﻿               SEQUENCE LISTING

<110> DSM IP Assets B.V.

<120> MUTANT LIPASE AND USE THEREOF

<160> 7

<170> BiSSAP 1.2

<210> 1
<211> 534
<212> PRT
<213> Candida rugosa 


<400> 1
Ala Pro Thr Ala Thr Leu Ala Asn Gly Asp Thr Ile Thr Gly Leu Asn 
1               5                   10                  15      
Ala Ile Ile Asn Glu Ala Phe Leu Gly Ile Pro Phe Ala Glu Pro Pro 
            20                  25                  30          
Val Gly Asn Leu Arg Phe Lys Asp Pro Val Pro Tyr Ser Gly Ser Leu 
        35                  40                  45              
Asp Gly Gln Lys Phe Thr Ser Tyr Gly Pro Ser Cys Met Gln Gln Asn 
    50                  55                  60                  
Pro Glu Gly Thr Tyr Glu Glu Asn Leu Pro Lys Ala Ala Leu Asp Leu 
65                  70                  75                  80  
Val Met Gln Ser Lys Val Phe Glu Ala Val Ser Pro Ser Ser Glu Asp 
                85                  90                  95      
Cys Leu Thr Ile Asn Val Val Arg Pro Pro Gly Thr Lys Ala Gly Ala 
            100                 105                 110         
Asn Leu Pro Val Met Leu Trp Ile Phe Gly Gly Gly Phe Glu Val Gly 
        115                 120                 125             
Gly Thr Ser Thr Phe Pro Pro Ala Gln Met Ile Thr Lys Ser Ile Ala 
    130                 135                 140                 
Met Gly Lys Pro Ile Ile His Val Ser Val Asn Tyr Arg Val Ser Ser 
145                 150                 155                 160 
Trp Gly Phe Leu Ala Gly Asp Glu Ile Lys Ala Glu Gly Ser Ala Asn 
                165                 170                 175     
Ala Gly Leu Lys Asp Gln Arg Leu Gly Met Gln Trp Val Ala Asp Asn 
            180                 185                 190         
Ile Ala Ala Phe Gly Gly Asp Pro Thr Lys Val Thr Ile Phe Gly Glu 
        195                 200                 205             
Ser Ala Gly Ser Met Ser Val Met Cys His Ile Leu Trp Asn Asp Gly 
    210                 215                 220                 
Asp Asn Thr Tyr Lys Gly Lys Pro Leu Phe Arg Ala Gly Ile Met Gln 
225                 230                 235                 240 
Ser Gly Ala Met Val Pro Ser Asp Ala Val Asp Gly Ile Tyr Gly Asn 
                245                 250                 255     
Glu Ile Phe Asp Leu Leu Ala Ser Asn Ala Gly Cys Gly Ser Ala Ser 
            260                 265                 270         
Asp Lys Leu Ala Cys Leu Arg Gly Val Ser Ser Asp Thr Leu Glu Asp 
        275                 280                 285             
Ala Thr Asn Asn Thr Pro Gly Phe Leu Ala Tyr Ser Ser Leu Arg Leu 
    290                 295                 300                 
Ser Tyr Leu Pro Arg Pro Asp Gly Val Asn Ile Thr Asp Asp Met Tyr 
305                 310                 315                 320 
Ala Leu Val Arg Glu Gly Lys Tyr Ala Asn Ile Pro Val Ile Ile Gly 
                325                 330                 335     
Asp Gln Asn Asp Glu Gly Thr Phe Phe Gly Thr Ser Ser Leu Asn Val 
            340                 345                 350         
Thr Thr Asp Ala Gln Ala Arg Glu Tyr Phe Lys Gln Ser Phe Val His 
        355                 360                 365             
Ala Ser Asp Ala Glu Ile Asp Thr Leu Met Thr Ala Tyr Pro Gly Asp 
    370                 375                 380                 
Ile Thr Gln Gly Ser Pro Phe Asp Thr Gly Ile Leu Asn Ala Leu Thr 
385                 390                 395                 400 
Pro Gln Phe Lys Arg Ile Ser Ala Val Leu Gly Asp Leu Gly Phe Thr 
                405                 410                 415     
Leu Ala Arg Arg Tyr Phe Leu Asn His Tyr Thr Gly Gly Thr Lys Tyr 
            420                 425                 430         
Ser Phe Leu Ser Lys Gln Leu Ser Gly Leu Pro Val Leu Gly Thr Phe 
        435                 440                 445             
His Ser Asn Asp Ile Val Phe Gln Asp Tyr Leu Leu Gly Ser Gly Ser 
    450                 455                 460                 
Leu Ile Tyr Asn Asn Ala Phe Ile Ala Phe Ala Thr Asp Leu Asp Pro 
465                 470                 475                 480 
Asn Thr Ala Gly Leu Leu Val Lys Trp Pro Glu Tyr Thr Ser Ser Ser 
                485                 490                 495     
Gln Ser Gly Asn Asn Leu Met Met Ile Asn Ala Leu Gly Leu Tyr Thr 
            500                 505                 510         
Gly Lys Asp Asn Phe Arg Thr Ala Gly Tyr Asp Ala Leu Phe Ser Asn 
        515                 520                 525             
Pro Pro Ser Phe Phe Val 
    530                 

<210> 2
<211> 1602
<212> DNA
<213> Artificial Sequence

<220> 
<221> source
<222> 1..1602
<223> /organism="Artificial Sequence"
      /note="Codon optimized nucleotide sequence for expression in
      Pichia pastoris of the mature coding sequence of Lip1 of Candida
      rugosa"
      /mol_type="unassigned DNA"

<400> 2
gccccaaccg ccactttggc taacggtgac accatcaccg gtttgaacgc catcatcaac      60

gaagccttct tgggtattcc atttgccgaa ccaccagttg gtaacttgag attcaaggac     120

ccagttccat actccggttc cttggatggt caaaagttca cttcttacgg tccatcttgt     180

atgcaacaaa acccagaagg tacctacgaa gaaaacttgc caaaggcagc tttagatctg     240

gttatgcaat ccaaagtttt cgaagctgtt tctccatctt ctgaagactg tttgaccatt     300

aatgttgtta gaccacccgg gacaaaggct ggtgccaact tgccagttat gttgtggatc     360

tttggtggtg gttttgaagt tggtggtact agtaccttcc ctccagccca aatgattacc     420

aagtctattg ctatgggtaa gccaatcatc cacgtttctg tcaactacag agtctccagc     480

tggggtttct tggctggtga cgaaatcaag gccgaaggtt ctgccaacgc cggtttgaag     540

gaccaaagat tgggtatgca atgggtggct gacaacattg ctgcttttgg tggtgatcca     600

actaaggtta ctatctttgg tgaatctgct ggttctatgt ccgtcatgtg tcacattttg     660

tggaacgacg gtgacaacac ttacaagggt aagccattgt tcagagctgg tatcatgcaa     720

tctggtgcta tggttccatc tgacgccgtc gacggtatct acggtaacga aatttttgac     780

ttgttggctt ccaacgctgg ttgtggttct gcctctgaca agttggcttg tttgagaggt     840

gtttcttctg acactttgga agacgccacc aacaacaccc ctggtttctt ggcttactcc     900

tccttaagat tgtcttactt gccaagacca gacggtgtta acatcaccga cgacatgtac     960

gctttggtta gagaaggtaa gtatgccaac atccctgtta tcatcggtga ccaaaacgac    1020

gaaggtacct tctttggtac ttcttctttg aacgttacca ctgatgccca agccagagaa    1080

tatttcaagc aatcttttgt ccacgctagc gacgctgaaa tcgacacttt gatgactgct    1140

tacccaggtg acatcactca aggttctcca tttgacactg gaattctaaa cgccttgacc    1200

ccacaattca agagaatctc tgctatcttg ggtgacttgg gttttacttt ggctcgtaga    1260

tacttcttga accactacac cggtggtacc aagtactctt tcttgtctaa gcaattgtct    1320

ggtttgccag ttttgggtac tttccactcc aacgatatcg tcttccaaga ctacttgttg    1380

ggttctggtt ccttgatcta caacaacgct ttcattgctt ttgccactga cttggaccca    1440

aacaccgccg gtttgttggt taagtggcca gaatacacct cttcttctca atctggtaac    1500

aacttgatga tgatcaacgc tttgggtttg tacaccggta aggacaactt cagaaccgcc    1560

ggttacgacg ctttgttctc caacccacca tctttctttg tt                       1602


<210> 3
<211> 2582
<212> DNA
<213> Komagataella phaffii

<220> 
<221> source
<222> 1..2582
<223> /organism="Komagataella phaffii"
      /note="strain ATCC 76273; HIS4 gene"
      /mol_type="unassigned DNA"

<400> 3
aatacggctt cagaatttct caagactaca ctcactgtcc gacttcaagt atgacatttc      60

ccttgctacc tgcatacgca agtgttgcag agtttgataa ttccttgagt ttggtaggaa     120

aagccgtgtt tccctatgct gctgaccagc tgcacaacct gatcaagttc actcaatcga     180

ctgagcttca agttaatgtg caagttgagt catccgttac agaggaccaa tttgaggagc     240

tgatcgacaa cttgctcaag ttgtacaata atggtatcaa tgaagtgatt ttggacctag     300

atttggcaga aagagttgtc caaaggatcc caggcgctag ggttatctat aggaccctgg     360

ttgataaagt tgcatccttg cccgctaatg ctagtatcgc tgtgcctttt tcttctccac     420

tgggcgattt gaaaagtttc actaatggcg gtagtagaac tgtttatgct ttttctgaga     480

ccgcaaagtt ggtagatgtg acttccactg ttgcttctgg tataatcccc attattgatg     540

ctcggcaatt gactactgaa tacgaacttt ctgaagatgt caaaaagttc cctgtcagtg     600

aaattttgtt ggcgtctttg actactgacc gccccgatgg tctattcact actttggtgg     660

ctgactcttc taattactcg ttgggcctgg tgtactcgtc caaaaagtct attccggagg     720

ctataaggac acaaactgga gtctaccaat ctcgtcgtca cggtttgtgg tataaaggtg     780

ctacatctgg agcaactcaa aagttgctgg gtatcgaatt ggattgtgat ggagactgct     840

tgaaatttgt ggttgaacaa acaggtgttg gtttctgtca cttggaacgc acttcctgtt     900

ttggccaatc aaagggtctt agagccatgg aagccacctt gtgggatcgt aagagcaatg     960

ctccagaagg ttcttatacc aaacggttat ttgacgacga agttttgttg aacgctaaaa    1020

ttagggagga agctgatgaa cttgcagaag ctaaatccaa ggaagatata gcctgggaat    1080

gtgctgactt attttatttt gcattagtta gatgtgccaa gtacggtgtg acgttggacg    1140

aggtggagag aaacctggat atgaagtccc taaaggtcac tagaaggaaa ggagatgcca    1200

agccaggata caccaaggaa caacctaaag aagaatccaa acctaaagaa gtcccttctg    1260

aaggtcgtat tgaattgtgc aaaattgacg tttctaaggc ctcctcacaa gaaattgaag    1320

atgcccttcg tcgtcctatc cagaaaacgg aacagattat ggaattagtc aaaccaattg    1380

tcgacaatgt tcgtcaaaat ggtgacaaag cccttttaga actaactgcc aagtttgatg    1440

gagtcgcttt gaagacacct gtgttagaag ctcctttccc agaggaactt atgcaattgc    1500

cagataacgt taagagagcc attgatctct ctatagataa cgtcaggaaa ttccatgaag    1560

ctcaactaac ggagacgttg caagttgaga cttgccctgg tgtagtctgc tctcgttttg    1620

caagacctat tgagaaagtt ggcctctata ttcctggtgg aaccgcaatt ctgccttcca    1680

cttccctgat gctgggtgtt cctgccaaag ttgctggtcg caaagaaatt gtttttgcat    1740

ctccacctaa gaaggatggt acccttaccc cagaagtcat ctacgttgcc cacaaggttg    1800

gtgctaagtg tatcgtgcta gcaggaggcg cccaggcagt agctgctatg gcttacggaa    1860

cagaaactgt tcctaagtgt gacaaaatat ttggtccagg aaaccagttc gttactgctg    1920

ccaagatgat ggttcaaaat gacacatcag ccctgtgtag tattgacatg cctgctgggc    1980

cttctgaagt tctagttatt gctgataaat acgctgatcc agatttcgtt gcctcagacc    2040

ttctgtctca agctgaacat ggtattgatt cccaggtgat tctgttggct gtcgatatga    2100

cagacaagga gcttgccaga attgaagatg ctgttcacaa ccaagctgtg cagttgccaa    2160

gggttgaaat tgtacgcaag tgtattgcac actctacaac cctatcggtt gcaacctacg    2220

agcaggcttt ggaaatgtcc aatcagtacg ctcctgaaca cttgatcctg caaatcgaga    2280

atgcttcttc ttatgttgat caagtacaac acgctggatc tgtgtttgtt ggtgcctact    2340

ctccagagag ttgtggagat tactcctccg gtaccaacca cactttgcca acgtacggat    2400

atgcccgtca atacagcgga gttaacactg caaccttcca gaagttcatc acttcacaag    2460

acgtaactcc tgagggactg aaacatattg gccaagcagt gatggatctg gctgctgttg    2520

aaggtctaga tgctcaccgc aatgctgtta aggttcgtat ggagaaactg ggacttattt    2580

aa                                                                   2582


<210> 4
<211> 34
<212> DNA
<213> Artificial Sequence

<220> 
<221> source
<222> 1..34
<223> /organism="Artificial Sequence"
      /note="Nucleotide sequence of an 34 bp FRT recombination site "
      /mol_type="unassigned DNA"

<400> 4
gaagttccta tactttctag agaataggaa cttc                                 34


<210> 5
<211> 4
<212> PRT
<213> Artificial Sequence

<220> 
<223> Glutamine Alanine repeat

<400> 5
Glu Ala Glu Ala 
1               

<210> 6
<211> 89
<212> PRT
<213> Artificial Sequence

<220> 
<223> α-mating factor from S. cerevisiae followed by a Kex2 processing
      site (KR) and Glutamine Alanine repeat

<400> 6
Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 
1               5                   10                  15      
Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln 
            20                  25                  30          
Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe 
        35                  40                  45              
Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu 
    50                  55                  60                  
Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val 
65                  70                  75                  80  
Ser Leu Glu Lys Arg Glu Ala Glu Ala 
                85                  

<210> 7
<211> 1650
<212> DNA
<213> Artificial Sequence

<220> 
<221> source
<222> 1..1650
<223> /organism="Artificial Sequence"
      /note="Nucleotide sequence encoding a Kex2 processing site
      followed by the Glutamine Alanine repeat and the codon optimized
      Candida rugosa 534 wild type lipase (LIP1) with an additional
      XhoI site and NotI site at the 5’ and 3’ ends, respectively"
      /mol_type="unassigned DNA"

<400> 7
ctcgagaaga gagaggccga agctgcccca accgccactt tggctaacgg tgacaccatc      60

accggtttga acgccatcat caacgaagcc ttcttgggta ttccatttgc cgaaccacca     120

gttggtaact tgagattcaa ggacccagtt ccatactccg gttccttgga tggtcaaaag     180

ttcacttctt acggtccatc ttgtatgcaa caaaacccag aaggtaccta cgaagaaaac     240

ttgccaaagg cagctttaga tctggttatg caatccaaag ttttcgaagc tgtttctcca     300

tcttctgaag actgtttgac cattaatgtt gttagaccac ccgggacaaa ggctggtgcc     360

aacttgccag ttatgttgtg gatctttggt ggtggttttg aagttggtgg tactagtacc     420

ttccctccag cccaaatgat taccaagtct attgctatgg gtaagccaat catccacgtt     480

tctgtcaact acagagtctc cagctggggt ttcttggctg gtgacgaaat caaggccgaa     540

ggttctgcca acgccggttt gaaggaccaa agattgggta tgcaatgggt ggctgacaac     600

attgctgctt ttggtggtga tccaactaag gttactatct ttggtgaatc tgctggttct     660

atgtccgtca tgtgtcacat tttgtggaac gacggtgaca acacttacaa gggtaagcca     720

ttgttcagag ctggtatcat gcaatctggt gctatggttc catctgacgc cgtcgacggt     780

atctacggta acgaaatttt tgacttgttg gcttccaacg ctggttgtgg ttctgcctct     840

gacaagttgg cttgtttgag aggtgtttct tctgacactt tggaagacgc caccaacaac     900

acccctggtt tcttggctta ctcctcctta agattgtctt acttgccaag accagacggt     960

gttaacatca ccgacgacat gtacgctttg gttagagaag gtaagtatgc caacatccct    1020

gttatcatcg gtgaccaaaa cgacgaaggt accttctttg gtacttcttc tttgaacgtt    1080

accactgatg cccaagccag agaatatttc aagcaatctt ttgtccacgc tagcgacgct    1140

gaaatcgaca ctttgatgac tgcttaccca ggtgacatca ctcaaggttc tccatttgac    1200

actggaattc taaacgcctt gaccccacaa ttcaagagaa tctctgctat cttgggtgac    1260

ttgggtttta ctttggctcg tagatacttc ttgaaccact acaccggtgg taccaagtac    1320

tctttcttgt ctaagcaatt gtctggtttg ccagttttgg gtactttcca ctccaacgat    1380

atcgtcttcc aagactactt gttgggttct ggttccttga tctacaacaa cgctttcatt    1440

gcttttgcca ctgacttgga cccaaacacc gccggtttgt tggttaagtg gccagaatac    1500

acctcttctt ctcaatctgg taacaacttg atgatgatca acgctttggg tttgtacacc    1560

ggtaaggaca acttcagaac cgccggttac gacgctttgt tctccaaccc accatctttc    1620

tttgtttaat aaggttaaag gggcggccgc                                     1650


