                         SEQUENCE LISTING

<110>  Covestro Deutschland AG
 
<120>  Novel monooxygenases for the manufacture of hydroxylated 
       hydrocarbons based on substitution of amino acids by alanine

<130>  14213

<160>  4     

<170>  PatentIn version 3.5

<210>  1
<211>  771
<212>  PRT
<213>  Rhodococcus ruber

<400>  1

Met Ser Ala Ser Val Pro Ala Ser Ala Cys Pro Val Asp His Ala Ala 
1               5                   10                  15      


Leu Ala Gly Gly Cys Pro Val Ser Thr Asn Ala Ala Ala Phe Asp Pro 
            20                  25                  30          


Phe Gly Pro Ala Tyr Gln Ala Asp Pro Ala Glu Ser Leu Arg Trp Ser 
        35                  40                  45              


Arg Asp Glu Glu Pro Val Phe Tyr Ser Pro Glu Leu Gly Tyr Trp Val 
    50                  55                  60                  


Val Thr Arg Tyr Glu Asp Val Lys Ala Val Phe Arg Asp Asn Leu Val 
65                  70                  75                  80  


Phe Ser Pro Ala Ile Ala Leu Glu Lys Ile Thr Pro Val Ser Glu Glu 
                85                  90                  95      


Ala Thr Ala Thr Leu Ala Arg Tyr Asp Tyr Ala Met Ala Arg Thr Leu 
            100                 105                 110         


Val Asn Glu Asp Glu Pro Ala His Met Pro Arg Arg Arg Ala Leu Met 
        115                 120                 125             


Asp Pro Phe Thr Pro Lys Glu Leu Ala His His Glu Ala Met Val Arg 
    130                 135                 140                 


Arg Leu Thr Arg Glu Tyr Val Asp Arg Phe Val Glu Ser Gly Lys Ala 
145                 150                 155                 160 


Asp Leu Val Asp Glu Met Leu Trp Glu Val Pro Leu Thr Val Ala Leu 
                165                 170                 175     


His Phe Leu Gly Val Pro Glu Glu Asp Met Ala Thr Met Arg Lys Tyr 
            180                 185                 190         


Ser Ile Ala His Thr Val Asn Thr Trp Gly Arg Pro Ala Pro Glu Glu 
        195                 200                 205             


Gln Val Ala Val Ala Glu Ala Val Gly Arg Phe Trp Gln Tyr Ala Gly 
    210                 215                 220                 


Thr Val Leu Glu Lys Met Arg Gln Asp Pro Ser Gly His Gly Trp Met 
225                 230                 235                 240 


Pro Tyr Gly Ile Arg Met Gln Gln Gln Met Pro Asp Val Val Thr Asp 
                245                 250                 255     


Ser Tyr Leu His Ser Met Met Met Ala Gly Ile Val Ala Ala His Glu 
            260                 265                 270         


Thr Thr Ala Asn Ala Ser Ala Asn Ala Phe Lys Leu Leu Leu Glu Asn 
        275                 280                 285             


Arg Pro Val Trp Glu Glu Ile Cys Ala Asp Pro Ser Leu Ile Pro Asn 
    290                 295                 300                 


Ala Val Glu Glu Cys Leu Arg His Ser Gly Ser Val Ala Ala Trp Arg 
305                 310                 315                 320 


Arg Val Ala Thr Thr Asp Thr Arg Ile Gly Asp Val Asp Ile Pro Ala 
                325                 330                 335     


Gly Ala Lys Leu Leu Val Val Asn Ala Ser Ala Asn His Asp Glu Arg 
            340                 345                 350         


His Phe Asp Arg Pro Asp Glu Phe Asp Ile Arg Arg Pro Asn Ser Ser 
        355                 360                 365             


Asp His Leu Thr Phe Gly Tyr Gly Ser His Gln Cys Met Gly Lys Asn 
    370                 375                 380                 


Leu Ala Arg Met Glu Met Gln Ile Phe Leu Glu Glu Leu Thr Thr Arg 
385                 390                 395                 400 


Leu Pro His Met Glu Leu Val Pro Asp Gln Glu Phe Thr Tyr Leu Pro 
                405                 410                 415     


Asn Thr Ser Phe Arg Gly Pro Asp His Val Trp Val Gln Trp Asp Pro 
            420                 425                 430         


Gln Ala Asn Pro Glu Arg Thr Asp Pro Ala Val Leu Gln Arg Gln His 
        435                 440                 445             


Pro Val Thr Ile Gly Glu Pro Ser Thr Arg Ser Val Ser Arg Thr Val 
    450                 455                 460                 


Thr Val Glu Arg Leu Asp Arg Ile Val Asp Asp Val Leu Arg Val Val 
465                 470                 475                 480 


Leu Arg Ala Pro Ala Gly Asn Ala Leu Pro Ala Trp Thr Pro Gly Ala 
                485                 490                 495     


His Ile Asp Val Asp Leu Gly Ala Leu Ser Arg Gln Tyr Ser Leu Cys 
            500                 505                 510         


Gly Ala Pro Asp Ala Pro Thr Tyr Glu Ile Ala Val Leu Leu Asp Pro 
        515                 520                 525             


Glu Ser Arg Gly Gly Ser Arg Tyr Val His Glu Gln Leu Arg Val Gly 
    530                 535                 540                 


Gly Ser Leu Arg Ile Arg Gly Pro Arg Asn His Phe Ala Leu Asp Pro 
545                 550                 555                 560 


Asp Ala Glu His Tyr Val Phe Val Ala Gly Gly Ile Gly Ile Thr Pro 
                565                 570                 575     


Val Leu Ala Met Ala Asp His Ala Arg Ala Arg Gly Trp Ser Tyr Glu 
            580                 585                 590         


Leu His Tyr Cys Gly Arg Asn Arg Ser Gly Met Ala Tyr Leu Glu Arg 
        595                 600                 605             


Val Ala Gly His Gly Asp Arg Ala Ala Leu His Val Ser Ala Glu Gly 
    610                 615                 620                 


Thr Arg Val Asp Leu Ala Ala Leu Leu Ala Thr Pro Val Ser Gly Thr 
625                 630                 635                 640 


Gln Ile Tyr Ala Cys Gly Pro Gly Arg Leu Leu Ala Gly Leu Glu Asp 
                645                 650                 655     


Ala Ser Arg His Trp Pro Asp Gly Ala Leu His Val Glu His Phe Thr 
            660                 665                 670         


Ser Ser Leu Thr Ala Leu Asp Pro Asp Val Glu His Ala Phe Asp Leu 
        675                 680                 685             


Asp Leu Arg Asp Ser Gly Leu Thr Val Arg Val Glu Pro Thr Gln Thr 
    690                 695                 700                 


Val Leu Asp Ala Leu Arg Ala Asn Asn Ile Asp Val Pro Ser Asp Cys 
705                 710                 715                 720 


Glu Glu Gly Leu Cys Gly Ser Cys Glu Val Thr Val Leu Glu Gly Glu 
                725                 730                 735     


Val Asp His Arg Asp Thr Val Leu Thr Lys Ala Glu Arg Ala Ala Asn 
            740                 745                 750         


Arg Gln Met Met Thr Cys Cys Ser Arg Ala Cys Gly Asp Arg Leu Thr 
        755                 760                 765             


Leu Arg Leu 
    770     


<210>  2
<211>  7672
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Expression construct

<400>  2
tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg       60

cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc      120

ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg      180

gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc      240

acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt      300

ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc      360

ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta      420

acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt      480

tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta      540

tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat      600

tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa      660

actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc      720

gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga      780

aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc      840

agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac      900

cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac      960

aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat     1020

tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag     1080

tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca     1140

taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac     1200

ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg     1260

tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca     1320

tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac     1380

cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa     1440

cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga     1500

gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg     1560

gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc     1620

agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag     1680

aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc     1740

agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg     1800

cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac     1860

accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga     1920

aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt     1980

ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag     2040

cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg     2100

gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta     2160

tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc     2220

agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg     2280

tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta     2340

caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg     2400

ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct     2460

gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag     2520

gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc     2580

gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag     2640

aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt     2700

ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa     2760

acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg     2820

ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg     2880

tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc     2940

tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta     3000

cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca     3060

gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc     3120

ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc     3180

catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa     3240

ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc     3300

gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac     3360

gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca     3420

ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta     3480

atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa     3540

cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat     3600

tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca     3660

ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa     3720

aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt     3780

atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg     3840

cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca     3900

gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta     3960

tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg     4020

agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat     4080

gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct     4140

ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg     4200

catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat     4260

tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc     4320

tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca     4380

gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg     4440

ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt     4500

tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg     4560

catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct     4620

cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga     4680

tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg     4740

ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc     4800

ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg     4860

cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg     4920

gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga     4980

aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa     5040

ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac     5100

agcagcggcc tggtgccgcg cggcagccat atggctagca tgactggtgg acagcaaatg     5160

ggtcgcggat ccgaattcat gagtgcatca gttccggcgt cggcgtgtcc cgtcgatcac     5220

gcggccctgg ccggcggctg tccggtgtcg acgaacgccg cggcgttcga tccgttcggg     5280

cccgcgtacc aggccgatcc ggccgagtcg ctgcgctggt cccgcgacga ggagccggtg     5340

ttctacagcc ccgaactcgg ctactgggtg gtcacccgct acgaggatgt gaaggcggtg     5400

ttccgcgaca acctcgtgtt ctcaccggcc atcgccctcg agaagatcac cccggtctcc     5460

gaggaggcca ccgccaccct cgcccgctac gactacgcca tggcccggac cctcgtgaac     5520

gaggacgagc ccgcccacat gccgcgccgc cgcgcactca tggacccgtt caccccgaag     5580

gaactggcgc accacgaggc gatggtgcga cggctcacgc gcgaatacgt cgaccgcttc     5640

gtcgaatccg gcaaggccga cctggtggac gagatgctgt gggaggtacc gctcaccgtc     5700

gccctgcact tcctcggcgt gccggaggag gacatggcga cgatgcgcaa gtactcgatc     5760

gcccacaccg tgaacacctg gggccgcccc gcgcccgagg agcaggtcgc cgtcgccgag     5820

gcggtcggca ggttctggca gtacgcgggc acggtgctcg agaagatgcg ccaggacccc     5880

tcggggcacg gctggatgcc ctacgggatc cgcatgcagc agcagatgcc ggacgtcgtc     5940

accgactcct acctgcactc gatgatgatg gccggcatcg tcgccgcgca cgagaccacg     6000

gccaacgcgt ccgcgaacgc gttcaagctg ctgctcgaga accgcccggt gtgggaggag     6060

atctgcgcgg atccgtcgct gatccccaac gccgtcgagg agtgcctgcg ccactcggga     6120

tcggtcgcgg cgtggcgacg ggtggccacc accgacaccc gcatcggcga cgtcgacatc     6180

cccgccggcg caaagctgct cgtcgtcaac gcctccgcca accatgacga gcggcacttc     6240

gaccgtcccg acgagttcga catccggcgc ccgaactcga gcgaccacct caccttcggg     6300

tacggcagcc atcagtgcat gggcaagaac ctggcccgca tggagatgca gatcttcctc     6360

gaggaactga ccacgcggct tccccacatg gaactcgtac ccgatcagga gttcacctac     6420

ctgccgaaca cctcgttccg cggtcccgat cacgtgtggg tgcagtggga tccgcaggcg     6480

aaccccgagc gcaccgaccc ggccgtgctg caacggcagc atcccgtcac catcggcgag     6540

ccctccaccc ggtcggtgtc acgcaccgtc accgtcgagc gcctggaccg gatcgtcgac     6600

gacgtgctgc gcgtcgtcct acgggctcct gcaggaaatg cgttgcccgc gtggactcct     6660

ggcgcccaca tcgatgtcga cctcggtgcg ctgtcgcggc agtactccct gtgcggtgcg     6720

cccgacgcgc ccacctacga gatcgccgtt ctgctggacc ccgagagccg cggtggctcg     6780

cgctacgtcc acgaacagct ccgggtgggg ggatcgctcc ggattcgcgg gccccggaac     6840

cacttcgcgc tcgaccccga cgccgagcac tacgtgttcg tggccggcgg catcggcatc     6900

acccccgtcc tggccatggc cgaccacgcc cgcgcccggg ggtggagcta cgaactgcac     6960

tactgcggcc ggaaccgttc cgggatggcc tatctcgagc gggtcgccgg gcacggggac     7020

cgcgccgccc tgcacgtctc ggcggaaggc acccgggtcg acctcgccgc cctcctcgcg     7080

acgccggtgt ccggcaccca gatctacgcg tgcgggcccg gacggctgct cgccggactc     7140

gaggacgcga gccggcactg gcccgacggt gcgctgcacg tcgagcactt cacctcgtcc     7200

ctcacggcac tcgacccgga cgtcgagcac gccttcgacc tcgacctgcg cgactcggga     7260

ctcaccgtgc gggtcgagcc cacccagacc gtcctcgacg cgttgcgcgc caacaacatc     7320

gacgtgccca gcgactgcga ggaaggcctc tgcggctcct gcgaggtcac cgtcctcgaa     7380

ggcgaggtcg accaccgcga caccgtgctc accaaggccg agcgggcggc gaaccggcag     7440

atgatgacct gctgctcgcg tgcctgcggc gaccgactga ccctccgact ctgaaagctt     7500

gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga     7560

aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc     7620

tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg at             7672


<210>  3
<211>  31
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  PCR primer

<400>  3
ctggaattca tgagtgcatc agttccggcg t                                      31


<210>  4
<211>  27
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  PCR primer

<400>  4
catcaagctt tcagagtcgc agggcca                                           27


