               SEQUENCE LISTING

<110> BASF SE

<120> Protease resistant against natural inhibitors

<130> 0000078823

<150> EP 16193375.9
<151> 11.10.2016

<160> 14

<170> BiSSAP 1.3

<210> 1
<211> 269
<212> PRT
<213> Bacillus lentus

<220> 
<223> wildtype

<400> 1
Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala 
1               5                   10                  15      
His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp 
            20                  25                  30          
Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser 
        35                  40                  45              
Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr 
    50                  55                  60                  
His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu 
65                  70                  75                  80  
Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala 
                85                  90                  95      
Asp Gly Arg Gly Ala Ile Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala 
            100                 105                 110         
Gly Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser 
        115                 120                 125             
Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly 
    130                 135                 140                 
Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Ser Ser Ile Ser 
145                 150                 155                 160 
Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln 
                165                 170                 175     
Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu Asp Ile 
            180                 185                 190         
Val Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Gly Ser Thr Tyr 
        195                 200                 205             
Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala 
    210                 215                 220                 
Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile 
225                 230                 235                 240 
Arg Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu 
                245                 250                 255     
Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg 
            260                 265                 

<210> 2
<211> 275
<212> PRT
<213> Bacillus amyloliquefaciens

<220> 
<223> wildtype

<400> 2
Ala Gln Ser Val Pro Tyr Gly Val Ser Gln Ile Lys Ala Pro Ala Leu 
1               5                   10                  15      
His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp 
            20                  25                  30          
Ser Gly Ile Asp Ser Ser His Pro Asp Leu Lys Val Ala Gly Gly Ala 
        35                  40                  45              
Ser Met Val Pro Ser Glu Thr Asn Pro Phe Gln Asp Asn Asn Ser His 
    50                  55                  60                  
Gly Thr His Val Ala Gly Thr Val Ala Ala Leu Asn Asn Ser Ile Gly 
65                  70                  75                  80  
Val Leu Gly Val Ala Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 
                85                  90                  95      
Gly Ala Asp Gly Ser Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu 
            100                 105                 110         
Trp Ala Ile Ala Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly 
        115                 120                 125             
Pro Ser Gly Ser Ala Ala Leu Lys Ala Ala Val Asp Lys Ala Val Ala 
    130                 135                 140                 
Ser Gly Val Val Val Val Ala Ala Ala Gly Asn Glu Gly Thr Ser Gly 
145                 150                 155                 160 
Ser Ser Ser Thr Val Gly Tyr Pro Gly Lys Tyr Pro Ser Val Ile Ala 
                165                 170                 175     
Val Gly Ala Val Asp Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Val 
            180                 185                 190         
Gly Pro Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln Ser Thr 
        195                 200                 205             
Leu Pro Gly Asn Lys Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Ser 
    210                 215                 220                 
Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn 
225                 230                 235                 240 
Trp Thr Asn Thr Gln Val Arg Ser Ser Leu Glu Asn Thr Thr Thr Lys 
                245                 250                 255     
Leu Gly Asp Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala 
            260                 265                 270         
Ala Ala Gln 
        275 

<210> 3
<211> 269
<212> PRT
<213> Artificial Sequence

<220> 
<223> BLAP 101E

<400> 3
Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala 
1               5                   10                  15      
His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp 
            20                  25                  30          
Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser 
        35                  40                  45              
Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr 
    50                  55                  60                  
His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu 
65                  70                  75                  80  
Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala 
                85                  90                  95      
Asp Gly Glu Gly Ala Ile Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala 
            100                 105                 110         
Gly Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser 
        115                 120                 125             
Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly 
    130                 135                 140                 
Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Ser Ser Ile Ser 
145                 150                 155                 160 
Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln 
                165                 170                 175     
Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu Asp Ile 
            180                 185                 190         
Val Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Gly Ser Thr Tyr 
        195                 200                 205             
Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala 
    210                 215                 220                 
Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile 
225                 230                 235                 240 
Arg Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu 
                245                 250                 255     
Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg 
            260                 265                 

<210> 4
<211> 269
<212> PRT
<213> Artificial Sequence

<220> 
<223> BLAP 3T, 4I, 101E, 205I

<400> 4
Ala Gln Thr Ile Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala 
1               5                   10                  15      
His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp 
            20                  25                  30          
Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser 
        35                  40                  45              
Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr 
    50                  55                  60                  
His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu 
65                  70                  75                  80  
Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala 
                85                  90                  95      
Asp Gly Glu Gly Ala Ile Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala 
            100                 105                 110         
Gly Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser 
        115                 120                 125             
Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly 
    130                 135                 140                 
Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Ser Ser Ile Ser 
145                 150                 155                 160 
Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln 
                165                 170                 175     
Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu Asp Ile 
            180                 185                 190         
Val Ala Pro Gly Val Asn Ile Gln Ser Thr Tyr Pro Gly Ser Thr Tyr 
        195                 200                 205             
Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala 
    210                 215                 220                 
Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile 
225                 230                 235                 240 
Arg Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu 
                245                 250                 255     
Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg 
            260                 265                 

<210> 5
<211> 269
<212> PRT
<213> Artificial Sequence

<220> 
<223> Subtilisin 309, Savinase

<400> 5
Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala 
1               5                   10                  15      
His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp 
            20                  25                  30          
Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser 
        35                  40                  45              
Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr 
    50                  55                  60                  
His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu 
65                  70                  75                  80  
Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala 
                85                  90                  95      
Ser Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala 
            100                 105                 110         
Gly Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser 
        115                 120                 125             
Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly 
    130                 135                 140                 
Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile Ser 
145                 150                 155                 160 
Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln 
                165                 170                 175     
Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu Asp Ile 
            180                 185                 190         
Val Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Gly Ser Thr Tyr 
        195                 200                 205             
Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala 
    210                 215                 220                 
Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile 
225                 230                 235                 240 
Arg Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu 
                245                 250                 255     
Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg 
            260                 265                 

<210> 6
<211> 269
<212> PRT
<213> Artificial Sequence

<220> 
<223> BLAP variant 3T, 4I, 205I

<400> 6
Ala Gln Thr Ile Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala 
1               5                   10                  15      
His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp 
            20                  25                  30          
Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser 
        35                  40                  45              
Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr 
    50                  55                  60                  
His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu 
65                  70                  75                  80  
Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala 
                85                  90                  95      
Asp Gly Arg Gly Ala Ile Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala 
            100                 105                 110         
Gly Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser 
        115                 120                 125             
Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly 
    130                 135                 140                 
Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Ser Ser Ile Ser 
145                 150                 155                 160 
Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln 
                165                 170                 175     
Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu Asp Ile 
            180                 185                 190         
Val Ala Pro Gly Val Asn Ile Gln Ser Thr Tyr Pro Gly Ser Thr Tyr 
        195                 200                 205             
Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala 
    210                 215                 220                 
Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile 
225                 230                 235                 240 
Arg Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu 
                245                 250                 255     
Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg 
            260                 265                 

<210> 7
<211> 269
<212> PRT
<213> Artificial Sequence

<220> 
<223> subtilisin 309 variant described in WO9820115A1

<400> 7
Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala 
1               5                   10                  15      
His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp 
            20                  25                  30          
Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser 
        35                  40                  45              
Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr 
    50                  55                  60                  
His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu 
65                  70                  75                  80  
Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala 
                85                  90                  95      
Ser Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala 
            100                 105                 110         
Gly Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser 
        115                 120                 125             
Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly 
    130                 135                 140                 
Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile Ser 
145                 150                 155                 160 
Ala Pro Ala Ser Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln 
                165                 170                 175     
Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Pro Gly Leu Asp Ile 
            180                 185                 190         
Val Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Gly Ser Thr Tyr 
        195                 200                 205             
Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala 
    210                 215                 220                 
Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile 
225                 230                 235                 240 
Arg Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu 
                245                 250                 255     
Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg 
            260                 265                 

<210> 8
<211> 270
<212> PRT
<213> Artificial Sequence

<220> 
<223> Subtilisin variant S99AD

<400> 8
Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala 
1               5                   10                  15      
His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp 
            20                  25                  30          
Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser 
        35                  40                  45              
Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr 
    50                  55                  60                  
His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu 
65                  70                  75                  80  
Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala 
                85                  90                  95      
Ala Asp Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp 
            100                 105                 110         
Ala Gly Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro 
        115                 120                 125             
Ser Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg 
    130                 135                 140                 
Gly Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile 
145                 150                 155                 160 
Ser Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp 
                165                 170                 175     
Gln Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu Asp 
            180                 185                 190         
Ile Val Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Gly Ser Thr 
        195                 200                 205             
Tyr Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly 
    210                 215                 220                 
Ala Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln 
225                 230                 235                 240 
Ile Arg Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn 
                245                 250                 255     
Leu Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg 
            260                 265                 270 

<210> 9
<211> 7
<212> PRT
<213> Artificial Sequence

<220> 
<223> Variant of loop sequence 98-104 of SEQ ID NO: 1

<400> 9
Ala Asp Gly Glu Gly Ala Ile 
1               5           

<210> 10
<211> 7
<212> PRT
<213> Artificial Sequence

<220> 
<223> Variant of loop sequence 98-104 of SEQ ID NO: 1

<400> 10
Ala Asp Gly Asp Gly Ala Ile 
1               5           

<210> 11
<211> 7
<212> PRT
<213> Artificial Sequence

<220> 
<223> Variant of loop sequence 98-104 of SEQ ID NO: 1

<400> 11
Ala Asp Gly Asp Gly Ser Val 
1               5           

<210> 12
<211> 7
<212> PRT
<213> Artificial Sequence

<220> 
<223> Variant of loop sequence 98-104 of SEQ ID NO: 1

<400> 12
Ala Asp Gly Glu Gly Ser Val 
1               5           

<210> 13
<211> 8
<212> PRT
<213> Artificial Sequence

<220> 
<223> Variant of loop sequence 98-104 of SEQ ID NO: 1

<400> 13
Ala Ala Asp Gly Glu Gly Ser Val 
1               5               

<210> 14
<211> 8
<212> PRT
<213> Artificial Sequence

<220> 
<223> Variant of loop sequence 98-104 of SEQ ID NO: 1

<400> 14
Ala Ser Glu Gly Glu Gly Ser Val 
1               5               

