METHOD FOR PRODUCING OXALATE OXIDASES HAVING ACTIVITY OPTIMUM NEAR PHYSIOLOGICAL PH AND USE OF SUCH RECOMBINANT OXALATE OXIDASES IN THE TREATMENT OF OXALATE-RELATED DISEASES

Abstract

Novel oxalate oxidases are provided, which have suitable oxalate degrading activity near physiological pH (7.4). The properties of these OxOx make them potential drug candidates for use in reducing oxalate concentration in patients suffering from excess of oxalate. Especially due to the high activity at physiological pH, the OxOx's are suitable drug candidates for parenteral administration, i.e. to reduce the oxalate concentration in the plasma.

Claims

1. A recombinant oxalate oxidase having at least 60% sequence identity to the polypeptides of SEQ ID NO: 2, SEQ ID NO: 4,or SEQ ID NO: 6, or at least 99% sequence identity to the polypeptide of SEQ ID NO: 8.

2. A recombinant oxalate oxidase according to claim 1 having at least 70% at least 75% at least 80%, at least 85%, least 90%, at least 95%, at least 98%, at least 99% or at least 100% sequence identity to the polypeptides of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, or at least 100% sequence identity to the polypeptide of SEQ ID NO: 8.

3. A recombinant oxalate oxidase having a relative activity of at least 20% at pH 7.5.

4. A recombinant oxalate oxidase according to claim 3 having a relative activity of at least 60%.

5. A recombinant oxalate oxidase according to claim 3 or 4 having a relative activity of at least 80%.

6. A recombinant oxalate oxidase according to any of the preceding claims having at least 60% sequence identity to the polypeptides of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, or at least 99% sequence identity to the polypeptide of SEQ ID NO: 8.

7. A recombinant oxalate oxidase according to any of the preceding claims having at least 70% at least 75% at least 80%, at least 85%, least 90%, at least 95%, at least 98%, at least 99% or at least 100% sequence identity to the polypeptides of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, or at least 100% sequence identity to the polypeptide of SEQ ID NO: 8.

8. An oxalate oxidase characterized in that it is isolated from Beta vulgaris (beet) stem, Bougainvillea spectabilis leaves, Mirabilis jalapa young leaf, Telosma cordatum (Brum. f.) Merr leaf, Jatropha gossypiifolia Linn. var. elegans Mueller leaf or Sauropus androgynus(L.) Merr leaf.

9. An oxalate oxidase according to claim 8 characterized in that it degrades oxalate at pH 7.4 and 37 C.

10. An oxalate oxidase according to claim 8 or 9 characterized in that the relative oxidase activity at pH 7.4and 37 C. is at least 80%.

11. A recombinant oxalate oxidase having at least 60% sequence identity with the oxalate oxidase defined in any one of claims 8-10.

12. A recombinant oxalate oxidase according to any of the preceding claims having at least 70% at least 75% at least 80%, at least 85%, least 90%, at least 95%, at least 98%, at least 99% or at least 100% sequence identity with the oxalate oxidase defined in any one of claims 8-10.

13. A recombinant oxalate oxidase according to any of the preceding claims in pegylated form.

14. A recombinant oxalate oxidase according to any of the preceding claims for use as a medicament.

15. A recombinant oxalate oxidase according to any of the preceding claims for use in the treatment or prevention of diseases associated with excess oxalate.

16. An isolated polynucleotide encoding the oxalate oxidase of any of claims 1-15.

17. A polynucleotide according having at least 60% sequence identity to the nucleotides of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, or at least 99% identity to the nucleotides of SEQ ID NO: 7.

18. A polynucleotide according to claim 16 or 17 having at least 70% at least 75% at least 80% or at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 100% sequence identity to the nucleotides of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, or at least 100% sequence identity to the nucleotides of SEQ ID NO: 7.

19. A recombinant expression vector comprising a polynucleotide as defined in any of claims 16-18.

20. A recombinant host cell comprising a polynucleotide as defined in any of claims 16-18.

21. A method for producing an oxalate oxidase as defined in any of claims 1-15 comprising cultivating the recombinant host cell of claim 20 under conditions suitable for expression of the oxalate oxidase.

22. A transgenic plant, plant part or plant cell transformed with a polynucleotide as deined in any of claim 16-18.

23. A pharmaceutical composition comprising a recombinant oxalate oxidase as defined in any of claims 1-15.

24. A recombinant oxalate oxidase for use in the treatment of primary hyperoxaluria, secondary hyperoxaluria, Zellweger spectrum disorder or chronic renal failure by administering the recombinant oxalate oxidase parenterally to the systemic circulation to degrade oxalate in the body.

25. A recombinant oxalate oxidase for use according to claim 24, wherein the recombinant oxalate oxidase is as defined in any one of claims 1-15

26. A method for treating a subject suffering from primary hyperoxaluria, secondary hyperoxaluria, Zellweger spectrum disorder or chronic renal failure, the treatment comprises administering the recombinant oxalate oxidase parenterally to the systemic circulation to degrade oxalate in the body.

27. A method according to claim 26, wherein the recombinant oxalate oxidase is as defined in any one of claims 1-15

Description

LEGENDS TO FIGURES

[0113] FIG. 1. Modifications of the pET-32 vector to generate the pAT plasmid. The DNA sequence highlighted in yellow was deleted in a pET-32 vector (SEQ ID NO. 23) to generate the pAT plasmid (SEQ ID NO. 24).

[0114] FIG. 2 Schematic diagram of the Pichia expression plasmid Z-5102.

[0115] FIG. 3 Schematic diagram of the Pichia expression plasmid GAPZ-5102.

[0116] FIG. 4 Schematic diagram of the Pichia expression plasmid Z-5601.

[0117] FIG. 5 Schematic diagram of the Pichia expression plasmid GAPZ-5601.

[0118] FIG. 6 Schematic diagram of the Pichia expression plasmid Z-30640.

[0119] FIG. 7 Schematic diagram of the Pichia expression plasmid GAPZ-5102.

[0120] FIG. 8 SDS-PAGE analysis of the expression of beet OxO in P. pastoris. M: protein marker; M: protein marker; lanes 1-10, X-33 (ZA-5102).

[0121] FIG. 9 SDS-PAGE analysis of the expression of beet OxO in P. pastoris. M: protein marker; lanes 1-9, X-33(Z-5601).

[0122] FIG. 10 SDS-PAGE analysis of the expression of banana OxO in P. pastoris. M: protein marker; lanes 1-10, X-33(ZA-30640).

[0123] FIG. 11 SDS-PAGE analysis of the expression of beet OxO in P. pastoris. M: protein marker; lanes 1-9, X-33(GAPZ-5601).

[0124] FIG. 12 PCR amplification of the OxO genes from cDNA library of sugar beet. M. DNA Maker; Lane1-4, 5601; Lane5-8, 5102

[0125] FIG. 13 PCR amplification of the OxO gene from cDNA library of banana. M. DNA Maker; Lane 1-3, 30640

[0126] FIG. 14 Schematic diagram of the plant expression vector

[0127] FIG. 15 Histochemical assay of oxalate oxidase activity after overnight incubation in OxOx histological buffer

[0128] FIG. 16 Detection of OxO activity of the purified cell wall fraction from pea plants by colorimetric assay, pH 7.4. The left tube containing 2 mM oxalic acid; The right tube containing no oxalic acid.

[0129] FIG. 17 Detection of OxOx activity of the purified 5102 fractions from pea plants.

[0130] FIG. 18 Purification of recombinant beet OxOx 5102. M, Protein Maker; lane 1, Sample before purified by Q Sepharose column; Lane 2, the washing sample; Lane 3, eluted sample 13#; Lane 4, eluted sample 14#; Lane 5, eluted sample 15#.

[0131] FIG. 19 Relative activity of recombinant oxalate oxidases.

[0132] FIG. 21 Serum oxalate concentration curve from Example 12.

[0133] FIG. 22 24 h total urine oxalate curve from Example 12.

[0134] FIG. 23 OxOx-B5102 antibody titer detection of different PEG-OxOx-B5102s from Example 12.

REFERENCES

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EXAMPLES

Strains and Plants

[0169] E. coli BL21(DE3), E. coli Origami B(DE3), P. pastoris, Wild-type N. benthamiana plants, P. sativum plants.

Example 1

Screening of Plants and Testing for Oxalate Oxidase Activity

[0170] Collection of plants species: more than 1000 plants species were collected for testing. Here is a list of the plants which have been tested.

TABLE-US-00001 Vitis vinifera Lanrus nobilis Linn Fragaria ananassa Duchesne Parakmeria Latungensis Eriobotrya japonica Parakmeria Yunnanensis Coriandrum sativum L. Eucommia ulmoides Oliver Chrysanthemum coronarium Loropetalum chinensis (R. Br.) Oliv Brassica rapa chinensis Celtis sinensis Pers Brassica rapa pekinensis Tulipa gesneriana Brassica campestris pekinensis Sinojackia xylocarpa Hu Brassica pekinensis Osmanthus armatus Diels Lactuca sativa Malus hupehensis(Pamp.)Rehd Vicia faba Linn Sloanea hemsleyana Pisum sativum Linn ligustrum japonicum Solanum tuberosum L Thunb. var rvtundifolium B1 Solanum tuberosum L Pittosporum tobira Colocasia esculenta Acer palmatum Lactuca sativa L. var. capitata L. E. pungens Thunb Bamboo Shoot Prunus ceraifera Brassica parachinensis Taiwania cryptomerioides Hayata Toona sinensis. A. Juss. Chaenomeles sinensis Koehne Pisum sativum Linn Taxus wallichiana Zucc Allium sativum L. 0rmosia henryi Prain Apium graveolens L. Pyracantha fortuneana Hordeum vulgare L. Mallotus repandus (Willd.) Muell. Arg Hordeum vulgare L. Distylium racemosum Sieb. et Zucc Hordeum vulgare L. Viburnum setigerum Hance Cruciferae Brassica Jasminum mesnyi Allium epa L. Chimonanthus praecox(L.)Link Alliaceae Allium A. tuberosum Hovenia acerba Lindl. Dioscorea opposita Mahonia fortunei (Lindl.)Fedde Vigna radiata Cercis chinensis Bge Momordica charantia Chionanthus retusus Cucumis sativus Linn Fraxinus hupehensis Benincasa hispida Alpinia zerumbet Ananas comosus Asparagus myriocladus Ananas comosus Cerasus conradinae (Koehne) Prunus ceraifera cv. Pissardii Y et Li Alliaceae Allium A. tuberosum Juniperus chinensis cv. kaizuka Pisum sativum Linn Brunfelsia acuminata(Pohl.)Benth Benincasa hispida Bambusa ventricosa McClure Benincasa hispida Hedera nepalensis Vigna radiata Dendrobeaathamia capitata(Wall.) Cruciferae Brassica Hutoh. var. emeiensis(Fang et Hsi- Momordica charantia eh)Fang et. W. K. Hu Ananas comosus Cyperus papyrus L. Alliaceae Allium A. tuberosum Pilea nummulariifollia(Sw.)Wedd. Alliaceae Allium A. tuberosum Syngonium podophyllum Benincasa hispida :Iresine herbstii Hook. f Vigna radiata Simlax China L Giycine max(L)Merrill Gleditsia japonica Eleocharis dulcis Euonymus grandiflorus Wall. Alliaceae Allium A. tuberosum Planch. cv. Fenghongpeier Sorghum bicolor Ilex cornuta Lindl. et Paxt. Brassic campestris Berchemia sinica Scheid Galium aparine Symplocos sununtia Buch.- Geranium carolinianumL. Ham. ex. D. Don Euphorbia helioscopia Dianthus chinensis L. Portulaca oleracea L. Olea europaea L Oxalis corniculata Begonia masoniana Vicia sepium linn. Marsilea quadrifolia Eugenia javanica Hibiscus rosa-sinensis L. Lam leaves Dracaena cochinchinen Eugenia javanica Common Rush Lam fruit Manilkara zapota van Royen Reineckea carnea Ligustrum lucidum Ait Bougainvillea spectabilis wind Begonia thurstonin Bougainvillea spectabilis wind Nephrolepis biserrata (Sw.) Schott Sinojackta huanamelensis Quisqualis indica Neolitsea sericea Asystasia gangetica Heptacodium Miconiodes rehd Podocarpus macrophyllus banana Artabotrys hexapetalus (L.f.) Bhandari Populus bonatii Levl Telosma cordatum (Brum. f.) Merr Vitis vinifera Forsythia viridissima Prunus armeniaca L. Osmanthus matsumuranus Hayata Mirabilis jalapa Linn Phoebe zhennan S. Lee et F. N. Wei Mirabilis jalapa Linn Davidia involucrata Baill Gordonia axillaris Neolepisorus ovatus Lindera aggregata (Sims) Setcreasea pallida Kosterm Clerodendron japonicum (Thunb.) Lllicium henryi Sweet PhoebechekiangensisC. B. Shang Hydrangea macrophylla (Thunb.)Ser Fokienia hodginsii (Dunn) Henry Psidium littorale Raddi et Thomas Savia miltiorrhiza Bunge Viburnum macrocephalum Miq Fagraea ceilanica f. keteleeri Paeonia suffruticosa Zanthoxylum simullans Hance Sycopsis sinensis Oliver Cinnamomum camphora Aesculus wilsonii Rehd Acer albopurpurascens Hayata Edyeworthia chrysantha Lindl Daphniphyllum macropodum Pilea peperomioides Diels Camellia sasanqua Helleborus thibetanus Franch. Sinocalycanthus chinensis Euonymus fortunei Hand.-Mazz. Bulbus Fritillaria Dicliptera chinensis Costus tonkinensis Gagnep Stevia rebaudiana Disporum bodinieri (Levi. et Rhizoma Alpiniae Officinarum Vaniot.) Asarum forbesii Maxim Podocarpus fleuryi Sarcandra glabra Tetracentron sinense Peristrophe baphica Reinwardtia trigyna Peperomia tetraphylla Valeriana hardwickii Erythrina crista-galli L Fagopyrum dibotrys (D. Don) Rumex japonicus Houtt Hara Melodinus hemsleyanus Diels. Thalia dealbata Tibouchina aspera Muscari botryoides Mill. Rumex hastatus Fagus longipetiolata Seem Hibiscus Sabdariffa Linn Jussiaea reppens L. Catharanthus roseus Podocarpus fleuryi Tragopogon porrifolius Linn Ardisia crenata Sim Mussaenda pubescens Ait. f. Torreya fargesii Franch Sedum sarmentosum Bunge Aesculus wangii Hu ex Fang Pilea cavaleriei Levl Nerium oleander Sauropus rostrata Miq Strobilanthes cusia Acorus gramineus Drynaria fortunei Euphorbia neriifolia L. Belamcanda chinensis Euphorbia tirucalli Linn Kalimeris indica (Linn.) Sch.) Dianthus serotinus Rhoeo discolor Codariocalyx motorius Fiveleaf Gynostemma Herb Acalypha hispida Sterculia nobililis Acalypha hispida Choerospondias axillaris Garcinia oblongifolia Citrus medica L. var. sarcodactylis Cynanchum stauntonii Gynostemma compressum Aloe vera var. chinensis Chrysanthemum indicum Polygonum capitatum SpilsheS acmella (Linn) Murr Iris japonica Ilex kudmcha C. J. Tseng Acanthopanax gracilistylus Cinnamomum cassia Dicranostigma leptopodum Rumex crispus L. var. japonicus Iris wilsonii Clerodendranthus spicatus Solanum mammosum Linn. (Thunb.) Rheum officinale Glechoma longituba Rabdosia nervosa (Hemsl.) HymenocallisSpciosa Plumbago zeylanica L Agrimonia pilosa Ledeb Desmodium styracifolium Dimocarpus longgana Lour. Salvia substolonifera Stib. Lxora chinensis Aspidistra elatior Bl. Rhapis excelsa (Thunb.) Achyranthes bidentata Bl Eranthemum pulchellum Andrews Achyranthes bidentata Bl Hypericum perforatum L Pandanus tectorius Hippeastrum rutilum(Ker- Lygodium scandens (L.) Sw. Gawl.)Herb. Asparagus cochinchinensis Pogonatherum crinitum (Thunb.) Lonicera dasystyla Kunth Zanthoxylum nitidum Echinacea purpurea Moench Billbergia pyramidalis Pseudocalymma Ficus sarmentosa alliaceum(Lam.)Sandwith Pyrrosia drakeana Sanguisorba officinalis L. Ardisia pusilla A. DC. desmodium triquetrum Boehmeria nivea Rabdosia japonica (Burm. f.) Hara Artemisia japonica Thunb Tadehagi pseudotriquetrum (DC.) Forsythia suspensa Houttuynia cordata Thunb. Ardisia gigantifolia stapf Gynura procumbens (Lour.) Merr. Tacca chantrieri Andre Vinca major Linn Morinda officinalis How. Zephyranthes carinata Herb Ardisia villosa Platycodon grandiflorus Hemerocallis citrina Baroni Aquilaria sinensis Goodyera procera Salvia cavaleriei Fallopia multiflora Datura innoxia Mill. Campsis radicans (L.) Seem. Codariocalyx gyroides Piper longum Linn. Aerva sanguinolenta Passionfora edulis Aster novi-belgii Prunella vulgaris Colocasia antiquorum Schott Jasminum nudiflorum Heteropanax fragrans Kadsura longipedunculata Thunbergia laurifolia Lindl. Mentha haplocalyx Iris germanica Evodia lepta (Spreng.) Merr Alocasia macrorrhiza Acacia confusa Merr Gelsemium elegans Stephania kwangsiensis Cocculus laurifolius DC. Tetrastigma hemsleyanum Podocarous macrophyllus Herba Cayratica Jeponicae Syzygium jambos (L.) Alston Asarum maximum Hemsl. Buddleja asiatica Lour. Argyreia acuta Lour. Buddleja davidii Solallum nigrum L. ver Caesalpinia decapetala Curculigo capitulata Jatropha gossypiifolia Fructus Amomi Peucedanum praeruptorum Dunn Polygonum chinense L. Carpesium abrotanoides Linn. Rosa chinensis Euphorbia heberophylla L selaginella moellendorfii hieron Coleus amboinicus Lour Herba Aristolochiae Lycoris aurea Rubus cochinchinensis Tratt Sauropus androgynus(L.)Merr Rosa multiflora Thunb. Houttuynia emeiensis Rhododendron pulchrum Sweet Sophora flavescens Dichotomanthus tristaniaecarpa Kurz Salvia coccinea Linn Cyclobalanopsis glaucoides Schotky a plant in Tadehagi Rhododendron anthopogonoides Rhinacanthus nasutus (L.) Kurz Maxim Ardisia japonica (Thunb) Blume Malus halliana (Voss.) Koehne Trachelospermum jasminoides Pistacia weinmannifolia Wisteria sinensis Ginkgo biloba Duchesnea indica Melaleuca bracteata Ajuga decumbens Thunb Rhododendron fuyuanense Z. H. Yang Potentilla kleiniana Ligustrumx Vicaryi Pteris semipinnata L. Euonymus Japonicus Senecio cannabifolius Less Cerasus cerasoides Ophitopogin japonicum Rhododendron nanhai Polygonum hydropiper L. Rhododendron suimohua Pueraria lobata Rhododendron delavayi Franch Mentha spicata Linn. Exochorda racemosa (Lindl.) Rehd Lonicera fulvotomentosa Rosmarinus officinalis Acacia confusa Merr Cotoneaster franchetii Erythropalum scandens Bl. Prunus serrulata Caryota mitis Lour. Pinus armandii Franch Cirsium segetum Pittosporum brevicalyx (Oliv.) Gagnep Curcuma kwangsiensis Osyris wightiana Wall. ex Wight Artemisia argyi Rhododendron mayingtao Limnophila sessiliflora Rhododendron hancockii Hemsl. Alpinia katsumadai Hayata Rhododendron ciliicalyx Franch. Blumea balsamifera DC Amygdalus persica Ficus pumila L Abelia parvifolia Hemsl. Bryophyllum pinnatum Lonicera nitida Baggesens Agastache rugosa Rhodedenron schlippenbachii Polygonum chinense L Amygdalus persica f. atropurpurea Jasminum amplexicaule Pyracarltha fortuneana Gnetum montanum Markgr Trigonobalanus verticillata Rhododendron pachypodum Rhododendron kunming Parakmeria yunnanensis Acer cappadocicum Sinocalycanthus chinensis Rhododendron mucronatum Cerasus campanulata Rhododendron fenpao Quercus variabilis Blume Dianthus plumarius Rhododendron Wanxia Musella lasiocarpa Spiraea japonica L.f. Rhododendron irroratum Franch Eucommia ulmoides Oliver Pistacia chinensis Hovenia acerba Hovenia acerba Lindl. Rhododendron fortunei Michelia lacei W. W. Smith Rhododendron leptothrium Distylium pingpienense (Hu) Walk. magnolia grandiflora linn Sedum Nicaeense All. Rhododendron pachypodum Aglaia odorata Barleria cristata Ilex crenata cv. Convexa Makino Vitex negundo Linn Rhododendron wanxia Viburnum opulus L Spiraea cantoniensis Spiraea japonica L.f. Lorpetalum chindensevar.rubrum Cotoneaster salicifolius Photinia serrulata Rhododendron albersenianum Rhododendron rigidum Franch Eriobotrya bengalensis Rhododendron annae Franch. Cerasus clarofolia Rhododendron sinogrande Rhododendron vialii Cotoneaster horizontalis Dcne Amygdalus persica Linn Rhododendron aberconwayi Cowan Ailanthus altissima Podocarpus macrophyllus var. maki Rhododendron taronense Rhododendron decorum Fr Rhododendrin davidii Itea yunnanensis Franch. Sapiumsebiferum (L.) Roxb Lonicera nitidaMaigrun Michelia maudiae Dunn Michelia figo Rhododendron spinuliferum Liriodendron chinensis (Hemsl.) Machilus yunnanensis Sarg Liquidambar formosana Hance Olea ferruginea Royle Davidia involucrata Baill Eriobotrya japonica Prunus salicina Lindl. Luculia intermedia Hutch Sinomanglietia glauca Rhododendron jinzhizhu Liquidambar formosana Hance Sect. Dacrycarpus Endl Acer negundo L. Ophiopogon japonicus cv. Nanus Magnolia soulangeana Soul. Ligustrum japonicumHowardii Vinca minor Linn. Rhododendron pulchrum Rhododendron ciliatum Hook. f. Pvracantha angustifolia magnolia grandiflora linn Chamaecyparis lawsoniana Trigonobalanus doichangensis Cedrus deodara Hedera nepalensis Thujopsis dolabrata Quercus variabilis Ehretia dicksonii Hance Rhododendron simsii Acer cappadocicum Ilex cornuta Camellia sasanqua Fatsia japonica Araucaria araucana Lindera communis Ficus curtipes Corner Pistacia weinmannifolia Manglietia duclouxii Alstonia yunnanensis Diels Stranvaesia davidiana Machilus thunbergii Rhamnus gilgiana Heppl. Bischofia polycarpa Agrimonia pilosa Ledeb. Cercidiphyllum japonicum Caragna fruten (L) koch Viburnum odoratissimum Euonymus fortunei Lamium galeobdolon Pieris formosa Chaenomele japonica Fraxinus retusifoliolata Spiraea blumei Sapindus delavayi Agapanthus africanus Elaeocarpus sylvestris Musella lasiocarpa Carayaillinoensis Cryptomeria fortunei Micheliamacclurel Weigela florida Eurya loquaiana Dunn Paeonia lactiflora Ficus virens Parthenocissus tricuspidata Buxus microphylla Acer palmatum Dissectum Lithocarpus henryi Bougainvillea spectabilis Ficus religiosa Linn Aesculus wangii Ligustrum lucidum Mahonia fortunei Disporopsis pernyi Osmanthus fragrans Manglietiainsignis Exbucklandia populnea Olea cuspidata Cinnamomum bodinieri Hypoestes sanguinolenta Machilus longipedicellata Cornus officinalis Ficus virens Ait. Daphniphyllum longeracemosum Duranta repens Linn. Reineckia carnea Manglietia grandis Acorus gramineus Olea europaea L. Cerasus cerasoides Parakmeria yunnanensis Dipteronia dyeriana Henry Tamarix chinensis Lindera megaphylla pyrus pyrifolia Liquidambar formosana Hance Prunus serrulata Podocarpus fleuryi Iris confusa Morus alba L Iris wilsonii Zanthoxylum acanthopodium Bambusa multiplex Ficus altissima Bl. Senecio scandens Iresine herbstii Neolitsea sericea Cedrus deodara Fokienia hodginsii Thujopsis dolabrata Viburnum odoratissimum Ehretia dicksonii Hance Cerasus pseudocerasus Acer cappadocicum Ilex cornuta Camellia sasanqua Fatsia japonica Araucaria araucana Lindera communis Ficus curtipes Corner Pistacia weinmannifolia Manglietia duclouxii Alstonia yunnanensis Diels Stranvaesia davidiana Machilus thunbergii Rhamnus gilgiana Heppl. Bischofia polycarpa Agrimonia pilosa Ledeb. Cercidiphyllum japonicum Caragna fruten (L) koch Viburnum odoratissimum Euonymus fortunei Lamium galeobdolon Pieris formosa Chaenomele japonica Fraxinus retusifoliolata Spiraea blumei Sapindus delavayi Agapanthus africanus Elaeocarpus sylvestris Musella lasiocarpa Carayaillinoensis Cryptomeria fortunei Micheliamacclurel Weigela florida Eurya loquaiana Dunn Paeonia lactiflora Ficus virens Parthenocissus tricuspidata Buxus microphylla Acer palmatum Dissectum Lithocarpus henryi Hypoestes sanguinolenta Ficus religiosa Linn Aesculus wangii Ligustrum lucidum Mahonia fortunei Disporopsis pernyi Osmanthus fragrans Manglietiainsignis Exbucklandia populnea Olea cuspidata Cinnamomum bodinieri Rhododendren simsii Machilus longipedicellata Strawberry Ficus virens Ait. Pineapple leaves Duranta repens Linn. White gourd Manglietia grandis Young garlic shoot Grape Purple cabbage Loquat Purple Chinese cabbage flower Pineapple sarcocarp Cabbage lettuce Cucumber Potato sprout Chives flower Horse bean Tender leaf of Chinese toon Taro Bamboo shoots Pakchoi cabbage Potato Coriander herb Chinese lettuce Pea shoots Pakchoi cabbage flower Crowndaisy chrysanthemum

[0171] OxOx Activity Assay (HPLC method): The different parts of plant materials such as leaf, root, stem, fruit and flower were analyzed separately. Plant materials were homogenized with water. The soluble and insoluble fractions were collected by centrifugation. The insoluble part was re-suspended with DI water for OxOx activity analysis. 40 l of solution or suspension was incubated with 360 l of 12 mM oxalate in 50 mM phosphate buffer, pH 7.4, at 37 C. for 48 hours. The reaction was quenched by the addition of 100 L 1.5 N H.sub.2SO.sub.4 (sufficient to lower the pH below 1.0, a pH at which that the enzyme is inactive). The reaction mixture was immediately centrifuged and the clear supernatant was analyzed by an HPLC method to detect oxalate. One unit of activity is defined as the amount of enzyme required to degrade 1 mole of oxalate in one minute under the above conditions.

[0172] OxOx activity assay (colorimetric method): 20 L solution sample is mixed into 580 L solution containing 10 mM oxalic acid, 4 mM hydroxybenzenesulphonic acid sodium, 2 units horseradish peroxidase and 1 mM 4-anti-aminoantipyrine in 50 mM potassium phosphate buffer, pH 6.0 or 7.4, and reacted at 37 C. for 10-30 minutes. The color is monitored at 492 nm. One unit of activity is defined as the amount of enzyme required to degrade 1 mole of oxalate in one minute under the above conditions.

[0173] This assay is specific for oxalate oxidase as an oxidase generates hydrogen peroxide which can be detected by development of color in the presence of peroxidase, while decarboxylase does not produce hydrogen peroxide.

[0174] Results

[0175] OxOx from banana peel, beet stem, Bougainvillea spectabilis leaves, Mirabilis jalapa young leaf, Telosma cordatum (Brum. f.) Merr leaf, Jatropha gossypiifolia Linn. var. elegans Mueller leaf and Sauropus androgynus (L.) Merr leaf shows significant activity at pH 7.4. All others either contain oxalate oxidase activity, but at acid pH, or no oxalate oxidase activity.

[0176] OxOx from banana peels (Clinical Chemistry, 1985; 31(4):649), beet Stems (Clinical Chemistry, 1983; 29(10):1815-1819) has been reported in literature, but no activity around pH 7.4. It is suggested that the OxOx reported in literature may be different from these described here. As demonstrated in examples, three OxOx genes (5100, 5102, and 5601) have been isolated from sweet beet and they all have different amino acid sequence and DNA sequence, and show different properties as well. Thus, it is likely to assume that none of these OxOx is the same as the one reported in literature. Although only one OxOx gene has been cloned from banana in this work, there are dozens of similar proteins with more than 50% similarity called germin or germin-like proteins in one single plant (Critical Reviews in Plant Sciences, 2008; 27(5): 342-375). OxOx from Bougainvillea spectabilis leaves (Biochem. J.,1962, 85, 33) has been reported in literature and we have also identified an OxOx in our experiments. OxOx from the other four plants are first discovered in this work.

Example 2

Genes Encoding OxOx from Banana and Sweet Beet

Cloning of Oxalate Oxidase Genes from Beet Sugar and Banana

[0177] Several approaches to clone the OxOx genes from these plants have been tried. The first one is to search for public database to find if any OxOx gene has been published in the plant. Genes claimed to encode OxOx in sweet beet (GenBank: AAG36665.1) and a germin-like protein gene from banana (GenBank: AAL05886.1) are found. These genes have been synthesized and expressed by E. coli and yeast, and found that these genes encode SOD, not OxOx. It is not surprised, because germin SOD and OxOx (a germin as well) often share more than 50% similarity. The second approach is to design degeneration primers according to the conserved sequences of these reported OxOx to clone the targeted gene from all above 7 plants with OxOx activity at neutral pH. However, no gene encoding protein with OxOx activity was cloned. The third approach is to search the genome of banana and sweet beet that was just available in draft text. All germin and germin like protein genes were analyzed and collected from the genome data of banana and sweet beet. It has been reported that one amino acid, the Asparagine at position 75 in barley OxOx, is the key for OxOx activity (see reference J BIOL CHEM VOL. 281, NO. 10, pp. 6428-6433, 2006). There is only one such gene from banana (the gene1013, SEQ ID. 15) and sweet beet (the gene108, SEQ ID. 9) found, respectively. Thus, the germin genes containing this key amino acid at that corresponding position were clone and expressed by E. coli, yeast and plant, but no OxOx activity was detected either. After these experiments, the question is raised if these OxOx with some activity at neutral pH are germin or germin-like proteins. Thus, the forth approach is to obtain a small amount of OxOx by purification and use the protein to help finding the genes. Various efforts have been made to purify milligram levels of OxOx from these plant materials: banana peel, beet Stem, Bougainvillea spectabilis leaves, Mirabilis jalapa young leaf, Telosma cordatum (Brum. f.) Merr leaves, Jatropha gossypiifolia Linn. var. elegans Mueller leaves and Sauropus androgynus (L.) Merr leaves, which show significant activity at pH 7.4. However, only OxOx from banana peel and beet stem has been purified. The OxOx from the two plants shows similar size and subunit compositions as other germin, which indicates they are still possible germin or germin-like proteins. The purified OxOx was used to analyze amino acid sequence of peptides generated from the enzymes by mass spectrometry. Then, using the amino acid sequences to search for public database, but it did not produce any meaningful results. However, using these amino acid sequences to design degenerated primers to clone genes from sweet beet and banana. The gene 303 (SEQ ID 13) and 122 (SEQ ID 11) were cloned from sweet beet, but none from banana. The two genes have cloned and expressed by E. coli, yeast and plant, but no OxOx activity was detected from anyone. Then the purified OxOx from banana and sweet beet was used for determination of the N-terminal sequences of the proteins. The N-terminal sequences were used to search for draft genomes of sweet beet and banana to find possible genes, no meaningful gene was found. Then all matched or partly matched segments and the following DNA sequence up to several thousands base pairs were analyzed by deleting any possible introns or sequence mistakes. One gene from each plant (5601 from sweet beet and 30640 from banana) was finally found. Primers were designed to clone the genes from mRNA. One gene has been cloned from banana, but three similar genes have been cloned from sweet beet. The cloned genes were inserted in pMD19T simple vector (Takara) and sequenced to confirm their accuracy.

[0178] Results

[0179] DNA and protein sequences of beet OxOx 5100, 5102, 5601 and banana OxOx 30640.

TABLE-US-00002 SEQID1-5100DNA: ATGGTCTTTGCAATGAGCTTTACTTCTCATATTTAC- GTGGCTTCGGCCTCTGATCCTGGTCTCCTACAGGATTTTTGTGTGGGTG- TAAATGACCCTGATTCAGCAGTGTTTGTAAATGGAAAATTCTGCAAGAACCCAAAA- GACGTGACAATCGACGATTTCTTATACAAAGGGTTTAATATTCCCTCAGA- CACAAACAACACTCAAAGAGCAGAAGCCACACTAGTAGATGTCAATCGAT- TTCCAGCACTTAACACATTAGGTGTAGCCATGGCTCGTGTAGACTTT- GCGTCCTTTGGCCTAAACACACCTCATTTGCACCCTCGTGGTTCTGAGA- TATTCGCGGTCCTAGAGGGGACTTTATATGCCGGCATTGTCACCACCGATAATAA- GCTTTTCGACACGGTGTTGA- GAAAGGGTGACATGATTGTTTTCCCTCAAGGCTTAATCCACTTCCAGCTTAATCTT- GGCAAGACAGATGCTCTTGCTATTGCCTCTTTTGGGAGCCAATTTCCTGGACGAG- TTAATGTTGCTAATGGTGTCTTTGGAACTACGCCACAAATTTT- GGATGATGTACTTACCCAAGCGTTTCAGGTAGATAAGATGGTGATTGAG- CAACTTCGATCTCAGTTTTCAGGTCCAAACACATCAATCAACACTGGAA- GATCTATTCTTAAACTCTTAACTGATGTTGCT SEQID2-5100protein: MVFAMSFTSHIYVASASDPGLLQDFCVGVNDPD- SAVFVNGKFCKNPKDVTIDDFLYKGFNIPSDTNNTQRAEATLVDVNRFPAL- NTLGVAMARVDFASFGLNTPHLHPRGSEIFAVLEGTLYAGIVTT- DNKLFDTVLRKGDMIVFPQGLIHFQLNLGKT- DALAIASFGSQFPGRVNVANGVFGTTPQILDDVLTQAFQVDKMVIEQLRSQFSGPN- TSINTGRSILKLLTDVA SEQID3-5102DNASequence(w/oN-terminalsignalpeptidesequence,648bp): TCTGATCCTGGTCTCCTACAGGATTTTTGTGTGGGTGTAAATGACCCTGATTCAG- CAGTGTTTGTAAATGGAAAATTCTGCAAGAACCCAAAAGACGTGACAATCGAC- GATTTCTTATACAAAGGGTTTAATATTCCCTCAGACACAAACAACACTCAAAGAG- CAGAAGCCACACTAGTAGATGTCAATCGATTTCCAGCACTTAACACATTAGGTG- TAGCCATGGCTCGTGTAGACTTTGCGTCCTTTGGCCTAAACACACCTCATTT- GCACCCTCGTGGTTCTGAGATATTCGCGGTGCTAGAGGGGACTTTATATGCCGG- CATTGTCACCACCGATTACAAGCTTTTCGACACGGTGTTGA- GAAAGGGTGACATGATTGTTTTCCCTCAAGGCTTAATCCACTTCCAGCTTAATCTT- GGCAAGACAGATGCTCTTGCTATTGCCTCTTTTGGGAGCCAATTTCCTGGACGAG- TTAATGTTGCTAATGGTGTCTTTGGAACTACGCCACAAATTTT- GGATGATGTACTTACCCAAGCGTTTCAGGTAGATGAGATGGTGATTCAG- CAACTTCGATCTCAGTTTTCAGGTCAAAACATATCAATCAACACTGGAA- GATCTATTOTTAAACTOTTAACTGATGTTGCT SEQID4-5102ProteinSequence(w/oN-terminalsignalpeptidesequence,216aa): SDPGLLQDFCVGVNDPDSAVFVNGKFCKNPKDVTIDDFIYKGF- NIPSDTNNTQRAEATLVDVNRFPALNTLGVAMARVDFASFGLNTPHLHPRGSEI- FAVLEGTLYAGIVTTDNKLFDTVLRKGDMIVFPQGLIHFQLNLGKT- DALAIASFGSQFPGRVNVANGVFGTTPQILDDVLTQAFQVDEMVIQQLRSQFSGPN- TSINTGRSILKLLTDVA SEQID5-5601DNASequence(w/oN-terminalsignalpeptidesequence,648bp): TCCGATCCTGCACCCCTTCAAGATTTTTGTATTGCTGTAAATGATCCCAATTCTG- CAGTGCTTGTGAATGGAAAGCTTTGTAAGAACCCAAAAGAAGTGACAA- TAGATGATTTCTTGTACAAAGGGTTTAATATACCTGCAGACACAAACAACAC- TCAAGGAGCAAGTGCCACACTAGTGGACATTACTCTATTCCCTGCAG- TTAACACACAAGGAGTCTCCATGGCTCGTGTGGACTTTGCGCCATTT- GGACTAAACACCCCTCATTTACATCCTCGTGGCTCAGAGGTTTTCGCAG- TGATGGAAGGGATTATGTATGCTGGTTTTGTGACCACTGATTATAAGCTCTATGATA- CAATTATAAAAAAGGGTGA- TATTATTGTGTTTCCACAAGGTCTAATTCATTTCCAACTTAATCTTGGGAAGA- CAGATGCTTTAGCAATTGCCTCATTTGGGAGCCAAAATCCAGGGAGAATTAA- TATCGCTGACAGTGTGTTTGGTACTACTCCGCGTGTTCTAGATGATGTGCTTAC- CAAAGGATTTCAAATCGATGAGTTGTTGGTCAAGCAACTTCGTTCTCAGTTTTC- TACTGATAATATATCAACAAGCACTGGAAGGTCATTTTTGAAATTGC- TATCTGAAACTTAT SEQID6-5601ProteinSequence(w/oN-terminalsignalpeptidesequence,216aa): SDPAPLQDFCIAVNDPNSAVLVNGKLCKNPKEVTIDDFLYKGFNIPADTNNTQGA- SATLVDITLFPAVNTQGVSMARVDFAPYGLNTPHLHPRGSEVFAVMEGIMYAGFVTT- DYKLYDTIIKKGDIIVFPQGLIHFQLNLGKTDALAIASFGSQNPGRINI- ADSVFGTTPRVLDDVLTKGFQ1DELLVKQLRSQFSTDNISTSTGRSFLKLLSETY SEQID7-30640DNASequence(w/oN-terminalsignalpeptidesequence,642bp): TTTGATCCGAGTCCTCTCCAAGACTTTTGCGTTGCTGACTACGACTCCAAC- GTGTTTGTGAACGGATTCGCCTGCAAGAAAGCTAAGGATGTCACGG- CAGATGACTTCTACTTCACCGGCTTAGACAAGCCCGCGAGCACCGCCAAC- GAGCTTGGCGCAAACATCACTCTCGTCAACGTGGAAC- GACTCCCAGGCCTCAACTCCCTTGGCGTCGCCATGTCTCGCATCGACTAC- GCGCCCTTCGGTCTCAACCCTCCTCACTCGCATCCACGATCGTCGGAGATACTG- CACGTGGCGGAAGGAACGCTCTACGCCGGCTTCGTCACCTCCAACAC- GGAAAACGGCAACCTTCTCTTCGCTAAGAAGCTGAAGAAGGGCGACGCGTTT- GTGTTCCCCAGGGGCCTCATACACTTCCAGTTCAACATCGGGGACAC- CGATGCGGTGGCGTTCGCTACCTTCGGCAGCCAGAGCCCGGGTCTCGTCAC- CACCGCCAACGCACTGTTCGGATCGAAGCCGCCCATCGCTGATTACATTCTT- GCCCAGGCCGTGCAGCTTAGCAAGACGACCGTGGGCTGGCTTCAGCAGCAG- CAGTGGTTGGACATCGCTCAAGAATATGGACAACGCTTAGTTCAAGCTAAT SEQID8-30640ProteinSequence(w/oN-terminalsignalpeptidesequence,213aa): FDPSPLQDFCVADYDSNVFVNGFACKKAKDVTADDFYFTGLDKPASTANELGA- NITLVNVERLPGLNSLGVAMSRIDYAPFGLNPPHSHPRSSEILHVAEGTLYAGFV- TSNTENGNLLFAKKLKKGDAFVFPRGLIHFQFNIGDTDAVAFATFGSQSPGLVT- TANALFGSKPPIADYILAQAVQLSKTTVGWLQQQQWLDIAQEYGQRLVQAN

[0180] DNA and protein sequences of beet germin-like proteins 108, 122, 303 and banana germin-like protein 1013.

TABLE-US-00003 SEQIDNO:9108DNAsequence ATGGCTCCCCTACTCTACCTTGTAGTATTCTTGCTT- GCTCCTTTTCTCTCCCATGCTGCGGATCCCGATCCTTTGCTAGATTTTTGTG- TAGCGGACCTTAATGCCTCTCCCTCATTTGCTAATTTCCCTTGCAAACAAAC- CTCAAATGTGACCTCTGAAGATTTCTTCTTT- GATGGGTTTATGAATGAGGGAAACACATCAAACTCGTTT- GGATCAAGGGTCACACCCGGAAACGTCCTCACATTTCCTGCCCTTAA- TATGCTCGGGATTTCAATGAATCGGGTTGATCTTGCTGTGGATGG- GATGAACCCGCCCCATTCCCACCCACGAGCAAGTGA- GAGCGGTGTGGTGATGAAGGGGAGAGTTCTAGTAGGGTTCGTAACCACGGG- GAATGTGTACTATTCAAAGGTGTTGGTTCCAGGACAGATGTTT- GTAATCCCAAGGGGGTTGGTTCATTTTCAAAAGAATGTTGGACAAAATAAGGCAC- TCATCATTACAGCTTTCAATAGTCAGAATCCAGGAGTAG- TGTTATTATCCTCAACCCTGTTTGGTACAAACCCTTCAATTCCAGATGATGTTTTAA- GCCAAACTTTCCTAGTGGACCAGAGCATTGTCGAAGGAATAAAATCCAACTTTTGA SEQIDNO:10108proteinsequence MAPLLYLVVFLLAPFLSHAADPDPLLDFCVADLNASPSFANFPCKQTSNV- TSEDFFFDGFMNEGNTSNSFGSRVTPGNVLTFPALNMLGISMNRVDLAVDGMNP- PHSHPRASESGVVMKGRVLVGFVTTGNVYYSKVLVPGQMFVIPR- GLVHFQKNVGQNKALIITAFNS- QNPGVVLLSSTLFGTNPSIPDDVLSQTFLVDQSIVEGIKSNF* SEQIDNO:11122DNAsequence: Atggaagtcgtcgcagctgtatcttttctggccgtgttattggctctggtttcccctgccctcgccaatgatcctga- tatcttttctggccgtgttattggctctggtttcccctgccctcgccaatgatcctga- tatgctccaagatgtttgtgtcgctgattccacctctggagtgaaattgaatggattt- gcatgcaaggatgcagcaagcattacaccagaagacttcttctttgctggaa- tatccaaacccggaatgacaaacaatacaatgaaatccctagtaaccggagctaac- gtcgaaaagataccgggtttaaacacactcggagtgtccatgggtcg- tatcgacttcggcccaggtggtcttaacccacctcacactcacccac- gagccacagaaatggtctttgtgttatatggagaattggacgttggtttcctaac- tacttctaataagctcatttctaagcatattaaaactggtgaaacttttgtttttccta- gagggttagtccactttcagaaaaataatggggataaacctgctgctttagtcac- tgcttttaatagtcagttgcctggcacccaatcaatagctgccacgttgtttac- gtcgaccccacctgttccagataatgttttaactatgactttccaagtcggtacta- aacaagtccagaagatcaaggctaggctcgctcctaagaagtaa SEQIDNO:12122proteinsequence: mevvaaysflavllalvspalandpdmlqdvcvadstsgvklngfackdaasitped- fffagiskpgmtnntmkslvtganvekipglntlgvsmgridfgpgglnp- phthpratemvfvlygeldvgflttsnkliskhiktgetfvfprglvhfqknngdk- paalvtafnsqlpgtqsiaatlftstppvpdnvltmtf SEQIDNO:13303DNAsequence: Atggcggctgtttgggtagtcttggtggtgctagcggcggcttttgctgttggggtcttt- gccagcgatcctgatatgcttcaagatgtttgtgttgctgatcgtacatctggaatattag- tgaatggattcacatgtaaaaatatgaccatgataacccctgaagacttcttcttcaccg- gaatttcacaaccaggccaaatcacaaataaaatccttggttctcgagtcaccg- gagcgaatgtgcaggacatccctggtctcaacaccttgggag- tctcgatggctcgtgtcgactttactccctacggtctaaacccacctcacattcacccta- gaatcgtccaccctcgtgccactgaaatgatctatgttcttaagggtgaattgtacgtt- ggttttataacgaccgacaataagctcatttccaaggttgttaaagctggagaagtattt- gttttccctagaggtttggctcactttcagaaaaacatgttgaaatatccagctgctgcatt- agctgccttcaacagccaacttccaggcactcaacaattt- gcagctgctctctttacttccaatcctcctgtgtctaatgatgtgtt- ggctcaggcttttaacattgacgaacacaatgtcaaaaagattagggctg SEQIDNO:14303proteinsequence: MAAVWVVLVVLAAAFAVGVFASDPDMLQDVCVADRTSGILVNGFTCKNMTMITPED- FFFTGISQPGQITNKILGSRVTGANVQDIPGLNTLGVSMARVDFTPYGLNP- PHIHPRATEMIYVLKGELYVGFITTDNKLISKVVKAGEVFVFPRGLAH- FQKNMLKYPAAALAAFNSQLPGTQQFAAALFTSNPPVSNDVLAQAFNIDEHNVK- KIRAGLTP SEQIDNO:151013DNAsequenc ATGGAGTCGCACTACACGAAGAGACCATTCCTCCTCTTTCTCTCCTTCAC- CGTCCTCCTCGTGTTGATCCGCGCTGACCCTGATCCTCTCCAG- GACTTCTGCGTCGCCGACCTCGGAGCTACTGTGGTCGTCAATGGGTTCCCGTG- CAAGCCCGCGTCCGGAGTCACGTCCGACGACTTCTTCTTCGCCG- GACTGTCCAGGGAGGGCAACACCAGCAATATCTTCGGGTCCAACGTGACCAAC- GCCAACATGCTCAGCTTCCCGGGGCTCAACACCCTCGGCGTCTCCATGAAC- CGCGTCGACGTCGCCCCCGGCGGCAC- CAACCCGCCCCACAGCCACCCGAGGGCTAC- CGAGCTCATCATCCTCCTCAAGGGCCGGCTGCTGGTGGGGTTCATCAGCACCAG- TAACCAGTTCTTCTCCAAGGTCTTGAACCCCGGCGA- GATGTTCGTGGTGCCCAAGGGGCTCATCCACTTCCAGTACAACGTCGGCAAGGA- GAAGGCGCTCGCCATCACCACCTTCGACAGCCAGCTCCCCGGAGTAG- TGATCGCCTCCACCACCCTGTTCGCATCGAATCCGGCGATTCCCGAC- GATGTGCTGGCCAAAGCTTTTCAGGTGGAC- GCGAAGGTCGTCGCTCTCATCAAGTCCAAGTTTGAGAGATAA SEQIDNO:161013proteinsequence MESHYTKRPFLLFLSFTVLLVLIRADPDPLQDFCVADLGATVVVNGFPCKPASGV- TSDDFFFAGLSREGNTSNIFGSNVTNANMLSFPGLNTLGVSMNRVDVAPGGTNP- PHSHPRATELIILLKGRLLVGFISTSNQFF- SKVLNPGEMFVVPKGLIHFQYNVGKEKALAITTFDSQLPGVVIASTTL- FASNPAIPDDVLAKAFQVDAKVVALIKSKFER*

Example 3

Recombinant Expression of Oxalate Oxidase by E. coli

[0181] Part 1. Plasmid Construction and Protein Expression

[0182] Expression Plasmid Construction:

[0183] The pAT plasmid was produced by deleting the DNA sequence between the 212.sup.th bp and the 729.sup.th bp of the pET-32 vector. The 5102, 5100, 5601 and 30640 genes were then ligated into the modified pET-32 vector (pAT) using the Ncol and Xhol restriction sites. The resulting plasmids were then transformed into E. coli Origami B competent cells, which are designed to be favorable for disulfide bond formation of expressed proteins, since there is one disulfide bond within each OxOx monomer and it is essential for the protein native structure as well as enzyme activity. See FIG. 1.

[0184] Protein Production in Small Scale:

[0185] Cells were grown at 37 C. in 200 mL LB medium (10 g tryptone, 5 g yeast extract, and 10 g NaCl in 950 mL deionized water.) supplemented with 100 g/mL ampicillin. When OD.sub.600 reached 0.6-0.8, expression was induced for 4 h at 37 C. after addition of IPTG and MnCl.sub.2 to a final concentration at 0.6 mM and 1 mM, respectively. Cells were collected by centrifugation at 9,500 rpm for 10 min and suspended in 50 mM arginine buffer and then sonicated on ice. The insoluble matter was washed twice with 50 mM arginine, and collected by centrifugation for 15 min at 9,500 rpm. The insoluble material contains about 80% of OxOx inclusion body.

[0186] Protein Production in Large Scale:

[0187] For large-scale production of OxOx proteins, E. coli Origami B cells were grown in a 7 L fermenter (3.5-L working volume) in LB medium supplemented with 100 g/mL ampicillin and 5 mM MnCl2. The initial glycerol and yeast extract concentrations were 12 g/L. Fermentation was carried out at 30-37 C. with vigorous aeration and agitation and the pH of the medium was maintained at 6.85 by addition of 10% ammonia. After 8 h of batch growth, the cells were grown in a fed-batch mode with a continuous supply of glycerol and yeast extract. The culture at OD.sub.600 of 20 was induced with 0.8 mM IPTG, cultivated for another 20 h, and then harvested.

[0188] Part 2. Protein Refolding

[0189] E. coli cells were broken by homogenizer or sonication. OxOx inclusion body was obtained by washing the broken cells and collected by slow speed centrifugation, which is well known by scientists in the field. The purified inclusion body was dissolved in 8M urea, pH 8.0. The soluble fraction was obtained following incubation at room temperature for 20 min followed by centrifugation.

[0190] Proteins were refolded by rapid dilution:

[0191] Inclusion bodies were dissolved in 8 M urea, pH 8.0. Rapid dilution was achieved by adding the 8 M urea OxOx solution drop-wise into refolding buffer with rapid stirring.

[0192] The refolding buffers we have used: [0193] 1. 20 mM Tris, 300 mM NaCl, 1 mM MnCl.sub.2, 1 mM GSH/0.2 mM GSSG, pH8.0 [0194] 2. 20 mM Tris, 300 mM NaCl, 1 mM MnCl.sub.2, 1 mM GSH/0.2 mM GSSG, 400 mM arginine, pH8.0 [0195] 3. 20 mM Tris, 300 mM NaCl, 1 mM MnCl.sub.2, 1 mM GSH/0.2 mM GSSG, 100 mM acyclodextrin, pH8.0 [0196] 4. 20 mM Tris, 300 mM NaCl, 1 mM MnCl.sub.2, 1 mM GSH/0.2 mM GSSG, 2% bcyclodextrin, pH8.0 [0197] 5. 20 mM Tris, 300 mM NaCl, 1 mM MnCl.sub.2, 1 mM GSH/0.2 mM GSSG, 40% sucrose, pH8.0 [0198] 6. 20 mM Tris, 300 mM NaCl, 1 mM MnCl.sub.2, 1 mM GSH/0.2mM GSSG, 40% glucose, pH 8.0 [0199] 7. 20 mM Tris, 1 mM MnCl.sub.2, 1 mM GSH/0.2 mM GSSG, pH8.0 [0200] 8. 20 mM Tris, 1 mM MnCl.sub.2, 1 mM GSH/0.2 mM GSSG, 400 mM arginine, pH8.0 [0201] 9. 20 mM Tris, 1 mM MnCl.sub.2, 1 mM GSH/0.2 mM GSSG, 50 mM betaine, pH8.0 [0202] 10. 20 mM Tris, 1 mM MnCl.sub.2, 50 mM betaine, pH8.0 [0203] 11. 20 mM Tris, 0.5-5 mM MnCl.sub.2, 1 mM GSH/0.2 mM GSSG, 50 mM betaine, pH8.0 [0204] 12. 20 mM Tris, 1 mM MnCl.sub.2, 1 mM GSH/0.2 mM GSSG, 50 mM betaine, pH4.0-10.0 [0205] 13. 20 mM Tris, 1 mM MnCl.sub.2, 50 mM betaine, 0.05 mM 1-hexanol, 50 mM acetamide, 300 mM KCl, pH8.0

[0206] Results:

[0207] 1. OxOx from banana is expressed as inclusion body with a yield in the range of 0.2-0.6 gram per liter of culture in a flask, 2-6 gram per liter of culture in a fermentor. The production yield can be improved after optimization of conditions. The inclusion bodies usually show a little OxOx activity, but after refolding, a specific activity in the range of 0.1 to 10 units per mg of protein is readily obtained after purification with Phenylsepharose column.

[0208] 2. OxOx 5100 from sweet beet is expressed as inclusion bodies with a yield in the range of 0.1-0.5 gram per liter of culture in a flask, 1-5 gram per liter of culture in a fermentor. The production yield can be improved after optimization of conditions. The inclusion bodies usually show a little OxOx activity, but after refolding, a specific activity in the range of 0.1 to 15 units per mg of protein is readily obtained after purification with Phenyl-sepharose column.

[0209] 3. OxOx 5102 from sweet beet is expressed as inclusion bodies with a yield in the range of 0.1-0.5 gram per liter of culture in a flask, 1-5 gram per liter of culture in a fermentor. The production yield can be improved after optimization of conditions. The inclusion bodies usually show a little OxOx activity, but after refolding, a specific activity in the range of 0.1 to 10 units per mg of protein is readily obtained after purification with Phenyl-sepharose column.

[0210] 4. OxOx 5601 from sweet beet is expressed as inclusion bodies with a yield in the range of 0.1-1 gram per liter of culture in a flask, 1-10 gram per liter of culture in a fermentor. The production yield can be improved after optimization of conditions. The inclusion bodies usually show a little OxOx activity, but after refolding, a specific activity in the range of 0.1 to 10 units per mg of protein is readily obtained after purification with Phenyl-sepharose column.

[0211] 5. The refolding conditions have not been optimized, but it has been observed that refolding buffer pH at 8.0, stabilizers or solubility enhancers including betaine, NaCl or KCl, acetamide, and 1-hexanol, and the concentration of MnCl.sub.2around 1 mM. are important for effective refolding.

Example 4

Recombinant Expression of Oxalate Oxidase by P. pastoris

[0212] Expression Vector Construction

[0213] The genes of beet 5102 and 5601 and banana 30640 were amplified from a pMD18-T simple vector containing these genes using a pair of primers designed to introduce an Xho I followed by a Kex2 protease cleavage site at the 5 and an Not I restriction site at the 3 (Table 1). The PCR products were digested with Xho I and Not I and cloned into the pPICZB and pGAPZA vectors digested with the same restriction enzymes, resulting in the recombinant plasmids of Z-5102, GAPZ-5102, Z-5601, GAPZ-5601, Z-30640 and GAPZ-30640 (FIGS. 2-7). The recombinant plasmids of Z-5102, Z-5601 and Z-30640 were linearized with Pme I (Mss I) and GAPZ-5102, GAPZ-5601 and GAPZ-30640 were linearized with Bln I (Avr II). Then, all the linearized vectors were electro-transformed into P. pastoris X-33 according to the methods of high efficiency transformation of P. pastoris pretreated with lithium acetate and dithiothreitol, which recommended by Wu S and Letchworth GJ (2004

TABLE-US-00004 TABLE1 Primersusedtoconstructexpressionvectors SEQ Oligo- IDNO: nucleotides Sequence(5 to3) 17 5102F CCGCTCGAGAAAAGATCTGATCCTGGTCTC CTACAG 18 5102R AAATATGCGGCCGCTCAAGCAACATCAGTT AAGAGTT 19 5601F CCGCTCGAGAAAAGATCCGATCCTGCACCC CTT 20 5601R AAATATGCGGCCGCTCAATAAGTTTCAGAT AGCAATTTC 21 30640F CCGCTCGAGAAAAGATTTGATCCGAGTCCT CTCCA 22 30640R AAATATGCGGCCGCTCAATTAGCTTGAACT AAGCGTTG

[0214] Protein Expression

[0215] Positive clones were initially selected by YPDS medium plates (10 g/l yeast extract, 20 g/l peptone, 20 g/l dextrose, 1 M sorbitol, and 20 g/l agar) containing 100 g/ml Zeocin. Then, multiple-copy transformants were further screened by YPDS resistance plates containing Zeocin at a final concentration of 1 mg/ml. The selected colonies were checked first with PCR and then sequencing to verify a right gene inserted into yeast chromosome. The high Zeocin resistance clones were selected to check their expression by shaking flask fermentation.

[0216] The growth and induction media in shaking flask were BMGY (yeast extract 1% (w/v), peptone 2% (w/v), 100 mM potassium phosphate buffer at pH 6.0, yeast nitrogen base with no amino acids 1.34% (w/v), glycerol 1% (w/v), biotin 0.04% (w/v)) and BMMY (its composition is similar to BMGY, but with 1.0% (v/v) methanol instead of glycerol). A single colony of a selected strain was first inoculated into a 20 ml bottle with 4 mL YPD medium and grew at 28 C. for 18-20 h. Then, 4% (v/v) of the culture was inoculated into a 500 ml flask containing 50 ml (or 250 ml flask containing 25 ml) BMGY. The cells were grown at 28 C., shaking at 220 rpm, for 18-20 h to reach an OD.sub.600 of 3.0-6.0, then harvested by centrifugation and re-suspended in 50 mL BMMY medium containing 5 mM MnCl.sub.2 for methanol induction. The induction temperature was set at 28 C., and 100% methanol was added daily to reach methanol concentrations at 1.0% (v/v). After 96-120 h methanol induction, the supernatant of the culture was collected by centrifugation at 9500 rpm for 5 min at 4 C., and used for OxO activity assay and SDS-PAGE. Proteins were stained with Coomassies Brilliant Blue R-250. All the supernatant samples were concentrated by TCA precipitation. All the samples used for SDS-PAGE analysis are 30 l of concentrated supernatant. For the constructive expression of GAP promoter, the growth was BYPD medium (10 g/l yeast extract, 20 g/l peptone, 20 g/l glucose, biotin 400 g/l, MnCl.sub.2 5 mM and 100 mM potassium phosphate buffer, pH 6.0) and the cells were grown at 28 C., shaking at 220 rpm. Add 2% glucose into BYPD medium after 48 h of culture. After 72 h of culture, the supernatant of the culture was collected and analyzed as above conditions

[0217] Fed-batch fermentation: Inoculum (any recombinant yeast culture described above) was produced at 30 C. in a 2 l flask containing 400 ml YPD medium shaken at 220 rpm for 18 h. Then, 10% (v/v) of the inoculum was inoculated into the 7 l fermentor containing 2.8 l FM22 medium (KH.sub.2PO.sub.4, 42.9 g/l; (NH.sub.4).sub.2SO.sub.4, 5 g/l; CaSO.sub.4.2H.sub.2O, 1.0 g/l; K.sub.2SO.sub.4, 14.3 g/l; MgSO.sub.4.7H.sub.2O, 11.7 g/l; glycerol, 40 g/l and 2.5 ml/l PTM4 trace salt solution). PTM4 trace salt solution. The PMT4 solution was composed of (g/l): CuSO.sub.4.5H.sub.2O, 2.0; Nal, 0.08; MnSO.sub.4.H.sub.2O, 3.0; Na.sub.2MoO.sub.4.2H.sub.2O, 0.2; H.sub.3BO.sub.3, 0.02; CaSO.sub.4.2H.sub.2O, 0.5; CoCl.sub.2, 0.5; ZnCl.sub.2, 7; FeSO.sub.4.7H.sub.2O, 22; biotin, 0.2 and 1 ml/l concentrated H.sub.2SO.sub.4 [18]. The glycerol fed-batch solution contained (I.sup.1): 500 g glycerol (100%) and 4 ml PTM4 stock solution. The methanol fed-batch solution consisted of 4 ml PTM4 stock solution and 1 l of pure methanol. Once glycerol was depleted from culture broth, an 8 h glycerol exponential fed-batch phase was started at a growth rate of 0.16 h.sup.1. The methanol limited fed-batch strategy was carried out at the end of glycerol transition phase. Methanol feeding rate was regulated to maintain the DO above the set point in the culture broth according to the recommendations of Pichia Fermentation Process Guidelines (Invitrogen). The temperature was set at 30 C. at the glycerol batch and fed-batch phases, and then decreased to 25 C. at the beginning of the methanol induction phase. The pH value was kept at 5.5 by addition of 28% (w/w) ammonium hydroxide. The DO level was maintained at 20-50% of air saturation by a cascaded control of the agitation rate at 500-800 rpm with an airflow rate of 150-400 l/h. Foaming was controlled through addition of antifoaming agent (Dowfax DF103, USA). The fed-batch feeding medium was pumped into the fermentor according to a predetermined protocol.

[0218] Purification of Recombinant Protein

[0219] The purification procedure was basically done as follow: the cell-free fermentation broth containing the secreted enzyme was concentrated and dialyzed overnight against 50 mM potassium phosphate and citric acid buffer pH 3.5 and then loaded into a cation exchange column, or dialyzed overnight against 50 mM potassium phosphate and citric acid buffer pH7.5 and then loaded into an anion exchange column. If not sufficient, a phenyl-sepharose column is applied. The flow-through fractions were pooled and concentrated by ultra-filtration for SDS-PAGE and activity assay.

[0220] Results

[0221] The three oxalate oxidase genes of 5102, 5601 and 30640 have been successfully expressed in P. pastoris with a yield in a range of 0.01-1 mg per liter of culture, as shown by SDS-PAGE with a band at molecular weight of 24 kD (FIGS. 8-11). Oxalate oxidase purified from the broth, all of them showed oxalate degradation activity at pH 7.4.

Example 5

Production of Oxalate Oxidases in Plants by Transient Expression

[0222] Construction of Expression Vectors

[0223] The genes for transient expression (5601 and 5102 from sugar beet, 30640 from banana) were amplified by gene-specific primers flanked by restriction sites Xbal and Kpnl. After Xbal and Kpnl digestion, the amplified fragments (FIGS. 12-13) were cloned into pHTE to produce pHTE-5601, pHTE-5102 and pHTE-30640 (FIG. 14), respectively. All positive recombinant plasmids were selected by colony PCR or by enzyme digestion and separately introduced into the A. tumefaciens strain GV3101 by the freeze/thaw method

[0224] 5.1 Transient Expression in Tobacco Leaves

[0225] Plant Materials

[0226] Wild-type N. benthamiana plants were grown in a greenhouse with a 16/8 hr light/dark cycle at 25 C. for 5 to 8 weeks.

[0227] 5.1.1 Bacterial Culture and Suspension Preparation

[0228] 1. Pre-cultures are prepared 2 days before infiltration by inoculating 3 mL of LB medium (10 g tryptone, 5 g yeast extract, and 10 g NaCl in 950 mL deionized water.) containing 25 mg/L rifampicin and 50 mg/L kanamycin with isolated colonies of Agrobacterium strains harbouring expression plasmids, overnight at 28 C. under constant agitation at 220 rpm to grow preferentially to an OD (600 nm)>1.2.

[0229] 2. Inoculate fresh LB medium containing 25 mg/L rifampicin, 50 mg/L kanamycin and 20 M acetosyringone with pre-culture at 1:100 ratio. For each plant to be infiltrated, 20 mL of each strain should be prepared. The cultures were incubated at 28 C. under constant agitation at 220 rpm to an OD (600 nm) of 0.8-1.2 (18 h).

[0230] 3. Centrifuge cultures (5000 rpm; 5 min) and discard supernatant.

[0231] 4. Resuspend the bacterial pellets in 5 volume of bacteria resuspension solution (10 mM 2-N-morpholinoethanesulfonic acid (MES)) pH 5.5, 10 mM MgSO.sub.4, and 100 mM acetosyringone), and incubate for 4 h at room temperature before use.

[0232] 5.1.2 Syringe Infiltration and Plant Incubation

[0233] 1. Fill a 1 mL- or 3 mL-syringe (without needle) with bacterial suspension, and hold the leaf to be infiltrated between the index and the syringe, the syringe being on the abaxial side of the leaf. Gently push the piston to force the bacterial suspension enter into the leaf and maintain an even pressure during the infiltration. Wetting of the leaf surrounding the infiltration point is observed as the suspension enters the tissue in the apoplastic space. For each point of infiltration, a surface of 7 cm.sup.2 should be filled. Several points of infiltration may be necessary to completely inoculate each leaf.

[0234] 2. Infiltrate a maximum number of leaves on each plant and remove all uninfiltrated leaves as well as apical and axillary buds to avoid growth of non-infiltrated leaves during the incubation period.

[0235] 3. Incubate infiltrated plants in the greenhouse for 7 days, watering the plants as needed and continuing nitrogen fertilization.

[0236] 5.1.3 Leaf Disk OxOx Activity Assay

[0237] Agroinfiltrated tobacco leaves were harvested 7 days post infiltration (dpi) for OxOx activity assays. Histochemical assay of OxOx activity was carried out. The histochemical buffer contains 40mM succinate, 2.5 mM oxalic acid, 5 U/ml horseradish peroxidase, 0.6 mg/ml 4-chloro-1-naphthol, 60% (V/V) ethanol, pH 5.0. Leaf discs were added to the buffer and viewed following incubation at room temperature overnight. The control is the leaf discs made from the same way as the tested leaf discs except that no OxOx gene agroinfiltrated into tobacco leaves.

[0238] The results are given below together with the results from the experiments relating to transient expression in pea

[0239] 5.2 Transient Expression in Pea

[0240] 5.2.1 Plant Materials

[0241] The seeds of pea plant (P. sativum) were obtained from the local market. The seeds were sowed and plants were grown in a plant growth chamber at 25 C. under a 16 h cool fluorescent light/8 h dark cycle.

[0242] 5.2.2 Bacterial Culture, Suspension Preparation and Vacuum-infiltration

[0243] Agrobacterium GV3101 cultures, bearing binary vectors, were grown in modified YEB media (6 g/L yeast extract, 5 g/L peptone, 5 g/L sucrose, 2 mM MgSO.sub.4) with antibiotics (100 mg/mL of kanamycin, 50 mg/mL of rifampicin,) for 2 days at 28 C. For final scaledup growth, initial 2-day cultures were diluted 1:100 in the same YEB medium supplemented with antibiotics, 10 mM MES, pH 5.6, 20 M of acetosyringone and allowed to grow 18-24 h to an OD.sub.595 nm of about 2.4. Bacterial cells were supplemented with 55 g/L of sucrose and 200 M acetosyringone and the suspension was incubated for 1 h at 22 C. Tween20 were added to final concentrations of 0.005% and the suspension was used for vacuum-infiltration. An amount of 1.2 L of pretreated suspension of Agrobacterium was placed into a 2 L glass beaker inside a vacuum. The whole pea plants were immersed into the suspension and held for 1 min under vacuum (0.07-0.1 MPa) and the vacuum was rapidly released. The pea plant roots were rinsed in water and left for 5-7 days at 20-22 C. with 16 h light every day. After 5-6 days of incubation, the pea plants were cut out from the base and homogenized for protein extraction.

[0244] 5.2.3 Enzyme Assay

[0245] For analysis of protein expression levels, protein was extracted with extraction buffer (50 mM citric acid-phosphate, pH 7.0). The extraction buffer and harvested pea plants were in a 1:1 (v/w) ratio. The tissues were cut from the base and homogenized in a blender at high speed for 1 min. Homogenate was centrifuged for 15 min at 7000 g. The supernatant was centrifuged for 15 min at 14,000 g and the resulting supernatants were analyzed by 12% SDS-PAGE gel. The pellet was washed twice by re-suspension in 10 vol. of the homogenization medium containing 1% (w:v) Triton X-100 and four times with 30 vol. of the same medium without Triton X-100. After each wash the pellet was collected by centrifugation at 1000 g for 10 min. The final pellet was considered to be the purified cell wall fraction, and was used for testing the OxOx activity by colorimetric assay. For purification of OxOx, the supernatant of centrifuged homogenate was precipitated with 80% saturated ammonium sulfate. The pellet was re-dissolved in 50 ml buffer A (50 mM citric acid-phosphate, pH 6.0, 2M NaCl), and the resulting suspension was centrifuged for 20 min at 14,000 g. The resulting supernatant was loaded onto Phenyl Sepharose HP column previously equilibrated with buffer A. The protein was eluted with a linear gradient of NaCl (2-0 M) in the same buffer and fractions were collected at a rate of 0.5 ml/min. The collection fractions were detected the OxOx activity by colorimetric assay. Then the fractions having OxOx activity were mixed together and reloaded on a Q Sepharose column. OxOx was eluted with a liner gradient of NaCl (0-2 M) in buffer A. All the collection fractions were tested OxO activity by colorimetric method.

[0246] Results

[0247] 1. The genes of OxOx 5102, 5601 and 30640 have been amplified (FIGS. 12-13)

[0248] 2. The plant expression vector for OxOx 5102, 5601 and 30640 (FIG. 14)

[0249] 3. The genes for OxOx 5102, 5601 and 30640 are expressed with OxOx activity in tobacco leaves (FIG. 15)

[0250] 4. The genes for OxOx 5102, 5601 and 30640 are expressed with OxOx activity in pea leaves (FIG. 16)

[0251] 5. OxOx 5102 expressed by pea leaves has been purified and shows activity (FIGS. 17 and 18)

[0252] 6. The expression levels of OxOx 5102, 5601 and 30640 by pea leaves in the range of 0.01-5 mg per gram of fresh leaves, but majority of the expressed OxOx is associated with the solid material (FIG. 16)

Example 6

The Activity of Oxalate Oxidases Expressed by E. coli at Different pH

[0253] Activity Assay: the enzyme solution 10 l is added to 190 l of 800 mg/L 4-Aminoantipyrine, 4.8 mM sodium 3,5-dichloro-2-hydroxybenzenesulfonate, 10 unit per ml horseradish peroxidase, 5 mM oxalate, 50 mM phosphate.

[0254] The mixture is placed at 37 C. for 10-60 min in a plate reader to read OD.sub.600. One unit of activity is defined as the enzyme amount required to produce 1 mole of formate from oxalate under the above conditions.

[0255] Activity pH profile: samples were tested as described earlier using a series of buffers within a pH range of 4.5-8.0 (50 mM citrate for pH 4.5-6.0 and 50 mM potassium phosphate for pH 6.0-8.0).

[0256] This test is used to test the oxalate oxidases for oxalate degrading activity as referred to in the claims.

[0257] Results

[0258] The maximum activity is 18.2 units per mg for 5100, 19.5 units per mg for 5102, 18.7 units per mg for 5601, and 14 units per mg for 30640. For easy comparison, the activity for each enzyme at different pH is given in the table 1 and FIG. 19 as relative activity, which is calculated by the activity divided by the maximum activity.

TABLE-US-00005 TABLE 1 The relative activity of OxOx at pH 4.5-8 pH 5100 5102 5601 30640 4.5 69.23 83.42 45.10 45.52 5 88.08 91.23 79.34 69.11 5.5 92.95 94.30 100.00 80.37 6.0 99.99 96.17 77.60 97.92 6.5 92.60 100.00 49.20 99.99 7.0 90.69 95.10 36.73 92.56 7.5 82.00 92.60 21.78 65.33 8.0 81.75 87.50 18.17 42.17

Example 7

Inclusion Body WashingOxOx-B5102

[0259] The harvested cell pellets were resuspended in a ratio of 30 g cell paste per 1L wash buffer (50 mM Tris-HCl, 2M urea, 50 mM NaCl, 5 mM EDTA, 5 mM DTT, pH8.0). Benzonase nuclease (100 units per liter with 0.5 mM MgCl.sub.2) was added to cell suspension to digest E. coli nucleic acid (including DNA and RNA), incubated at 37 C. for 15 min. The pre-treated cells were passed through a pre-cooled homogenizer (NTI, USA) 4 times at 1100 bar pressure, then centrifuged at 8000g for 10min in 500 ml bottles at 4 C. The pellets were resuspended and washed with wash buffer 3 times and deionized water twice. The purity of inclusion body was analyzed by SDS-PAGE. The purity of the inclusion body usually reached above 80%.

Example 8

Q-Sepharose Purification of Inclusion Body

[0260] The washed inclusion body from Example 7 with purity>80% was dissolved in urea buffer (20 mM Tris-HCl, 8M urea, pH 8.0), and centrifuged at 13000 g for 10 min. The supernatant was loaded on pre-balanced Q-sepharose column with the urea buffer and further washed 5 column volumes (CV) with the same buffer. The impurity was eluted by elution buffer B (8M urea, 30 g/L NaCl, Tris-HCl, pH8.0) with 6 CV. The OxOx-B5102 protein was eluted by elution buffer C (8M urea, 160 g/L NaCl, Tris-HCl, pH8.0). The purity of OxOx-B5102 inclusion body after Q-sephrose column purification usually reached above 95%. The purified OxOx-B5102 inclusion body solution was concentrated to 5 mg/mL for protein refolding by 10K ultra-filtration tubes.

Example 9

Refolding of Inclusion Body

[0261] Refolding was performed as described herein before.

Example 10

Phenyl Sepharose Purification of OxOx-B5102

[0262] NaCl was added to the refolded OxOx-B5102 mixture to a final concentration of 500 mM, and then passed through 0.45 m membrane. The clarified OxOx-B5102 solution was loaded into Phenyl sepharose column pre-balanced with 5 CV of balance buffer (10 mM Tris-HCl, 500 mM NaCl, pH 8.0). The column was further washed with 1.5 CV of wash buffer (10 mM Tris-HCl, 250 mM NaCl, pH 8.0), and then OxOx-B5102 was eluted by elution buffer (10 mM Tris-HCl, 20% Isopropanol, pH 7.0). The target protein was precipitated by adding NaCl to the solution up to 50 mM and collected by centrifugation at 12000 g for 10 min, 4 C. The pellets were re-dissolved in borate buffer (10 mM Na.sub.2B.sub.4O.sub.7H.sub.3BO.sub.3, pH 9.0) for PEGylation. The purity of collected OxOx-B5102 sample was analyzed by SDS-PAGE, the activity of OxOx-B5102 was analyzed by activity assay.

Example 11

PEGylation and Purification of PEG-OxOx

[0263] The concentration of OxOx-B5102 in borate buffer was adjusted to 2 mg/mL for pegylation. A ratio of 10 times of PEG molecules over the number of lysine residues on OxOx surface was used for pegylation reaction. Different sizes of Methoxy PEG Succinimidyl Carboxymethyl Ester (mPEG-SC) at 2 kD, 5 kD, 10 kD, 20 kD, 4 armed-20 kD, 30 kD, 40 kD and 4 armed-40 kD were tested one by one. The mPEG-SC was gradually added into OxOx-B5102 solution and gently mixed by using magnetic stirrer. The reaction was maintained for 6 h at 28 C., and then was stopped by adding glycine.

[0264] The pegylated OxOx-B5102 sample was loaded on size exclusion chromatography (GE HiLoad Superdex 16/600GL, USA) pre-balanced with phosphate buffer (10 mM K.sub.2HPO.sub.4KH.sub.2PO.sub.4, pH 7.4) and eluted by the same buffer at an elution rate of 1 mL/min. The target protein was collected.

Example 12

Reduction of Plasma Oxalate and Urine Oxalate in Rat Model

[0265] 30 male Sprague Dawley (SD) rats, weighing approximately about 130-150 g and less than 5 week were purchased from local animal experimental center. Rats were housed in a plastic individual ventilated cage (IVC) system (temperature 1826 C., moisture 4070%) and fed with distilled water and regular rat food every day. After 1 week of acclimatization, Rats were randomly divided into control group and 4 experimental groups (six rats per group) and transferred to metabolic cages with single occupation. Rats in control group were fed regular food and water; the experimental groups were fed with regular food and 1% ethylene glycol as drinking water. Blood sample (about 0.3 ml) was collected from tail-vein and heparin was added into blood as anticoagulant. Serum was obtained immediately from fresh blood sample by centrifugation at 5000 g for 5 min at 4 C. Urine sample was collected from urine collection tube of metabolic cage every 12 h (8:30 and 20:30) and acidified by using 2M HCl immediately to pH 1.5-2.0. Serum and urine oxalate of all rats was detected and monitored until oxalate level was stable, then PEG-OxOx-B5102 was injected through vein.

[0266] Serum oxalate was analyzed by using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex red) fluorescence method. The procedure: fresh serum samples were acidified to pH2.0 and serum proteins were removed by filtration with 10K ultra-filtration tube. The filtrate (10 l) was added to 96 well multi-plate for 6 wells. The reaction buffer containing oxalate oxidase (100 mM citrate buffer, pH5.4, 10 M Amplex red, 1U/mL HRP, 0.1U/mL OxOx-B5102) were added into 3 wells and the background reaction buffer without OxOx (100 mM citrate buffer, pH5.4, 10 M Amplex red, 1U/mL HRP) was added into the other 3 wells. All reactions were incubated at 25 C. for 30 min, then fluorescence of each well was detected by using fluorescence multi-plate reader (excitation wavelength: 538 nm; emission wavelength: 590 nm). The fluorescence of a sample is the average value of the three wells after minus the average of the three background control wells. Oxalate concentration was calculated by fluorescence value, which was calibrated by oxalate standard curve.

[0267] Urine oxalate concentration was detected by using colorimetric assay (adopted commercially available Trinity oxalate kit). The operation procedure was done according to the manual of the kit.

[0268] Following the acclimation period, OxOx-B5102 pegylated with 3 different molecular weights of PEG (20 kD, 30 kD, and 40 kD PEG) was administered to rats in 3 experiment groups, respectively, 0.2 mg OxOx each time per rat, for consecutive 3 days. Saline was administered to rats in the fourth experiment group, recorded as placebo control group at same dose. Rats in regular control were fed as usual without any treatment.

[0269] Serum oxalate and urine oxalate was monitored every day. The results showed that PEG-OxOx-B5102 could reduce 4050% serum oxalate and 2040% urine oxalate compared with placebo control (FIG. 21 and FIG. 22).

Example 13

Immunogenicity Evaluation of PEG-OxOx-B5102s

[0270] OxOx-B5102 and OxOx-B5102 pegylated with different molecular weights of PEG (2 kD, 5 kD, 10 kD, 20 kD, 30 kD and 40 kD) were injected intraveneously into SD rats for immunogenicity evaluation, respectively, through tail veins every week for 4 weeks. The dose was 0.2 mg OxOx each time. Serum samples were collected and detected antibody titer by using ELISA method.

[0271] The ELISA method procedure: (1) rat serum samples were collected at different timepoints (0 d, 7 d, 14 d, 21 d, 28 d, 45 d, 60 d) post injecting of PEG-OxOx-B5102 and stored at 20 C. until use. (2) Dilute OxOx-B5102 to a final concentration of 10 g/ml in coating buffer (50 mM carbonate/bicarbonate buffer, pH9.6) and transfer 100 l to each well of a high affinity, protein-binding ELISA plate. Cover the plate with a tinfoil and incubated at 4 C. overnight. (3) Bring the plate to room temperature, flick off the capture antibody solution, wash 3 times with PBS-T buffer (1.5 mM KH.sub.2PO.sub.4; 8.1 mM Na.sub.2HPO.sub.4.12H.sub.2O; 136 mM NaCl; 2.7 mM KCl; 0.05% Tween-20), and block non-specific binding sites by adding 300 l of blocking solution (1.5 mM KH.sub.2PO.sub.4; 8.1 mM Na.sub.2HPO.sub.4.12H.sub.2O; 136 mM NaCl; 2.7 mM KCl; 5% non-fat dry milk) to each well. (4) Seal plate and incubate at 37 C. for 12 hour. (5) Wash 3 times with PBS-T buffer and firmly blot plate against clean paper towels. (6) Dilute serum samples using PBS-T buffer to 50 time, 100 time, 200 time, 400 time, 800 time (perform dilutions in polypropylene tubes) and add 100l per well to the ELISA plate. (7) Seal the plate and incubate at 37 C. temperature for 1 hours or at 4 C. overnight. Wash3 times with PBS-T buffer. Washes can be effectively accomplished by filling wells with multichannel pipettor. For increased stringency, after each wash, let the plate stand briefly, flick off the buffer, and blot plates on paper towels before refilling. (8) Dilute the HRP labeled goat anti-rat antibody to its pre-determined optimal concentration in PBS-T buffer (usually between 1/5000-1/20000). Add 100 l per well. (9) Seal the plate and incubate at room temperature for 1 hour. Wash5 times with PBS-T buffer. (10) For each plate, mix 6 ml of TMB Reagent A (0.5 mM EDTA-Na; 5 mM citric acid; 10% glycerol; 0.04% tetramethyl benzidine) with 6 ml TMB Reagent B (165 mM sodium acetate; 8.3 mM citric acid; 0.06% 30%-H.sub.2O.sub.2) immediately prior to use. Transfer 100 L into each well and incubate at room temperature for 30 min. To stop the reaction, add 100 l of stopping solution (2M H.sub.2SO.sub.4). (11) Read the optical density (OD) for each well with a micro-plate reader set to 450 nm.

[0272] The OxOx antibody titer reached peak at 28 day post-injection (data not shown). The results on 28 days (FIG. 23) showed that OxOx-B5102 antibody titer dropped significantly when OxOx was pegylated, and dropped further when pegylated with large size of PEG. There was little antibody was detected to against OxOx-B5102 when OxOx was pegylated with 10K, 20K, 30K, and 40K PEG.