Proteases

Abstract

The invention relates to a novel class of serine proteases of peptidase family S2A or S1E that are stable in the presence of copper (Cu 2+ ) and/or inhibited by copper only to a limited extent. Structural features of potential relevance for this effect are also disclosed. This class of proteases includes proteases derived from Brachysporiella gayana, Nocardiopsis dassonvillei subsp. dassonvillei, Nocardiopsis prasina, and Nocardiopsis alba, but excludes the known proteases derived from Metarhizium anisopliae and Nocardiopsis sp. NRRL 18262. The invention also relates to DNA encoding such proteases, the expression thereof in a host cell, including animal and plant cells, as well as to the use thereof, e.g., in animal feed and in detergents. In particular, the invention relates to animal feed and animal feed additives, such as premix, incorporating these proteases together with 1-500 ppm Cu (in-feed-concentration).

1 . A protease of peptidase family S2A and/or peptidase family S1E which
 (a) has a residual activity of at least 0.80 after incubation for 164 hours, at pH 7 and 25° C., in assay buffer supplemented with 0.1% K-Sorbate, and in the presence of 1 mM Cu 2+ , the residual activity being measured relative to the activity after 0 hours of incubation; and/or   (b) has a relative activity of at least 0.66 in the presence of 1 mM Cu 2+ , relative to a control without Cu 2+ ;   wherein the activity measurements of (a) and (b) are on the substrate Suc-AAPF-pNA, in assay buffer at pH 7.0 and 25° C.; and   wherein for the measurements of i), the protease has a purity by SDS-PAGE of at least 90%.   
     
     
         2 - 9 . (canceled) 
     
     
         10 . The protease of any of the preceding claims selected from the group consisting of:
 (a) a polypeptide having an amino acid sequence which has a degree of identity of at least 40% to amino acids 1 to 186 of SEQ ID NO: 14, and/or to amino acids 1-188 of (SEQ ID NOs: 12, 10, 8, 6, 4 or 2);   (b) a polypeptide which is encoded by a nucleic acid sequence which hybridizes under low stringency conditions with
 (i) the mature protease encoding part of the plasmid contained in  Escherichia coli  DSM 15509, 
 (ii) nucleotides 726-1283 of SEQ ID NO: 13, nucleotides 499-1062 of SEQ ID NO: 11, nucleotides 502-1065 of SEQ ID NO: 9, nucleotides 496-1059 of SEQ ID NO: 7, nucleotides 496-1059 of SEQ ID NO: 5, nucleotides 559-1122 of SEQ ID NO: 3, and/or nucleotides 900-1466 of SEQ ID NO: 1, 
 (iii) a subsequence of (i) or (ii) of at least 100 nucleotides, or 
 (iv) a complementary strand of (i), (ii) or (iii); 
   (c) a variant of the polypeptide having an amino acid sequence of amino acids 1 to 186 of SEQ ID NO: 14, and/or amino acids 1-188 of (SEQ ID NOs: 12, 10, 8, 6, 4 or 2) comprising a substitution, deletion, 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.   
     
     
         11 - 24 . (canceled) 
     
     
         25 . An isolated protease of peptidase family S2A and/or peptidase family S1E, selected from the group consisting of:
 (a) a polypeptide comprising an amino acid sequence which has a degree of identity of at least 90% to the sequence of amino acids 1-188 of SEQ ID NO: 2, the sequence of amino acids 1-188 of SEQ ID NO: 4, the sequence of amino acids 1-188 of SEQ ID NO: 10, the sequence of amino acids 1-188 of SEQ ID NO: 12, and/or the sequence of amino acids 1-186 of SEQ ID NO: 14; and   (b) a fragment of the sequence of amino acids 1-188 of SEQ ID NO: 2, the sequence of amino acids 1-188 of SEQ ID NO: 4, the sequence of amino acids 1-188 of SEQ ID NO: 10, the sequence of amino acids 1-188 of SEQ ID NO: 12, and/or the sequence of amino acids 1-186 of SEQ ID NO: 14 which has protease activity.   
     
     
         26 . The protease of  claim 25 , which comprises an amino acid sequence which has a degree of identity of at least 92% to the sequence of amino acids 1-188 of SEQ ID NO: 2, the sequence of amino acids 1-188 of SEQ ID NO: 4, the sequence of amino acids 1-188 of SEQ ID NO: 10, the sequence of amino acids 1-188 of SEQ ID NO: 12, and/or the sequence of amino acids 1-186 of SEQ ID NO: 14. 
     
     
         27 . The protease of  claim 25 , which comprises an amino acid sequence which has a degree of identity of at least 95% to the sequence of amino acids 1-188 of SEQ ID NO: 2, the sequence of amino acids 1-188 of SEQ ID NO: 4, the sequence of amino acids 1-188 of SEQ ID NO: 10, the sequence of amino acids 1-188 of SEQ ID NO: 12, and/or the sequence of amino acids 1-186 of SEQ ID NO: 14. 
     
     
         28 . The protease of  claim 25 , which comprises an amino acid sequence which has a degree of identity of at least 97% to the sequence of amino acids 1-188 of SEQ ID NO: 2, the sequence of amino acids 1-188 of SEQ ID NO: 4, the sequence of amino acids 1-188 of SEQ ID NO: 10, the sequence of amino acids 1-188 of SEQ ID NO: 12, and/or the sequence of amino acids 1-186 of SEQ ID NO: 14. 
     
     
         29 . The protease of  claim 25 , which comprises the sequence of amino acids 1-188 of SEQ ID NO: 2, the sequence of amino acids 1-188 of SEQ ID NO: 4, the sequence of amino acids 1-188 of SEQ ID NO: 10, the sequence of amino acids 1-188 of SEQ ID NO: 12, and/or the sequence of amino acids 1-186 of SEQ ID NO: 14. 
     
     
         30 . The protease of  claim 25 , which is a fragment of the sequence of amino acids 1-188 of SEQ ID NO: 2, the sequence of amino acids 1-188 of SEQ ID NO: 4, the sequence of amino acids 1-188 of SEQ ID NO: 10, the sequence of amino acids 1-188 of SEQ ID NO: 12, and/or the sequence of amino acids 1-186 of SEQ ID NO: 14. 
     
     
         31 . An animal feed additive comprising at least one protease of  claim 25  and
 (a) at least one fat soluble vitamin, and/or 
 (b) at least one water soluble vitamin, and/or 
 (c) at least one trace mineral. 
 
     
     
         32 . The animal feed additive of  claim 31 , which further comprises Cu in an amount as to provide an in-feed-concentration of Cu of 1-500 ppm. 
     
     
         33 . The animal feed additive of  claim 31 , which comprises Cu in a concentration of up to 100,000 ppm. 
     
     
         34 . An animal feed composition having a crude protein content of 50 to 800 g/kg and comprising at least one protease of  claim 25 . 
     
     
         35 . The animal feed composition of  claim 34 , which further comprises Cu at a concentration of 1-500 ppm. 
     
     
         36 . A detergent composition comprising at least one protease of  claim 25  and at least one surfactant. 
     
     
         37 . An isolated nucleic acid sequence comprising a nucleic acid sequence which encodes the protease of  claim 25 . 
     
     
         38 . A nucleic acid construct comprising the nucleic acid sequence of  claim 37  operably linked to one or more control sequences that direct the production of the protease in a suitable expression host. 
     
     
         39 . A recombinant expression vector comprising the nucleic acid construct of  claim 38 . 
     
     
         40 . A recombinant host cell comprising the nucleic acid construct of  claim 38 . 
     
     
         41 . A method for producing a protease, comprising
 (a) cultivating a recombinant host cell of  claim 40  to produce a supernatant comprising the protease; and   (b) recovering the protease.