(19) & (11) EP 2 258 838 A1 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 08.12.2010 Bulletin 2010/49 C12N 9/52 (2006.01) C12N 15/57 (2006.01) C12N 5/10 (2006.01) C12N 15/82 (2006.01) (2006.01) (2006.01) (21) Application number: 10180249.4 A23K 1/165 A23L 1/305 C11D 3/386 (2006.01) (22) Date of filing: 17.06.2005 (84) Designated Contracting States: (72) Inventors: AT BE BG CH CY CZ DE DK EE ES FI FR GB GR • Lassen, Soeren Flensted HU IE IS IT LI LT LU MC NL PL PT RO SE SI SK TR 3520, Farum (DK) • Sjoeholm, Carsten (30) Priority: 21.06.2004 DK 200400969 3450, Alleroed (DK) • Oestergaard, Peter Rahbek (83) Declaration under Rule 32(1) EPC (expert 2830, Virum (DK) solution) • Fischer, Morten 2950, Vedbæk (DK) (62) Document number(s) of the earlier application(s) in accordance with Art. 76 EPC: Remarks: 05748647.4 / 1 766 001 This application was filed on 27-09-2010 as a divisional application to the application mentioned (71) Applicant: Novozymes A/S under INID code 62. 2880 Bagsvaerd (DK) (54) Nocardiopsis proteases (57) Proteases derived from Nocardiopsis dasson- their recombinant production in various hosts, including villei subsp. dassonvillei DSM 43235,Nocardiopsis transgenic plants and non-human animals, and their use prasina DSM 15649, Nocardiopsis prasina (previously in animal feed and detergents. The proteases are acid- alba) DSM 14010 Nocardiopsis sp. DSM 16424, Nocar- stable, alkali-stable, and/or thermostable. diopsis alkaliphila DSM 44657 and Nocardiopsis lucen- tensis DSM 44048, as well as homologous proteases; EP 2 258 838 A1 Printed by Jouve, 75001 PARIS (FR) EP 2 258 838 A1 Description Field of the Invention 5 [0001] The present invention relates to isolated polypeptides having protease activity and isolated nucleic acid se- quences encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells, including plant and animal cells, comprising the nucleic acid sequences, as well as methods for producing and using the polypep- tides, in particular the use of the polypeptides in animal feed, and detergents. 10 Background of the Invention [0002] Proteases derived from Nocardiopsis sp. NRRL 18262 and Nocardiopsis dassonvillei NRRL 18133 are dis- closed in WO 88/03947. The DNA and amino acid sequences of the protease derived fromNocardiopsis sp. NRRL 18262 are shown in DK application no. 1996 00013. WO 01/58276 discloses the use in animal feed of acid-stable 15 proteases related to the protease derived fromNocardiopsis sp. NRRL 18262, as well as a protease derived from Nocardiopsis alba DSM 14010. [0003] JP 2-255081-A discloses a protease derived from Nocardiopsis sp. strain OPC- 210 (FERM P- 10508), however without sequence information. The strain is no longer available, as the deposit was withdrawn. [0004] DD 200432|8 discloses a proteolytic preparation derived from Nocardiopsis dassonvillei strain ZIMET 43647, 20 however without sequence information. The strain appears to be no longer available. [0005] JP 2003284571-A discloses the amino acid sequence and the corresponding DNA sequence of a protease derived from Nocardiopsis sp. TOA-1 (FERM P-18676). The sequence has been entered in GENESEQP with no. ADF43564. [0006] It is an object of the present invention to provide alternative proteases, in particular for use in animal feed and/or 25 detergents. Summary of the Invention [0007] A number of proteases were cloned, purified and characterized. These proteases are designated as follows: 30 Protease L1a derived from Nocardiopsis dassonvillei subsp. dassonvillei DSM 43235 (see SEQ ID NOs. 1 and 2); protease L1b derived from Nocardiopsis prasina DSM 15649 (see SEQ ID NOs: 3 and 4); protease L1c derived from Nocardiopsis prasina (previously alba) DSM 14010 (see SEQ ID NOs: 5 and 6); protease L2a derived from Nocardiopsis sp. DSM 16424 (see SEQ ID NOs: 7 and 8); protease L2b derived from Nocardiopsis alkaliphila DSM 44657 (see SEQ ID NOs: 9 and 10); and protease L2c derived from Nocardiopsis lucentensis DSM 44048 (see SEQ ID NOs: 11 and 12). 35 [0008] In a first aspect, the invention relates to an isolated polypeptide having protease activity, selected from the group consisting of: (a) a polypeptide having an amino acid sequence which has a degree of identity to amino acids 1-192 of SEQ ID NO: 6 of at least 71.5%; (b) a polypeptide which is encoded by a nucleic acid sequence which hybridizes under very high stringency conditions with (i) nucleotides 574-1149 of SEQ ID NO:5, (ii) a subsequence of (i) of at least 100 nucleotides, and/or (iii) a complementary strand of (i), or (ii); (c) a variant of the polypeptide having an amino acid 40 sequence of amino acids 1-192 of SEQ ID NO: 6 comprising a substitution, deletion, extension, and/or insertion of one or more amino acids; (d) an allelic variant of (a), or (b); and (e) a fragment of (a), (b), or (d) that has protease activity. [0009] In five alternative aspects, corresponding to the five sets of particular embodiments set forth at the end of the present description, the present invention also relates to: 45 An isolated polypeptide having protease activity, selected from the group consisting of: (a) a polypeptide having an amino acid sequence which has a degree of identity to amino acids 1-192 of SEQ ID NO: 4 of at least 69.9%; (b) a polypeptide which is encoded by a nucleic acid sequence which hybridizes under low stringency conditions with (i) nucleotides 574-1149 of SEQ ID NO:3, (ii) a subsequence of (i) of at least 100 nucleotides, and/or (iii) a comple- mentary strand of (i), or (ii); (c) a variant of the polypeptide having an amino acid sequence of amino acids 1-192 50 of SEQ ID NO: 4 comprising a substitution, deletion, extension, and/or insertion of one or more amino acids; (d) an allelic variant of (a), or (b); and (e) a fragment of (a), (b), or (d) that has protease activity. An isolated polypeptide having protease activity, selected from the group consisting of: (a) a polypeptide having an amino acid sequence which has a degree of identity to amino acids 1-192 of SEQ ID NO: 2 of at least 75.1%; (b) a polypeptide which is encoded by a nucleic acid sequence which hybridizes under low stringency conditions with (i) 55 nucleotides 568-1143 of SEQ ID NO: 1, (ii) a subsequence of (i) of at least 100 nucleotides, and/or (iii) a comple- mentary strand of (i), or (ii); (c) a variant of the polypeptide having an amino acid sequence of amino acids 1-192 of SEQ ID NO: 2 comprising a substitution, deletion, extension, and/or insertion of one or more amino acids; (d) an allelic variant of (a), or (b); and (e) a fragment of (a), (b), or (d) that has protease activity. 2 EP 2 258 838 A1 An isolated polypeptide having protease activity, selected from the group consisting of: (a) a polypeptide having an amino acid sequence which has a degree of identity to amino acids 1-189 of SEQ ID NO: 8 of at least 92.2%; (b) a polypeptide which is encoded by a nucleic acid sequence which hybridizes under low stringency conditions with (i) nucleotides 586-1152 of SEQ ID NO: 7, (ii) a subsequence of (i) of at least 100 nucleotides, and/or (iii) a comple- 5 mentary strand of (i), or (ii); (c) a variant of the polypeptide having an amino acid sequence of amino acids 1-189 of SEQ ID NO: 8 comprising a substitution, deletion, extension, and/or insertion of one or more amino acids; (d) an allelic variant of (a), or (b); and (e) a fragment of (a), (b), or (d) that has protease activity. An isolated polypeptide having protease activity, selected from the group consisting of: (a) a polypeptide having an amino acid sequence which has a degree of identity to amino acids 1-189 of SEQ ID NO: 10 of at least 93.2%; (b) 10 a polypeptide which is encoded by a nucleic acid sequence which hybridizes under low stringency conditions with (i) nucleotides 586-1149 of SEQ ID NO: 9, (ii) a subsequence of (i) of at least 100 nucleotides, and/or (iii) a com- plementary strand of (i), or (ii); (c) a variant of the polypeptide having an amino acid sequence of amino acids 1-189 of SEQ ID NO: 10 comprising a substitution, deletion, extension, and/or insertion of one or more amino acids; (d) an allelic variant of (a), or (b); and (e) a fragment of (a), (b), or (d) that has protease activity. 15 An isolated polypeptide having protease activity, selected from the group consisting of: (a) a polypeptide having an amino acid sequence which has a degree of identity to amino acids 1-189 of SEQ ID NO: 12 of at least 83.3%; (b) a polypeptide which is encoded by a nucleic acid sequence which hybridizes under low stringency conditions with (i) nucleotides 586-1152 of SEQ ID NO: 11, (ii) a subsequence of (i) of at least 100 nucleotides, and/or (iii) a complementary strand of (i), or (ii); (c) a variant of the polypeptide having an amino acid sequence of amino acids 20 1-189 of SEQ ID NO: 12 comprising a substitution, deletion, extension, and/or insertion of one or more amino acids; (d) an allelic variant of (a), or (b); and (e) a fragment of (a), (b), or (d) that has protease activity. [0010] The invention also relates to isolated nucleic acid sequences encoding the above polypeptides and to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and 25 using the polypeptides, in particular within animal feed, and detergents.