Novel asparaginases and uses thereof

Abstract

The present invention relates to an asparaginase having the width of the pH activity profile which is at least 3.5. Furthermore the invention relates to newly identified asparaginase polypeptide according to any one of SEQ ID NO: 2 or SEQ ID NO: 4 and to variants thereof and to polynucleotide sequences that encode such novel asparaginase variants. Furthermore the invention relates to the use of these novel asparaginase variants in industrial processes.

1 . An isolated nucleic acid comprising:
 (a) a nucleotide sequence encoding an aspiraginase, wherein said aspiraginase comprises an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4; or   (b) a nucleotide sequence encoding an aspiraginase, wherein said aspiraginase comprises an amino acid sequence which has at least 90% identity with SEQ ID NO: 2 or SEQ ID NO: 4;   (c) a nucleotide sequence encoding an aspiraginase, wherein the nucleotide sequence comprises SEQ ID NO: 1 or SEQ ID NO: 3;   (d) a nucleotide sequence which has at least 80% homology with a nucleotide sequence according to (a), (b), or (c);   (e) a nucleotide sequence which hybridizes at high stringency with a complementary strand of a nucleotide sequence according to (a), (b), (c), or (d);   (f) a subsequence of a nucleotide sequence according to (a), (b), (c), (d), or (e) having at least 100 nucleotides; or   (g) a complementary strand of a nucleotide sequence according to (a), (b), (c), (d), (e), or (f).   
     
     
         2 . The isolated nucleic acid according to  claim 1 , comprising a nucleotide sequence encoding an aspiraginase, wherein said aspiraginase comprises an amino acid sequence which has at least 95% identity with SEQ ID NO: 2 or SEQ ID NO: 4. 
     
     
         3 . The isolated nucleic acid according to  claim 1 , comprising a nucleotide sequence encoding an aspiraginase, wherein said aspiraginase comprises an amino acid sequence which has at least 90% identity with SEQ ID NO: 2 and wherein the amino acid sequence comprises one or more of: Ala at position 25, Thr at position 28, Tyr at position 30, Ser at position 35, Leu at position 53, Ser at position 63, Ala at position 64, Asp at position 65, Asn at position 66, Ala at position 74, Ile at position 77, Ile at position 79, Gln at position 80, Thr at position 81, Glu at position 88, Pro at position 106, Val at position 108, Ser at position 111, Leu at position 114, Ala at position 117, Thr at position 119, Glu at position 122, Asp at position 126, Ser at position 131, Val at position 161, Ala at position 168, Gln at position 181, Pro at position 189, Ala at position 190, Leu at position 197, Val at position 205, Phe at position 208, Ala at position 210, Ser at position 211, Val at position 224, Asn at position 228, Ala at position 231, Ile at position 232, Val at position 233, Lys at position 236, Tyr at position 238, Tyr at position 240, Thr at position 250, Thr at position 251, Val at position 252, Val at position 254, Arg at position 255, Ser at position 259, Tyr at position 267, Gln at position 270, Gln at position 273, Ser at position 279, Asp at position 282, Asn at position 283, Lys at position 286, Ser at position 293, Ser at position 297, Ser at position 299, Gly at position 301, Ser at position 303, Asp at position 304, Asp at 307, Ile at position 309, Ala at position 310, Ser at position 311, Lys at position 312, His at position 313, Ser at position 314, Ile at position 317, Leu at position 319, Thr at position 321, Gly at position 324, Thr at position 330, Phe at position 345, Ala at position 351, Ala at position 360, Glu at position 361, Gly at position 364, Phe at position 365, Lys at position 366, Arg at position 369, Glu at position 370, Lys at position 374, Val at position 375 or Val at position 377, said positions being defined with reference to SEQ ID NO: 2. 
     
     
         4 . The isolated nucleic acid according to  claim 1 , comprising a nucleotide sequence encoding an aspiraginase, wherein said aspiraginase comprises an amino acid sequence which has at least 90% identity with SEQ ID NO: 4 and wherein the amino acid sequence comprises one or more of: Ser at position 35, Leu at position 53, Ser at position 63, Ala at position 64, Asp at position 65, Asn at position 66, Ala at position 74, Ile at position 77, Ile at position 79, Gln at position 80, Thr at position 81, Glu at position 88, Pro at position 106, Val at position 108, Ser at position 111, Leu at position 114, Ala at position 117, Thr at position 119, Glu at position 122, Asp at position 126, Val at position 161, Ala at position 168, Gln at position 181, Pro at position 189, Ala at position 190, Leu at position 197, Val at position 205, Phe at position 208, Ser at position 211, Val at position 224, Asn at position 228, Ala at position 231, Ile at position 232, Val at position 233, Lys at position 236, Tyr at position 238, Thr at position 250, Thr at position 251, Val at position 254, Arg at position 255, Ser at position 259, Tyr at position 267, Gln at position 270, Asp at position 279, Asp at position 282, Asn at position 283, Lys at position 286, Ser at position 293, Ser at position 297, Ser at position 299, Ser at position 300, Gly at position 301, Ser at position 303, Asp at position 304, Ile at position 309, Ser at position 311, Thr at position 312, His at position 313, Ser at position 314, Ile at position 317, Thr at position 321, Gly at position 324, Pro at position 330, Glu at position 333, Gln at position 336, Phe at position 341, Ala at position 356, Gly at position 360, Glu at position 362, Arg at position 365, Glu at position 366, Lys at position 370, Val at position 371, Gly at position 372 or Val at position 373, said positions being defined with reference to SEQ ID NO: 4. 
     
     
         5 . The isolated nucleic acid according to  claim 1 , wherein the isolated nucleic acid comprises a nucleotide sequence encoding an aspiraginase having a pH optimum between 6 and 7 and/or which has a specific activity at pH of at least 5 which is higher in comparison to the specific activity of a wild-type asparaginase from  A. niger  as disclosed in WO 2004/030468 measured under the same conditions. 
     
     
         6 . An isolated nucleic acid according to  claim 1 , wherein the isolated nucleic acid comprises a nucleotide sequence encoding an aspiraginase having a pH activity profile which is at least 3.5 pH units wide. 
     
     
         7 . An isolated nucleic acid according to  claim 1 , wherein the isolated nucleic acid comprises a nucleotide sequence encoding an aspiraginase having a pH activity profile which is at least 4 pH units wide. 
     
     
         8 . An isolated nucleic acid according to  claim 1 , wherein the isolated nucleic acid comprises a nucleotide sequence encoding an aspiraginase having a pH activity profile which is at least 5 pH units wide. 
     
     
         9 . The isolated nucleic acid according to  claim 1 , wherein the isolated nucleic acid encodes an aspiraginase that is a variant comprising an amino acid sequence that has one or more truncation(s), and/or at least one substitution, deletion and/or insertion of an amino acid as compared to the amino acid sequence of any one of SEQ ID NO: 2 or, SEQ ID NO: 4. 
     
     
         10 . The isolated nucleic acid according to  claim 1 , wherein the isolated nucleic acid is operably linked to one or more control sequences capable of directing the expression of an asparaginase in a suitable expression host. 
     
     
         11 . A recombinant expression vector comprising the isolated nucleic acid of  claim 10 . 
     
     
         12 . A recombinant host cell comprising the expression vector of  claim 11 . 
     
     
         13 . A method for producing an asparaginase, the method comprising cultivating the host cell of  claim 12  under conditions conducive to production of the asparaginase and recovering the asparaginase.