                         SEQUENCE LISTING

<110>  JILIN ASYMCHEM LABORATORIES CO.,LTD
 
<120>  Immobilized Enzyme,Preparation method and Use thereof

<130>  PN110371KLY

<160>  9     

<170>  PatentIn version 3.5

<210>  1
<211>  253
<212>  PRT
<213>  Ketoreductase(Acetobacter sp. )


<220>
<221>  VARIANT
<222>  (1)..(253)
<223>  KRED-Ac-V2

<220>
<221>  MUTAGEN
<222>  (1)..(253)
<223>  E144S+A94T+N156T

<400>  1

Met Ala Arg Val Ala Gly Lys Val Ala Ile Val Ser Gly Ala Ala Asn 
1               5                   10                  15      


Gly Ile Gly Lys Ala Thr Ala Gln Leu Leu Ala Lys Glu Gly Ala Lys 
            20                  25                  30          


Val Val Ile Gly Asp Leu Lys Glu Glu Asp Gly Gln Lys Ala Val Ala 
        35                  40                  45              


Glu Ile Lys Ala Ala Gly Gly Glu Ala Ala Phe Val Lys Leu Asn Val 
    50                  55                  60                  


Thr Asp Glu Ala Ala Trp Lys Ala Ala Ile Gly Gln Thr Leu Lys Leu 
65                  70                  75                  80  


Tyr Gly Arg Leu Asp Ile Ala Val Asn Asn Ala Gly Ile Thr Tyr Ser 
                85                  90                  95      


Gly Ser Val Glu Ser Thr Ser Leu Glu Asp Trp Arg Arg Val Gln Ser 
            100                 105                 110         


Ile Asn Leu Asp Gly Val Phe Leu Gly Thr Gln Val Ala Ile Glu Ala 
        115                 120                 125             


Met Lys Lys Ser Gly Gly Gly Ser Ile Val Asn Leu Ser Ser Ile Ser 
    130                 135                 140                 


Gly Leu Ile Gly Asp Pro Met Leu Ala Ala Tyr Thr Ala Ser Lys Gly 
145                 150                 155                 160 


Gly Val Arg Leu Phe Thr Lys Ser Ala Ala Leu His Cys Ala Lys Ser 
                165                 170                 175     


Gly Tyr Lys Ile Arg Val Asn Ser Val His Pro Gly Tyr Ile Trp Thr 
            180                 185                 190         


Pro Met Val Ala Gly Leu Thr Lys Glu Asp Ala Ala Ala Arg Gln Lys 
        195                 200                 205             


Leu Val Asp Leu His Pro Ile Gly His Leu Gly Glu Pro Asn Asp Ile 
    210                 215                 220                 


Ala Tyr Gly Ile Leu Tyr Leu Ala Ser Asp Glu Ser Lys Phe Val Thr 
225                 230                 235                 240 


Gly Ser Glu Leu Val Ile Asp Gly Gly Tyr Thr Ala Gln 
                245                 250             


<210>  2
<211>  253
<212>  PRT
<213>  Ketoreductase(Acetobacter sp. )


<220>
<221>  VARIANT
<222>  (1)..(253)
<223>  KRED-Ac-V1

<220>
<221>  MUTAGEN
<222>  (1)..(253)
<223>  E144S+A94N+N156V

<400>  2

Met Ala Arg Val Ala Gly Lys Val Ala Ile Val Ser Gly Ala Ala Asn 
1               5                   10                  15      


Gly Ile Gly Lys Ala Thr Ala Gln Leu Leu Ala Lys Glu Gly Ala Lys 
            20                  25                  30          


Val Val Ile Gly Asp Leu Lys Glu Glu Asp Gly Gln Lys Ala Val Ala 
        35                  40                  45              


Glu Ile Lys Ala Ala Gly Gly Glu Ala Ala Phe Val Lys Leu Asn Val 
    50                  55                  60                  


Thr Asp Glu Ala Ala Trp Lys Ala Ala Ile Gly Gln Thr Leu Lys Leu 
65                  70                  75                  80  


Tyr Gly Arg Leu Asp Ile Ala Val Asn Asn Ala Gly Ile Asn Tyr Ser 
                85                  90                  95      


Gly Ser Val Glu Ser Thr Ser Leu Glu Asp Trp Arg Arg Val Gln Ser 
            100                 105                 110         


Ile Asn Leu Asp Gly Val Phe Leu Gly Thr Gln Val Ala Ile Glu Ala 
        115                 120                 125             


Met Lys Lys Ser Gly Gly Gly Ser Ile Val Asn Leu Ser Ser Ile Ser 
    130                 135                 140                 


Gly Leu Ile Gly Asp Pro Met Leu Ala Ala Tyr Val Ala Ser Lys Gly 
145                 150                 155                 160 


Gly Val Arg Leu Phe Thr Lys Ser Ala Ala Leu His Cys Ala Lys Ser 
                165                 170                 175     


Gly Tyr Lys Ile Arg Val Asn Ser Val His Pro Gly Tyr Ile Trp Thr 
            180                 185                 190         


Pro Met Val Ala Gly Leu Thr Lys Glu Asp Ala Ala Ala Arg Gln Lys 
        195                 200                 205             


Leu Val Asp Leu His Pro Ile Gly His Leu Gly Glu Pro Asn Asp Ile 
    210                 215                 220                 


Ala Tyr Gly Ile Leu Tyr Leu Ala Ser Asp Glu Ser Lys Phe Val Thr 
225                 230                 235                 240 


Gly Ser Glu Leu Val Ile Asp Gly Gly Tyr Thr Ala Gln 
                245                 250             


<210>  3
<211>  253
<212>  PRT
<213>  Ketoreductase(Acetobacter sp. )


<220>
<221>  SITE
<222>  (1)..(253)
<223>  KRED-Ac-mather line

<400>  3

Met Ala Arg Val Ala Gly Lys Val Ala Ile Val Ser Gly Ala Ala Asn 
1               5                   10                  15      


Gly Ile Gly Lys Ala Thr Ala Gln Leu Leu Ala Lys Glu Gly Ala Lys 
            20                  25                  30          


Val Val Ile Gly Asp Leu Lys Glu Glu Asp Gly Gln Lys Ala Val Ala 
        35                  40                  45              


Glu Ile Lys Ala Ala Gly Gly Glu Ala Ala Phe Val Lys Leu Asn Val 
    50                  55                  60                  


Thr Asp Glu Ala Ala Trp Lys Ala Ala Ile Gly Gln Thr Leu Lys Leu 
65                  70                  75                  80  


Tyr Gly Arg Leu Asp Ile Ala Val Asn Asn Ala Gly Ile Ala Tyr Ser 
                85                  90                  95      


Gly Ser Val Glu Ser Thr Ser Leu Glu Asp Trp Arg Arg Val Gln Ser 
            100                 105                 110         


Ile Asn Leu Asp Gly Val Phe Leu Gly Thr Gln Val Ala Ile Glu Ala 
        115                 120                 125             


Met Lys Lys Ser Gly Gly Gly Ser Ile Val Asn Leu Ser Ser Ile Glu 
    130                 135                 140                 


Gly Leu Ile Gly Asp Pro Met Leu Ala Ala Tyr Asn Ala Ser Lys Gly 
145                 150                 155                 160 


Gly Val Arg Leu Phe Thr Lys Ser Ala Ala Leu His Cys Ala Lys Ser 
                165                 170                 175     


Gly Tyr Lys Ile Arg Val Asn Ser Val His Pro Gly Tyr Ile Trp Thr 
            180                 185                 190         


Pro Met Val Ala Gly Leu Thr Lys Glu Asp Ala Ala Ala Arg Gln Lys 
        195                 200                 205             


Leu Val Asp Leu His Pro Ile Gly His Leu Gly Glu Pro Asn Asp Ile 
    210                 215                 220                 


Ala Tyr Gly Ile Leu Tyr Leu Ala Ser Asp Glu Ser Lys Phe Val Thr 
225                 230                 235                 240 


Gly Ser Glu Leu Val Ile Asp Gly Gly Tyr Thr Ala Gln 
                245                 250             


<210>  4
<211>  541
<212>  PRT
<213>  Cyclohexanone monooxygenase(Rhodococcus sp. Phi1)


<220>
<221>  VARIANT
<222>  (1)..(541)
<223>  CHMO-Rs-V2

<220>
<221>  MUTAGEN
<222>  (1)..(541)
<223>  F508Y+F435N+L438A+T436S+F280V+S441V+L510V

<400>  4

Met Thr Ala Gln Ile Ser Pro Thr Val Val Asp Ala Val Val Ile Gly 
1               5                   10                  15      


Ala Gly Phe Gly Gly Ile Tyr Ala Val His Lys Leu His Asn Glu Gln 
            20                  25                  30          


Gly Leu Thr Val Val Gly Phe Asp Lys Ala Asp Gly Pro Gly Gly Thr 
        35                  40                  45              


Trp Tyr Trp Asn Arg Tyr Pro Gly Ala Leu Ser Asp Thr Glu Ser His 
    50                  55                  60                  


Leu Tyr Arg Phe Ser Phe Asp Arg Asp Leu Leu Gln Asp Gly Thr Trp 
65                  70                  75                  80  


Lys Thr Thr Tyr Ile Thr Gln Pro Glu Ile Leu Glu Tyr Leu Glu Ser 
                85                  90                  95      


Val Val Asp Arg Phe Asp Leu Arg Arg His Phe Arg Phe Gly Thr Glu 
            100                 105                 110         


Val Thr Ser Ala Ile Tyr Leu Glu Asp Glu Asn Leu Trp Glu Val Ser 
        115                 120                 125             


Thr Asp Lys Gly Glu Val Tyr Arg Ala Lys Tyr Val Val Asn Ala Val 
    130                 135                 140                 


Gly Leu Leu Ser Ala Ile Asn Phe Pro Asp Leu Pro Gly Leu Asp Thr 
145                 150                 155                 160 


Phe Glu Gly Glu Thr Ile His Thr Ala Ala Trp Pro Glu Gly Lys Asn 
                165                 170                 175     


Leu Ala Gly Lys Arg Val Gly Val Ile Gly Thr Gly Ser Thr Gly Gln 
            180                 185                 190         


Gln Val Ile Thr Ala Leu Ala Pro Glu Val Glu His Leu Thr Val Phe 
        195                 200                 205             


Val Arg Thr Pro Gln Tyr Ser Val Pro Val Gly Asn Arg Pro Val Thr 
    210                 215                 220                 


Lys Glu Gln Ile Asp Ala Ile Lys Ala Asp Tyr Asp Gly Ile Trp Asp 
225                 230                 235                 240 


Ser Val Lys Lys Ser Ala Val Ala Phe Gly Phe Glu Glu Ser Thr Leu 
                245                 250                 255     


Pro Ala Met Ser Val Ser Glu Glu Glu Arg Asn Arg Ile Phe Gln Glu 
            260                 265                 270         


Ala Trp Asp His Gly Gly Gly Val Arg Phe Met Phe Gly Thr Phe Gly 
        275                 280                 285             


Asp Ile Ala Thr Asp Glu Ala Ala Asn Glu Ala Ala Ala Ser Phe Ile 
    290                 295                 300                 


Arg Ser Lys Ile Ala Glu Ile Ile Glu Asp Pro Glu Thr Ala Arg Lys 
305                 310                 315                 320 


Leu Met Pro Thr Gly Leu Tyr Ala Lys Arg Pro Leu Cys Asp Asn Gly 
                325                 330                 335     


Tyr Tyr Glu Val Tyr Asn Arg Pro Asn Val Glu Ala Val Ala Ile Lys 
            340                 345                 350         


Glu Asn Pro Ile Arg Glu Val Thr Ala Lys Gly Val Val Thr Glu Asp 
        355                 360                 365             


Gly Val Leu His Glu Leu Asp Val Leu Val Phe Ala Thr Gly Phe Asp 
    370                 375                 380                 


Ala Val Asp Gly Asn Tyr Arg Arg Ile Glu Ile Arg Gly Arg Asn Gly 
385                 390                 395                 400 


Leu His Ile Asn Asp His Trp Asp Gly Gln Pro Thr Ser Tyr Leu Gly 
                405                 410                 415     


Val Thr Thr Ala Asn Phe Pro Asn Trp Phe Met Val Leu Gly Pro Asn 
            420                 425                 430         


Gly Pro Asn Ser Asn Ala Pro Pro Val Ile Glu Thr Gln Val Glu Trp 
        435                 440                 445             


Ile Ser Asp Thr Val Ala Tyr Ala Glu Arg Asn Glu Ile Arg Ala Ile 
    450                 455                 460                 


Glu Pro Thr Pro Glu Ala Glu Glu Glu Trp Thr Gln Thr Cys Thr Asp 
465                 470                 475                 480 


Ile Ala Asn Ala Thr Leu Phe Thr Arg Gly Asp Ser Trp Ile Phe Gly 
                485                 490                 495     


Ala Asn Val Pro Gly Lys Lys Pro Ser Val Leu Tyr Tyr Val Gly Gly 
            500                 505                 510         


Leu Gly Asn Tyr Arg Asn Val Leu Ala Gly Val Val Ala Asp Ser Tyr 
        515                 520                 525             


Arg Gly Phe Glu Leu Lys Ser Ala Val Pro Val Thr Ala 
    530                 535                 540     


<210>  5
<211>  541
<212>  PRT
<213>  Cyclohexanone monooxygenase(Rhodococcus sp. Phi1)


<220>
<221>  VARIANT
<222>  (1)..(541)
<223>  CHMO-Rs-V1

<220>
<221>  MUTAGEN
<222>  (1)..(541)
<223>  F508Y+F435N+L438A+T436S+F280V+S441V

<400>  5

Met Thr Ala Gln Ile Ser Pro Thr Val Val Asp Ala Val Val Ile Gly 
1               5                   10                  15      


Ala Gly Phe Gly Gly Ile Tyr Ala Val His Lys Leu His Asn Glu Gln 
            20                  25                  30          


Gly Leu Thr Val Val Gly Phe Asp Lys Ala Asp Gly Pro Gly Gly Thr 
        35                  40                  45              


Trp Tyr Trp Asn Arg Tyr Pro Gly Ala Leu Ser Asp Thr Glu Ser His 
    50                  55                  60                  


Leu Tyr Arg Phe Ser Phe Asp Arg Asp Leu Leu Gln Asp Gly Thr Trp 
65                  70                  75                  80  


Lys Thr Thr Tyr Ile Thr Gln Pro Glu Ile Leu Glu Tyr Leu Glu Ser 
                85                  90                  95      


Val Val Asp Arg Phe Asp Leu Arg Arg His Phe Arg Phe Gly Thr Glu 
            100                 105                 110         


Val Thr Ser Ala Ile Tyr Leu Glu Asp Glu Asn Leu Trp Glu Val Ser 
        115                 120                 125             


Thr Asp Lys Gly Glu Val Tyr Arg Ala Lys Tyr Val Val Asn Ala Val 
    130                 135                 140                 


Gly Leu Leu Ser Ala Ile Asn Phe Pro Asp Leu Pro Gly Leu Asp Thr 
145                 150                 155                 160 


Phe Glu Gly Glu Thr Ile His Thr Ala Ala Trp Pro Glu Gly Lys Asn 
                165                 170                 175     


Leu Ala Gly Lys Arg Val Gly Val Ile Gly Thr Gly Ser Thr Gly Gln 
            180                 185                 190         


Gln Val Ile Thr Ala Leu Ala Pro Glu Val Glu His Leu Thr Val Phe 
        195                 200                 205             


Val Arg Thr Pro Gln Tyr Ser Val Pro Val Gly Asn Arg Pro Val Thr 
    210                 215                 220                 


Lys Glu Gln Ile Asp Ala Ile Lys Ala Asp Tyr Asp Gly Ile Trp Asp 
225                 230                 235                 240 


Ser Val Lys Lys Ser Ala Val Ala Phe Gly Phe Glu Glu Ser Thr Leu 
                245                 250                 255     


Pro Ala Met Ser Val Ser Glu Glu Glu Arg Asn Arg Ile Phe Gln Glu 
            260                 265                 270         


Ala Trp Asp His Gly Gly Gly Val Arg Phe Met Phe Gly Thr Phe Gly 
        275                 280                 285             


Asp Ile Ala Thr Asp Glu Ala Ala Asn Glu Ala Ala Ala Ser Phe Ile 
    290                 295                 300                 


Arg Ser Lys Ile Ala Glu Ile Ile Glu Asp Pro Glu Thr Ala Arg Lys 
305                 310                 315                 320 


Leu Met Pro Thr Gly Leu Tyr Ala Lys Arg Pro Leu Cys Asp Asn Gly 
                325                 330                 335     


Tyr Tyr Glu Val Tyr Asn Arg Pro Asn Val Glu Ala Val Ala Ile Lys 
            340                 345                 350         


Glu Asn Pro Ile Arg Glu Val Thr Ala Lys Gly Val Val Thr Glu Asp 
        355                 360                 365             


Gly Val Leu His Glu Leu Asp Val Leu Val Phe Ala Thr Gly Phe Asp 
    370                 375                 380                 


Ala Val Asp Gly Asn Tyr Arg Arg Ile Glu Ile Arg Gly Arg Asn Gly 
385                 390                 395                 400 


Leu His Ile Asn Asp His Trp Asp Gly Gln Pro Thr Ser Tyr Leu Gly 
                405                 410                 415     


Val Thr Thr Ala Asn Phe Pro Asn Trp Phe Met Val Leu Gly Pro Asn 
            420                 425                 430         


Gly Pro Asn Ser Asn Ala Pro Pro Val Ile Glu Thr Gln Val Glu Trp 
        435                 440                 445             


Ile Ser Asp Thr Val Ala Tyr Ala Glu Arg Asn Glu Ile Arg Ala Ile 
    450                 455                 460                 


Glu Pro Thr Pro Glu Ala Glu Glu Glu Trp Thr Gln Thr Cys Thr Asp 
465                 470                 475                 480 


Ile Ala Asn Ala Thr Leu Phe Thr Arg Gly Asp Ser Trp Ile Phe Gly 
                485                 490                 495     


Ala Asn Val Pro Gly Lys Lys Pro Ser Val Leu Tyr Tyr Leu Gly Gly 
            500                 505                 510         


Leu Gly Asn Tyr Arg Asn Val Leu Ala Gly Val Val Ala Asp Ser Tyr 
        515                 520                 525             


Arg Gly Phe Glu Leu Lys Ser Ala Val Pro Val Thr Ala 
    530                 535                 540     


<210>  6
<211>  541
<212>  PRT
<213>  Cyclohexanone monooxygenase(Rhodococcus sp. Phi1)


<220>
<221>  SITE
<222>  (1)..(541)
<223>  CHMO-Rs-mather line

<400>  6

Met Thr Ala Gln Ile Ser Pro Thr Val Val Asp Ala Val Val Ile Gly 
1               5                   10                  15      


Ala Gly Phe Gly Gly Ile Tyr Ala Val His Lys Leu His Asn Glu Gln 
            20                  25                  30          


Gly Leu Thr Val Val Gly Phe Asp Lys Ala Asp Gly Pro Gly Gly Thr 
        35                  40                  45              


Trp Tyr Trp Asn Arg Tyr Pro Gly Ala Leu Ser Asp Thr Glu Ser His 
    50                  55                  60                  


Leu Tyr Arg Phe Ser Phe Asp Arg Asp Leu Leu Gln Asp Gly Thr Trp 
65                  70                  75                  80  


Lys Thr Thr Tyr Ile Thr Gln Pro Glu Ile Leu Glu Tyr Leu Glu Ser 
                85                  90                  95      


Val Val Asp Arg Phe Asp Leu Arg Arg His Phe Arg Phe Gly Thr Glu 
            100                 105                 110         


Val Thr Ser Ala Ile Tyr Leu Glu Asp Glu Asn Leu Trp Glu Val Ser 
        115                 120                 125             


Thr Asp Lys Gly Glu Val Tyr Arg Ala Lys Tyr Val Val Asn Ala Val 
    130                 135                 140                 


Gly Leu Leu Ser Ala Ile Asn Phe Pro Asp Leu Pro Gly Leu Asp Thr 
145                 150                 155                 160 


Phe Glu Gly Glu Thr Ile His Thr Ala Ala Trp Pro Glu Gly Lys Asn 
                165                 170                 175     


Leu Ala Gly Lys Arg Val Gly Val Ile Gly Thr Gly Ser Thr Gly Gln 
            180                 185                 190         


Gln Val Ile Thr Ala Leu Ala Pro Glu Val Glu His Leu Thr Val Phe 
        195                 200                 205             


Val Arg Thr Pro Gln Tyr Ser Val Pro Val Gly Asn Arg Pro Val Thr 
    210                 215                 220                 


Lys Glu Gln Ile Asp Ala Ile Lys Ala Asp Tyr Asp Gly Ile Trp Asp 
225                 230                 235                 240 


Ser Val Lys Lys Ser Ala Val Ala Phe Gly Phe Glu Glu Ser Thr Leu 
                245                 250                 255     


Pro Ala Met Ser Val Ser Glu Glu Glu Arg Asn Arg Ile Phe Gln Glu 
            260                 265                 270         


Ala Trp Asp His Gly Gly Gly Phe Arg Phe Met Phe Gly Thr Phe Gly 
        275                 280                 285             


Asp Ile Ala Thr Asp Glu Ala Ala Asn Glu Ala Ala Ala Ser Phe Ile 
    290                 295                 300                 


Arg Ser Lys Ile Ala Glu Ile Ile Glu Asp Pro Glu Thr Ala Arg Lys 
305                 310                 315                 320 


Leu Met Pro Thr Gly Leu Tyr Ala Lys Arg Pro Leu Cys Asp Asn Gly 
                325                 330                 335     


Tyr Tyr Glu Val Tyr Asn Arg Pro Asn Val Glu Ala Val Ala Ile Lys 
            340                 345                 350         


Glu Asn Pro Ile Arg Glu Val Thr Ala Lys Gly Val Val Thr Glu Asp 
        355                 360                 365             


Gly Val Leu His Glu Leu Asp Val Leu Val Phe Ala Thr Gly Phe Asp 
    370                 375                 380                 


Ala Val Asp Gly Asn Tyr Arg Arg Ile Glu Ile Arg Gly Arg Asn Gly 
385                 390                 395                 400 


Leu His Ile Asn Asp His Trp Asp Gly Gln Pro Thr Ser Tyr Leu Gly 
                405                 410                 415     


Val Thr Thr Ala Asn Phe Pro Asn Trp Phe Met Val Leu Gly Pro Asn 
            420                 425                 430         


Gly Pro Phe Thr Asn Leu Pro Pro Ser Ile Glu Thr Gln Val Glu Trp 
        435                 440                 445             


Ile Ser Asp Thr Val Ala Tyr Ala Glu Arg Asn Glu Ile Arg Ala Ile 
    450                 455                 460                 


Glu Pro Thr Pro Glu Ala Glu Glu Glu Trp Thr Gln Thr Cys Thr Asp 
465                 470                 475                 480 


Ile Ala Asn Ala Thr Leu Phe Thr Arg Gly Asp Ser Trp Ile Phe Gly 
                485                 490                 495     


Ala Asn Val Pro Gly Lys Lys Pro Ser Val Leu Phe Tyr Leu Gly Gly 
            500                 505                 510         


Leu Gly Asn Tyr Arg Asn Val Leu Ala Gly Val Val Ala Asp Ser Tyr 
        515                 520                 525             


Arg Gly Phe Glu Leu Lys Ser Ala Val Pro Val Thr Ala 
    530                 535                 540     


<210>  7
<211>  603
<212>  PRT
<213>  Cyclohexanone monooxygenase(Rhodococcus ruber-SD1)


<220>
<221>  VARIANT
<222>  (1)..(603)
<223>  CHMO-Rr-V2

<220>
<221>  MUTAGEN
<222>  (1)..(603)
<223>  Y559M+P190L+P504V

<400>  7

Met Thr Thr Ser Ile Asp Arg Glu Ala Leu Arg Arg Lys Tyr Ala Glu 
1               5                   10                  15      


Glu Arg Asp Lys Arg Ile Arg Pro Asp Gly Asn Asp Gln Tyr Ile Arg 
            20                  25                  30          


Leu Asp His Val Asp Gly Trp Ser His Asp Pro Tyr Met Pro Ile Thr 
        35                  40                  45              


Pro Arg Glu Pro Lys Leu Asp His Val Thr Phe Ala Phe Ile Gly Gly 
    50                  55                  60                  


Gly Phe Ser Gly Leu Val Thr Ala Ala Arg Leu Arg Glu Ser Gly Val 
65                  70                  75                  80  


Glu Ser Val Arg Ile Ile Asp Lys Ala Gly Asp Phe Gly Gly Val Trp 
                85                  90                  95      


Tyr Trp Asn Arg Tyr Pro Gly Ala Met Cys Asp Thr Ala Ala Met Val 
            100                 105                 110         


Tyr Met Pro Leu Leu Glu Glu Thr Gly Tyr Met Pro Thr Glu Lys Tyr 
        115                 120                 125             


Ala His Gly Pro Glu Ile Leu Glu His Cys Gln Arg Ile Gly Lys His 
    130                 135                 140                 


Tyr Asp Leu Tyr Asp Asp Ala Leu Phe His Thr Glu Val Thr Asp Leu 
145                 150                 155                 160 


Val Trp Gln Glu His Asp Gln Arg Trp Arg Ile Ser Thr Asn Arg Gly 
                165                 170                 175     


Asp His Phe Thr Ala Gln Phe Val Gly Met Gly Thr Gly Leu Leu His 
            180                 185                 190         


Val Ala Gln Leu Pro Gly Ile Pro Gly Ile Glu Ser Phe Arg Gly Lys 
        195                 200                 205             


Ser Phe His Thr Ser Arg Trp Asp Tyr Asp Tyr Thr Gly Gly Asp Ala 
    210                 215                 220                 


Leu Gly Ala Pro Met Asp Lys Leu Ala Asp Lys Arg Val Ala Val Ile 
225                 230                 235                 240 


Gly Thr Gly Ala Thr Ala Val Gln Cys Val Pro Glu Leu Ala Lys Tyr 
                245                 250                 255     


Cys Arg Glu Leu Tyr Val Val Gln Arg Thr Pro Ser Ala Val Asp Glu 
            260                 265                 270         


Arg Gly Asn His Pro Ile Asp Glu Lys Trp Phe Ala Gln Ile Ala Thr 
        275                 280                 285             


Pro Gly Trp Gln Lys Arg Trp Leu Asp Ser Phe Thr Ala Ile Trp Asp 
    290                 295                 300                 


Gly Val Leu Thr Asp Pro Ser Glu Leu Ala Ile Glu His Glu Asp Leu 
305                 310                 315                 320 


Val Gln Asp Gly Trp Thr Ala Leu Gly Gln Arg Met Arg Ala Ala Val 
                325                 330                 335     


Gly Ser Val Pro Ile Glu Gln Tyr Ser Pro Glu Asn Val Gln Arg Ala 
            340                 345                 350         


Leu Glu Glu Ala Asp Asp Glu Gln Met Glu Arg Ile Arg Ala Arg Val 
        355                 360                 365             


Asp Glu Ile Val Thr Asp Pro Ala Thr Ala Ala Gln Leu Lys Ala Trp 
    370                 375                 380                 


Phe Arg Gln Met Cys Lys Arg Pro Cys Phe His Asp Asp Tyr Leu Pro 
385                 390                 395                 400 


Ala Phe Asn Arg Pro Asn Thr His Leu Val Asp Thr Gly Gly Lys Gly 
                405                 410                 415     


Val Glu Arg Ile Thr Glu Asn Gly Val Val Val Ala Gly Val Glu Tyr 
            420                 425                 430         


Glu Val Asp Cys Ile Val Tyr Ala Ser Gly Phe Glu Phe Leu Gly Thr 
        435                 440                 445             


Gly Tyr Thr Asp Arg Ala Gly Phe Asp Pro Thr Gly Arg Asp Gly Val 
    450                 455                 460                 


Lys Leu Ser Glu His Trp Ala Gln Gly Thr Arg Thr Leu His Gly Met 
465                 470                 475                 480 


His Thr Tyr Gly Phe Pro Asn Leu Phe Val Leu Gln Leu Met Gln Gly 
                485                 490                 495     


Ala Ala Leu Gly Ser Asn Ile Val His Asn Phe Val Glu Ala Ala Arg 
            500                 505                 510         


Val Val Ala Ala Ile Val Asp His Val Leu Ser Thr Gly Thr Ser Ser 
        515                 520                 525             


Val Glu Thr Thr Lys Glu Ala Glu Gln Ala Trp Val Gln Leu Leu Leu 
    530                 535                 540                 


Asp His Gly Arg Pro Leu Gly Asn Pro Glu Cys Thr Pro Gly Met Tyr 
545                 550                 555                 560 


Asn Asn Glu Gly Lys Pro Ala Glu Leu Lys Asp Arg Leu Asn Val Gly 
                565                 570                 575     


Tyr Pro Ala Gly Ser Ala Ala Phe Phe Arg Met Met Asp His Trp Leu 
            580                 585                 590         


Ala Ala Gly Ser Phe Asp Gly Leu Thr Phe Arg 
        595                 600             


<210>  8
<211>  603
<212>  PRT
<213>  Cyclohexanone monooxygenase(Rhodococcus ruber-SD1)


<220>
<221>  VARIANT
<222>  (1)..(603)
<223>  CHMO-Rr-V1

<220>
<221>  MUTAGEN
<222>  (1)..(603)
<223>  P190L+Y559M+C249V+C393V+C257A+M45T

<400>  8

Met Thr Thr Ser Ile Asp Arg Glu Ala Leu Arg Arg Lys Tyr Ala Glu 
1               5                   10                  15      


Glu Arg Asp Lys Arg Ile Arg Pro Asp Gly Asn Asp Gln Tyr Ile Arg 
            20                  25                  30          


Leu Asp His Val Asp Gly Trp Ser His Asp Pro Tyr Thr Pro Ile Thr 
        35                  40                  45              


Pro Arg Glu Pro Lys Leu Asp His Val Thr Phe Ala Phe Ile Gly Gly 
    50                  55                  60                  


Gly Phe Ser Gly Leu Val Thr Ala Ala Arg Leu Arg Glu Ser Gly Val 
65                  70                  75                  80  


Glu Ser Val Arg Ile Ile Asp Lys Ala Gly Asp Phe Gly Gly Val Trp 
                85                  90                  95      


Tyr Trp Asn Arg Tyr Pro Gly Ala Met Cys Asp Thr Ala Ala Met Val 
            100                 105                 110         


Tyr Met Pro Leu Leu Glu Glu Thr Gly Tyr Met Pro Thr Glu Lys Tyr 
        115                 120                 125             


Ala His Gly Pro Glu Ile Leu Glu His Cys Gln Arg Ile Gly Lys His 
    130                 135                 140                 


Tyr Asp Leu Tyr Asp Asp Ala Leu Phe His Thr Glu Val Thr Asp Leu 
145                 150                 155                 160 


Val Trp Gln Glu His Asp Gln Arg Trp Arg Ile Ser Thr Asn Arg Gly 
                165                 170                 175     


Asp His Phe Thr Ala Gln Phe Val Gly Met Gly Thr Gly Leu Leu His 
            180                 185                 190         


Val Ala Gln Leu Pro Gly Ile Pro Gly Ile Glu Ser Phe Arg Gly Lys 
        195                 200                 205             


Ser Phe His Thr Ser Arg Trp Asp Tyr Asp Tyr Thr Gly Gly Asp Ala 
    210                 215                 220                 


Leu Gly Ala Pro Met Asp Lys Leu Ala Asp Lys Arg Val Ala Val Ile 
225                 230                 235                 240 


Gly Thr Gly Ala Thr Ala Val Gln Val Val Pro Glu Leu Ala Lys Tyr 
                245                 250                 255     


Ala Arg Glu Leu Tyr Val Val Gln Arg Thr Pro Ser Ala Val Asp Glu 
            260                 265                 270         


Arg Gly Asn His Pro Ile Asp Glu Lys Trp Phe Ala Gln Ile Ala Thr 
        275                 280                 285             


Pro Gly Trp Gln Lys Arg Trp Leu Asp Ser Phe Thr Ala Ile Trp Asp 
    290                 295                 300                 


Gly Val Leu Thr Asp Pro Ser Glu Leu Ala Ile Glu His Glu Asp Leu 
305                 310                 315                 320 


Val Gln Asp Gly Trp Thr Ala Leu Gly Gln Arg Met Arg Ala Ala Val 
                325                 330                 335     


Gly Ser Val Pro Ile Glu Gln Tyr Ser Pro Glu Asn Val Gln Arg Ala 
            340                 345                 350         


Leu Glu Glu Ala Asp Asp Glu Gln Met Glu Arg Ile Arg Ala Arg Val 
        355                 360                 365             


Asp Glu Ile Val Thr Asp Pro Ala Thr Ala Ala Gln Leu Lys Ala Trp 
    370                 375                 380                 


Phe Arg Gln Met Cys Lys Arg Pro Val Phe His Asp Asp Tyr Leu Pro 
385                 390                 395                 400 


Ala Phe Asn Arg Pro Asn Thr His Leu Val Asp Thr Gly Gly Lys Gly 
                405                 410                 415     


Val Glu Arg Ile Thr Glu Asn Gly Val Val Val Ala Gly Val Glu Tyr 
            420                 425                 430         


Glu Val Asp Cys Ile Val Tyr Ala Ser Gly Phe Glu Phe Leu Gly Thr 
        435                 440                 445             


Gly Tyr Thr Asp Arg Ala Gly Phe Asp Pro Thr Gly Arg Asp Gly Val 
    450                 455                 460                 


Lys Leu Ser Glu His Trp Ala Gln Gly Thr Arg Thr Leu His Gly Met 
465                 470                 475                 480 


His Thr Tyr Gly Phe Pro Asn Leu Phe Val Leu Gln Leu Met Gln Gly 
                485                 490                 495     


Ala Ala Leu Gly Ser Asn Ile Pro His Asn Phe Val Glu Ala Ala Arg 
            500                 505                 510         


Val Val Ala Ala Ile Val Asp His Val Leu Ser Thr Gly Thr Ser Ser 
        515                 520                 525             


Val Glu Thr Thr Lys Glu Ala Glu Gln Ala Trp Val Gln Leu Leu Leu 
    530                 535                 540                 


Asp His Gly Arg Pro Leu Gly Asn Pro Glu Cys Thr Pro Gly Met Tyr 
545                 550                 555                 560 


Asn Asn Glu Gly Lys Pro Ala Glu Leu Lys Asp Arg Leu Asn Val Gly 
                565                 570                 575     


Tyr Pro Ala Gly Ser Ala Ala Phe Phe Arg Met Met Asp His Trp Leu 
            580                 585                 590         


Ala Ala Gly Ser Phe Asp Gly Leu Thr Phe Arg 
        595                 600             


<210>  9
<211>  603
<212>  PRT
<213>  Cyclohexanone monooxygenase(Rhodococcus ruber-SD1)


<220>
<221>  SITE
<222>  (1)..(603)
<223>  CHMO-Rr-mather line

<400>  9

Met Thr Thr Ser Ile Asp Arg Glu Ala Leu Arg Arg Lys Tyr Ala Glu 
1               5                   10                  15      


Glu Arg Asp Lys Arg Ile Arg Pro Asp Gly Asn Asp Gln Tyr Ile Arg 
            20                  25                  30          


Leu Asp His Val Asp Gly Trp Ser His Asp Pro Tyr Met Pro Ile Thr 
        35                  40                  45              


Pro Arg Glu Pro Lys Leu Asp His Val Thr Phe Ala Phe Ile Gly Gly 
    50                  55                  60                  


Gly Phe Ser Gly Leu Val Thr Ala Ala Arg Leu Arg Glu Ser Gly Val 
65                  70                  75                  80  


Glu Ser Val Arg Ile Ile Asp Lys Ala Gly Asp Phe Gly Gly Val Trp 
                85                  90                  95      


Tyr Trp Asn Arg Tyr Pro Gly Ala Met Cys Asp Thr Ala Ala Met Val 
            100                 105                 110         


Tyr Met Pro Leu Leu Glu Glu Thr Gly Tyr Met Pro Thr Glu Lys Tyr 
        115                 120                 125             


Ala His Gly Pro Glu Ile Leu Glu His Cys Gln Arg Ile Gly Lys His 
    130                 135                 140                 


Tyr Asp Leu Tyr Asp Asp Ala Leu Phe His Thr Glu Val Thr Asp Leu 
145                 150                 155                 160 


Val Trp Gln Glu His Asp Gln Arg Trp Arg Ile Ser Thr Asn Arg Gly 
                165                 170                 175     


Asp His Phe Thr Ala Gln Phe Val Gly Met Gly Thr Gly Pro Leu His 
            180                 185                 190         


Val Ala Gln Leu Pro Gly Ile Pro Gly Ile Glu Ser Phe Arg Gly Lys 
        195                 200                 205             


Ser Phe His Thr Ser Arg Trp Asp Tyr Asp Tyr Thr Gly Gly Asp Ala 
    210                 215                 220                 


Leu Gly Ala Pro Met Asp Lys Leu Ala Asp Lys Arg Val Ala Val Ile 
225                 230                 235                 240 


Gly Thr Gly Ala Thr Ala Val Gln Cys Val Pro Glu Leu Ala Lys Tyr 
                245                 250                 255     


Cys Arg Glu Leu Tyr Val Val Gln Arg Thr Pro Ser Ala Val Asp Glu 
            260                 265                 270         


Arg Gly Asn His Pro Ile Asp Glu Lys Trp Phe Ala Gln Ile Ala Thr 
        275                 280                 285             


Pro Gly Trp Gln Lys Arg Trp Leu Asp Ser Phe Thr Ala Ile Trp Asp 
    290                 295                 300                 


Gly Val Leu Thr Asp Pro Ser Glu Leu Ala Ile Glu His Glu Asp Leu 
305                 310                 315                 320 


Val Gln Asp Gly Trp Thr Ala Leu Gly Gln Arg Met Arg Ala Ala Val 
                325                 330                 335     


Gly Ser Val Pro Ile Glu Gln Tyr Ser Pro Glu Asn Val Gln Arg Ala 
            340                 345                 350         


Leu Glu Glu Ala Asp Asp Glu Gln Met Glu Arg Ile Arg Ala Arg Val 
        355                 360                 365             


Asp Glu Ile Val Thr Asp Pro Ala Thr Ala Ala Gln Leu Lys Ala Trp 
    370                 375                 380                 


Phe Arg Gln Met Cys Lys Arg Pro Cys Phe His Asp Asp Tyr Leu Pro 
385                 390                 395                 400 


Ala Phe Asn Arg Pro Asn Thr His Leu Val Asp Thr Gly Gly Lys Gly 
                405                 410                 415     


Val Glu Arg Ile Thr Glu Asn Gly Val Val Val Ala Gly Val Glu Tyr 
            420                 425                 430         


Glu Val Asp Cys Ile Val Tyr Ala Ser Gly Phe Glu Phe Leu Gly Thr 
        435                 440                 445             


Gly Tyr Thr Asp Arg Ala Gly Phe Asp Pro Thr Gly Arg Asp Gly Val 
    450                 455                 460                 


Lys Leu Ser Glu His Trp Ala Gln Gly Thr Arg Thr Leu His Gly Met 
465                 470                 475                 480 


His Thr Tyr Gly Phe Pro Asn Leu Phe Val Leu Gln Leu Met Gln Gly 
                485                 490                 495     


Ala Ala Leu Gly Ser Asn Ile Pro His Asn Phe Val Glu Ala Ala Arg 
            500                 505                 510         


Val Val Ala Ala Ile Val Asp His Val Leu Ser Thr Gly Thr Ser Ser 
        515                 520                 525             


Val Glu Thr Thr Lys Glu Ala Glu Gln Ala Trp Val Gln Leu Leu Leu 
    530                 535                 540                 


Asp His Gly Arg Pro Leu Gly Asn Pro Glu Cys Thr Pro Gly Tyr Tyr 
545                 550                 555                 560 


Asn Asn Glu Gly Lys Pro Ala Glu Leu Lys Asp Arg Leu Asn Val Gly 
                565                 570                 575     


Tyr Pro Ala Gly Ser Ala Ala Phe Phe Arg Met Met Asp His Trp Leu 
            580                 585                 590         


Ala Ala Gly Ser Phe Asp Gly Leu Thr Phe Arg 
        595                 600             


