Performance-enhanced and temperature-resistant protease variants

Abstract

Proteases that comprise an amino acid sequence that is at least 70% identical to the amino acid sequence indicated in SEQ ID NO. 1 over its entire length and that comprise, in the count in accordance with SEQ ID NO. 1, the amino acid substitution R99E or R99D in combination with at least two further amino acid substitutions that are selected from the group consisting of S3T, V4I, and V199I, display very good cleaning performance in particular on blood-containing stains, as well as very good temperature stability.

What is claimed is: 
     
         1 . A protease comprising an amino acid sequence that is at least 70% identical to the amino acid sequence indicated in SEQ ID NO. 1 over its entire length and comprises, in the count in accordance with SEQ ID NO. 1, the amino acid substitution R99E or R99D in combination with at least two further amino acid substitutions that are selected from the group consisting of S3T, V4I, and V199I. 
     
     
         2 . A protease, selected from the group consisting of proteases obtainable from:
 (a.) protease according to  claim 1  as a starting molecule by single or multiple conservative amino acid substitution, wherein the protease comprises, in the count in accordance with SEQ ID NO. 1, the amino acid substitution R99E or R99D in combination with at least two further amino acid substitutions that are selected from the group consisting of S3T, V4I, and V199I;   (b.) protease according to  claim 1  as a starting molecule by fragmentation, deletion mutagenesis, insertion mutagenesis, or substitution mutagenesis, and comprises an amino acid sequence that corresponds to the starting molecule over a length of at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 265, or 266 continuously connected amino acids, wherein the amino acid substitution R99E or R99D contained in the starting molecule, in combination with at least two further amino acid substitutions that are selected from the group consisting of S3T, V4I, and V199I, is still present;   (c.) protease according to  claim 1  as a starting molecule by means of one or more amino acid substitutions in positions that are associated in an alignment with the positions 36, 42, 47, 56, 61, 69, 87, 96, 101, 102, 104, 114, 118, 120, 130, 139, 141, 142, 154, 157, 188, 193, 205, 211, 224, 229, 236, 237, 242, 243, 255, and 268 of the protease from  Bacillus lentus  in accordance with SEQ ID NO. 1, wherein the protease comprises, in the count in accordance with SEQ ID NO. 1, the amino acid substitution R99E or R99D in combination with at least two further amino acid substitutions that are selected from the group consisting of S3T, V4I, and V199I.   
     
     
         3 . A method for manufacturing a protease, comprising the introduction of an amino acid substitution R99E or R99D, in combination with at least two further amino acid substitutions that are selected from the group consisting of S3T, V4I, and V199I, in the count according to SEQ ID NO. 1, into a starting protease that is at least 70% identical to the amino acid sequence indicated in SEQ ID NO. 1 over its entire length. 
     
     
         4 . The method according to  claim 3 , further comprising one or more of the following method steps:
 (a.) introducing a single or multiple conservative amino acid substitution, wherein the protease comprises, in the count in accordance with SEQ ID NO. 1, the amino acid substitution R99E or R99D in combination with at least two further amino acid substitutions that are selected from the group consisting of S3T, V4I, and V199I;   (b.) modifying the amino acid sequence by fragmentation, deletion mutagenesis, insertion mutagenesis, or substitution mutagenesis, in such a way that the protease comprises an amino acid sequence that corresponds to the starting molecule over a length of at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 265, or 266 continuously connected amino acids, wherein the amino acid substitution R99E or R99D, in combination with at least two further amino acid substitutions that are selected from the group consisting of S3T, V4I, and V199I, is still present;   (c.) introducing a single or multiple amino acid substitution into one or more of the positions that are associated in an alignment with the positions 36, 42, 47, 56, 61, 69, 87, 96, 101, 102, 104, 114, 118, 120, 130, 139, 141, 142, 154, 157, 188, 193, 205, 211, 224, 229, 236, 237, 242, 243, 255, and 268 of the protease from  Bacillus lentus  in accordance with SEQ ID NO. 1, wherein the protease comprises, in the count in accordance with SEQ ID NO. 1, the amino acid substitution R99E or R99D in combination with at least two further amino acid substitutions that are selected from the group consisting of S3T, V4I, and V199I.   
     
     
         5 . A nucleic acid coding for a protease according to one of  claims 1 . 
     
     
         6 . A vector containing a nucleic acid according to  claim 5 . 
     
     
         7 . A non-human host cell that contains a nucleic acid according to  claim 5 . 
     
     
         8 . A method for manufacturing a protease, comprising
 (a.) cultivating a host cell in accordance with  claim 7 ,   (b.) isolating the protease from the culture medium or from the host cell.   
     
     
         9 . A washing or cleaning agent, comprising at least one protease according to  claim 1 . 
     
     
         10 . A method for cleaning textiles or hard surfaces, wherein that in at least one method step an agent according to  claim 9  is utilized.