing 
                 50 mM tris/HCl, 20 mM KCl, pH 
               
               
                   
                   
                 8.0 
               
               
                   
                 Elution 
                 Linear gradient with rising salt 
               
               
                   
                   
                 content 
               
               
                   
                   
                 200 ml 50 mM tris, 20 mM KCl, pH 
               
               
                   
                   
                 8.0 
               
               
                   
                   
                 20 ml 50 mM tris, 200 mM KCl, pH 
               
               
                   
                   
                 8.0 
               
               
                   
                   
               
             
          
         
       
     
     The detection took place at 280 nm, the fractions were trapped at 10 ml each. The peptide amidase was able to be eluted at a salt content of 80-120 mM KCl from the anion exchanger. The active fractions were pooled and concentrated with the aid of an Amicon ultrafiltration cell with a YM 10 membrane to 2 ml for the following gel filtration. 
     1.3.2 Gel filtration 
     The active fractions of the anion exchange chromatography were concentrated as described and subjected to a gel filtration on Superdex G 75 material (Pharmacia, Uppsala). The following conditions applied for the gel filtration: 
     
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 Column 
                 60 cm * π * 0.8 * 0.8 * cm 2  = 120.6 ml 
               
               
                   
                 Travel rate 
                 1 ml/min 
               
               
                   
                 Equilibration 
                 50 mM tris, 150 mM KCl, pH 7.5 
               
               
                   
                 Specimen 
                 2 ml à 4.55 mg BSAeq/ml of the 
               
               
                   
                   
                 concentrated active fractions of the 
               
               
                   
                   
                 anion exchange chromatography 
               
               
                   
                 Elution 
                 50 mM tris, 150 mM KCl, pH 7.5 
               
               
                   
                 Fraction size 
                 1 ml 
               
               
                   
                 Detection 
                 280 nm 
               
               
                   
                   
               
             
          
         
       
     
     The active fractions of the gel filtration were pooled and concentrated via a YM 10 membrane in an Amicon ultrafiltration cell to 1 ml (1.47 mg BSAeq/ml) and were available in this manner for the following isoelectric focusing. 
     1.3.3 Isoelectric Focusing 
     The active fractions of the gel filtration and those of the gel filtration evaporated to 1 ml as described under 1.3.2 were purified with the aid of isoelectric focusing. The conditions for this are presented in the following. 
     
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 Column 
                 5.1 cm * 0.25 * 0.25 * cm 2  * π = 1 ml 
               
               
                   
                   
                 Mono P (Pharmacia, Uppsala) 
               
               
                   
                 Travel rate 
                 1 ml/min 
               
               
                   
                 Equilibration 
                 25 mM triethanol amine, pH 8.0 
               
               
                   
                 Specimen 
                 1 ml of the concentrated active 
               
               
                   
                   
                 fractions of the gel filtration (re- 
               
               
                   
                   
                 buffered with 25 mM triethanol amine, 
               
               
                   
                   
                 pH 8.0, 1.47 mg BSAeq/ml) 
               
               
                   
                 Washing 
                 25 mM triethanol amine, pH 8.0 
               
               
                   
                 Elution 
                 10 mM linear pH gradient from A to B 
               
               
                   
                   
                 Eluent A: 25 mM triethanol amine, pH 
               
               
                   
                   
                 8.0 
               
               
                   
                   
                 Eluent B: Polybuffer 74 (diluted 
               
               
                   
                   
                 1:10, Pharmacia), pH 5.0 
               
               
                   
                 Fractions 
                 0.5 ml 
               
               
                   
                 Detection 
                 280 nm 
               
               
                   
                   
               
             
          
         
       
     
     In the first two steps of the purification (anion exchange chromatography and gel filtration) interfering protease/peptidase activities were completely removed. After the isoelectric focusing, a preparation enriched 533-fold was obtained. A main band which is enzymatically active is observed in the native gel. The N-terminal sequence was determined by means of a liquid-phase sequenator (Applied Biosystems 470 with on-line HPLC coupling) after elution of the band out of the native gel. 
     EXAMPLE 2 
     Characterization of the Enzyme 
     pH Optimum and pH Stability 
     FIG. 1 shows the dependency of the conversion rate in % on the pH. 50 mM Mc-Ilvain buffer were used in the pH range of 3.0-7.25, 50 mM tris/HCl buffer between pH 7.0-9.0 and for the basic pH range of 9.5-10.5 50 mM Na 2 CO 3  buffer. The test batch of 1 ml was composed as follows: 
     
       
         
               
               
             
           
               
                   
               
             
             
               
                 100 μl 
                 100 mM Z-Gly-Tyr-NH 2  (dissolved in 
               
               
                   
                 H 2 O/dimethylformamide in a 1:1 ratio) 
               
               
                 200 μl 
                 Enzyme solution à 63 μg BSAeq/ml of the 
               
               
                   
                 purification stage after the gel 
               
               
                   
                 filtration 
               
               
                 700 μl 
                 Buffer. 
               
               
                   
               
             
          
         
       
     
     A pre-incubation took place without the addition of substrate for 5 min at 30° C. The reaction was then started by adding 100 μl substrate and incubated 4 hours at 30° C. In order to stop the reaction, 100 μl of the reaction batch were removed and compounded with 100 μl glacial acetic acid as well as 1.4 ml HPLC mobile solvent (see 1.4 standard assay of peptide amidase). 
     FIG. 1 shows the results of the analysis of the pH optimum. The pH optimum of the peptide amidase peptide amidase from  Xanthomonas maltophilia  results at 6.0±0.5. 
     The relative activity (%) as a function of the time (h) was determined for the pH stability of peptide amidase from  Xanthomonas maltophilia . 50 mM Kp i  buffer was used for the pH range of 5.0-7.0 and 50 mM tris/HCl buffer between 7.0-9.0. For the analyses of pH stability 100 μl enzyme solution a 0.73 mg BSAeq/ml (purification stage after the gel filtration) were incubated with 900 μl buffer of differing pH at 30° C. Specimens were drawn at different times and the activity determined with Z-Gly-Tyr-NH 2  as substrate. The results are shown in FIG. 2. A good stability results in the range of pH 7-pH 8. 
     EXAMPLE 3 
     Temperature Optimum and Temperature Stability 
     The temperature optimum of peptide amidase from  Xanthomonas maltophilia  was determined. To this end 200 μl enzyme solution à 63 μg BSAeq/ml of the purification stage after the gel filtration were compounded with 700 μl of the 50 mM tris/HCl buffer pre-tempered to the particular temperature and the reaction started with 100 μl Z-Gly-Tyr-NH 2  (10 mM in the test batch). Aliquots of 100 [mycro]l were stopped after 2 h with 100 μl glacial acetic acid and the activity determined corresponding to 1.4. The result is shown in FIG.  3 . The temperature optimum was approximately 35-40° C. at a pH of 7.5. 
     The relative activity (%) as a function of the time (h) was determined for the temperature stability of peptide amidase from  Xanthomonas maltophilia . To this end 100 μl enzyme solution &amp; 0.73 mgBSAeq/ml were compounded with 900 μl pre-tempered 50 mM tris/HCl buffer and incubated at 20, 30, 37 and 56° C. Specimens were drawn at different times and the activity determined with Z-Gly-Tyr-NH 2  as substrate. The results are shown in FIG.  4 . Whereas the enzyme is inactivated at 56° C. after only a few minutes, it proves to be extremely stable at 20, 30 and 37° C. 
     EXAMPLE 4 
     Enzyme Kinetics of Peptide Amidase 
     The dependency of the reaction rate of peptide amidase from  Xanthomonas maltophilia  on the substrate concentration (Z-Gly-Tyr-NH 2 ) was determined and the kinetic parameters determined from the data according to Marquardt. To this end, 50 μl enzyme solution à 27 μg BSAeq/ml were compounded with 400 μl 50 mM tris/HCl buffer, pH 7.5. The reaction was started by adding Z-Gly-Tyr-NH 2  as substrate. The substrate was present in the reaction batches in a concentration range of 0.4 to 20 mM. The incubation took place for 2 hours at 30° C. The enzyme activity was then determined as described under 1.4. The K m  value was determined with 0.82 mM and v max  with 0.53 U/mg BSAeq. 
     FIG. 5 shows the reaction rate of the deamidation of Z-Gly-Tyr-NH 2  as a function of the substrate concentration. 
     1.4 Standard Assay of Peptide Amidase 
     The reaction conditions were selected with a view to the pH stability of peptide amidase from  Xanthomonas maltophilia.    
     Standard Assay of Peptide Amidase 
     
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 50 mM tris/HCl, pH 7.5 
                 350-700 μl 
               
               
                   
                 Enzyme solution 
                 100-200 μl 
               
               
                   
                 100 mM Z-Gly-Tyr-NH 2  dissolved in 
                  50-100 μl 
               
               
                   
                 buffer/DMF 1:1 (10 mM in the test) 
               
               
                   
                 Test batch 
                 500-1000 μl 
               
               
                   
                 Temperature 
                 30° C. 
               
               
                   
                 Incubation time 
                 variable 
               
               
                   
                   
               
             
          
         
       
     
     In order to stop the reaction 100 μl of the reaction solution was removed, compounded with 100 μl glacial acetic acid and filled with 1.4 ml HPLC mobile solvent. An aliquot of 20 μl was analyzed by HPLC. The conditions for the HPLC analysis of Z-Gly-Tyr-NH 2  were as follows: 
     
       
         
               
               
               
             
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 Mobile solvent: 
                 65% 10 mM TBA-sulfate 
               
               
                   
                   
                 (tetrabutyl ammonium 
               
               
                   
                   
                 sulfate) 
               
               
                   
                   
                 35% acetonitrile 
               
               
                   
                 Travel rate 
                 1 ml/min 
               
               
                   
                 Travel time 
                 10 min 
               
               
                   
                 Detection 
                 280 nm 
               
               
                   
                 Elution 
                 isocratic 
               
               
                   
                 Column 
                 RP-18, ODS Hypersil (5 μm) 
               
             
          
           
               
                   
                 Retention times 
                 Z-Gly-Tyr-NH 2   
                 4.5 min 
               
               
                   
                   
                 Z-Gly-Tyr-OH 
                 5.9 min 
               
               
                   
                 Example 5 
               
               
                   
                 Substrate spectrum 
               
               
                   
                   
               
             
          
         
       
     
     18 mU of peptide amidase (purification state after the gel filtration) per ml test batch was used for the substrate spectrum of peptide amidase from  Xanthomonas maltophilia . The concentration of the tested substrates was 10 mM in the reaction batch. Unless otherwise indicated, L-amino acid derivatives were used. The incubation took place in 50 mM tris/HCl, pH 7.5 at 30° C. for 3 hours. The reaction was stopped by heating for 5 minutes at 95° C. The determination of activity took place using enzymatic determination of the released ammonia (Bergmeyer, U. (1985): Methods of Enzymatic Analysis, p. 459, VCH Verlagsgesellschaft, Weinheim). Table 4a shows the conversion of dipeptide amides, table 4b the conversion of N-acetylamino acid amides and table 4c the influence of the N-terminal protective groups and of the amino acid adjacent to the C terminus. 
     The following thin-layer chromatographic methods were used for the analysis of the substrates and products cited in table 4d. To this end 1 μl of the stopped reaction batch was applied. 
     
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 Stationary phase 
                 DC aluminum foil 60 F 254   
               
               
                   
                   
                 (20 * 10 cm 2 ) 
               
               
                   
                 Mobile phase 
                 Pyridine/butanol/glacial acetic 
               
               
                   
                   
                 acid/water (12:15:3:5) 
               
               
                   
                 Detection 
                 Ninhydrin (0.3% in propan-1-ol) 
               
               
                   
                   
                 The color development took place 
               
               
                   
                   
                 for approximately 3 min at 
               
               
                   
                   
                 100° C.) 
               
               
                   
                   
               
             
          
         
       
     
     The following HPLC separating conditions applied for the analysis of the conversion of longer-chain peptides shown in table 4e: 
     
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                   
                   
                   
                 Retention 
                   
               
               
                   
                   
                   
                   
                 time 
               
             
          
           
               
                   
                   
                 Mobile 
                 amide 
                 acid 
                 Wavelength 
               
             
          
           
               
                   
                 Substrate 
                 solvent* 
                   
                 (min) 
                 (nm) 
               
               
                   
                   
               
             
          
           
               
                   
                 Z-Pro-Leu- 
                 60/40 
                 6.0 
                 7.3 
                 256 
               
               
                   
                 Gly-NH 2   
               
               
                   
                 Z-Gly-Gly- 
                 60/40 
                 4.9 
                 6.0 
                 220 
               
               
                   
                 Leu-NH 2   
               
               
                   
                 Gly-D-Phe- 
                 80/20 
                 3.8 
                 4.4 
                 280 
               
               
                   
                 Tyr-NH 2   
               
               
                   
                 Leu- 
                 80/20 
                 6.1 
                 7.6 
                 220 
               
               
                   
                 encephaline 
               
               
                   
                 amide 
               
               
                   
                   
               
               
                   
                 *10 mM tetrabutylammonium sulfate/acetonitrile  
               
             
          
         
       
     
     In addition, the presence of possible protease- or peptidase activity was tested for with the aid of thin-layer chromatography. No release of amino acids could be detected for any of the peptides. 
     
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 4a 
               
             
             
               
                   
               
               
                 Conversion of dipeptide amides 
               
             
          
           
               
                   
                 Substrate 
                 Relative conversion 
               
               
                   
                 (10 mM) 
                 (%) 
               
               
                   
                   
               
             
          
           
               
                   
                 Ala-Gly-NH 2   
                 39 
               
               
                   
                 Phe-Gly-NH 2   
                 51 
               
               
                   
                 Tyr-Gly-NH 2   
                 54 
               
               
                   
                 Leu-Gly-NH 2   
                 54 
               
               
                   
                 Gly-Gly-NH 2   
                 8 
               
               
                   
                 Val-Gly-NH 2   
                 22 
               
               
                   
                 Pro-Gly-NH 2   
                 66 
               
               
                   
                 Gly-Tyr-NH 2   
                 174 
               
               
                   
                 Phe-Ala-NH 2   
                 203 
               
               
                   
                 Ala-Ala-NH 2   
                 211 
               
               
                   
                 Leu-Ala-NH 2   
                 217 
               
               
                   
                 Gly-Phe-NH 2   
                 214 
               
               
                   
                 Tyr-Phe-NH 2   
                 190 
               
               
                   
                 Ser-Phe-NH 2   
                 217 
               
               
                   
                 Ala-Phe-NH 2   
                 218 
               
               
                   
                 Leu-Phe-NH 2   
                 203 
               
               
                   
                 Val-Phe-NH 2   
                 140 
               
               
                   
                 His-Phe-NH 2   
                 137 
               
               
                   
                 Pro-Phe-NH 2   
                 158 
               
               
                   
                 Tyr-Pro-NH 2   
                 0 
               
               
                   
                 Ala-Asn-OH 
                 0 
               
               
                   
                 Gly-Asn-OH 
                 0 
               
               
                   
                 Ala-Gln-OH 
                 0 
               
               
                   
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 4b 
               
             
             
               
                   
               
               
                 Conversion of N-acetyl amino acid amides 
               
             
          
           
               
                   
                 Substrate (10 mM) 
                 Relative conversion (%) 
               
               
                   
                   
               
             
          
           
               
                   
                 Ac-Ala-NH 2   
                 182 
               
               
                   
                 Ac-Val-NH 2   
                 1 
               
               
                   
                 Ac-Ile-NH 2   
                 6 
               
               
                   
                 Ac-D,L-neopentylglycine 
                 131 
               
               
                   
                 amide 
               
               
                   
                 Ac-Pro-NH 2   
                 0 
               
               
                   
                 Ac-Cys-NH 2   
                 82 
               
               
                   
                 Ac-D,L-Met-NH 2   
                 102 
               
               
                   
                 Ac-Ser-NH 2   
                 58 
               
               
                   
                 Ac-Thr-NH 2   
                 21 
               
               
                   
                 Ac-Phe-NH 2   
                 134 
               
               
                   
                 Ac-Tyr-NH 2   
                 123 
               
               
                   
                 Ac-Phg-NH 2 * 
                 28 
               
               
                   
                 Ac-Trp-NH 2   
                 157 
               
               
                   
                 Ac-Nal-NH 2 * 
                 84 
               
               
                   
                 Ac-Lys-NH 2   
                 175 
               
               
                   
                 Ac-Arg-NH 2   
                 110 
               
               
                   
                 Ac-His-NH 2   
                 177 
               
               
                   
                 Ac-Glu-NH 2   
                 162 
               
               
                   
                   
               
               
                   
                 *Phg = phenylglycine Nal = naphthylalanine  
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 4c 
               
             
             
               
                   
               
               
                 Influence of the protective group and of adjacent 
               
               
                 amino acid on the deamidation of the C terminus 
               
             
          
           
               
                   
                 Substrate (10 mM) 
                 Relative conversion (%) 
               
               
                   
                   
               
             
          
           
               
                   
                 Ac-Tyr-NH 2   
                 123 
               
               
                   
                 Bz-Tyr-NH 2   
                 173 
               
               
                   
                 Gly-Tyr-NH 2   
                 174 
               
               
                   
                 Phe-Ala-NH 2   
                 203 
               
               
                   
                 Ala-Ala-NH 2   
                 211 
               
               
                   
                 Leu-Ala-NH 2   
                 217 
               
               
                   
                 Z-Ala-NH 2   
                 62 
               
               
                   
                 Z-D-Ala-NH 2   
                 0 
               
               
                   
                 N-Methoxy-D,L-Met-NH 2   
                 109 
               
               
                   
                 N-Ethoxy-D,L-Met-NH 2   
                 124 
               
               
                   
                 N-Carbamoyl-D,L-Met-NH 2   
                 77 
               
               
                   
                 N-Acetyl-D,L-Met-NH 2   
                 102 
               
               
                   
                 Arg-Met-NH 2   
                 125 
               
               
                   
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
           
               
                 TABLE 4d 
               
             
             
               
                   
               
               
                 Enzymatic conversion of unprotected amino acid amides 
               
             
          
           
               
                   
                 Substrate (10 mM) 
                 Conversion 
               
               
                   
                   
               
               
                   
                 H-Ala-NH 2   
                 0 
               
               
                   
                 H-arg-NH 2   
                 0 
               
               
                   
                 H-Leu-NH 2   
                 0 
               
               
                   
                 H-Tyr-NH 2   
                 0 
               
               
                   
                 H-Met-NH 2   
                 0 
               
               
                   
                 H-Phe-NH 2   
                 0 
               
               
                   
                 H-Lys-NH 2   
                 0 
               
               
                   
                 H-Val-NH 2   
                 0 
               
               
                   
                 H-Ile-NH 2   
                 0 
               
               
                   
                 H-Thr-NH 2   
                 0 
               
               
                   
                 H-Trp-NH 2   
                 0 
               
               
                   
                 H-Ser-NH 2   
                 0 
               
               
                   
                 H-D-Ala-NH 2   
                 0 
               
               
                   
                 H-D-Leu-NH 2   
                 0 
               
               
                   
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 4e 
               
             
             
               
                   
               
               
                 Enzymatic conversion of longer-chain peptides 
               
             
          
           
               
                   
                 Substrate (10 mM) 
                 Relative conversion (%) 
               
               
                   
                   
               
             
          
           
               
                   
                 Z-Pro-Leu-Gly-NH 2   
                 50 
               
               
                   
                 Z-Gly-Gly-Leu-NH 2   
                 6 
               
               
                   
                 Gly-Phe-D-Phe-NH 2   
                 0 
               
               
                   
                 Gly-D-Phe-Tyr-NH 2   
                 33 
               
               
                   
                 Leu-encephaline amide 
                 52 
               
               
                   
                   
               
             
          
         
       
     
     The results show that with the exception of L-Pro, all proteinogenous, protected amino acid amides and peptide amides are deamidated. Even a few protected, non-proteinogenous amino acid amides are hydrolyzed (phenylglycine, naphthylalanine, neopentylglycine). D-amino acids in the C-terminal position are not converted. The amide function in the side chains of asparagine and glutamine are not attacked. The peptide bonds of longer-chain oligopeptides are not split, unprotected amino acid amides are not converted. 
     EXAMPLE 6 
     Splitting of Racemic Mixtures of N-protected Zmino Acid Amides 
     The tests for the splitting [separation] of racemic mixtures took place in 50 mM triethylammonium carbonate buffer, pH 7.5. The test batches a 50 ml with 20 U peptide amidase each were incubated at 30° C. Z-D,L-Ala-NH 2 , Ac-D,L-Met-NH 2  and Ac-D,L-neopentylglycine amide (Ac-D,L-Npg-NH 2 ) were available as substrates. The concentration of the substrates in the reaction batch was 10 mM. Specimens were taken at different times, the reaction stopped by heating for 5 minutes at 95° C. and the enantiomeric purity examined with HPLC. The results are shown in the following: 
     
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 Z-D-Ala-OH 
                  0.4% 
               
               
                   
                 Z-L-Ala-OH 
                 99.6% 
               
               
                   
                 Ac-D-Met-OH 
                 0.2-0.3% 
               
               
                   
                 Ac-L-Met-OH 
                 99.7-99.8% 
               
               
                   
                 Ac-D-Npg-OH 
                  0.2% 
               
               
                   
                 Ac-L-Npg-OH 
                  99.8%. 
               
               
                   
                   
               
             
          
         
       
     
     The results are also shown in FIG. 6, which refers to the splitting of racemic mixtures of Z-D, L-Ala-NH 2 , and FIG. 7, which shows the enantioselective deamidation of N-Ac-D,L-Met-NH 2 . 
     EXAMPLE 7 
     Influence of Enzyme Effectors on the Peptide Amidase Activity 
     The tests took place in 50 mM tris/HCl, pH 7.5. After a preincubation of the enzyme (38 mU per batch, purification stage after the gel filtration) with the effector (10 mM in the test batch) for 1 h at 30° C. the reaction was started by adding substrate (Z-Gly-Tyr-NH 2 , 10 mM in the test batch). The reaction batches were incubated for 2.5 h at 30° C. Table 5 shows the results of the influence of enzyme effectors on the peptide amidase activity. It is apparent that peptide amidase belongs neither to the classic serine hydrolases which are inhibited at a concentration of PMSF of 1 mM or of Pefabloc of 0.2 mM nor to the metalloenzymes which are inhibited by EDTA or 1.10-phenanthroline in the concentration range indicated here. 
     
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Enzymatic activity in the presence of potential inhibitors 
               
             
          
           
               
                   
                 Potential inhibitor 
                 residual 
               
               
                   
                 (10 mM in the test) 
                 activity (%) 
               
               
                   
                   
               
             
          
           
               
                   
                 CuSO 4 *5 H 2 O 
                 103 
               
               
                   
                 CoCl 2 *6 H 2 O 
                 80 
               
               
                   
                 NiCl 2 *6 H 2 O 
                 93 
               
               
                   
                 HgCl 2 *6 H 2 O 
                 67 
               
               
                   
                 EDTA 
                 96 
               
               
                   
                 1,10-phenanthroline 
                 92 
               
               
                   
                 2,2′-dipyridyl 
                 97 
               
               
                   
                 D-cycloserine 
                 100 
               
               
                   
                 Semicarbazide 
                 99 
               
               
                   
                 1,4-dithio-D,L-threitol (DDT) 
                 96 
               
               
                   
                 Actinonine 
                 106 
               
               
                   
                 Phenylmethane sulfonyl fluoride 
                 58 
               
               
                   
                 (PMSF) 
               
               
                   
                 4-(2-aminoethyl)-benzylsulfonyl 
               
               
                   
                 fluoride 
               
               
                   
                 (Pefabloc) 10.0 mM 
                 0 
               
               
                   
                 0.3 mM 
                 62 
               
               
                   
                 0.1 Mm 
                 82 
               
               
                   
                   
               
             
          
         
       
     
     EXAMPLE 8 
     Influence of Solvents on the Peptide Amidase Activity and Stability of Peptide Amidase During Incubation in Solvent 
     200 μl enzyme solution (59 μg BSAeq/ml, purification stage after the gel filtration) were compounded in each instance with 700 μl of an appropriate mixture of 50 mM tris/solvent, pH 7.5 for the examinations of the influence of solvents on the peptide amidase activity. Dimethylformamide (DMF), acetone, ethanol and propan-1-ol, which were present in the reaction batch up to 70% by volumetric amount, were tested as solvents. The reaction was started by adding substrate Z-Gly-Tyr-NH 2 . The incubation took place for 90 minutes at 30° C. Then, aliquots of 100 μl of the reaction batches were provided with 100 μl glacial acetic acid and the enzyme activity determined as described in 1.4. FIG. 8 shows the results of the influence of solvents on the enzyme activity. It is apparent that at 20% DMF, 94% residual activity is still present and even at 30%, 59% is still present. In the presence of other solvents such as propan-2-ol the enzyme distinctly loses activity but is nevertheless not completely inactivated at a content of 10% (residual activity 30%). 
     In order to determine the solvent stability of peptide amidase from  Xanthomonas maltophilia , 600 μl enzyme solution à 57 μg BSAeq/ml (purification state after the gel filtration) was compounded with 150 μl solvent and, as control, 150 μl 50 mM tris/HCl, pH 7.5. The incubation took place at 30° C. Specimens for the determination of activity were drawn at different times. Dimethylformamide (DMF), acetone, ethanol and propan-1-ol were tested as solvent. FIG. 9 shows the solvent stability of peptide amidase from  Xanthomonas maltophilia . After 26 hours, a residual activity of 32% still results at a solvent content of 20% DMF. 
     EXAMPLE 9 
     Determination of the Molar Mass 
     The relative molar mass of peptide amidase from  Xanthomonas maltophilia  was determined by gel filtration (travel conditions see 1.3.2). The following calibration proteins were used. 
     
       
         
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Molecular 
                   
                   
               
               
                   
                 weight 
                 V e   
                 K AV   
               
               
                   
                 (daltons) 
                 (ml) 
                 (−) 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Ribonuclease A 
                 13700 
                 85.6 
                 0.53 
               
               
                   
                 Chymotrypsinogen 
                 25000 
                 77.9 
                 0.42 
               
               
                   
                 Ovalbumin 
                 43000 
                 65.7 
                 0.26 
               
               
                   
                 Bovine serum 
                 67000 
                 58.6 
                 0.16 
               
               
                   
                 albumin 
               
               
                   
                 Blue dextran 
                 2000000 
                 26.4 
                 0.00 
               
               
                   
                 2000 
               
               
                   
                   
               
             
          
         
       
     
     The detection took place at 280 nm. The elution volume of the peptide amidase was determined by determination of activity. The relative molar mass was determined from the elution volume, using the calibration curve, at 38000±1000 daltons. 
     EXAMPLE 10 
     Inducibility of Peptide Amidase from  Xanthomonas Maltophilia    
     1 l Erlenmeyer flasks were provided with 200 ml of the nutrient medium used in the second part of the screening for the examinations of the inducibility of peptide amidase from  Xanthomonas maltophilia . The amount of yeast extract was raised to 0.1% thereby and only one amide derivative added to 0.5% of the medium in each instance. In addition, media without the addition of amides and with 1.5% yeast extract were examined. Ac-DL-Met-NH 2 , leucine amide and carnitine amide were used as amides. The flasks were inoculated to 0.5% and agitated 2 days at 30° C. and 120 rpm. The obtention of the raw extracts took place as already described above. Table 6 shows the results. It is apparent from the data that the enzyme is formed even without the addition of Ac-DL-Met-NH 2  as inductor in the cell, but that the specific activity can be doubled to tripled by the addition of this inductor. On the other hand, the addition of other amides shows no influence. 
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Induction of peptide amidase from Xanthomonas maltophilia 
               
             
          
           
               
                   
                   
                   
                 Protein 
                 Spec. 
               
               
                   
                   
                   
                 content 
                 activity 
               
               
                   
                   
                 Activity 
                 (mg 
                 (mU/mg 
               
               
                   
                 Additives 
                 (mU/ml) 
                 (BSAeq/ml) 
                 BSAeq) 
               
               
                   
                   
               
             
          
           
               
                   
                 Without 
                 29 
                 6.62 
                 4 
               
               
                   
                 additive 
               
               
                   
                 1.5 yeast 
                 58 
                 8.29 
                 7 
               
               
                   
                 extract 
               
               
                   
                 Ac-DL-Met-NH 2   
                 88 
                 6.65 
                 13 
               
               
                   
                 Leu-NH 2   
                 15 
                 5.22 
                 3 
               
               
                   
                 Carnitine amide 
                 30 
                 7.13 
                 7 
               
               
                   
                   
               
             
          
         
       
     
     EXAMPLE 11 
     Preparation of Racemic, N α -protected Amino Acid Amides for the Enzymatic Separation with Peptide Amidase 
     A) N α -formyl-DL-methionine amide 
     30 g (0.20 mole) DL-methionine amide was agitated with 250 ml methyl formate for two days at room temperature, during which fine, slightly yellow crystals developed. They were filtered off and recrystallized out of a mixture of 150 ml hexane and 160 ml ethanol. After cooling off, filtering, washing and drying, 23 g (55%) N 60  -formyl-DL-methionine amide was obtained in the form of colorless crystals with a melting point of 121-122° C. 
     B) N 60  methylaminocarbonyl-DL-methionine Amide 
     18 ml (0.3 mole) methylisocyanate was dripped into a solution of 30 g (0.2 mole) DL-methionine amide in 350 ml water at 5-10° C., during which a colorless, crystalline precipitate formed. After 30 min of subsequent agitation at 5-10° C. and 30 min at room temperature, the crystals were filtered off, washed with water and dried, yielding 25 g product. A concentrating of the mother liquor yielded a further 10 g. 29 g (70%) N α methylaminocarbonyl-DL-methionine amide in the form of light, flaky crystals with a melting point of 156-157° C. were obtained by recrystallization out of methanol/acetic ester. 
     C) N α -methoxycarbonyl-DL-methionine Amide 
     85 ml (1.11 moles) methylchloroformate were dripped into a solution of 150 g (1.02 moles) DL-methionine amide in 200 ml water at 5-10° C., during which the pH was maintained at 8-10 by the addition of sodium hydroxide solution. After 30 min of subsequent reaction, 60 ml water was added, whereupon crystals formed which were filtered off, washed with water and dissolved in 250 ml water. This solution was extracted twice and the crystallization mother liquor extracted once with 500 ml methylene chloride in each instance. After drying in a vacuum, 162 g yellowish, crystalline residue remained. It was dissolved in 200 ml hot ethanol. Then, 400 ml MTBE was added and after the appearance of the first colorless crystals another 200 ml MTBE. After cooling in an ice bath, filtering off, washing with 200 ml MTBE and drying, 127 g (60k) N α -methoxycarbonyl-DL-methionine amide with a melting point of 93-94° C. was obtained. 
     D) N α -ethoxycarbonyl-DL-methionine amide 
     106 ml (1.10 moles) ethylchloroformate were dripped into a solution of 150 g (1.02 moles) DL-methionine amide in 200 ml water at 5-10° C., during which the pH was maintained at 7-9 by adding sodium hydroxide solution. A thick, finely crystalline precipitate rapidly formed. A suspension which was just able to still be agitated was obtained by the addition of 800 ml water, which suspension was agitated 3 hours further at room temperature. Then the colorless crystals were filtered off, washed with water and recrystallized out of 900 ml water. After cooling to 0-5° C., filtering off, washing with water and drying, 129 g (57%) N α ethoxycarbonyl-DL-methionine amide with a melting point of 110-112° C. was obtained. 
     E) N α -benzyloxycarbonyl-DL-methionine Amide 
     51 ml benzyloxycarbonyl chloride was dripped into a solution of 50 g (0.34 mole) DL-methionine amide in 200 ml water at 5-20° C., during which the pH was maintained at 7-9 with sodium hydroxide solution. A slimy precipitate formed immediately which became finely crystalline upon the addition of 200 ml MTBE. After the end of the addition the mixture was agitated 30 min further at room temperature. The precipitated, colorless crystals were then filtered off, washed with a little MTBE and recrystallized out of 700 ml toluene. After filtering off, washing with toluene and drying, 67 g (69%) N α benzyloxycarbonyl-DL-methionine amide with a melting point of 120-122° C. was obtained. 
     F) N α -acetyl-DL-neopentylglycine Amide 
     12 ml (0.13 mole) acetic anhydride was dripped into a solution of 17 g (0.12 mole) DL-neopentylglycine in 200 ml water under ice cooling in approximately 30 min, during which the pH was maintained at approximately 8 with sodium hydroxide solution. After 1 h agitation at room temperature, an extraction was performed three times with 100 ml methylene chloride and the organic phase was evaporated after drying over sodium sulfate. 20 g (89%) N-acetyl-DL-neopentylglycine remained as colorless solid. 
     Then, 14 ml (0.1 mole) triethylamine was dripped into 19 g (0.1 mole) N-acetyl-DL-neopentylglycine in 150 ml THF at −10° C. in 15 min. After 10 min a solution of 10 ml (0.1 mole) ethylchloroformate in 10 ml THF was added dropwise in such a manner that the temperature did not exceed −5° C. Then, 70 ml 25% ammonia solution were added all at once, then 50 ml THF, following which the mixture was agitated 2 h at −5° C. and overnight at room temperature. The batch was then evaporated to dryness, thoroughly agitated with 100 ml water, the solid filtered off and recrystallized out of 70 ml methanol. A total of 15 g (78%) N α -acetyl-DL-neopentylglycine amide was obtained in the form of colorless crystals with a melting point &gt;205° C. 
     G) N α -benzyloxycarbonyl-DL-neopentylglycine amide 
     15 ml (0.11 mole) benzyloxycarbonylchloride was dripped into a solution of 15 g (0.10 ml) DL-neopentylglycine in 150 ml water under ice cooling in approximately 30 min, during which the pH was maintained at approximately 9 with sodium hydroxide solution. After 1 h agitation at room temperature an extraction was carried out three times with 75 ml methylene chloride and the organic phase dried in a rotary vacuum after drying over sodium sulfate. 25 g (90t) N-benzyloxycarbonyl-DL-neopentylglycine remained as a colorless oil which crystallized upon standing. 
     Then, 12 ml (0.90 mole) triethylamine was dripped into 24 g (0.09 mole) N-benzyloxycarbonyl-DL-neopentylglycine in 150 ml THF at −10° C. in 15 min. After 10 min a solution of 8 ml (0.09 mole) ethylchloroformate in 10 ml THF was added dropwise in approximately 30 min in such a manner that the temperature did not exceed −5° C. After 30 min agitation at −5° C., 60 ml 25% ammonia solution were added all at once and the mixture then agitated 2 h at −5° C and overnight at room temperature. The batch was then evaporated to dryness, taken up with 50 ml water and extracted with 150 ml methylene chloride. After drying of the organic phase over sodium sulfate and drying in a rotary vacuum, 26 g residue remained which was dissolved in 50 ml methanol and then compounded with 70 ml water within approximately 2 h under ice cooling. After filtration, washing with methanol/water and drying, 13 g (54%) N α benzyloxycarbonyl-DL-neopentylglycine amide were obtained as a colorless solid with a melting range of 132-137° C. 
     EXAMPLE 12 
     Preparation of D-neopentylglycine from N-acetyl-D,L-neopentylglycine Amide 
     10 mM N-acetyl-D,L-neopentylglycine amide were incubated with 20 U peptide amidase in a 50 ml Erlenmeyer flask in 50 mM triethylammonium carbonate buffer at pH 7.5 and 30° C. The conversion was controlled by HPLC. After 24 hours the conversion was 50% and did not change any more as time passed. The enzyme was now deactivated by five minutes of heating and the enantiomeric purity examined. The purity of N-acetyl-L-neopentylglycine (determined by ligand exchange chromatography) was 99.8% and the purity of the N-acetyl-D-neopentylglycine amide (determined by inclusion chromatography) was 99.7%. The two products were separated by anion exchange chromatography on Amberlite M 500 and the N-acetyl-D-neopentylglycine amide converted by several hours of hydrolysis in 6-normal, boiling hydrochloric acid into the free D-amino acid, during which the demonstration of the complete conversion by HPLC or DC takes place. The amount of rotation of the D-neopentylglycine [α 25   D ] (c=0.5, 6N HCl) was −16.2°. 
     Methods Used for the Separation of Enantiomers 
     The enantiomers of the remaining N-acyl-amino acid amide were separated from each other by inclusion chromatography and the two enantiomers of the N-acyl amino acid product by ligand exchange chromatography. 
     
       
         
           
             1 
           
           
             
               17 amino acids 
               amino acid 
               single 
               linear 
             
             
               peptide 
             
             
               not provided 
             
              1
Xaa Arg Asn Val Pro Phe Pro Tyr Ala Glu Thr Asp Val Ala Asp
1               5                   10                  15
Leu Gln

Technology Category: 8