# Patent

## Title
Surfactant-nutrients for bioremediation of hydrocarbon contaminated

## Abstract

A gene encoding a hydrogen peroxide-generating NADH oxidase and a method for preparing a large amount of the NADH oxidase with the use of the gene and gene recombinant techniques are disclosed.

## Background
1. Field of the Invention

The present invention relates to a DNA fragment encoding an enzyme that catalyzes the reaction in which NADH is oxidized by oxygen molecules to generate hydrogen peroxide (Japanese Laid-Open Publication 2-407889) and a method for preparing the enzyme with the use of a microorganism including a plasmid containing the fragment. The enzyme is useful for detection of a very small amount of substance in vivo with the use of the reaction generating NADH.

2. Description of the Prior Art

NADH oxidases are divided into two groups, one group which generates hydrogen peroxide (hydrogen peroxide-generating enzymes) and the other group which generates water (water-generating enzymes) by the reaction with oxygen molecules.

Hydrogen peroxide-generating enzymes can be found in a culture of Streptococus mutans. However, it is difficult to obtain a stable culture condition in which water-generating enzymes can be obtained in a much higher level than the level of hydrogen peroxide-generating enzymes, which can also be found in the culture. Moreover, it is necessary to separate hydrogen peroxide generating enzymes from water-generating enzymes in the culture. Therefore, a method for preparing a large amount of hydrogen peroxide-generating NADH oxidases stably in a rational way, i.e., a method for preparing NADH oxidases with the use of gene recombinant techniques is desired.

The conventional method for preparing NADH oxidases with a culture of Streptococus mutans is not suitable for obtaining a large amount of a hydrogen peroxide-generating NADH oxidase. Therefore, one of the objectives of the present invention is to provide a gene encoding a NADH oxidase, which can be used in a method for preparing a large amount of a hydrogen peroxide-generating NADH oxidase by gene recombinant techniques.

As the result of our researches to develop a method for preparing a hydrogen peroxide-generating NADH oxidase, we have eventually cloned a gene encoding hydrogen peroxide-generating NADH oxidase derived from Streptococus mutans and sequenced the gene.

The present invention, provides a DNA fragment encoding the hydrogen peroxide-generating NADH oxidase.

The present invention also provides a DNA fragment that encodes a protein containing a NADH binding site of the NADH oxidase having the following amino acid sequence: Val Leu Val Ile Gly Gly Gly Pro Ala Gly Asn Ser Ala Ala Ile Tyr Ala Ala Arg Lys Gly Val Lys Thr __________________________________________________________________________

The present invention provides a DNA fragment of following base sequence that encodes a protein containing a NADH binding site of the NADH oxidase: GTCCTTGTTA TTGGTGGGGG TCCTGCTGGT AATAGCGCGG CTATCTATGC TGCAAGAAAG GGAGTTAAAA CA __________________________________________________________________________

The present invention also provides a DNA fragment encoding a protein containing a FAD binding site of the NADH oxidase having the following amino acid sequence: Val Ala Val Ile Gly Gly Gly Asn Ser Gly Leu Glu Ala Ala Ile Asp Leu Ala Gly Leu Ala Ser His Val Tyr __________________________________________________________________________

The present invention provides a DNA fragment of the following base sequence which encodes a protein containing a FAD binding site of the NADH oxidase: GTCGCTGTCA TTGGCGGTGG AAACTCAGGT TTAGAAGCAG TCATTGATTT GGCTGGGTTA GCTAGCCATG TCTAT __________________________________________________________________________

The present invention also provides a DNA fragment of following base sequence which encodes the NADH oxidase: AT GGCATTAGAC GCAGAAATCA AAGAGCAGTT AGGACAGTAT CTTCAATTAC TTGAGTGTGA GATTGTTTTA CAAGCTCAAT TAAAAGACGA TGCTAATTCT CAAAAAGTTA AGGAATTTCT CCAAGAAATC GTTGCAATGT CTCCTATGAT TTCTTTAGAC GAAAAGGAAC TTCCGCGAAC ACCTAGTTTT CGCATAGCTA AAAAGGGGCA AGAATCTGGT GTTGAATTTG CTGGCTTACC CCTTGGTCAC GAATTTTACT TCGTTTATCT TGGCTCTGTT ACAGGTTTCA GGGCGTCCGC TAAGGTAGAG ACTGATATTG TCAAACGCAT TCAAGCTGTT GATGAACCTA TGCATTTTGA AACCTATGTT AGTTTGACTT GTCATAATTG TCCAGATGTT GTTCAGGCTT TCAATATCAT GTCAGTTGTT AATCCCAACA TTTCACATAC AATGGTGGAA GGTGGCATGT TTAAAGATGA AATTGAAGCT AAGGGAATTA TGTCTGTGCC AACTGTCTAT AAAGATGGAA CAGAATTTAC CTCAGGGCGT GCTAGCATAG AGCAATTACT AGACTTGATA GCAGGTCCTC TTAAAGAAGA TGCTTTTGAT GATAAAGGTG TTTTTGATGT CCTTGTTATT GGTGGGGGTC CTGCTGGTAA TAGCGCGGCT ATCTATGCTG CAAGAAAGGG AGTTAAAACA GGACTTTTAG CTGAAACCAT GGGTGGTCAA GTTATGGAAA CCGTGGGTAT TGAAAATATG ATCGGTACCC CATATGTTGA AGGACCCCAA TTAATGGCTC AGGTGGAAGA GCATACCAAG TCTTATTCTG TTGACATCAT GAAGGCACCG CGTGCTAAGT CTATTCAAAA GACAGACTTG GTTGAAGTTG AACTTGATAA TGGAGCTCAT TTGAAAGCAA AGACAGCTGT TTTGGCCTTA GGTGCCAAGT GGCGTAAAAT CAATGTACCA GGAGAAAAAG AATTCTTTAA TAAAGGTGTT ACTTACTGTC CGCACTGTGA TGGTCCTCTT TTCACAGACA AAAAAGTCGC TGTCATTGGC GGTGGAAACT CAGGTTTAGA AGCAGCTATT GATTTGGCTG GGTTAGCTAG CCATGTCTAT ATTTTAGAAT TTTTACCTGA GTTAAAAGCT GATAAGATCT TACAAGATCG TGCGGAAGCT CTTGATAATA TTACCATTCT AACTAATGTT GCGACTAAAG AAATTATTGG CAATGACCAC GTAGAAGGTC TTCGTTACAG TGATCGTACG ACCAATGAAG AGTACTTGCT TGATTTAGAA GGTGTTTTTG TTCAAATTGG ATTGGTACCT AGTACTGACT GGTTAAAGGA TAGTGGACTA GCACTCAATG AAAAAGGTGA AATCATTGTT GCTAAAGATG GCGCAACTAA TATTCCTGCT ATTTTTGCAG CTGGTGATTG CACAGATAGT GCCTACAAAC AAATTATCAT TTCCATGGGT TCTGGAGCTA CTGCGGCTTT AGGTGCCTTT GATTATTTGA TTAGAAATT// __________________________________________________________________________

The present invention also provides a DNA fragment encoding the NADH oxidase having the following amino acid sequence: Met Ala Leu Asp Ala Glu Ile Lys Glu Gln Leu Gly Gln Tyr Leu 15 Gln Leu Leu Glu Cys Glu Ile Val Leu Gln Ala Gln Leu Lys Asp 30 Asp Ala Asn Ser Gln Lys Val Lys Glu Phe Leu Gln Glu Ile Val 45 Ala Met Ser Pro Met Ile Ser Leu Asp Glu Lys Glu Leu Pro Arg 60 Thr Pro Ser Phe Arg Ile Ala Lys Lys Gly Gln Glu Ser Gly Val 75 Glu Phe Ala Gly Leu Pro Leu Gly His Glu Phe Tyr Phe Val Tyr 90 Leu Gly Ser Val Thr Gly Phe Arg Ala Ser Ala Lys Val Glu Thr 105 Asp Ile Val Lys Arg Ile Gln Ala Val Asp Glu Pro Met His Phe 120 Glu Thr Tyr Val Ser Leu Thr Cys His Asn Cys Pro Asp Val Val 135 Gln Ala Phe Asn Ile Met Ser Val Val Asn Pro Asn Ile Ser His 150 Thr Met Val Glu Gly Gly Met Phe Lys ASp Glu Ile Glu Ala Lys 165 Gly Ile Met Ser Val Pro Thr Val Tyr Lys Asp Gly Thr Glu Phe 180 Thr Ser Gly Arg Ala Ser Ile Glu Gln Leu Leu Asp Leu Ile Ala 195 Gly Pro Leu Lys Glu Asp Ala Phe Asp Asp Lys Gly Val Phe Asp 210 Val Leu Val Ile Gly Gly Gly Pro Ala Gly Asn Ser Ala Ala Ile 225 Tyr Ala Ala Arg Lys Gly Val Lys Thr Gly Leu Leu Ala Glu Thr 240 Met Gly Gly Gln Val Met Glu Thr Val Gly Ile Glu Asn Met Ile 255 Gly Thr Pro Tyr Val Glu Gly Pro Gln Leu Met Ala Gln Val Glu 270 Glu His Thr Lys Ser Tyr Ser Val Asp Ile Met Lys Ala Pro Arg 285 Ala Lys Ser Ile Gln Lys Thr Asp Leu Val Glu Val Glu Leu Asp 300 Asn Gly Ala His Leu Lys Ala Lys Thr Ala Val Lue Ala Lue Gly 315 Ala Lys Trp Arg Lys Ile Asn Val Pro Gly Glu Lys Glu Phe Phe 330 Asn Lys Gly Val Thr Tyr Cys Pro His Cys Asp Gly Pro Leu Phe 345 Thr Asp Lys Lys Val Ala Val Ile Gly Gly Gly Asn Ser Gly Leu 360 Glu Ala Ala Ile Asp Leu Ala Gly Leu Ala Ser His Val Tyr Ile 375 Leu Glu Phe Leu Pro Glu Leu Lys Ala Asp Lys Ile Leu Gln Asp 390 Arg Ala Glu Ala Lue Asp Asn Ile Thr Ile Leu Thr Asn Val Ala 405 Thr Lys Glu Ile Ile Gly Asn Asp His Val Glu Gly Leu Arg Tyr 420 Ser Asp Arg Thr Thr Asn Glu Glu Tyr Leu Leu Asp Leu Glu Gly 435 Val Phe Val Gln Ile Gly Leu Val Pro Ser Thr Asp Trp Leu Lys 450 Asp Ser Gly Leu Ala Leu Asn Glu Lys Gly Glu Ile Ile Val Ala 465 Lys Asp Gly Ala Thr Asn Ile Pro Ala Ile Phe Ala Ala Gly Asp 480 Cys Thr Asp Ser Ala Tyr Lys Gln Ile Ile Ile Ser Met Gly Ser 495 Lys Ala Thr Ala Ala Leu Gly Ala Phe Asp Tyr Leu Ile Arg Asn 510 __________________________________________________________________________

In the upstream region of the DNA fragment of the present invention may be a non-translational region having the following base sequence: GGATCCCTTC TCATGTTCTC TCACAAGGAT TTGAGGTTTT AGGTGAAGAT GGTTTAGCAC 60 AACGTGGAAC CTTTATTGTA GATCCGGATG GTATCATTCA AATGATGGAA GTCAATGCAG 120 ATGGTATTGG TCGTGATGCT AGTACCTTGA TTGATAAAGT TCGTGCAGCT CAATCTATTC 180 GCCAACATCC AGGAGAAGTT TGCCCTGCCA AATGGAAAGA GGGAGCTGAA ACTTTAAAAC 240 CAAGTTTGGT ACTTGTCGGT AAAATTTAAG GAGAAACT __________________________________________________________________________

The gene encoding the NADH oxidase of the present invention may be cloned by standard methods. For example, first, genomic DNA is prepared from a microorganism having an ability to produce a hydroxy peroxide-generating NADH oxidase. The prepared genomic DNA is digested with an appropriate restriction enzyme to obtain DNA fragments. A vector is also digested with the appropriate restriction enzymes. One of the obtained DNA fragments and the digested vector are ligated by the use of a T4 DNA ligase to obtain recombinant DNA.

The obtained recombinant DNAs are further treated with appropriate restriction enzymes. Then each of the treated DNA fragments is ligated into a cloning vector and the obtained recombinant vector is introduced into a host microorganism to prepare a transformant. The transformants may be screened for desired transformants by a standard method in order to clone a gene encoding the NADH oxidase.

In more detail, Streptococus mutans NCIB11723 is cultured in a medium containing appropriate amounts of carbon, nitrogen, inorganic salts, and other nutrient sources and the resulting culture is centrifuged to collect the cells. Examples of preferable mediums include a brain-heart infusion medium and the like. The temperature for culture is in the range from 20.degree. to 40.degree. C., preferably, from 30.degree. to 37.degree. C. The culture starts with pH6 to 8, preferably about 7. The culture continues until about the middle of the log growth phase by a stand culture. The DNA can be prepared from lytic cells.

Methods for lysing cells include cell treatments with cell wall-lytic enzymes, such as a lysozyme and an .alpha.-glucanase, and optionally together with other enzymes or surfactant, such as sodium lauryl sulfate.

DNA can be isolated and purified from lytic cells according to standard techniques, such as appropriate combination of phenol extraction, protein removing, protease treatment, ribonuclease treatment, alcohol precipitation, centrifugation, and the like.

Methods for cleaving DNAs include ultrasonication, restriction enzyme treatment and the like. After being cleaved, a base sequence of the terminal of a DNA fragment can be altered by the treatment with modifying enzymes, such as phosphatases and DNA polymerases.

To select DNA fragments having a base sequence encoding the desired protein, cleaved DNAs can be gel electrophoresed and subjected to Southern hybridization (Southern, E. M., J. Mol. Biol., 98, 503 (1975)) with the use of synthetic probes based on amino acid sequence of the desired protein. Positive DNA fragment(s) can be extracted from a gel to obtain appropriate length of fragment(s).

As for vectors, those derived from phages or plasmids, which can autonomically propagate in a host cell can be used. For example, .lambda. phages, M13 phages, pBR322, pUC118, and pUC119 can be used for Escherichia coli (E. coli) host cell.

Method for ligating a DNA fragment and a vector fragment can use any methods using known DNA ligase and the like. For example, a DNA fragment and a vector fragment can be ligated in vitro with the action of an appropriate DNA ligase to prepare recombinant DNA.

As for host cell, any microorganism in which recombinant DNAs can be stably maintained and autonomically propagate, and be expressed, can be used.

Methods for introducing recombinant DNAs into host microorganism include known methods, for example, when a host microorganism is E. coli, a calcium method can be used (Lederberg. E. M., & Cohen. S. N., J. Bacteriol., 119, 1072 (1974)).

When the vector is derived from .lambda. phage, .lambda. phage particles are first formed by an in vitro packaging method (Horn, B., Methods in Enzymol., 68, 299 (1979)). Then the particles are added to a culture suspension of E. coli to obtain transduced phages with an ability to produce the NADH oxidase.

Methods for selecting transformed microorganisms containing recombinant DNA include standard methods, such as colony hybridization method for obtaining positive clones. The obtained transformants are cultured in a liquid at 37.degree. C. and plasmids in the transformants can be obtained by known methods, such as an alkaline extraction method (Birnboim, H. C. & Doly, J., Nucleic Acids Res., 7, 1513 (1979)). The sequence of inserts in the plasmids can be determined by a dideoxy method (Sanger, F., Nickelen, S., & Colusion, A. R., Proc. Natl. Acad. Sci. 74, 5493 (1977)). A large amount of the desired hydrogen peroxide-generating NADH oxidase can be obtained by culturing the selected transformants.

Streptococus mutans NCIB11723 (The National Collection of Industrial and Marin Bacteria, 23 Street, Machar Drive, Aberdeen, U.K.) was cultured in a brain-heart infusion liquid medium at 37.degree. C. for 6 hours and then centrifuged to collect the cells. The obtained cells were washed and treated with a lysozyme, N-acetylmuramidase (2000 U/ml). Then genomic DNA was isolated by a method of Saito-Miura (Saito, H. & Miura, K., Biochem. Biophys. Acta, 72, 619 (1963)). The isolated genomic DNA was dissolved in a Tris-Hcl/EDTA buffer and partially digested with Sau3A1. The digested fragments were separated by a 10 to 40% sucrose density-gradient centrifugation to obtain DNA fragments with 9 to 23 kb. The genomic DNA fragments were ligated with arms of .lambda.EMBL3 phage (Stratagene Cloning Systems, 11099 North Torry, Pines Road, La Jolla Calif. 92037, U.S.A.) by the use of T4 DNA ligase. The ligated fragments were packed in phage particles by using in vitro packaging kit (Stratagene Cloning Systems, 11099 North Torry, Pines Road, La Jolla Calif. 92037, U.S.A.). E. coli P2392 (Stratagene Cloning Systems, 11099 North Torry, Pines Road, La Jolla Calif. 92037, U.S.A.) was infected with the phage particles.

The infected plaques of E. coli were screened by a plaque hybridization method with the use of synthetic probes based on the N terminal amino acid sequence of hydrogen peroxide-generating NADH oxidase. A positive clone hybridizing with the synthetic probes was selected. peroxide-generating NADH oxidase

The positive clone obtained in Example 1 was also reacted with an antibody directed to the hydrogen peroxide-generating NADH oxidase. DNA was extracted from the positive clone and digested with BamH1 to obtain a DNA fragment with 4 kbp. The DNA fragment and plasmid pMW (a plasmid with an unique BamH1 site and an ampicillin resistance gene; Nippon-Gene, 1-29, Tonyacho, Toyama 930, Japan) digested with BamH1 were mixed and ligated by adding a T4 DNA ligase in the mixture. The mixture was used to introduce the recombinant plasmid into E. coli JM109 (Takara Shuzo Co., LTD., Shijo-Higashinotoin, Shimogyo-ku, Kyoto 600-91, Japan). The clone that reacted with the above-mentioned antibody was selected from the transformants.

The positive clone was cultured and centrifuged to collect the cells. After the cells were washed, the plasmid was extracted with an alkaline method. The obtained plasmid was designated as pHS19. encoding hydrogen peroxide-generating NADH oxidase

The plasmid PHS19 obtained in Example 2 was digested with EcoR1 to obtain DNA fragments with 1 kbp and 3 kbp. The DNA fragments were ligated with plasmid PUC118 or PUC119 and used for cloning of a gene encoding hydrogen peroxide-generating NADH oxidase. A transformant including the 1 kbp DNA fragment and a transformant including the 3 kbp DNA fragment were obtained. Plasmids were extracted from both of the transformants. Deletion variants including inserts with a variety of sizes necessary for the determination of base sequences were prepared with the use of kilo sequencing deletion kit (Takara Shuzo Co., Ltd., Shijo-Higashinotoin, Shimogyo-ku, Kyoto 600-91, Japan). A DNA base sequence was determined by a dideoxy nucleotide chain termination method (Messing, J. & Vieira, J., Gene, 19, 269 (1982).

The obtained base sequence is given in SEQ ID NO: 1. In this base sequence, 1-278 is a non-translational region containing promoter region, and 279-1810 is a structural DNA. An amino acid sequence deduced from the structural DNA is given in SEQ ID NO: 2. The obtained amino acid sequence was shown a 54.6% homology with the NADH oxidase encoding DNA fragment derived from Amphibacillus xylanus(GENETYX: Amino Acid Homology Data).

In an amino acid sequence of a NADH oxidase, it is presumed that binding sites for NADH, the substrate of the NADH oxidase and FAD (flavin coenzyme) contain amino acid sequence -GXXXXG-, wherein G is glycine and X may be any amino acid residue (Ho-Jin PARK, et. al., Eur. J. Biochem., 205, 875-879(1992)). The amino acid sequence given in SEQ ID NO: 2, obtained in above Example 3 was analyzed and find two sites having the sequences corresponding to -GXXXXG-. Therefore, we assumed one of which is a NADH binding site and the another is a FAD binding site.

A NADH binding site of the NADH oxidase derived from Amphibacilius xylanus contains an amino acid sequence of -GGGPAG- (Gly Gly Gly Pro Ala Gly), and a FAD binding site contains -GGGNSG- (Gly Gly Gly Asn Ser Gly)(Nimura, Y. et. al.: Annual Meeting of Japanese Society of Agricultural Chemistry (1992)).

Compared with both of the NADH and FAD binding sites of the NADH oxidase of Amphibacillus xylanus, 215-220 of SEQ ID NO: 2 is same as the sequence of the NADH binding site, and 354-359 is same as that of FAD binding site. Therefore, it is concluded that in the SEQ ID NO. 2, the regions of 215-220 and 354-359 are each corresponding to a NADH binding site and a FAD binding site of the NADH oxidase.

Accordingly, it is concluded that 921-938 of SEQ ID NO:1 is the region encoding the NADH binding site and 1338-1355 is that encoding the FAD binding site. SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 2 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1809 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: doubule (D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (v) ORIGINAL SOURCE: (B) STRAIN:Streptococcus mutans NCIB11723 (vi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GGATCCCTTCTCATGTTCTCTCACAAGGATTTGAGGTTTTAGGTGAAGATGGTTTAGCAC60 AACGTGGAACCTTTATTGTAGATCCGGATGGTATCATTCAAATGATGGAAGTCAATGCAG120 ATGGTATTGGTCGTGATGCTAGTACCTTGATTGATAAAGTTCGTGCAGCTCAATCTATTC180 GCCAACATCCAGGAGAAGTTTGCCCTGCCAAATGGAAAGAGGGAGCTGAAACTTTAAAAC240 CAAGTTTGGTACTTGTCGGTAAAATTTAAGGAGAAACTATGGCATTAGACGCAGAAATCA300 AAGAGCAGTTAGGACAGTATCTTCAATTACTTGAGTGTGAGATTGTTTTACAAGCTCAAT360 TAAAAGACGATGCTAATTCTCAAAAAGTTAAGGAATTTCTCCAAGAAATCGTTGCAATGT420 CTCCTATGATTTCTTTAGACGAAAAGGAACTTCCGCGAACACCTAGTTTTCGCATAGCTA480 AAAAGGGGCAAGAATCTGGTGTTGAATTTGCTGGCTTACCCCTTGGTCACGAATTTTACT540 TCGTTTATCTTGGCTCTGTTACAGGTTTCAGGGCGTCCGCTAAGGTAGAGACTGATATTG600 TCAAACGCATTCAAGCTGTTGATGAACCTATGCATTTTGAAACCTATGTTAGTTTGACTT660 GTCATAATTGTCCAGATGTTGTTCAGGCTTTCAATATCATGTCAGTTGTTAATCCCAACA720 TTTCACATACAATGGTGGAAGGTGGCATGTTTAAAGATGAAATTGAAGCTAAGGGAATTA780 TGTCTGTGCCAACTGTCTATAAAGATGGAACAGAATTTACCTCAGGGCGTGCTAGCATAG840 AGCAATTACTAGACTTGATAGCAGGTCCTCTTAAAGAAGATGCTTTTGATGATAAAGGTG900 TTTTTGATGTCCTTGTTATTGGTGGGGGTCCTGCTGGTAATAGCGCGGCTATCTATGCTG960 CAAGAAAGGGAGTTAAAACAGGACTTTTAGCTGAAACCATGGGTGGTCAAGTTATGGAAA1020 CCGTGGGTATTGAAAATATGATCGGTACCCCATATGTTGAAGGACCCCAATTAATGGCTC1080 AGGTGGAAGAGCATACCAAGTCTTATTCTGTTGACATCATGAAGGCACCGCGTGCTAAGT1140 CTATTCAAAAGACAGACTTGGTTGAAGTTGAACTTGATAATGGAGCTCATTTGAAAGCAA1200 AGACAGCTGTTTTGGCCTTAGGTGCCAAGTGGCGTAAAATCAATGTACCAGGAGAAAAAG1260 AATTCTTTAATAAAGGTGTTACTTACTGTCCGCACTGTGATGGTCCTCTTTTCACAGACA1320 AAAAAGTCGCTGTCATTGGCGGTGGAAACTCAGGTTTAGAAGCAGCTATTGATTTGGCTG1380 GGTTAGCTAGCCATGTCTATATTTTAGAATTTTTACCTGAGTTAAAAGCTGATAAGATCT1440 TACAAGATCGTGCGGAAGCTCTTGATAATATTACCATTCTAACTAATGTTGCGACTAAAG1500 AAATTATTGGCAATGACCACGTAGAAGGTCTTCGTTACAGTGATCGTACGACCAATGAAG1560 AGTACTTGCTTGATTTAGAAGGTGTTTTTGTTCAAATTGGATTGGTACCTAGTACTGACT1620 GGTTAAAGGATAGTGGACTAGCACTCAATGAAAAAGGTGAAATCATTGTTGCTAAAGATG1680 GCGCAACTAATATTCCTGCTATTTTTGCAGCTGGTGATTGCACAGATAGTGCCTACAAAC1740 AAATTATCATTTCCATGGGTTCTGGAGCTACTGCGGCTTTAGGTGCCTTTGATTATTTGA1800 TTAGAAATT1809 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 510 amino acids (B) TYPE: amino acids (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (v) ORIGINAL SOURCE: (B) STRAIN:Streptococcus mutans NCIB11723 (vi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: MetAlaLeuAspAlaGluIleLysGluGlnLeuGlyGlnTyrLeu 151015 GlnLeuLeuGluCysGluIleValLeuGlnAlaGlnLeuLysAsp 202530 AspAlaAsnSerGlnLysValLysGluPheLeuGlnGluIleVal 354045 AlaMetSerProMetIleSerLeuAspGluLysGluLeuProArg 505560 ThrProSerPheArgIleAlaLysLysGlyGlnGluSerGlyVal 657075 GluPheAlaGlyLeuProLeuGlyHisGluPheTyrPheValTyr 808590 LeuGlySerValThrGlyPheArgAlaSerAlaLysValGluThr 95100105 AspIleValLysArgIleGlnAlaValAspGluProMetHisPhe 110115120 GluThrTyrValSerLeuThrCysHisAsnCysProAspValVal 125130135 GlnAlaPheAsnIleMetSerValValAsnProAsnIleSerHis 140145150 ThrMetValGluGlyGlyMetPheLysAspGluIleGluAlaLys 155160165 GlyIleMetSerValProThrValTyrLysAspGlyThrGluPhe 170175180 ThrSerGlyArgAlaSerIleGluGlnLeuLeuAspLeuIleAla 185190195 GlyProLeuLysGluAspAlaPheAspAspLysGlyValPheAsp 200205210 ValLeuValIleGlyGlyGlyProAlaGlyAsnSerAlaAlaIle 215220225 TyrAlaAlaArgLysGlyValLysThrGlyLeuLeuAlaGluThr 230235240 MetGlyGlyGlnValMetGluThrValGlyIleGluAsnMetIle 245250255 GlyThrProTyrValGluGlyProGlnLeuMetAlaGlnValGlu 260265270 GluHisThrLysSerTyrSerValAspIleMetLysAlaProArg 275280285 AlaLysSerIleGlnLysThrAspLeuValGluValGluLeuAsp 290295300 AsnGlyAlaHisLeuLysAlaLysThrAlaValLeuAlaLeuGly 305310315 AlaLysTrpArgLysIleAsnValProGlyGluLysGluPhePhe 320325330 AsnLysGlyValThrTyrCysProHisCysAspGlyProLeuPhe 335340345 ThrAspLysLysValAlaValIleGlyGlyGlyAsnSerGlyLeu 350355360 GluAlaAlaIleAspLeuAlaGlyLeuAlaSerHisValTyrIle 365370375 LeuGluPheLeuProGluLeuLysAlaAspLysIleLeuGlnAsp 380385390 ArgAlaGluAlaLeuAspAsnIleThrIleLeuThrAsnValAla 395400405 ThrLysGluIleIleGlyAsnAspHisValGluGlyLeuArgTyr 410415420 SerAspArgThrThrAsnGluGluTyrLeuLeuAspLeuGluGly 425430435 ValPheValGlnIleGlyLeuValProSerThrAspTrpLeuLys 440445450 AspSerGlyLeuAlaLeuAsnGluLysGlyGluIleIleValAla 455460465 LysAspGlyAlaThrAsnIleProAlaIlePheAlaAlaGlyAsp 470475480 CysThrAspSerAlaTyrLysGlnIleIleIleSerMetGlySer 485490495 LysAlaThrAlaAlaLeuGlyAlaPheAspTyrLeuIleArgAsn 500505510

## Claims

1. A DNA molecular comprising nucleotides 279 through 1809 of sequence I.D. No. 1, wherein the molecule has the following base sequence: AT        GGCATTAGAC  GCAGAAATCA AAGAGCAGTT AGGACAGTAT  CTTCAATTAC TTGAGTGTGA GATTGTTTTA  CAAGCTCAAT TAAAAGACGA TGCTAATTCT  CAAAAAGTTA AGGAATTTCT CCAAGAAATC  GTTGCAATGT CTCCTATGAT TTCTTTAGAC  GAAAAGGAAC TTCCGCGAAC ACCTAGTTTT  CGCATAGCTA AAAAGGGGCA AGAATCTGGT  GTTGAATTTG CTGGCTTACC CCTTGGTCAC  GAATTTTACT TCGTTTATCT TGGCTCTGTT  ACAGGTTTCA GGGCGTCGGC TAAGGTAGAG  ACTGATATTG TCAAACGCAT TCAAGCTGTT  GATGAACCTA TGCATTTTGA AACCTATGTT  AGTTTGACTT GTCATAATTG TCCAGATGTT  GTTCAGGCTT TCAATATCAT GTCAGTTGTT  AATCCCAACA TTTCACATAC AATGGTGGAA  GGTGGCATGT TTAAAGATGA AATTGAAGCT  AAGGGAATTA TGTCTGTGCC AACTGTCTAT  AAAGATGGAA CAGAATTTAC CTGAGGGCGT  GCTAGCATAG AGCAATTACT AGACTTGATA  GCAGGTCCTG TTAAAGAAGA TGCTTTTGAT  GATAAAGGTG TTTTTGATGT CCTTGTTATT  GGTGGGGGTC CTGCTGGTAA TAGCGCGGCT  ATCTATGCTG CAAGAAAGGG AGTTAAAACA  GGACTTTTAG CTGAAACCAT GGGTGGTCAA  GTTATGGAAA CCGTGGGTAT TGAAAATATG  ATCGGTACCC CATATGTTGA AGGACCCCAA  TTAATGGCTC AGGTGGAAGA GCATACCAAG  TCTTATTCTG TTGACATCAT GAAGGCACCG  CGTGCTAAGT CTATTCAAAA GACAGACTTG  GTTGAAGTTG AACTTGATAA TGGAGCTCAT  TTGAAAGCAA AGACAGCTGT TTTGGCCTTA  GGTGCCAAGT GGCGTAAAAT CAATGTACCA  GGAGAAAAAG AATTCTTTAA TAAAGGTGTT  ACTTACTGTC CGCACTGTGA TGGTCCTCTT  TTCACAGACA AAAAAGTCGC TGTCATTGGC  GGTGGAAACT CAGGTTTAGA AGCAGCTATT  GATTTGGCTG GGTTAGCTAG CCATGTCTAT  ATTTTAGAAT TTTTACCTGA GTTAAAAGCT  GATAAGATCT TACAAGATCG TGCGGAAGCT  CTTGATAATA TTACCATTGT AACTAATGTT  GCGACTAAAG AAATTATTGG CAATGACCAC  GTAGAAGGTC TTCGTTACAG TGATCGTACG  ACCAATGAAG AGTACTTGCT TGATTTAGAA  GGTGTTTTTG TTCAAATTGG ATTGGTACCT  AGTACTGACT GGTTAAAGGA TAGTGGACTA  GGACTCAATG AAAAAGGTGA AATCATTGTT  GCTAAAGATG GCGCAACTAA TATTCCTGCT  ATTTTTGCAG CTGGTGATTG CACAGATAGT  GCCTACAAAC AAATTATCAT TTCCATGGGT  TCTGGAGCTA CTGCGGCTTT AGGTGCCTTT  GATTATTTGA TTAGAAATT/. ______________________________________

2. A DNA molecule comprising the amino acid sequence I.D. No. 2, wherein the molecule encodes the following amino acid sequence: Met Ala Leu Asp Ala Glu Ile Lys Glu Gln Leu Gly Gln Tyr Leu 15 Gln Leu Leu Glu Cys Glu Ile Val Leu Gln Ala Gln Leu Lys Asp 30 Asp Ala Asn Ser Gln Lys Val Lys Glu Phe Leu Gln Glu Ile Val 45 Ala Met Ser Pro Met Ile Ser Leu Asp Glu Lys Glu Leu Pro Arg 60 Thr Pro Ser Phe Arg Ile Ala Lys Lys Gly Gln Glu Ser Gly Val 75 Glu Phe Ala Gly Leu Pro Leu Gly His Glu Phe Tyr Phe Val Tyr 90 Leu Gly Ser Val Thr Gly Phe Arg Ala Ser Ala Lys Val Glu Thr 105 Asp Ile Val Lys Arg Ile Gln Ala Val Asp Glu Pro Met His Phe 120 Glu Thr Tyr Val Ser Leu Thr Cys His Asn Cys Pro Asp Val Val 135 Gln Ala Phe Asn Ile Met Ser Val Val Asn Pro Asn Ile Ser His 150 Thr Met Val Glu Gly Gly Met Phe Lys ASp Glu Ile Glu Ala Lys 165 Gly Ile Met Ser Val Pro Thr Val Tyr Lys Asp Gly Thr Glu Phe 180 Thr Ser Gly Arg Ala Ser Ile Glu Gln Leu Leu Asp Leu Ile Ala 195 Gly Pro Leu Lys Glu Asp Ala Phe Asp Asp Lys Gly Val Phe Asp 210 Val Leu Val Ile Gly Gly Gly Pro Ala Gly Asn Ser Ala Ala Ile 225 Tyr Ala Ala Arg Lys Gly Val Lys Thr Gly Leu Leu Ala Glu Thr 240 Met Gly Gly Gln Val Met Glu Thr Val Gly Ile Glu Asn Met Ile 255 Gly Thr Pro Tyr Val Glu Gly Pro Gln Leu Met Ala Gln Val Glu 270 Glu His Thr Lys Ser Tyr Ser Val Asp Ile Met Lys Ala Pro Arg 285 Ala Lys Ser Ile Gln Lys Thr Asp Leu Val Glu Val Glu Leu Asp 300 Asn Gly Ala His Leu Lys Ala Lys Thr Ala Val Lue Ala Lue Gly 315 Ala Lys Trp Arg Lys Ile Asn Val Pro Gly Glu Lys Glu Phe Phe 330 Asn Lys Gly Val Thr Tyr Cys Pro His Cys Asp Gly Pro Leu Phe 345 Thr Asp Lys Lys Val Ala Val Ile Gly Gly Gly Asn Ser Gly Leu 360 Glu Ala Ala Ile Asp Leu Ala Gly Leu Ala Ser His Val Tyr Ile 375 Leu Glu Phe Leu Pro Glu Leu Lys Ala Asp Lys Ile Leu Gln Asp 390 Arg Ala Glu Ala Lue Asp Asn Ile Thr Ile Leu Thr Asp Val Ala 425 Thr Lys Glu Ile Ile Gly Asn Asp His Val Glu Gly Leu Arg Tyr 420 Ser Asp Arg Thr Thr Asn Glu Glu Tyr Leu Leu Asp Leu Glu Gly 435 Val Phe Val Gln Ile Gly Leu Val Pro Ser Thr Asp Trp Leu Lys 450 Asp Ser Gly Leu Ala Leu Asn Glu Lys Gly Glu Ile Ile Val Ala 465 Lys Asp Gly Ala Thr Asn Ile Pro Ala Ile Phe Ala Ala Gly Asp 480 Cys Thr Asp Ser Ala Tyr Lys Gln Ile Ile Ile Ser Met Gly Ser 495 Lys Ala Thr Ala Ala Leu Gly Ala Phe Asp Tyr Leu Ile Arg Asn 510. __________________________________________________________________________

3. A DNA molecule wherein the 5' terminal non-translated region comprises the following base sequence: GGATCCCTTC TCATGTTCTC TCACAAGGAT TTGAGGTTTT AGGTGAAGAT GGTTTAGCAC 60 AACGTGGAAC CTTTATTGTA GATCCGGATG GTATCATTCA AATGATGGAA GTCAATGCAG 120 ATGGTATTGG TCGTGATGCT ACTACCTTGA TTGATAAAGT TGTGCAGCT CAATCTATTC 180 GCCAACATCC AGGAGAAGTT TGCCCTGCCA AATGGAAAGA GGGAGCTGAA ACTTTAAAAC 240 CAAGTTTGGT ACTTGTCGGT AAAATTTAAG GAGAAACT. __________________________________________________________________________

4. A plasmid including a DNA molecule encoding an NADH oxidase according to claim 1.

5. A plasmid including a DNA molecule encoding an NADH oxidase according to claim 2.