Abstract:
The present invention relates to lipolytic enzymes and DNA sequences encoding same. More specifically, the present invention relates to the lipolytic enzyme and DNA sequences encoding same isolated from  Fusarium sulphureum  and analogues thereof.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims under 35 U.S.C. 119 priority from Danish application no. PA 2000 00989, filed on Jun. 26, 2000, and the benefit of U.S. provisional application No. 60/215,462, filed Jun. 30, 2000, the contents of which are fully incorporated herein by reference. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to lipolytic enzymes, methods of using and producing lipolytic enzymes, as well as a nucleic acid sequence encoding lipolytic enzymes. 
     BACKGROUND OF THE INVENTION 
     Lipolytic enzymes (such as lipases and phospholipases) are capable of hydrolyzing carboxylic ester bonds in a substrate to release carboxylic acids. They are known to be useful, e.g., in baking and detergents. 
     A lipase/phospholipase from  Fusarium oxysporum  and its sequence are known. WO 98/26057. 
     SUMMARY OF THE INVENTION 
     The inventors have isolated a lipolytic enzyme from  Fusarium sulphureum . The inventors also isolated the gene encoding the novel lipolytic enzyme and cloned it into an  E. coli  strain. 
     Accordingly, the invention provides a lipolytic enzyme which may be a polypeptide having an amino acid sequence as the mature peptide shown in SEQ ID NO: 1. 
     Further, the lipolytic enzyme of the invention may be a polypeptide encoded by the lipolytic enzyme encoding part of the DNA sequence cloned into a plasmid present in  Escherichia coli  deposit number DSM 13539. 
     The lipolytic enzyme may also be an analogue of the polypeptide defined above which: 
     i) has at least 85% homology with said polypeptide, 
     ii) is immunologically reactive with an antibody raised against said polypeptide in purified form, 
     iii) is an allelic variant of said polypeptide, 
     Finally, the lipolytic enzyme of the invention may be a polypeptide which is encoded by a nucleic acid sequence which hybridizes under high stringency conditions with a complementary strand of the nucleic acid sequence of SEQ ID NO: 1 encoding the mature polypeptide or a subsequence thereof having at least 100 nucleotides. 
     The nucleic acid sequence of the invention may comprise a nucleic acid sequence which encodes the lipolytic enzyme described above, or it may encode a lipolytic enzyme and comprise: 
     a) the DNA sequence encoding a mature lipolytic enzyme cloned into a plasmid present in  Escherichia coli  DSM 13539, 
     b) the DNA sequence encoding a mature lipolytic enzyme shown in SEQ ID NO: 1, or 
     c) an analogue of the DNA sequence defined in a) or b) which 
     i) has at least 80% homology with said DNA sequence, or 
     ii) hybridizes at high stringency with said DNA sequence, its complementary strand or a subsequence thereof. 
     Other aspects of the invention provide a recombinant expression vector comprising the DNA sequence, and a cell transformed with the DNA sequence or the recombinant expression vector. 
     A comparison with full-length prior-art sequences shows that the mature amino acid sequence of the invention has 82% homology with the lipase/phospholipase from  Fusarium oxysporum  described above, and the corresponding DNA sequence of the invention shows 77% homology with that of the  F. oxysporum  enzyme. 
     DETAILED DESCRIPTION OF THE INVENTION 
     Genomic DNA Source 
     A lipolytic enzyme of the invention may be derived from a strain of Fusarium, particularly  F. sulphureum , using probes designed on the basis of the DNA sequences in this specification. 
     A strain of  Escherichia coli  containing a gene encoding lipolytic enzyme was deposited by the inventors under the terms of the Budapest Treaty with the DSMZ—Deutshe Sammmlung von Microorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig DE, Germany. The deposit date was Jun. 15, 2000, and the accession number was DSM 13539. 
     Properties of Lipolytic Enzyme 
     The lipolytic enzyme is able to hydrolyze carboxylic ester bonds and is classified as EC 3.1.1 according to Enzyme Nomenclature 1992, Academic Press, Inc. The enzyme has lipase (triacylglycerol lipase) activity (EC 3.1.1.3) and may also have phospholipase activity. 
     Recombinant Expression Vector 
     The expression vector of the invention typically includes control sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and, optionally, a selectable marker, a transcription terminator, a repressor gene or various activator genes. The vector may be an autonomously replicating vector, or it may be integrated into the host cell genome. 
     Production by Cultivation of Transformant 
     The lipolytic enzyme of the invention may be produced by transforming a suitable host cell with a DNA sequence encoding the lipolytic enzyme, cultivating the transformed organism under conditions permitting the production of the enzyme, and recovering the enzyme from the culture. 
     The host organism is preferably a eukaryotic cell, in particular a fungal cell, such as a yeast cell or a filamentous fungal cell, e.g. a strain of Aspergillus, Fusarium, Trichoderma or Saccharomyces, particularly  A. niger, A. oryzae, F. graminearum, F. sambucinum, F. cerealis  or  S. cerevisiae . The production of the lipolytic enzyme in such host organisms may be done by the general methods described in EP 238,023 (Novo Nordisk), WO 96/00787 (Novo Nordisk) or EP 244,234 (Alko). 
     Hybridization 
     The hybridization is used to indicate that a given DNA sequence is analogous to a nucleotide probe corresponding to a DNA sequence of the invention. The hybridization conditions are described in detail below. 
     Suitable conditions for determining hybridization between a nucleotide probe and a homologous DNA or RNA sequence involves presoaking of the filter containing the DNA fragments or RNA to hybridize in 5×SSC (standard saline citrate) for 10 min, and prehybridization of the filter in a solution of 5×SSC (Sambrook et al. 1989), 5×Denhardt&#39;s solution (Sambrook et al. 1989), 0.5% SDS and 100 μg/ml of denatured sonicated salmon sperm DNA (Sambrook et al. 1989), followed by hybridization in the same solution containing a random-primed (Feinberg, A. P. and Vogelstein, B. (1983)  Anal. Biochem.  132:6-13),  32 P-dCTP-labeled (specific activity &gt;1×10 9  cpm/μg) probe for 12 hours at approx. 45° C. The filter is then washed two times for 30 minutes in 2×SSC, 0.5% SDS at a temperature of at least 55° C., more preferably at least 60° C., more preferably at least 65° C., even more preferably at least 70° C., especially at least 75° C. 
     Molecules to which the oligonucleotide probe hybridizes under these conditions are detected using a x-ray film. 
     Alignment and Homology 
     The present invention also includes lipolytic enzymes and nucleotide sequences encoding same that have homology to the disclosed sequences. More preferably, the lipolytic enzymes and the nucleotide sequences of the invention may have homologies to the disclosed sequences of at least 85%, at least 90% or at least 95%, e.g. at least 96%, at least 97%, at least 98%. 
     For purposes of the present invention, alignments of sequences and calculation of homology scores were done using a Needleman-Wunsch alignment (i.e. global alignment), useful for both protein and DNA alignments. The default scoring matrices BLOSUM50 and the identity matrix are used for protein and DNA alignments respectively. The penalty for the first residue in a gap is −12 for proteins and −16 for DNA, while the penalty for additional residues in a gap is −2 for proteins and −4 for DNA. Alignment is from the FASTA package version v20u6 (W. R. Pearson and D. J. Lipman (1988), “Improved Tools for Biological Sequence Analysis”, PNAS 85:2444-2448, and W. R. Pearson (1990) “Rapid and Sensitive Sequence Comparison with FASTP and FASTA”, Methods in Enzymology, 183:63-98). 
     Lipase Activity (LU) 
     A substrate for lipase is prepared an emulsion of 5% by volume of tributyrin (glycerin tributyrate) using 0.1% gum Arabic as emulsifier. The hydrolysis of tributyrin at 30° C. at pH 7 is followed in a pH-stat titration experiment. One unit of lipase activity (1 LU) equals the amount of enzyme capable of releasing 1 μmol butyric acid/min at the standard conditions. 1 KLU=1000 LU. 
     Use of Lipolytic Enzyme 
     The lipolytic enzyme of the invention can be used in various industrial application of lipolytic enzymes, e.g. in baking, detergents, diglyceride synthesis (EP 307154), acidolysis, interesterification (WO 8802775), ester hydrolysis, oil degumming (JP-A 2-153997, U.S. Pat. No. 5,264,367), production of lysolecithin (JP patent 2794574, JP-B 6-087751) and in the process described in PCT/DK 00/00109. 
     Use in Baking 
     The lipolytic enzyme of the invention can be used in the preparation of dough, bread and cakes, e.g. to improve the elasticity of the bread or cake. Thus, the lipolytic enzyme can be used in a process for making bread, comprising adding the lipolytic enzyme to the ingredients of a dough, kneading the dough and baking the dough to make the bread. This can be done in analogy with WO 9404035 and EP 585988. 
     Use in Detergent 
     The variant may be used as a detergent additive, e.g. at a concentration (expressed as pure enzyme protein) of 0.001-10 (e.g. 0.01-1) mg per gram of detergent or 0.001-100 (e.g. 0.01-10) mg per liter of wash liquor. 
     The detergent composition of the invention may for example be formulated as a hand or machine laundry detergent composition including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use in general household hard surface cleaning operations. In a laundry detergent, the variant may be effective for the removal of fatty stains, for whiteness maintenance and for dingy cleanup. A laundry detergent composition may be formulated as described in WO 97/04079, WO 97/07202, WO 97/41212, PCT/DK WO 98/08939 and WO 97/43375. 
     The detergent composition of the invention may particularly be formulated for hand or machine dishwashing operations. e.g. as described in GB 2,247,025 (Unilever) or WO 99/01531 (Procter &amp; Gamble). In a dishwashing composition, the variant may be effective for removal of greasy/oily stains, for prevention of the staining/discoloration of the dishware and plastic components of the dishwasher by highly colored components and the avoidance of lime soap deposits on the dishware. 
     MATERIALS AND METHODS 
     Methods 
     Unless otherwise stated, DNA manipulations and transformations were performed using standard methods of molecular biology as described in Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, N.Y.; Ausubel, F. M. et al. (eds.) “Current protocols in Molecular Biology”, John Wiley and Sons, 1995; Harwood, C. R., and Cutting, S. M. (eds.) “Molecular Biological Methods for Bacillus”. John Wiley and Sons, 1990. 
     Enzymes 
     Enzymes for DNA manipulations (e.g. restriction endonucleases, ligases etc.) are obtainable from New England Biolabs, Inc. and were used according to the manufacturer&#39;s instructions. 
     Plasmids/vectors 
     pT7Blue (Invitrogen, Netherlands) 
     pCaHj483 is described in WO 9704079 and WO 9942566. 
     Cloning 
     LA PCR™ in vitro Cloning Kit (TaKaRa) was used for cloning and was used according to the manufacturer&#39;s instructions. 
     Microbial Strains 
       E. coli  JM109 (TOYOBO, Japan) 
       A. oryzae  BECh-2 is described in Danish patent application PA 1999 01726. It is a mutant of JaL 228 (described in WO 98/12300) which is a mutant of IFO 4177. 
     Media and Reagents 
     Cove: 342.3 g/L Sucrose, 20 ml/L COVE salt solution, 10 mM Acetamide, 30 g/L noble agar. 
     Cove-2: 30 g/L Sucrose, 20 ml/L COVE salt solution, 10 mM, Acetamide, 30 g/L noble agar. 
     Cove salt solution: per liter 26 g KCl, 26 g MgSO4-7aq, 76 g KH2PO4, 50 ml Cove trace metals. 
     Cove trace metals: per liter 0.04 g NaB407-10aq, 0.4 g CuSO4-5aq, 1.2 g FeSO4-7aq, 0.7 g MnSO4-aq, 0.7 g Na2MoO2-2aq, 0.7 g ZnSO4-7aq. 
     AMG trace metals: per liter 14.3 g ZnSO4-7aq, 2.5 g CuSO4-5aq, 0.5 g NiCl2, 13.8 g FeSO4, 8.5 g MnSO4, 3.0 g citric acid. 
     YPG: 4 g/L Yeast extract, 1 g/L KH2PO4, 0.5 g/L MgSO4-7aq, 5 g/L Glucose, pH 6.0. 
     STC: 0.8 M Sorbitol, 25 mM Tris pH 8, 25 mM CaCl2. 
     STPC: 40% PEG4000 in STC buffer. 
     Cove top agarose: 342.3 g/L Sucrose, 20 ml/L COVE salt solution, 10 mM Acetamide, 10 g/L low melt agarose. 
     MS-9: per liter 30 g soybean powder, 20 g glycerol, pH 6.0. 
     MDU-pH5: per liter 45 g maltose-1aq, 7 g yeast extract, 12 g KH2PO4, 1 g MgSO4-7aq, 2 g K2SO4, 0.5 ml AMG trace metal solution and 25 g 2-morpholinoethanesulfonic acid, pH 5.0. 
    
    
     EXAMPLES 
     Example 1 
     Cloning and Expression of Lipase Gene from  Fusarium sulphureum    
     Transformation in Aspergillus Strain 
       Aspergillus oryzae  strain BECh-2 was inoculated to 100 ml of YPG medium and incubated for 16 hrs at 32° C. at 120 rpm. Pellets were collected and washed with 0.6 M KCl, and resuspended 20 ml 0.6 M KCl containing a commercial β-glucanase product (Glucanex, product of Novo Nordisk A/S) at the concentration of 30 μl/ml. Cultures were incubated at 32° C. at 60 rpm until protoplasts formed, then washed with STC buffer twice. The protoplasts were counted with a hematometer and resuspended in an 8:2:0.1 solution of STC:STPC:DMSO to a final concentration of 2.5×10e7 protoplasts/ml. About 3 μg of DNA was added to 100 μl of protoplasts solution, mixed gently and incubated on ice for 30 min. One ml of SPTC was added and incubated 30 min at 37° C. After the addition of 10 ml of 50° C. Cove top agarose, the reaction was poured onto Cove agar plate. Transformation plates were incubated at 32° C. for 5 days. 
     PCR Screening of Lipase 
     PCR reactions on  Fusarium sulphureum  genomic DNA was done with two following primer sets: lip3/lip15 and lip10/lip17 designed based upon the alignment of 3 lipases from Fusarium. 
     
       
         lip3: 5′-carcayggigcigcigcitaytg-3′  (SEQ ID NO: 3) 
       
     
     
       
         lip15: 5′-ccicciariswrtgiccigt-3′  (SEQ ID NO: 4) 
       
     
     
       
         lip10: 5′-ggitgyggigticayiiiggitt-3′  (SEQ ID NO: 5) 
       
     
     
       
         lip17: 5′-ggrtcityiscrtkigtiac-3′  (SEQ ID NO: 6) 
       
     
     Reaction components (2.6 ng/μl of genomic DNA, 250 mM dNTP each, primer 250 nM each, 0.1 U/μl of Taq polymerase in 1×buffer (Roche Diagnostics, Japan)) were mixed and submitted for PCR under the following conditions. 
     
       
         
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Step 
                 Temperature 
                 Time 
               
               
                   
                   
               
             
             
               
                   
                 1 
                 94° C. 
                  1 min 
               
               
                   
                 2 
                 50° C. 
                  1 min 
               
               
                   
                 3 
                 72° C. 
                  2 min 
               
               
                   
                 4 
                 72° C. 
                 10 min 
               
               
                   
                 5 
                  4° C. 
                 forever 
               
               
                   
                   
               
             
          
         
       
     
     Steps 1 to 3 were repeated 30 times. 
     450 bp of fragment and 280 bp of fragment were amplified from primer sets of lip3/lip15 and lip10/lip17, respectively. They were gel-purified with GFX™ PCR DNA and Gel Band Purification kit (amersham pharmacia biotech) and ligated into a pT7Blue vector with ligation high (TOYOBO, Japan). The ligation mixtures were transformed into  E. coli  JM109. The resultant plasmids, pT27-0315 and pT27-1017, were sequenced and compared to the  Fusarium oxysporum  lipase, showing that a clone encodes the internal part of the lipase. 
     Cloning of Lipase Gene 
     In order to clone the missing part of the lipase gene, adaptor PCR was done. A cassette was made by mixing of adaptor L and adaptor S. 
     
       
         adaptor L: 5′-ctaatacgactcactatagggctcgagcggccgcccgggcaggt=3′  (SEQ ID NO:7) 
       
     
     
       
         adaptor S: 5′-acctgccc-3′  (SEQ ID NO: 8) 
       
     
     3′ and 5′ of adaptor S are dephosphorylated and amidation, respectively. 
     1.3 μg of Eco RV digested genome was ligated to the cassette and it was used as a PCR template. Reaction components (7 ng/μl of genomic DNA ligated to cassette, 250 mM dNTP each, primer 250 nM each, 0.05 U/μl of Expand high fidelity polymerase in 1×buffer (Roche Diagnostics, Japan)) were mixed and submitted for PCR under the following conditions. 
     
       
         
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Step 
                 Temperature 
                 Time 
               
               
                   
                   
               
             
             
               
                   
                 1 
                 94° C. 
                  2 min 
               
               
                   
                 2 
                 94° C. 
                 10 sec 
               
               
                   
                 3 
                 55° C. 
                 30 sec 
               
               
                   
                 4 
                 68° C. 
                 45 sec 
               
             
          
           
               
                 step 2-4 repeat 10 times 
               
             
          
           
               
                   
                 5 
                 94° C. 
                 10 sec 
               
               
                   
                 6 
                 55° C. 
                 30 sec 
               
               
                   
                 7 
                 68° C. 
                 45 sec + 
               
               
                   
                   
                   
                 20 sec/cycle 
               
             
          
           
               
                 step 5-7, repeat 20 times 
               
             
          
           
               
                   
                 8 
                 68° C. 
                  7 min 
               
               
                   
                 7 
                  4° C. 
                 forever 
               
               
                   
                   
               
             
          
         
       
     
     500 bp of DNA fragment corresponding to N-terimal region was obtained with 27N1long primer and 200 bp of DNA fragment corresponding to C-terminal region was obtained with 27C1long primer, 
     
       
         27N1long: 5′-tggacaaccgttccttgcgca-3′  (SEQ ID NO: 9) 
       
     
     
       
         27C1long: 5′-tacacgtacggtgctcctcgagtgg-3′  (SEQ ID NO: 10) 
       
     
     Obtained fragments were purified by GFX™ PCR DNA and Gel Band Purification kit (amersham pharmacia biotech) and sequenced with each primers which amplified the fragment. 
     The missing C-terminal part was cloned with LA PCR™ in vitro Cloning Kit (TaKaRa) following to the manufacturer&#39;s instructions. 350 bp of DNA fragment corresponding to C-terminal region was obtained from Xho I digested genome ligated to Sal I cassette of the kit with 27C2 primer. 
     
       
         27C2: 5′-tatctggcggcggtggcgac-3′  (SEQ ID NO: 11) 
       
     
     Obtained fragments were purified by GFX™ PCR DNA and Gel Band Purification kit (amersham pharmacia biotech) and sequenced with 27C2 primer. 
     The fidelity of taq polymerase is not good so in order to get the right sequence whole gene was amplified the following primers. 
     
       
         27N(Bam): 5′-cgcggatccatgctcctcctaccactcctctcagcc-3′  (SEQ ID NO: 12) 
       
     
     
       
         27C(Sal): 5′-acgcgtcgacttatgatgaacgattcttatggctatccacatactcct-3′  (SEQ ID NO 13) 
       
     
     Reaction components (6 ng/μl of genomic DNA, 250 mM dNTP each, primer 250 nM each, 0.05 U/μl of Expand high fidelity polymerase in 1×buffer (Roche Diagnostics, Japan)) were mixed and submitted for PCR under the following conditions. 
     
       
         
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Step 
                 Temperature 
                 Time 
               
               
                   
                   
               
             
             
               
                   
                 1 
                 94° C. 
                  2 min 
               
               
                   
                 2 
                 94° C. 
                 10 sec 
               
               
                   
                 3 
                 55° C. 
                 30 sec 
               
               
                   
                 4 
                 68° C. 
                 45 sec 
               
             
          
           
               
                 step 2-4 repeat 10 times 
               
             
          
           
               
                   
                 5 
                 94° C. 
                 10 sec 
               
               
                   
                 6 
                 55° C. 
                 30 sec 
               
               
                   
                 7 
                 68° C. 
                 45 sec + 
               
               
                   
                   
                   
                 20 sec/cycle 
               
             
          
           
               
                 step 5-7, repeat 20 times 
               
             
          
           
               
                   
                 8 
                 68° C. 
                  7 min 
               
               
                   
                 7 
                  4° C. 
                 forever 
               
               
                   
                   
               
             
          
         
       
     
     Amplified DNA fragment was gel-purified with GFX™ PCR DNA and Gel Band Purification kit (amersham pharmacia biotech) and ligated into a pT7Blue vector with ligation high (TOYOBO, Japan). The ligation mixtures were transformed into  E. coli  JM109. Four plasmids, pT27w-1, pT27w-2, pT27w-3, and pT27w-4, were sequence and their sequence were compared. pT27w-3 has no PCR error and it is defined as  Fusarium sulphureum  lipase nucleotide sequence. 
     Expression of Lipase Gene in  Aspergillus oryzae    
     The lipase gene was digested from pT27w-3 with BamH I and Sal I and ligated into the BamH I and XhoI sites in the Aspergillus expression cassette pCaHj483 which has  Aspergillus niger  neutral amylase promoter,  Aspergillus nidulans  TPI leader sequences,  Aspergillus niger  glucoamylase terminator and  Aspergillus nidulans  amdS gene as a marker. The resultant plasmid was pNL27w-8. 
     pNL27w-8 was transformed into  Aspergillus oryzae  BECh-2. The selected transformants were inoculated in 100 ml of MS-9 media and cultivated at 30° C. for 1 day. 3 ml of grown cell in MS-9 medium was inoculated to 100 ml of MDU-2BP medium and cultivated at 32° C. for 3 days. The supernatant was obtained by centrifugation. 
     The lipase productivity of selected transformants was determined as LU activity. The productivity of the best transformant TNL27-75 was 130 LU/ml and BECh2 has no lipase activity. 
     Example 2 
     Immunological Characterization of Lipolytic Enzyme 
     A purified lipolytic enzyme sample having the amino acid sequence shown as amino acids 1-319 of SEQ ID NO: 1 was tested by immunodiffusion against a polyclonal antibody raised against the  Fusarium oxysporum  lipase. No immunological cross-reaction was found. 
     SEQUENCE LISTING 
      lip3: 5′-carcayggigcigcigcitaytg-3′  (SEQ ID NO: 3) 
     
       
         lip15: 5′-ccicciariswrtgiccigt-3′  (SEQ ID NO: 4) 
       
     
     
       
         lip10: 5′-ggitgyggigticayiiiggitt-3′  (SEQ ID NO: 5) 
       
     
     
       
         lip17: 5′-ggrtcityiscrtkigtiac-3′  (SEQ ID NO: 6) 
       
     
     
       
         adaptor L: 5′-ctaatacgactcactatagggctcgagcggccgcccgggcaggt=3′  (SEQ ID NO: 7) 
       
     
     
       
         adaptor S: 5′-acctgccc-3′  SEQ ID NO: 
       
     
     
       
         27N1long: 5′-tggacaaccgttccttgcgca-3′  (SEQ ID NO: 8) 
       
     
     
       
         27C1long: 5′-tacacgtacggtgctcctcgagtgg-3′  (SEQ ID NO: 9) 
       
     
     
       
         27C2: 5′-tatctggcggcggtggcgac-3′  (SEQ ID NO:10) 
       
     
     
       
         27N(Bam): 5′-cgcggatccatgctcctcctaccactcctctcagcc-3′  (SEQ ID NO: 11) 
       
     
     
       
         27C(Sal): 5′-acgcgtcgacttatgatgaacgattcttatggctatccacatactcct-3′  (SEQ ID NO:12) 
       
     
     
       
         
           
             12 
           
           
             1 
             1161 
             DNA 
             Fusarium sulphureum 
             
               CDS 
               (1)..(94) 
                 
             
           
            1
atg ctc ctc cta cca ctc ctc tca gcc gtc act ctc gcg gta gca agt       48
Met Leu Leu Leu Pro Leu Leu Ser Ala Val Thr Leu Ala Val Ala Ser
    -30                 -25                 -20
cca cta gct tcc gtc gaa gag tac gcc aag tct ctc gaa gac aga g         94
Pro Leu Ala Ser Val Glu Glu Tyr Ala Lys Ser Leu Glu Asp Arg
-15                 -10                 -5              -1
gtaagcacca aactctcctc catatcatgc tatatactca tcacactccc ag ct  gtg    151
                                                          Ala Val
act gtg tct tcg tca gac tac aac aac ttc aag ttc tac atc caa cat      199
Thr Val Ser Ser Ser Asp Tyr Asn Asn Phe Lys Phe Tyr Ile Gln His
        5                   10                  15
ggc gcc gca gca tac tgt aac tcc gaa gcc tca gct ggc gca aag atc      247
Gly Ala Ala Ala Tyr Cys Asn Ser Glu Ala Ser Ala Gly Ala Lys Ile
    20                  25                  30
acc tgc gca agc aac ggt tgt cca acc gtc cag tcc aac ggc gca acc      295
Thr Cys Ala Ser Asn Gly Cys Pro Thr Val Gln Ser Asn Gly Ala Thr
35                  40                  45                  50
atc gtg gca tcc ttc ct  gtaagtcacg cccagtcaca aacatctcat             342
Ile Val Ala Ser Phe Leu
                55
acctcatact tatatgactt cttcag t ggt tcc aag act ggc atc ggc ggt       393
                               Gly Ser Lys Thr Gly Ile Gly Gly
                                           60
tat gtc gca aca gat tca tcc cgc aag gaa atc gtc gtc tcg atc cgt      441
Tyr Val Ala Thr Asp Ser Ser Arg Lys Glu Ile Val Val Ser Ile Arg
65                  70                  75                  80
gga agc agc aac atc cgc aac tgg ctt aca aac ctc gac ttt gac cag      489
Gly Ser Ser Asn Ile Arg Asn Trp Leu Thr Asn Leu Asp Phe Asp Gln
                85                  90                  95
tcc gac tgc agc ttg acc tcc ggc tgc ggc gta cac tcg ggc ttc cag      537
Ser Asp Cys Ser Leu Thr Ser Gly Cys Gly Val His Ser Gly Phe Gln
            100                 105                 110
aac gcc tgg gac gag atc tcg gag aga gcc act gct gct gtg gcc aag      585
Asn Ala Trp Asp Glu Ile Ser Glu Arg Ala Thr Ala Ala Val Ala Lys
        115                 120                 125
gca cgc aag gca aac tct ggt ttc aag gtc att gct aca ggc cac tcc      633
Ala Arg Lys Ala Asn Ser Gly Phe Lys Val Ile Ala Thr Gly His Ser
    130                 135                 140
ctc ggt ggt gcg gtc gct aca ttg gct gct gcg aat ctg agg gtt ggt      681
Leu Gly Gly Ala Val Ala Thr Leu Ala Ala Ala Asn Leu Arg Val Gly
145                 150                 155                 160
ggc aca ccc gtg gac atc tac acg tac ggt gct cct cga gtg ggc aac      729
Gly Thr Pro Val Asp Ile Tyr Thr Tyr Gly Ala Pro Arg Val Gly Asn
                165                 170                 175
gcc cag ctt tca gcg ttc atc tcg aac caa gct ggc ggg gaa tat cgc      777
Ala Gln Leu Ser Ala Phe Ile Ser Asn Gln Ala Gly Gly Glu Tyr Arg
            180                 185                 190
gtt act cac gcc aga gac ccc gtg cct cgt ctg ccc cct ctg gtg ttt      825
Val Thr His Ala Arg Asp Pro Val Pro Arg Leu Pro Pro Leu Val Phe
        195                 200                 205
gga tac agg cac act tcg ccc gag tac tgg cta tct ggc ggc ggt ggc      873
Gly Tyr Arg His Thr Ser Pro Glu Tyr Trp Leu Ser Gly Gly Gly Gly
    210                 215                 220
gac aag att gat tat acc atc agc gat atc aag gtc tgt gag ggc gcc      921
Asp Lys Ile Asp Tyr Thr Ile Ser Asp Ile Lys Val Cys Glu Gly Ala
225                 230                 235                 240
gct aat ctc cag tgt aac ggt ggc acg ctg ggt ttg gac att gcg gct      969
Ala Asn Leu Gln Cys Asn Gly Gly Thr Leu Gly Leu Asp Ile Ala Ala
                245                 250                 255
cat ctg cat tac ttc cag cac act gat gct tgc tcg gcg gga ggc att     1017
His Leu His Tyr Phe Gln His Thr Asp Ala Cys Ser Ala Gly Gly Ile
            260                 265                 270
tct ttt aga cga tac agg agt gct aag cgt gaa ggt atc gcc aag agg     1065
Ser Phe Arg Arg Tyr Arg Ser Ala Lys Arg Glu Gly Ile Ala Lys Arg
        275                 280                 285
gct gat atg tcg gat gct gag ctg gag aag aag ctc aac tct tat gtt     1113
Ala Asp Met Ser Asp Ala Glu Leu Glu Lys Lys Leu Asn Ser Tyr Val
    290                 295                 300
gag atg gat aag gag tat gtg gat agc cat aag aat cgt tca tca taa     1161
Glu Met Asp Lys Glu Tyr Val Asp Ser His Lys Asn Arg Ser Ser
305                 310                 315
 
           
             2 
             350 
             PRT 
             Fusarium sulphureum 
           
            2
Met Leu Leu Leu Pro Leu Leu Ser Ala Val Thr Leu Ala Val Ala Ser
    -30                 -25                 -20
Pro Leu Ala Ser Val Glu Glu Tyr Ala Lys Ser Leu Glu Asp Arg Ala
-15                 -10                 -5              -1  1
Val Thr Val Ser Ser Ser Asp Tyr Asn Asn Phe Lys Phe Tyr Ile Gln
            5                   10                  15
His Gly Ala Ala Ala Tyr Cys Asn Ser Glu Ala Ser Ala Gly Ala Lys
        20                  25                  30
Ile Thr Cys Ala Ser Asn Gly Cys Pro Thr Val Gln Ser Asn Gly Ala
    35                  40                  45
Thr Ile Val Ala Ser Phe Leu Gly Ser Lys Thr Gly Ile Gly Gly Tyr
50                  55                  60                  65
Val Ala Thr Asp Ser Ser Arg Lys Glu Ile Val Val Ser Ile Arg Gly
                70                  75                  80
Ser Ser Asn Ile Arg Asn Trp Leu Thr Asn Leu Asp Phe Asp Gln Ser
            85                  90                  95
Asp Cys Ser Leu Thr Ser Gly Cys Gly Val His Ser Gly Phe Gln Asn
        100                 105                 110
Ala Trp Asp Glu Ile Ser Glu Arg Ala Thr Ala Ala Val Ala Lys Ala
    115                 120                 125
Arg Lys Ala Asn Ser Gly Phe Lys Val Ile Ala Thr Gly His Ser Leu
130                 135                 140                 145
Gly Gly Ala Val Ala Thr Leu Ala Ala Ala Asn Leu Arg Val Gly Gly
                150                 155                 160
Thr Pro Val Asp Ile Tyr Thr Tyr Gly Ala Pro Arg Val Gly Asn Ala
            165                 170                 175
Gln Leu Ser Ala Phe Ile Ser Asn Gln Ala Gly Gly Glu Tyr Arg Val
        180                 185                 190
Thr His Ala Arg Asp Pro Val Pro Arg Leu Pro Pro Leu Val Phe Gly
    195                 200                 205
Tyr Arg His Thr Ser Pro Glu Tyr Trp Leu Ser Gly Gly Gly Gly Asp
210                 215                 220                 225
Lys Ile Asp Tyr Thr Ile Ser Asp Ile Lys Val Cys Glu Gly Ala Ala
                230                 235                 240
Asn Leu Gln Cys Asn Gly Gly Thr Leu Gly Leu Asp Ile Ala Ala His
            245                 250                 255
Leu His Tyr Phe Gln His Thr Asp Ala Cys Ser Ala Gly Gly Ile Ser
        260                 265                 270
Phe Arg Arg Tyr Arg Ser Ala Lys Arg Glu Gly Ile Ala Lys Arg Ala
    275                 280                 285
Asp Met Ser Asp Ala Glu Leu Glu Lys Lys Leu Asn Ser Tyr Val Glu
290                 295                 300                 305
Met Asp Lys Glu Tyr Val Asp Ser His Lys Asn Arg Ser Ser
                310                 315
 
           
             3 
             20 
             DNA 
             Artificial Sequence 
             
               Primer 
             
           
            3
ccnccnarns wrtgnccngt                                                 20
 
           
             4 
             23 
             DNA 
             Artificial Sequence 
             
               Primer 
             
           
            4
ggntgyggng tncaynnngg ntt                                             23
 
           
             5 
             20 
             DNA 
             Artificial Sequence 
             
               Primer 
             
           
            5
ggrtcntyns crtkngtnac                                                 20
 
           
             6 
             44 
             DNA 
             Artificial Sequence 
             
               Primer 
             
           
            6
ctaatacgac tcactatagg gctcgagcgg ccgcccgggc aggt                      44
 
           
             7 
             8 
             DNA 
             seq 8 
           
            7
acctgccc                                                               8
 
           
             8 
             21 
             DNA 
             Artificial Sequence 
             
               Primer 
             
           
            8
tggacaaccg ttccttgcgc a                                               21
 
           
             9 
             25 
             DNA 
             Artificial Sequence 
             
               Primer 
             
           
            9
tacacgtacg gtgctcctcg agtgg                                           25
 
           
             10 
             20 
             DNA 
             Artificial Sequence 
             
               Primer 
             
           
            10
tatctggcgg cggtggcgac                                                 20
 
           
             11 
             36 
             DNA 
             Artificial Sequence 
             
               Primer 
             
           
            11
cgcggatcca tgctcctcct accactcctc tcagcc                               36
 
           
             12 
             48 
             DNA 
             Artificial Sequence 
             
               Primer 
             
           
            12
acgcgtcgac ttatgatgaa cgattcttat ggctatccac atactcct                  48