Abstract:
A recombinant β-amylase which is superior to the original recombinant β-amylase in thermostability has been obtained by a site-directed mutagenesis with the recombinant β-amylase gene coding 531 amino acid residues. Substitutions were MET 181  of said enzyme with Leu, Ser 291  with Ala, Ile 293  with Val, Ser 346  with Pro, Ser 347  with Pro, Gln 348  with Asp and Ala 372  with Ser.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     This invention relates to a β-amylase with an improved thermostability as well as an improved enzyme stability in the alkaline pH region, a gene coding the enzyme and an expression vector containing the gene. 
     2. Description of the Related Art 
     Barley β-Amylase 
     Barley β-amylase is a β-amylase (1,4-α-D-glucan maltohydrolase  EC 3.2.1.2!) obtained from barley seeds and is well known along with soybean β-amylase, as a useful enzyme for the industrial maltose production used for transfusional solutions and foodstuffs. 
     However, since barley is one of the principal agricultural products for the production of livestock feeds and beverages (such as beer and whisky), from the viewpoint of the global food situation in the future it is not advisable to consume the harvested barley as a source of β-amylase. 
     Therefore, the method for producing β-amylase in microorganisms using genetic engineering techniques has been given attention as an other source of this enzyme than the barley. If the efficient expression of the barley β-amylase gene in a microorganism is accomplished, the steady supply of inexpensive β-amylase will become possible, obviously contributing a great deal to the maltose production. 
     Gene of Barley β-Amylase 
     As to the barley β-amylase gene, the cDNA consisted of 1754 base pairs of cultivar Hiproly has been reported, and also the amino acid sequence consisted of 535 residues has been deduced (Eur. J. Biochem., 169, 517 (1987)). In addition, the cDNA consisted of 1775 base pairs of cultivar Haruna Nijo has been reported, and also the amino acid sequence consisted of 535 residues has been established (J. Biochem., 115, 47 (1994)). 
     In studies on β-amylase of cultivar Haruna Nijo, the expression vector (pBETA92) was already constructed by inserting a DNA fragment, which was prepared by deleting 55 base pairs of a full-length cDNA from its 5&#39;-terminus and linking a SmaI linker, into the SmaI site of plasmid pKK223-3 (Pharmacia Biotech). Also the production of recombinant β-amylase has been accomplished by transforming Escherichia coli JM109 (Toyobo) with said expression vector and expressing the recombinant β-amylase gene therein. Furthermore, it was reported that the recombinant β-amylase comprising 531 amino acids showed almost the same properties as barley β-amylase (JP Hei6-58119; JP Hei6-303988). 
     However, a production of recombinant β-amylase in microorganisms which shows almost the same properties to those of β-amylase from barley seeds is not sufficient for the purpose. It is because of the fact that, since soybean β-amylase is somewhat superior to barley β-amylase in thermostability, soybean β-amylase is more widely used in practice. Therefore, in order to improve the utility value of the barley β-amylase, it is necessary to provide it at least with the similar function (thermostability) to that of soybean β-amylase. 
     As to the barley recombinant β-amylases with improved thermostability by protein engineering, it has been proved that a double-mutant β-amylase wherein Ser 291  of the enzyme is replaced with Ala, and Ser 346  is replaced with Pro, by site-directed mutagenesis, is superior to the original recombinant β-amylase (JP Hei6-126151). 
     To further improve the utility value of recombinant β-amylase, it is necessary to construct β-amylase with a further improved thermostability by protein engineering. 
     SUMMARY OF THE INVENTION 
     The present invention aims to construct a gene encoding a recombinant β-amylase with a further improved thermostability site-directed mutagenesis, provide a recombinant vector containing the gene, transform microorganisms with the vector and eventually provide recombinant β-amylase with a further improved thermostability. 
     As a result of studies to further improve the thermostability of β-amylase without changing the enzymatic function thereof, the inventors of the present invention have found that a sevenfold-mutant enzyme comprising the substitutions of Met 181  by Leu, Ile 293  by Val, Ser 347  by Pro, Gln 348  by Asp and Ala 372  by Ser in addition to those of Ser 291  by Ala and Ser 346  by Pro (JP Hei6-126151) was much superior to the double-mutant enzyme in thermostability accomplishing the present invention. 
     That is, the recombinant β-amylase according to the present invention is that comprising the amino acid sequence denoted by SEQ ID NO: 1. 
     β-Amylase according to the present invention is a recombinant β-amylase which acts on polysaccharides having α-1,4-glucoside linkages such as soluble starch, amylose and amylopectin in addition to maltooligosaccharides with a degree of polymerization higher than 3 liberating successively a β-maltose unit from the non-reducing ends thereof, shows more than 80% of the maximum enzymatic activity at pH 3.5-7.0 (37° C.), retains more than 80% remaining activity after the treatment for 1 h at pH 3.5-12.5 (37° C.), shows the maximum activity toward soluble starch at 65° C. and 87% of the maximum activity at 70° C. (pH 7.0), and is stable after treatment for 30 min at up to 62.5° C. in the absence of a substrate at pH 7.0. 
     Furthermore, the gene of the present invention is the gene encoding recombinant β-amylase comprising the amino acid sequence of SEQ ID NO: 1. 
     The gene according to the present invention is the gene encoding recombinant β-amylase of Claim 1 having the nucleotide sequence of SEQ ID NO: 2. 
     The expression vector according to the present invention is the expression vector for β-amylase comprising any one of the genes described above. An Expression vector of this sort is exemplified by that having the nucleotide sequence of SEQ ID NO: 3. 
     Host cells according to the present invention are those containing the expression vectors. 
     In the following, there will be described the practical method for preparing recombinant β-amylase according to the present invention, a gene encoding the enzyme and an expression vector containing the gene. 
     1. Base Substitution of β-Amylase Expression Vector pBETA92 By Site-Directed Mutagenesis 
     The base substitution at the specific site of the gene sequence of β-amylase expression vector pBETA92 can be achieved by site-directed mutagenesis (Anal. Biochem., 200, 81 (1992)). 
     2. Transformation Host Microorganism With β-Amylase Expression Vector 
     Any microorganisms can be used as the host cell so far as the expression vector for β-amylase with the improved thermostability can proliferate stably and autonomously therein. 
     As to the method to transform the host microorganism with the expression vector for recombinant β-amylase, any published method, for example, the competent cell method (J. Mol. Biol., 58, 159 (1970)) may be used in the case where the host microorganism is Escherichia coli. 
     3. Confirmation of DNA Sequence 
     DNA sequence can be performed by the chemical modification method according to Maxam-Gilbert (Methods in Enzymology, 65, 499 (1980)) or the dideoxynucleotide chain termination method (Gene, 19, 269 (1982)) or the like. 
     Furthermore, the amino acid sequence of β-amylase according to the present invention can be deduced from the DNA sequence. 
     4. Production and Purification of Recombinant β-Amylase 
     After growing the host microorganism harboring the β-amylase expression vector for a certain period, the pure preparation of recombinant β-amylase can be obtained by cell lysis, if necessary, followed by a combination of ammonium sulfate fractionation and various chromatographies such as gel filtration or ion exchange. 
     β-Amylase activity may be assayed using 2.4-dichlorophenyl β-maltopentaoside (Ono Pharmaceutical) as the substrate. In this case, one unit of enzyme is defined as the amount of enzyme which produces 1 μmol of dichlorophenol per min at 37° C. 
     5. Estimation of Thermostability 
     An aliquot of enzyme preparation (30 μl each) in 1.5-ml Eppendorf tubes was incubated at temperatures ranging from 50°-72.5° C. (at 2.5° C.-intervals) in a water bath for 30 min. The remaining activity was assayed using 20 μl aliquot withdrawn from the tube. The remaining activity versus temperature curves were used to determine the temperature curves of enzyme relative at which 50% of the initial activity was lost during 30-min heating period and half-inactivation temperature values provided a parameter for the ranking of thermal stabilities of the enzyme. 
     Soybean β-amylase used as a control is one purchased from Amano Pharmaceutical (trade name, Biozyme M-5). The enzyme preparation was diluted using a solution of 50 mM Good&#39;s buffer (pH 7.0)/1% bovine serum albumin. 
     Studies of effects of temperature and pH on β-amylase activity were done by reacting the enzyme with soluble starch at pH 7.0. The amount of the reducing sugar produced was measured by the dinitrosalicylic acid method (Denpun Kagaku Handbook, Asakurashoten, p. 188-189 (1977)), and 1 unit of the enzyme was defined as the amount which liberates 1 μmol of maltose per min. 
     6. Determination of Optimum pH 
     The reaction mixture, 0.4 ml of 1% soluble starch solution, 0.2 ml of various buffers (described below) and 0.2 ml of enzyme preparation, was incubated at 37° C. The amount of reducing sugars produced was measured by the dinitrosalicylic acid method, and results were expressed as the value relative to the maximum activity (100%). As a result of measuring the optimum pH in this manner, the optimum pH at which the enzyme shows more than 80% of the maximum activity was found to be in the range of 3.5-7.0. 
     Buffers used were as follows: 
     
         ______________________________________pH 2.5 ≃ 3.0              Citrate bufferpH 3.5 ≃ 5.5              Acetate bufferpH 6.0 ≃ 8.0              Good&#39;s bufferpH 8.5             Tris-maleate bufferpH 9.0 ≃ 11.0              Glycine buffer______________________________________ 
    
     7. Determination of pH Stability 
     To the enzyme preparation (50 μl) was added 100 mM various buffers (50 μl) and the mixture was incubated at 37° C. for 1 h. Then 0.9 ml of 500 mM Good&#39;s buffer (pH 7.0)/1% bovine serum albumin solution was added. To 0.4 ml aliquot withdrawn was added 0.4 ml of 1% soluble starch solution (pH 7.0), and the mixture was incubated at 37° C. and the remaining enzymatic activity was measured. As a result of measuring pH stability in this manner, the pH range where more than 80% of the original activity was stably retained was found to be 3.5-12.5. 
     Buffers used were as follows: 
     
         ______________________________________pH 3.0             Citrate bufferpH 3.5 ≃ 5.5              Acetate bufferpH 6.0 ≃ 8.0              Good&#39;s bufferpH 8.5             Tris-maleate bufferpH 9.0 ≃ 11.5              Glycine bufferpH 12.0 ≃ 13.0              KCl--NaOH buffer______________________________________ 
    
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a drawing showing the optimum temperature of each preparation. In the figure, (□ . . . □) indicates sevenfold-mutant β-amylase according to the present invention, (-) barley β-amylase, (◯-◯) original recombinant β-amylase and (▪ . . . ▪) soybean β-amylase. 
     FIG. 2 is a drawing showing the thermostability of each preparation. In the figure, (□ . . . □) indicates sevenfold-mutant β-amylase according to the present invention, (-) barley β-amylase, (◯-◯) original recombinant β-amylase and (▪ . . . ▪) soybean β-amylase. 
     FIG. 3 is a drawing showing the pH stability of each preparation. In the figure, (□ . . . □) indicates sevenfold-mutant β-amylase according to the present invention, (-) barley β-amylase and (◯-◯) original recombinant β-amylase. 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENT 
     The invention will now be described in further detail with reference to specific examples, however, it is understood that the scope of the present invention is not to be construed as being limited to them in any way. 
     EXAMPLE 1 
     Base Substitution of the Recombinant Wild-Type β-Amylase Expression Vector By Site-Directed Mutagenesis. 
     The site-directed mutagenesis was done using a Transfer Site-directed Mutagenesis kit (Clontech Laboratories). 
     Using the following four mutagenesis primers 5&#39;-AGCTGGAGAGTTGAGGTACCC-3&#39; (for Met 181  to Leu; SEQ ID NO: 4), 5&#39;-AATCAAGATCGCTGGCGTTCACTGGTG-3&#39; (for Ser 291  to Ala and Ile 293  to Val; SEQ ID NO: 5), 5&#39;-TTCGGAGCAACCCCCGGACGCGATGAGCGCA-3&#39; (for Ser 346  to Pro, Ser 347  to Pro and Gln 348  to Asp; SEQ ID NO: 6) and 5&#39;-CCTAAATGTGTCATGCGAAAA-3&#39; (for Ala 372  to Ser; SEQ ID NO: 7) and the selection primer 5&#39;-GGTTGAGTATTCACCAGTC-3&#39; (SEQ ID NO: 8), the site-directed mutagenesis was done according to the manual provided with the kit to obtain the recombinant β-amylase (sevenfold-mutant β-amylase) expression vector (pBETA92/sevenfold-mutant) as shown in SEQ ID NO: 3. 
     EXAMPLE 2 
     Determination of DNA Sequence 
     DNA sequence confirmed that, as shown in SEQ ID NO: 2 in the sequence list, A 541  was substituted with T, T 871  with G, A 877  with G, AG 1036-1037  with CC, T 1039  with C, C 1042  with G, G 1044  with C and G 1114  with T. Consequently, it was confirmed that the expression vector pBETA92/sevenfold-mutant is encoding the recombinant β-amylase as shown in SEQ ID NO: 1 of the sequence list. 
     EXAMPLE 3 
     Production and Purification of Recombinant β-Amylase 
     Escherichia coli JM109 harboring the expression vector pBETA92/sevenfold-mutant was grown in a liquid medium (containing 1% Tryptone, 0.5% yeast extract, 1% NaCl, 0.005% Ampicillin Na and 0.1 mM isopropyl β-D-thiogalactopyranoside in 400 ml of water, pH 7.0) at 37° C. for 24 h. After centrifugation to remove the culture medium, packed cells were suspended in a lysozyme solution (0.025% lysozyme, 20 mM Tris-HCl and 30 mM NaCl, pH 7.5) for 30 min on ice, and disrupted by sonication (50 W, 30 sec) followed by centrifugation. 
     To the above crude extract was added solid ammonium sulfate to 30% saturation. After the precipitate was removed by centrifugation, the supernatant was loaded onto a Butyl Toyopearl 650S (Toso) column (2.5×18.5 cm). The active fractions which were eluted with 50 mM acetate buffer (pH 5.5) were collected and dialyzed against 15 mM Tris-HCl (pH 8.0). The dialyzed solution was centrifuged to remove insoluble materials and then loaded onto a DEAE-Toyopearl 650S (Toso) column (2.5×18.5 cm). The active fractions which were eluted with 15 mM Tris-HCl (pH 8.0)/50 mM NaCl were collected, and added solid ammonium sulfate to 70% saturation. The precipitate formed were collected by centrifugation, dissolved in 50 mM acetate buffer (pH 5.5) and then dialyzed against the same buffer. Then the dialyzed solution was loaded onto a Toyopearl HW-50S (Toso) column (1.5×48.5 cm). The active fractions which were eluted with 50 mM acetate buffer (pH 5.5) were combined as the purified preparation of the recombinant β-amylase. On SDS-polyacrylamide gel electrophoresis the purified preparation showed a single protein band at an apparent molecular weight of 56,000 which migrated to almost the same position as the original recombinant β-amylase. 
     EXAMPLE 4 
     Enzymatic Properties of Sevenfold-Mutant β-Amylase 
     Comparison of the enzymatic properties of the sevenfold-mutant β-amylase with those of the original recombinant β-amylase revealed that both enzymes were similar except for the optimum temperature, thermostability and pH stability. 
     Results of studies on the optimum temperature are shown in FIG. 1. In contrast to the barley β-amylase and the original recombinant β-amylase which showed the maximum activity at 55° C. and almost no activity at 65°-70° C. the sevenfold-mutant β-amylase was found to show the maximum activity at 65° C. and a significant activity even at 70° C. It was also confirmed that the sevenfold-mutant β-amylase was significantly improved in thermostability as compared with the soybean β-amylase which showed the maximum activity at 60° C. 
     From heat-inactivation curves shown in FIG. 2, temperatures at which 50% of the initial activity was lost during a 30 min heating time were found as follows: 
     
         ______________________________________barley β-amylase  → 56.8° C.original recombinant β-amylase                  → 57.4° C.sevenfold-mutant β-amylase                  → 69.0° C.soybean β-amylase → 63.2° C.______________________________________ 
    
     The results indicate that the thermostability of the sevenfold-β-amylase was improved by 11.6° C. than that of the original recombinant β-amylase, and furthermore by 5.8° C. than that of the soybean β-amylase. 
     A great deal improvement of the sevenfold-mutant β-amylase in the thermostability was confirmed by the fact that, while the original recombinant β-amylase was almost completely inactivated by treatment at 62.5° C. for 30 min, the sevenfold-mutant β-amylase was not inactivated at all by the same treatment. 
     As to the pH stability, as shown in FIG. 3, while the barley B-amylase and the original recombinant B-amylase were stable in the pH range of 3.5-9.5, the sevenfold-mutant β-amylase was stable in the pH range of 3.5-12.5, indicating a significant improvement in the stability of the latter β-amylase in the alkaline pH range. 
     The present invention has made it possible to produce a recombinant β-amylase with improved thermostability as well as improved enzyme stability in the alkaline pH range. 
     
         __________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 8(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 531 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:MetLysGlyAsnTyrValGlnValTyrValMetLeuProLeuAspAla151015ValSerValAsnAsnArgPheGluLysGlyAspGluLeuArgAlaGln202530LeuArgLysLeuValGluAlaGlyValAspGlyValMetValAspVal354045TrpTrpGlyLeuValGluGlyLysGlyProLysAlaTyrAspTrpSer505560AlaTyrLysGlnLeuPheGluLeuValGlnLysAlaGlyLeuLysLeu65707580GlnAlaIleMetSerPheHisGlnCysGlyGlyAsnValGlyAspAla859095ValAsnIleProIleProGlnTrpValArgAspValGlyThrArgAsp100105110ProAspIlePheTyrThrAspGlyHisGlyThrArgAsnIleGluTyr115120125LeuThrLeuGlyValAspAsnGlnProLeuPheHisGlyArgSerAla130135140ValGlnMetTyrAlaAspTyrMetThrSerPheArgGluAsnMetLys145150155160AspPheLeuAspAlaGlyValIleValAspIleGluValGlyLeuGly165170175ProAlaGlyGluLeuArgTyrProSerTyrProGlnSerHisGlyTrp180185190SerPheProGlyIleGlyGluPheIleCysTyrAspLysTyrLeuGln195200205AlaAspPheLysAlaAlaAlaAlaAlaValGlyHisProGluTrpGlu210215220PheProAsnAspAlaGlyGlnTyrAsnAspThrProGluArgThrGln225230235240PhePheArgAspAsnGlyThrTyrLeuSerGluLysGlyArgPhePhe245250255LeuAlaTrpTyrSerAsnAsnLeuIleLysHisGlyAspArgIleLeu260265270AspGluAlaAsnLysValPheLeuGlyTyrLysValGlnLeuAlaIle275280285LysIleAlaGlyValHisTrpTrpTyrLysValProSerHisAlaAla290295300GluLeuThrAlaGlyTyrTyrAsnLeuHisAspArgAspGlyTyrArg305310315320ThrIleAlaArgMetLeuLysArgHisArgAlaSerIleAsnPheThr325330335CysAlaGluMetArgAspSerGluGlnProProAspAlaMetSerAla340345350ProGluGluLeuValGlnGlnValLeuSerAlaGlyTrpArgGluGly355360365LeuAsnValSerCysGluAsnAlaLeuProArgTyrAspProThrAla370375380TyrAsnThrIleLeuArgAsnAlaArgProHisGlyIleAsnGlnSer385390395400GlyProProGluHisLysLeuPheGlyPheThrTyrLeuArgLeuSer405410415AsnGlnLeuValGluGlyGlnAsnTyrValAsnPheLysThrPheVal420425430AspArgMetHisAlaAsnLeuProArgAspProTyrValAspProMet435440445AlaProLeuProArgSerGlyProGluIleSerIleGluMetIleLeu450455460GlnAlaAlaGlnProLysLeuGlnProPheProPheGlnGluHisThr465470475480AspLeuProValGlyProThrGlyGlyMetGlyGlyGlnAlaGluGly485490495ProThrCysGlyMetGlyGlyGlnValLysGlyProThrGlyGlyMet500505510GlyGlyGlnAlaGluAspProThrSerGlyMetGlyGlyGluLeuPro515520525AlaThrMet530(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 1596 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:GTGAAAGGCAACTATGTCCAAGTCTACGTCATGCTCCCTCTGGACGCCGTGAGCGTGAAC60AACAGGTTCGAGAAGGGCGACGAGCTGAGGGCGCAATTGAGGAAGCTGGTAGAGGCCGGT120GTGGATGGTGTCATGGTAGACGTCTGGTGGGGCTTGGTGGAGGGCAAGGGCCCCAAGGCG180TATGACTGGTCCGCCTACAAGCAGTTGTTTGAGCTGGTGCAGAAGGCTGGGCTGAAGCTA240CAGGCCATCATGTCGTTCCACCAGTGTGGTGGCAACGTCGGCGACGCCGTCAACATCCCA300ATCCCACAGTGGGTGCGGGACGTCGGCACGCGTGATCCCGACATTTTCTACACCGACGGT360CACGGGACTAGGAACATTGAGTACCTCACTCTTGGAGTTGATAACCAGCCTCTCTTCCAT420GGAAGATCTGCCGTCCAGATGTATGCCGATTACATGACAAGCTTCAGGGAGAACATGAAA480GACTTCTTGGATGCTGGTGTTATCGTCGACATTGAAGTGGGACTTGGCCCAGCTGGAGAG540TTGAGGTACCCATCATATCCTCAGAGCCACGGATGGTCGTTCCCAGGCATCGGAGAATTC600ATCTGCTATGATAAATACCTACAAGCAGACTTCAAAGCAGCAGCAGCGGCGGTCGGCCAT660CCTGAGTGGGAATTTCCTAACGATGCCGGACAGTACAATGACACTCCCGAGAGAACTCAA720TTCTTCAGGGACAACGGGACATACCTAAGTGAGAAGGGGAGGTTTTTCCTTGCATGGTAC780TCCAACAATCTGATCAAGCACGGTGACAGGATCTTGGATGAAGCAAACAAGGTCTTCTTG840GGATACAAGGTGCAATTGGCAATCAAGATCGCTGGCGTTCACTGGTGGTACAAGGTTCCA900AGCCATGCAGCCGAGCTCACAGCTGGGTACTATAACTTACATGATAGAGACGGCTACAGA960ACCATAGCACGCATGCTCAAAAGGCACCGTGCTAGCATTAACTTCACTTGCGCGGAGATG1020AGGGATTCGGAGCAACCCCCGGACGCGATGAGCGCACCAGAAGAACTAGTCCAACAGGTG1080TTGAGTGCTGGATGGAGAGAGGGCCTAAATGTGTCATGCGAAAACGCGCTTCCACGATAT1140GATCCAACTGCTTACAACACCATACTCAGGAATGCGAGGCCTCATGGAATCAACCAGAGC1200GGCCCTCCTGAGCACAAGCTGTTTGGATTCACCTACCTTCGGCTGTCGAATCAGCTGGTG1260GAGGGACAAAACTATGTCAACTTCAAGACCTTTGTCGACAGAATGCATGCCAACCTGCCT1320CGTGACCCATATGTTGATCCAATGGCGCCCTTGCCAAGATCAGGGCCAGAAATATCGATT1380GAGATGATCCTACAAGCAGCACAGCCAAAACTGCAGCCATTCCCCTTCCAGGAGCACACC1440GACCTGCCAGTAGGCCCTACTGGTGGCATGGGTGGGCAGGCTGAAGGCCCCACCTGTGGC1500ATGGGTGGGCAAGTTAAAGGCCCTACTGGTGGCATGGGTGGGCAGGCTGAAGACCCTACT1560AGTGGCATGGGTGGGGAGCTCCCTGCCACCATGTAA1596(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6312 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: circular(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:TTCCGGGATGGAGGTGAACGTGAAAGGCAACTATGTCCAAGTCTACGTCATGCTCCCTCT60GGACGCCGTGAGCGTGAACAACAGGTTCGAGAAGGGCGACGAGCTGAGGGCGCAATTGAG120GAAGCTGGTAGAGGCCGGTGTGGATGGTGTCATGGTAGACGTCTGGTGGGGCTTGGTGGA180GGGCAAGGGCCCCAAGGCGTATGACTGGTCCGCCTACAAGCAGTTGTTTGAGCTGGTGCA240GAAGGCTGGGCTGAAGCTACAGGCCATCATGTCGTTCCACCAGTGTGGTGGCAACGTCGG300CGACGCCGTCAACATCCCAATCCCACAGTGGGTGCGGGACGTCGGCACGCGTGATCCCGA360CATTTTCTACACCGACGGTCACGGGACTAGGAACATTGAGTACCTCACTCTTGGAGTTGA420TAACCAGCCTCTCTTCCATGGAAGATCTGCCGTCCAGATGTATGCCGATTACATGACAAG480CTTCAGGGAGAACATGAAAGACTTCTTGGATGCTGGTGTTATCGTCGACATTGAAGTGGG540ACTTGGCCCAGCTGGAGAGTTGAGGTACCCATCATATCCTCAGAGCCACGGATGGTCGTT600CCCAGGCATCGGAGAATTCATCTGCTATGATAAATACCTACAAGCAGACTTCAAAGCAGC660AGCAGCGGCGGTCGGCCATCCTGAGTGGGAATTTCCTAACGATGCCGGACAGTACAATGA720CACTCCCGAGAGAACTCAATTCTTCAGGGACAACGGGACATACCTAAGTGAGAAGGGGAG780GTTTTTCCTTGCATGGTACTCCAACAATCTGATCAAGCACGGTGACAGGATCTTGGATGA840AGCAAACAAGGTCTTCTTGGGATACAAGGTGCAATTGGCAATCAAGATCGCTGGCGTTCA900CTGGTGGTACAAGGTTCCAAGCCATGCAGCCGAGCTCACAGCTGGGTACTATAACTTACA960TGATAGAGACGGCTACAGAACCATAGCACGCATGCTCAAAAGGCACCGTGCTAGCATTAA1020CTTCACTTGCGCGGAGATGAGGGATTCGGAGCAACCCCCGGACGCGATGAGCGCACCAGA1080AGAACTAGTCCAACAGGTGTTGAGTGCTGGATGGAGAGAGGGCCTAAATGTGTCATGCGA1140AAACGCGCTTCCACGATATGATCCAACTGCTTACAACACCATACTCAGGAATGCGAGGCC1200TCATGGAATCAACCAGAGCGGCCCTCCTGAGCACAAGCTGTTTGGATTCACCTACCTTCG1260GCTGTCGAATCAGCTGGTGGAGGGACAAAACTATGTCAACTTCAAGACCTTTGTCGACAG1320AATGCATGCCAACCTGCCTCGTGACCCATATGTTGATCCAATGGCGCCCTTGCCAAGATC1380AGGGCCAGAAATATCGATTGAGATGATCCTACAAGCAGCACAGCCAAAACTGCAGCCATT1440CCCCTTCCAGGAGCACACCGACCTGCCAGTAGGCCCTACTGGTGGCATGGGTGGGCAGGC1500TGAAGGCCCCACCTGTGGCATGGGTGGGCAAGTTAAAGGCCCTACTGGTGGCATGGGTGG1560GCAGGCTGAAGACCCTACTAGTGGCATGGGTGGGGAGCTCCCTGCCACCATGTAATGGAA1620CCTTTATGATTTACTACCCTTTATGTTGTGTGTGAGTGTGACAGAGAAACCTTTCTCTGC1680CTTATTAATAATAAATAAAGCACATCACTTGTGTGTGTTCTGAAAAGCCCGGGGATCCGT1740CGACCTGCAGCCAAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAG1800ATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCG1860GTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGT1920GTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCA1980GTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAG2040GACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGC2100AGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGG2160CCTTTTTGCGTTTCTACAAACTCTTTTGTTTATTTTTCTAAATACATTCAAATATGTATC2220CGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGA2280GTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTT2340TTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAG2400TGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAG2460AACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTG2520TTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTG2580AGTATTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCA2640GTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAG2700GACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATC2760GTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTG2820TAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCC2880GGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGG2940CCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCG3000GTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGA3060CGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCAC3120TGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAA3180AACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCA3240AAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAG3300GATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCAC3360CGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAA3420CTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCC3480ACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAG3540TGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTAC3600CGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGC3660GAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTC3720CCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCA3780CGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACC3840TCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACG3900CCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCT3960TTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATA4020CCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGC4080GCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCA4140CTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCT4200ACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACG4260GGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCAT4320GTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATC4380AGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAG4440TTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTT4500TTCCTGTTTGGTCACTTGATGCCTCCGTGTAAGGGGGAATTTCTGTTCATGGGGGTAATG4560ATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGG4620TTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAA4680ATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGC4740CAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTT4800TCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGAC4860GTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCA4920GTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACC4980CGTGGCCAGGACCCAACGCTGCCCGAGATGCGCCGCGTGCGGCTGCTGGAGATGGCGGAC5040GCGATGGATATGTTCTGCCAAGGGTTGGTTTGCGCATTCACAGTTCTCCGCAAGAATTGA5100TTGGCTCCAATTCTTGGAGTGGTGAATCCGTTAGCGAGGTGCCGCCGGCTTCCATTCAGG5160TCGAGGTGGCCCGGCTCCATGCACCGCGACGCAACGCGGGGAGGCAGACAAGGTATAGGG5220CGGCGCCTACAATCCATGCCAACCCGTTCCATGTGCTCGCCGAGGCGGCATAAATCGCCG5280TGACGATCAGCGGTCCAGTGATCGAAGTTAGGCTGGTAAGAGCCGCGAGCGATCCTTGAA5340GCTGTCCCTGATGGTCGTCATCTACCTGCCTGGACAGCATGGCCTGCAACGCGGGCATCC5400CGATGCCGCCGGAAGCGAGAAGAATCATAATGGGGAAGGCCATCCAGCCTCGCGTCGCGA5460ACGCCAGCAAGACGTAGCCCAGCGCGTCGGCCGCCATGCCGGCGATAATGGCCTGCTTCT5520CGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTC5580CGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGA5640AAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCA5700TAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGG5760CTCTCAAGGGCATCGGTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCA5820GTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGG5880CGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCA5940TGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAG6000CAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCCGGG6060CTTATCGACTGCACGGTGCACCAATGCTTCTGGCGTCAGGCAGCCATCGGAAGCTGTGGT6120ATGGCTGTGCAGGTCGTAAATCACTGCATAATTCGTGTCGCTCAAGGCGCACTCCCGTTC6180TGGATAATGTTTTTTGCGCCGACATCATAACGGTTCTGGCAAATATTCTGAAATGAGCTG6240TTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACA6300CAGGAAACAGAA6312(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: other nucleic acid(A) DESCRIPTION: /desc =&#34;SYNTHETIC DNA&#34;(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:AGCTGGAGAGTTGAGGTACCC21(2) INFORMATION FOR SEQ ID NO:5:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 27 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: other nucleic acid(A) DESCRIPTION: /desc =&#34;SYNTHETIC DNA&#34;(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:AATCAAGATCGCTGGCGTTCACTGGTG27(2) INFORMATION FOR SEQ ID NO:6:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 31 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: other nucleic acid(A) DESCRIPTION: /desc =&#34;SYNTHETIC DNA&#34;(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:TTCGGAGCAACCCCCGGACGCGATGAGCGCA31(2) INFORMATION FOR SEQ ID NO:7:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: other nucleic acid(A) DESCRIPTION: /desc =&#34;SYNTHETIC DNA&#34;(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:CCTAAATGTGTCATGCGAAAA21(2) INFORMATION FOR SEQ ID NO:8:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 19 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: other nucleic acid(A) DESCRIPTION: /desc =&#34;SYNTHETIC DNA&#34;(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:GGTTGAGTATTCACCAGTC19__________________________________________________________________________