Patent Abstract:
The present invention relates to the processes of racemization and deprotection of special N-protected amino acids in the acylase/racemase system for the total conversion of special N-protected racemic amino acids into optically pure amino acids.

Full Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     The present application claims priority to German Application DE 10050123.0, filed Oct. 11, 2000. The entire contents of the application is incorporated herein by reference. 
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to the processes of racemization and deprotection of special N-protected amino acids in the acylase/racemase system for the total conversion of special N-protected racemic amino acids into optically pure amino acids. 
     2. Discussion of Background 
     Optically pure enantiomers of both L- and D-amino acids are important starting compounds in chemical syntheses, as well as for parenteral nutrition. Many methods of producing oprtically pure amino acids are possible and known to a person skilled in the art. Suitable processes include relevant enzymatic processes since they take place catalytically and produce with very high enantiomer concentrations of amino acids. 
     A racemic mixture of amino acids is not optically pure and contains both L-amino acid and D-amino acid enantiomers. Both L-amino acid and D-amino acid enantiomers can be utilized through enzymatic catalysis of a racemic mixture of N-protected amino acids. For example, it is known that L-amino acids are prepared from a racemic mixture of N-acetylated amino acids by using amino acid acylases. However, it is thought that these acylases are specific only for the cleavage of N-acetyl-protected amino acids and amines/alcohols (EP99118844.2; A. S. Bommarius et al., Tetrahedron; Asymmetry, 1997, Vol. 8, 3197-3200). Further, various racemization processes have been developed to prepare L-amino acids from the remaining D-acetyl amino acid fraction of the racemic mixture. DE 19935268.2 discloses an acetylamino acid racemase in the acylase/acetylamino acid racemase system that can prepare optically pure L-methionine from a racemic mixture of N-acetylmethionine. 
     Less specific N-acetylamino acid racemases (AAR) have been described previously. These racemases can be found in the microorganisms  Streptomyces atratus  Y-53 (Tokuyama et al., Appl. Microbiol. Biotechnol. 1994, 40, 835-840) and  Amycolatopis  sp. TS-1-60 (Tokuyama et al., Appl. Microbiol. Biotechnol. 1995a, 42, 853-859). For example, the racemase of Amycolatopis sp. TS-1-60 can catalyze the racemization of N-carbamoylamino acids to L-amino acids, although with less than optimal activity. 
     Processes for complete conversion of amino acids other than acetyl-protected or carbamoyl-protected amino acids to optically enriched amino acids are not known. Further, racemases with the ability to convert amino acids other than acetyl-protected or carbamoyl-protected amino acids are not known. Therefore, there is a need for enzymatic processes that racemize N-protected amino acids in general, as well as those N-protected amino acids other than acetyl-protected or carbamoyl-protected amino acids. The N-protected amino acid products of such racemic converstions may then be converted into the optically enriched amino acid by a subsequent enzymatic cleavage of the protecting group(s). 
     SUMMARY OF THE INVENTION 
     One object of the present invention is a process for the racemization of N-protected amino acids, comprising contacting a compound of the general formula (I):                           
     wherein 
     X=O, NH, 
     R 1 =CH 3 , CH 3 CH 2 , tert-butyl, benzyl and R 2  denotes the α-radical of a natural or synthetic amino acid, with an N-acetylamino acid racemase. 
     Another object of the present invention is a process for the cleavage of the protective group from N-protected amino acids, comprising contacting a compound of the general formula (I):                           
     wherein 
     X=O, NH, 
     R 1 =CH 3 , CH 3 CH 2 , tert.-butyl, benzyl, wherein if X is NH, then R 1  may be H, and R 2  denotes the α-radical of a natural or synthetic amino acid, with an amino acid acylase. 
     Another object of the present invention is a process, comprising contacting a compound of the general formula (I):                           
     wherein 
     X=O, NH, 
     R 1 =CH 3 , CH 3 CH 2 , tert-butyl, benzyl, wherein if X is NH, then R 1  may be H, and 
     R 2  denotes the α-radical of a natural or synthetic amino acid, 
     with an N-acetylamino acid racemase (AAR) in the presence of an amino acid acylase. In one embodiment, the racemase contacts the compound first followed by the acylase. Either or both the racemase and the acylase may be in a homogeneous free form, a recombinant free form, a part of a host organism, a portion of a digested cell mass, an immobilised form. 
     Another object of the present invention is to provide a process for the production of optically enriched amino acids from a racemic mixture of amino acids that are N-protected. 
     Another object of the present invention is to provide a process for the production of optically enriched amino acids from a racemic mixture of amino acids that are N-protected by means of a urethane-protected or carbamoyl-protected amino acid. 
     DETAILED DESCRIPTION OF THE INVENTION 
     Unless specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled artisan of molecular biology and biochemistry. 
     Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting. 
     Reference is made to standard textbooks of molecular biology that contain definitions and methods and means for carrying out basic scientific techniques, encompassed by the present invention. See, for example, Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1982) and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989) and various references cited therein. 
     The term “α-radical of an amino acid” is understood to denote the radical located on the α-C atom of an α-amino acid. This radical may be derived from a natural amino acid, as described in Beyer-Walter, Lehrbuch der organischen Chemie, S. Hirzel Verlag Stuttgart, 22 nd  Edition, 1991, p. 822f. Furthermore, corresponding α-radicals of synthetic α-amino acids are also covered, as listed for example in DE19903268.8. 
     “Optically enriched”, or “enantiomer-enriched”, compounds within the scope of the present invention is understood to mean the presence of an optical antipode mixed with the other antipodes in a concentration of &gt;50 mole %. 
     “Polynucleotide” in general relates to polyribonucleotides and polydeoxyribonucleotides, it being possible for these to be non-modified RNA or DNA or modified RNA or DNA. 
     “Polypeptides” are understood as meaning peptides or proteins, which comprise two or more amino acids, bonded via peptide bonds. 
     “Free form” of a polynucleotide or polypeptide refers to a polynucleotide or polypeptide separated out of its natural environment and into an aqueous solution. 
     “Digested cell mass” is any solution of cellular components produced as a direct result of disrupting the integrity of a cell wall and/or cell membrane. The cell may be a unicellular organism and/or may be a portion of a multi-cellular organism. 
     “N-acetylamino acid racemase” denotes a class of enzymes that can racemise optically enriched N-acetylamino acids. On account of the great similarity to one another, all N-acetylamino acid racemases known to the person skilled in the art can be used for the present conversions. Preferably, the racemases to be used are those from  Streptomyces atratus  Y-53 as well as Amycolatopis sp. TS-1-60. A process that is particularly preferred is one comprising the N-acetylamino acid racemase from  Amycolatopsis orientalis  subspecies  lurida  (SEQ ID NO. 2), since this particular N-acetylamino acid racemase has advantages over other representatives of this class of compounds with regard to dependence on metal ions and activity (EP99118844.2) This enzyme is encoded by the polynucleotide of SEQ ID NO. 1. 
     Also included in the present invention are those racemases having amino acid sequences that are at least 70, 80, 85, 90, 95, and 98% identical to SEQ ID NO. 2 and which have racemase activity. 
     Homology, sequence similarity, or sequence identity of nucleotide or amino acid sequences may be determined conventionally by using known software or computer programs such as the BestFit or Gap pairwise comparison programs (GCG Wisconsin Package, Genetics Computer Group, 575 Science Drive, Madison, Wis. 53711). BestFit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981), to find the best segment of identity or similarity between two sequences. Gap performs global alignments, i.e. aligning all of one sequence with all of another similar sequence, using the method of Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970). When using a sequence alignment program such as BestFit, to determine the degree of sequence homology, similarity, or identity, the default setting may be used, or an appropriate scoring matrix may be selected to optimize identity, similarity, or homology scores. Similarly, when using a program such as BestFit to determine sequence identity, similarity, or homology between two different amino acid sequences, the default settings may be used, or an appropriate scoring matrix, such as blosum45 or blosum80, may be selected to optimize identity, similarity, or homology scores. 
     Further racemases that may be used according to the invention are those portions or fragments of the above-mentioned polypeptides exhibiting any enzymatic racemization of optically enriched N-acetylamino acids. 
     Methods for determining racemase activity of polypeptides have been described previously. The analysis can thus be carried out, for example, by reacting enantiomer-pure amino acid derivatives in the presence of a solution containing at least one polypeptide and following the formation of a corresponding racemate using a polarimeter (Perkin-Elmer 241) at various wavelengths. The reaction can be carried out at temperatures ranging from 15 to 55° C. (heatable cell) for time increments from 3 to 12 hours in reaction media deemed appropriate for optimizing racemase activity. For example, the reaction media can be buffered at a pH ranging from 5.0 to 9.0 and can include divalent metal ion salt concentrations ranging from 1 to 15 mM. 
     “Amino acid acylases” within the scope of the invention is understood to denote enzymes that deacetylate N-acylamino acids in a stereospecific manner. In principle all representatives of this class of compound known to the person skilled in the art are suitable for the reactions according to the invention and may be employed. It is preferred, however, to employ amino acid acylases such as the L-specific acylase I or D-specific acylase from  Aspergillus oryzae.  Both amino acid acylases can be obtained from Amano International Enzyme Company at 1157 N Main Street, Lombard, Ill. 60148. 
     Further acylases that may be used for the reaction are described in the following literature citations: Wakayama M, Yada H, Kanda S, Hayashi S, Yatsuda Y, Sakai K, Moriguchi M, Role of conserved histidine residues in D-aminoacylase from  Alcaligenes xylosoxydans  subsp.  xylosoxydans  A-6, Biosci. Biotechnol. Biochem. 2000 Jan;64(l):1-8; Wakayama M, Hayashi S, Yatsuda Y, Katsuno Y, Sakai K, Moriguchi M., Overproduction of D-aminoacylase from  Alcaligenes xylosoxydans  subsp.  xylosoxydans  A-6 in  Escherichia coli  and its purification, Protein Expr. Purif. 1996 Jun;7(4):395-9; Wakayama M, Katsuno Y, Hayashi S, Miyamoto Y, Sakai K, Moriguchi M., Cloning and sequencing of a gene encoding D-aminoacylase from  Alcaligenes xylosoxydans  subsp.  xylosoxydans  A-6 and expression of the gene in  Escherichia coli,  Biosci. Biotechnol. Biochem. 1995 Nov;59(11):2115-9; Wakayama M, Ashika T, Miyamoto Y, Yoshikawa T, Sonoda Y, Sakai K, Moriguchi M.; Primary structure of N-acyl-D-glutamate amidohydrolase from  Alcaligenes xylosoxydans  subsp.  xylosoxydans  A-6, J. Biochem. (Tokyo). 1995 Jul;118(1):204-9; Chen HP, Wu SH, Wang KT., D-Aminoacylase from  Alcaligenes faecalis  possesses activities on D-methionine, Bioorg. Med. Chem. 1994 Jan;2(1):1-5; Moriguchi M, Sakai K, Miyamoto Y, Wakayama M., Production, purification, and characterization of D-aminoacylase from  Alcaligenes xylosoxydans  subsp.  xylosoxydans  A-6, Biosci. Biotechnol. Biochem. 1993 Jul;57(7):1149-52; Yang YB, Hsiao KM, Li H, Yano H, Tsugita A, Tsai YC, Characterization of D-aminoacylase from  Alcaligenes denitrficans  DA181, Biosci. Biotechnol. Biochem. 1992 Sep;56(9):1392-5; Tsai YC, Lin CS, Tseng TH, Lee H, Wang YJ, Production and immobilization of D-aminoacylase of  Alcaligenes faecalis DA 1 for optical resolution of N-acyl-DL-amino acids, Enzyme Microb. Technol. 1992 May;14(5):384-9; Batisse N, Weigel P, Lecocq M, Sakanyan V., Two amino acid amidohydrolase genes encoding L-stereospecific carbamoylase and aminoacylase are organized in a common operon in  Bacillus stearothermophilus,  Appl. Environ. Microbiol. 1997 Feb;63(2):763-6; Yang YB, Hu HL, Chang MC, Li H, Tsai YC, Purification and characterization of L-aminoacylase from  Alcaligenes denitrficans  DA181, Biosci. Biotechnol. Biochem. 1994 Jan;58(l):204-5; Jakob M, Miller YE, Rohm KH, Cloning and sequence analyses of cDNAs encoding aminoacylase I from porcine kidney, Biol. Chem. Hoppe Seyler. 1992 Dec;373(12):1227-31; Mitta M, Ohnogi H, Yamamoto A, Kato I, Sakiyama F, Tsunasawa S., The primary structure of porcine aminoacylase 1 deduced from cDNA sequence, J. Biochem. (Tokyo). 1992 Dec;112(6):737-42; Bommarius AS, Drauz K, Klenk H, Wandrey C., Operational stability of enzymes. Acylase-catalyzed resolution of N-acetyl amino acids to enantiomerically pure L-amino acids, Ann. N Y Acad. Sci. 1992 Nov 30;672:126-36; Gentzen I, Loffler HG, Schneider F., Aminoacylase from  Aspergillus oryzae.  Comparison with the pig kidney enzyme, Z. Naturforsch. [C]. 1980 Jul-Aug;35(7-8):544-50. 
     Further acylases that may be used according to the invention are those portions of the above-mentioned polypeptides exhibiting any deacetylation of N-acylamino acids in a stereospecific manner. 
     Methods for determining acylase activity of polypeptides on various N-protected amino acids have been described previously. The analysis can thus be carried out, for example, by reacting various N-protected amino acids derivatives in the presence of a solution containing at least one polypeptide and following the increasing presence of the unmodified N-amino acid. The reaction can be carried out at temperatures ranging from 15 to 55° C. (heatable cell) for time increments from 3 to 12 hours in reaction media deemed appropriate for optimizing acylase activity. For example, the reaction media can be buffered at a pH ranging from 5.0 to 9.0 and can include divalent metal ion salt concentrations ranging from 1 to 15 mM. 
     The invention also provides host organisms which express one or both of the racemase and acylase described herein. These enzymes may be expressed from the chromosome, either as endogenous or inserted by recombinations, or from a vector or plasmid existing episomally. These organisms can be cultured continuously or discontinuously in the batch process (batch culture) or in the fed batch (feed process) or repeated fed batch process (repetitive feed process). A summary of known culture methods is described in the textbook by Chmiel (Bioprozesstechnik 1. Einfiihrung in die Bioverfahrenstechnik [Bioprocess Technology 1. Introduction to Bioprocess Technology (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen [Bioreactors and Peripheral Equipment] (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)). 
     The culture medium to be used must meet the requirements of the particular strains in a suitable manner. Descriptions of culture media for various microorganisms are contained in the handbook “Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D.C., USA, 1981). 
     Digested cell mass may be produced, for example, from cell lysis methods described in Glenney, Jr., J. R. and Zokas, L. (1989). J. Cell Biol.108:2401; Glenney, Jr., J. R. (1991). Meth. Enzymology 201:92; Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1982); Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989) and various references cited therein. Such cell lysis methods may be performed on unicellular organisms or cells from multicellular organisms which may or may not express one or both of the racemase and acylase described herein. 
     N-acetylamino acid racemases and amino acid acylases may be used together, or successively, in the free form as homogeneously purified compounds or as enzymes produced by recombinant techniques. In addition the enzymes may also be used as a constituent of a host organism (whole cell catalyst as in U.S. Ser. No. 09/407062) or in combination with the digested cell mass of the host organism. It is also possible to use the enzymes in immobilised form (Bhavender P. Sharma, Lorraine F. Bailey and Ralph A. Messing, “Immobilisierte Biomaterialiem—Techniken und Anwendungen”, Angew. Chem. 1982, 94, 836-852). The immobilisation is advantageously carried out by lyophilisation (Dordick et al. J. Am. Chem. Soc. 194, 116, 5009-5010; Okahata et al. Tetrahedron Lett. 1997, 38, 1971-1974; Adlercreutz et al. Biocatalysis 1992, 6, 291-305). The lyophilisation is preferably carried out in the presence of surfactants such as Aerosol OT, polyvinylpyrrolidone, polyethylene glycol (PEG), or Brij 52 (diethylene glycol monocetyl ether) (Goto et al. Biotechnol. Techniques 1997, 11, 375-378). 
     The reaction according to the invention is preferably carried out in an enzyme-membrane reactor (DE 199 10 691.6). 
     Methods for the determination of N-amino acids have been described previously. The analysis can thus be carried out, for example, as described in Spackman et al. (Analytical Chemistry, 30, (1958), 1190) by anion exchange chromatography with subsequent ninhydrin derivation, or it can be carried out by reversed phase HPLC, for example as described by Lindroth et al. (Analytical Chemistry (1979) 51: 1167-1174). 
     The present invention is explained in more detail with the aid of the following embodiment examples. The microorganism,  Amycolatopsis orientalis  subsp.  lurida,  has been filed at the German Collection for Microorganisms under Number DSM43134. 
    
    
     EXAMPLES 
     Example 1 
     Detection of the racemase activity of a recombinant N-acetylamino acid racemase AAR enzyme 
     The substrate spectrum of the N-acetylamino acid racemase (AAR) from  Amycolatopsis orientalis  subsp.  lurida  was tested with the enzyme assay described herein below. 
     The composition of the assay was as follows: 
     
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 Buffer Tris/HCl 
                 50 
                 mM (pH 8.0) 
               
               
                   
                 Substrate 
                 25 
                 mM 
               
               
                   
                 Cobalt chloride 
                 6 
                 mM 
               
               
                   
                 AAR 
                 ca. 150 
                 μg purified protein 
               
               
                   
                 Final volume 
                 1 
                 ml 
               
               
                   
                   
               
             
          
         
       
     
     Enantiomer-pure amino acid derivatives were used in the assay and the formation of the corresponding racemate was followed using a polarimeter (Perkin-Elmer 241). The incubation was carried out at 30° C. (heatable cell) for 3 to 12 hours. The measurements were made at a wavelength of λ=365 nm. 
     
       
         
               
             
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 List of the tested substrates and 
               
               
                 corresponding specific activity of the AAR. 
               
             
          
           
               
                   
                 Substrate 
                 Specific Activity 
               
               
                   
                   
               
               
                   
                 N-methyloxycarbonyl-L-Met 
                 42 mU/mg 
               
               
                   
                   
               
             
          
         
       
     
     Example 2 
     Producing D-Met and L-Met from Moc-L-Met 
     Moc-L-Met was used in an assay to determine the activity of the L-acylase from  Aspergillus oryzae.    
     A. Producing L-Met from Moc-L-Met 
     
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 Buffer Tris/HCl 
                 50 
                 mM (pH 8.0) 
               
               
                   
                 Moc-L-Met 
                 25 
                 mM 
               
               
                   
                 Cobalt chloride 
                 6 
                 mM 
               
               
                   
                 L-Acylase 
                 2.0 
                 U 
               
               
                   
                 ( Aspergillus oryzae ) 
               
               
                   
                 Final volume 
                 200 
                 μL 
               
               
                   
                 Volume activity: 
                 1.4 
                 U/ml 
               
               
                   
                   
               
             
          
         
       
     
     B. Producing D-Met from Moc-L-Met 
     Moc-L-Met was used in an assay to determine the activity of the D-acylase from  Aspergillus oryzae  in the presence of an N-acetylamino acid racemase (AAR) from  Amycolatopsis orientalis  subsp.  lurida . 
     
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 Buffer Tris/HCl 
                 50 
                 mM (pH 8.0) 
               
               
                   
                 Moc-L-Met 
                 25 
                 mM 
               
               
                   
                 Cobalt chloride 
                 6 
                 mM 
               
               
                   
                 D-Acylase 
                 2.4 
                 U 
               
               
                   
                 (Amano International 
               
               
                   
                 Enzyme Company at 
               
               
                   
                 1157 N Main Street, 
               
               
                   
                 Lombard, IL 60148) 
               
               
                   
                 AAR 
                 0.4 
                 U 
               
               
                   
                 Final volume 
                 200 
                 μL 
               
               
                   
                 Volume activity: 
                 0.6 
                 U/ml 
               
               
                   
                   
               
             
          
         
       
     
     D-Met and L-Met were detected by reverse phase HPLC (RP 18). The unit data for the enzymes refer to specific activity with N-Ac-L-Met and N-Ac-D-Met as substrate. 
     Numerous modifications and variations on the present invention are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the accompanying claims, the invention may be practiced otherwise than as specifically described herein. 
     
       
         
               
             
           
               
                   
               
             
             
               
                                    
               
               
                 #             SEQUENCE LISTING  
               
               
                   
               
               
                   
               
               
                 &lt;160&gt; NUMBER OF SEQ ID NOS: 2  
               
               
                   
               
               
                 &lt;210&gt; SEQ ID NO 1  
               
               
                 &lt;211&gt; LENGTH: 1107  
               
               
                 &lt;212&gt; TYPE: DNA  
               
               
                 &lt;213&gt; ORGANISM: Amycolatopsis orientalis  
               
               
                 &lt;220&gt; FEATURE:  
               
               
                 &lt;221&gt; NAME/KEY: CDS  
               
               
                 &lt;222&gt; LOCATION: (1)..(1107)  
               
               
                 &lt;223&gt; OTHER INFORMATION:  
               
               
                   
               
               
                 &lt;400&gt; SEQUENCE: 1  
               
               
                   
               
               
                 gtg aaa ctc agc ggt gtg gaa ctg cgc cgg gt  
               
               
                 #c cgg atg ccg ctc gtg       48  
               
               
                 Val Lys Leu Ser Gly Val Glu Leu Arg Arg Va  
               
               
                 #l Arg Met Pro Leu Val  
               
               
                 1               5    
               
               
                 #                10   
               
               
                 #                15  
               
               
                   
               
               
                 gcc ccg ttc cgg acg tcg ttc ggg acg cag tc  
               
               
                 #c gag cgg gaa ttg ctg       96  
               
               
                 Ala Pro Phe Arg Thr Ser Phe Gly Thr Gln Se  
               
               
                 #r Glu Arg Glu Leu Leu  
               
               
                             20       
               
               
                 #            25       
               
               
                 #            30  
               
               
                   
               
               
                 ctg gtc cgc gcg gtg acc ccg gcg ggc gag gg  
               
               
                 #c tgg ggc gaa tgt gtc      144  
               
               
                 Leu Val Arg Ala Val Thr Pro Ala Gly Glu Gl  
               
               
                 #y Trp Gly Glu Cys Val  
               
               
                         35           
               
               
                 #        40           
               
               
                 #        45  
               
               
                   
               
               
                 gcg atg gag gcg ccg ctc tac tcg tcg gag ta  
               
               
                 #c aac gac gcc gcc gag      192  
               
               
                 Ala Met Glu Ala Pro Leu Tyr Ser Ser Glu Ty  
               
               
                 #r Asn Asp Ala Ala Glu  
               
               
                     50               
               
               
                 #    55               
               
               
                 #    60  
               
               
                   
               
               
                 cac gtg ctg cgg aac cat ctg atc ccc gca ct  
               
               
                 #g ctg gcg gcc gag gac      240  
               
               
                 His Val Leu Arg Asn His Leu Ile Pro Ala Le  
               
               
                 #u Leu Ala Ala Glu Asp  
               
               
                 65                   
               
               
                 #70                   
               
               
                 #75                   
               
               
                 #80  
               
               
                   
               
               
                 gtg acc gcg cac aag gtg acg ccg ttg ctg gc  
               
               
                 #g aag ttc aag ggc cac      288  
               
               
                 Val Thr Ala His Lys Val Thr Pro Leu Leu Al  
               
               
                 #a Lys Phe Lys Gly His  
               
               
                                 85   
               
               
                 #                90   
               
               
                 #                95  
               
               
                   
               
               
                 cgg atg gcg aag ggc gcg ctg gag atg gcg gt  
               
               
                 #c ctc gac gcc gaa ctc      336  
               
               
                 Arg Met Ala Lys Gly Ala Leu Glu Met Ala Va  
               
               
                 #l Leu Asp Ala Glu Leu  
               
               
                             100       
               
               
                 #           105       
               
               
                 #           110  
               
               
                   
               
               
                 cgc gcg cat gac cgg tcc ttc gcg gcc gag ct  
               
               
                 #g ggg tcc act cgc gac      384  
               
               
                 Arg Ala His Asp Arg Ser Phe Ala Ala Glu Le  
               
               
                 #u Gly Ser Thr Arg Asp  
               
               
                         115           
               
               
                 #       120           
               
               
                 #       125  
               
               
                   
               
               
                 tcc gtg gcc tgc ggg gtc tcg gtc ggg atc at  
               
               
                 #g gac tcg atc ccg cac      432  
               
               
                 Ser Val Ala Cys Gly Val Ser Val Gly Ile Me  
               
               
                 #t Asp Ser Ile Pro His  
               
               
                     130               
               
               
                 #   135               
               
               
                 #   140  
               
               
                   
               
               
                 ctg ctc gac gtc gtc ggc ggc tac ctc gac ga  
               
               
                 #g ggc tac gtc cgg atc      480  
               
               
                 Leu Leu Asp Val Val Gly Gly Tyr Leu Asp Gl  
               
               
                 #u Gly Tyr Val Arg Ile  
               
               
                 145                 1  
               
               
                 #50                 1  
               
               
                 #55                 1  
               
               
                 #60  
               
               
                   
               
               
                 aag ctg aag atc gag ccc ggc tgg gac gtc ga  
               
               
                 #g ccg gtc cgg cag gtg      528  
               
               
                 Lys Leu Lys Ile Glu Pro Gly Trp Asp Val Gl  
               
               
                 #u Pro Val Arg Gln Val  
               
               
                                 165   
               
               
                 #               170   
               
               
                 #               175  
               
               
                   
               
               
                 cgt gag cgc ttc ggt gac gac gtg ctg ctg ca  
               
               
                 #g gtc gac gcg aac acc      576  
               
               
                 Arg Glu Arg Phe Gly Asp Asp Val Leu Leu Gl  
               
               
                 #n Val Asp Ala Asn Thr  
               
               
                             180       
               
               
                 #           185       
               
               
                 #           190  
               
               
                   
               
               
                 gcg tac acg ctg ggc gac gcg ccc ctg ctg tc  
               
               
                 #c cgg ctc gac ccg ttc      624  
               
               
                 Ala Tyr Thr Leu Gly Asp Ala Pro Leu Leu Se  
               
               
                 #r Arg Leu Asp Pro Phe  
               
               
                         195           
               
               
                 #       200           
               
               
                 #       205  
               
               
                   
               
               
                 gac ctg ctg ctg atc gag cag ccg ctc gaa ga  
               
               
                 #a gag gac gtg ctc ggc      672  
               
               
                 Asp Leu Leu Leu Ile Glu Gln Pro Leu Glu Gl  
               
               
                 #u Glu Asp Val Leu Gly  
               
               
                     210               
               
               
                 #   215               
               
               
                 #   220  
               
               
                   
               
               
                 cac gcc gag ctg gcc aag cgg atc cgg acg cc  
               
               
                 #g atc tgc ctc gac gag      720  
               
               
                 His Ala Glu Leu Ala Lys Arg Ile Arg Thr Pr  
               
               
                 #o Ile Cys Leu Asp Glu  
               
               
                 225                 2  
               
               
                 #30                 2  
               
               
                 #35                 2  
               
               
                 #40  
               
               
                   
               
               
                 tcg atc gtc tcg gcc aag gcc gcc gcg gac gc  
               
               
                 #g atc aag ctc ggc gcc      768  
               
               
                 Ser Ile Val Ser Ala Lys Ala Ala Ala Asp Al  
               
               
                 #a Ile Lys Leu Gly Ala  
               
               
                                 245   
               
               
                 #               250   
               
               
                 #               255  
               
               
                   
               
               
                 tgc cag atc gtc aac atc aaa ccg ggc cgg gt  
               
               
                 #c ggc gga tac ctc gaa      816  
               
               
                 Cys Gln Ile Val Asn Ile Lys Pro Gly Arg Va  
               
               
                 #l Gly Gly Tyr Leu Glu  
               
               
                             260       
               
               
                 #           265       
               
               
                 #           270  
               
               
                   
               
               
                 gcc cgc cgg gtg cac gac gtc tgc gcg gca ca  
               
               
                 #c ggg atc gcg gtg tgg      864  
               
               
                 Ala Arg Arg Val His Asp Val Cys Ala Ala Hi  
               
               
                 #s Gly Ile Ala Val Trp  
               
               
                         275           
               
               
                 #       280           
               
               
                 #       285  
               
               
                   
               
               
                 tgc ggc ggg atg atc gag acc ggg ctc ggc cg  
               
               
                 #g gcg gcc aac gtc gca      912  
               
               
                 Cys Gly Gly Met Ile Glu Thr Gly Leu Gly Ar  
               
               
                 #g Ala Ala Asn Val Ala  
               
               
                     290               
               
               
                 #   295               
               
               
                 #   300  
               
               
                   
               
               
                 ctg gcc tcg ctg ccc ggc ttc acg ctg ccg gg  
               
               
                 #g gac acc tcg gcg tcc      960  
               
               
                 Leu Ala Ser Leu Pro Gly Phe Thr Leu Pro Gl  
               
               
                 #y Asp Thr Ser Ala Ser  
               
               
                 305                 3  
               
               
                 #10                 3  
               
               
                 #15                 3  
               
               
                 #20  
               
               
                   
               
               
                 ggc cgg ttc tat cgc acc gac atc acc gag cc  
               
               
                 #g ttc gtg ctg gac gcc     1008  
               
               
                 Gly Arg Phe Tyr Arg Thr Asp Ile Thr Glu Pr  
               
               
                 #o Phe Val Leu Asp Ala  
               
               
                                 325   
               
               
                 #               330   
               
               
                 #               335  
               
               
                   
               
               
                 ggg cat ctg ccg gtg ccg acc ggg ccg ggc ct  
               
               
                 #c ggg gtg act ccg att     1056  
               
               
                 Gly His Leu Pro Val Pro Thr Gly Pro Gly Le  
               
               
                 #u Gly Val Thr Pro Ile  
               
               
                             340       
               
               
                 #           345       
               
               
                 #           350  
               
               
                   
               
               
                 ccg gat ctt ctg gac gag gtc acc acg gag aa  
               
               
                 #a gcg tgg atc ggt tcg     1104  
               
               
                 Pro Asp Leu Leu Asp Glu Val Thr Thr Glu Ly  
               
               
                 #s Ala Trp Ile Gly Ser  
               
               
                         355           
               
               
                 #       360           
               
               
                 #       365  
               
               
                   
               
               
                 tag                   
               
               
                 #                   
               
               
                 #                   
               
               
                 #           1107  
               
               
                   
               
               
                   
               
               
                 &lt;210&gt; SEQ ID NO 2  
               
               
                 &lt;211&gt; LENGTH: 368  
               
               
                 &lt;212&gt; TYPE: PRT  
               
               
                 &lt;213&gt; ORGANISM: Amycolatopsis orientalis  
               
               
                   
               
               
                 &lt;400&gt; SEQUENCE: 2  
               
               
                   
               
               
                 Val Lys Leu Ser Gly Val Glu Leu Arg Arg Va  
               
               
                 #l Arg Met Pro Leu Val  
               
               
                 1               5    
               
               
                 #                10   
               
               
                 #                15  
               
               
                   
               
               
                 Ala Pro Phe Arg Thr Ser Phe Gly Thr Gln Se  
               
               
                 #r Glu Arg Glu Leu Leu  
               
               
                             20       
               
               
                 #            25       
               
               
                 #            30  
               
               
                   
               
               
                 Leu Val Arg Ala Val Thr Pro Ala Gly Glu Gl  
               
               
                 #y Trp Gly Glu Cys Val  
               
               
                         35           
               
               
                 #        40           
               
               
                 #        45  
               
               
                   
               
               
                 Ala Met Glu Ala Pro Leu Tyr Ser Ser Glu Ty  
               
               
                 #r Asn Asp Ala Ala Glu  
               
               
                     50               
               
               
                 #    55               
               
               
                 #    60  
               
               
                   
               
               
                 His Val Leu Arg Asn His Leu Ile Pro Ala Le  
               
               
                 #u Leu Ala Ala Glu Asp  
               
               
                 65                   
               
               
                 #70                   
               
               
                 #75                   
               
               
                 #80  
               
               
                   
               
               
                 Val Thr Ala His Lys Val Thr Pro Leu Leu Al  
               
               
                 #a Lys Phe Lys Gly His  
               
               
                                 85   
               
               
                 #                90   
               
               
                 #                95  
               
               
                   
               
               
                 Arg Met Ala Lys Gly Ala Leu Glu Met Ala Va  
               
               
                 #l Leu Asp Ala Glu Leu  
               
               
                             100       
               
               
                 #           105       
               
               
                 #           110  
               
               
                   
               
               
                 Arg Ala His Asp Arg Ser Phe Ala Ala Glu Le  
               
               
                 #u Gly Ser Thr Arg Asp  
               
               
                         115           
               
               
                 #       120           
               
               
                 #       125  
               
               
                   
               
               
                 Ser Val Ala Cys Gly Val Ser Val Gly Ile Me  
               
               
                 #t Asp Ser Ile Pro His  
               
               
                     130               
               
               
                 #   135               
               
               
                 #   140  
               
               
                   
               
               
                 Leu Leu Asp Val Val Gly Gly Tyr Leu Asp Gl  
               
               
                 #u Gly Tyr Val Arg Ile  
               
               
                 145                 1  
               
               
                 #50                 1  
               
               
                 #55                 1  
               
               
                 #60  
               
               
                   
               
               
                 Lys Leu Lys Ile Glu Pro Gly Trp Asp Val Gl  
               
               
                 #u Pro Val Arg Gln Val  
               
               
                                 165   
               
               
                 #               170   
               
               
                 #               175  
               
               
                   
               
               
                 Arg Glu Arg Phe Gly Asp Asp Val Leu Leu Gl  
               
               
                 #n Val Asp Ala Asn Thr  
               
               
                             180       
               
               
                 #           185       
               
               
                 #           190  
               
               
                   
               
               
                 Ala Tyr Thr Leu Gly Asp Ala Pro Leu Leu Se  
               
               
                 #r Arg Leu Asp Pro Phe  
               
               
                         195           
               
               
                 #       200           
               
               
                 #       205  
               
               
                   
               
               
                 Asp Leu Leu Leu Ile Glu Gln Pro Leu Glu Gl  
               
               
                 #u Glu Asp Val Leu Gly  
               
               
                     210               
               
               
                 #   215               
               
               
                 #   220  
               
               
                   
               
               
                 His Ala Glu Leu Ala Lys Arg Ile Arg Thr Pr  
               
               
                 #o Ile Cys Leu Asp Glu  
               
               
                 225                 2  
               
               
                 #30                 2  
               
               
                 #35                 2  
               
               
                 #40  
               
               
                   
               
               
                 Ser Ile Val Ser Ala Lys Ala Ala Ala Asp Al  
               
               
                 #a Ile Lys Leu Gly Ala  
               
               
                                 245   
               
               
                 #               250   
               
               
                 #               255  
               
               
                   
               
               
                 Cys Gln Ile Val Asn Ile Lys Pro Gly Arg Va  
               
               
                 #l Gly Gly Tyr Leu Glu  
               
               
                             260       
               
               
                 #           265       
               
               
                 #           270  
               
               
                   
               
               
                 Ala Arg Arg Val His Asp Val Cys Ala Ala Hi  
               
               
                 #s Gly Ile Ala Val Trp  
               
               
                         275           
               
               
                 #       280           
               
               
                 #       285  
               
               
                   
               
               
                 Cys Gly Gly Met Ile Glu Thr Gly Leu Gly Ar  
               
               
                 #g Ala Ala Asn Val Ala  
               
               
                     290               
               
               
                 #   295               
               
               
                 #   300  
               
               
                   
               
               
                 Leu Ala Ser Leu Pro Gly Phe Thr Leu Pro Gl  
               
               
                 #y Asp Thr Ser Ala Ser  
               
               
                 305                 3  
               
               
                 #10                 3  
               
               
                 #15                 3  
               
               
                 #20  
               
               
                   
               
               
                 Gly Arg Phe Tyr Arg Thr Asp Ile Thr Glu Pr  
               
               
                 #o Phe Val Leu Asp Ala  
               
               
                                 325   
               
               
                 #               330   
               
               
                 #               335  
               
               
                   
               
               
                 Gly His Leu Pro Val Pro Thr Gly Pro Gly Le  
               
               
                 #u Gly Val Thr Pro Ile  
               
               
                             340       
               
               
                 #           345       
               
               
                 #           350  
               
               
                   
               
               
                 Pro Asp Leu Leu Asp Glu Val Thr Thr Glu Ly  
               
               
                 #s Ala Trp Ile Gly Ser  
               
               
                         355           
               
               
                 #       360           
               
               
                 #       365

Technology Classification (CPC): 2