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 Field of the invention

The present invention relates to the treatment of oxalate-related diseases and provides oxalate oxidases (OxOx) that have suitable 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.

Background of the invention

Kidney/urinary tract stone disease (urolithiasis) is a major health problem throughout the world. Most of the stones associated with urolithiasis are composed of calcium oxalate alone or calcium oxalate plus calcium phosphate.

Many disease states are associated with an excess quantity of oxalate in the body including: primary hyperoxaluria, secondary hyperoxaluria, autism, vulvodynia, oxalosis associated with end-stage renal disease, cardiac conductance disorders, Crohn' s disease, inflammatory bowel disease, colitis, urolithiasis, oxalosis associated with end- stange renal disease, sarcoidosis, asthma, COPD, fibromyalgia, Zellweger syndrome, bariatric surgery and other enteric disease states. Oxalate may be absorbed through the whole gastrointestinal tract including the stomach, and the small and large intestines. Therefore removal of dietary oxalate in these organs is effective in preventing oxalate absorption. Absorption of dietary oxalate contributes to 10-70% of urinary oxalate secretion. It is believed that excess of concentrations of calcium oxalate is responsible for stone formation. Thus, by reducing the oxa- late concentration the risk of stone formation will be decreased.

There are very few, if any, treatment strategies known to significantly decrease the risk of stone formation. The main approaches have been to limit dietary oxalate absorption by orally administering oxalate degrading enzymes or bacteria, which come into con- tact with the content of the stomach and the intestines. The challenge in providing such treatment is the harsh acidic stomach environment, which may degrade the enzyme so that it becomes ineffective at low pH and in a high pepsin activity environment. The intestines are also challenging due to the presence of trypsin and chymotrypsin and due to a pH approaching neutral pH. Most of the known oxalate degrading enzymes have activity optimum at pH at an acidic pH. The challenge when oxalate degrading bacteria are used is to pass the gastric environment without losing the activity of the bacteria and to have the bacteria colonized in the intestines. An attempt has been to orally administer an enteric coated composition.

All germ-like oxalate oxidases reported in the literature are acid active and oxalate oxi- dase reported with activity at pH greater than 7 has not been confirmed as a single purified protein with gene sequence. WO 2011/066282 describes oxalate-degrading enzymes derived from fungi. Example 5 of WO 2011/066282 has not isolated the enzyme and no information about the protein sequence is given. The present inventors have tried to purify some of this enzyme, but failed.

Makoto K. et al. in Journal of the Institute of Brewing, vol 115, 2009, 232-237 describes enzymes derived from barley and malt, but conclude that the enzyme seems to be a flavoprotein (see page 235, 1 ^st column). The present inventors have repeated the experiment described in Makoto et al. using exactly the same barley seed, but failed to find any oxidase activity at pH 7.4.

However, there is still a need for treatment strategies, which effectively reduces the oxalate concentration in the body. Description of the invention

The present invention provides a novel treatment strategy for decreasing the concentration of oxalate in the body. The strategy is based on the finding of oxalate oxidases that have optimum or sufficient activity at pH 7.4 and thus, are capable of reducing oxalate concentration in the blood after parenteral administration. By using the parenteral administration route problems relating to degradation of the enzymes in the gastrointestinal tract combined with the necessity of having a pH, where the enzyme is active are avoided. However, a prerequisite for the treatment strategy is that the enzyme(s) is (are) active at physiological pH (pH 7.4). Until now only one oxalate oxidase has been described, which has some activity at pH 7.4 and that is an oxalate oxidase activity as- sociated with plant solid tissue from Bougainvillea spectabilis leaves (Biochem.

J. ,1962, 85, 33). The present invention also relates to oxalate oxidases (or variants thereof) with sufficient activity about pH 7.5 as well as the polynucleotides encoding the variants. The invention also relates to a recombinant host cell comprising the polynucleotides as well as a method for producing the oxalate oxidases variants. Especially, the invention relates to recombinant oxalate oxidases which have a sufficient activity at pH 7.5, i.e. they are capable of degrading oxalate at pH 7.4 such that the relative activity is at least 20%. The relative activity is calculated as (observed activity (pH 7.5)/max. activity) ^* 100%. One unit of activity is defined as the enzyme amount required to produce 1 μη-iole of formate from oxalate under the conditions described in Example 6 herein.

As seen from the examples herein recombinant oxalate oxidases have been produced, which have a relative activity at pH 7.5 of at least 60%. In particular, oxalate oxidases with a relative activity of at least 80% or at least 90% have been produced. To this end it is important to note that this finding is unique as no oxalate oxidase has been dis- closed with such an activity at pH 7.5 (range pH 7.0 - 8.0). Most oxalate oxidases described have max. activity at acidic pH. The oxalate oxidases having a suitable relative activity at pH 7.5 or in the range of pH7.0 - 8.0 are all based on the finding of oxalate oxidases from specific plant material, where the oxalate oxidases surprisingly are found to have activity at neutral/basic pH. As seen from the table in the Examples, many dif- ferent plants from a variety of plant families have been tested and only a few of them gave a positive result with respect to activity at pH 7-8.

An important consequence of this finding is the possibility of developing a therapeutic recombinant oxalate oxidase suitable for use in the treatment of diseases relating to excess of oxalate as described herein. Especially the finding opens up for a novel approach, namely i) administration by the parenteral route (i.e. degradation of oxalate in the plasma/blood) or ii) administration by the oral route using enteric coated material and delivery the recombinant enzymes to the intestines, where pH is about 6-8, i.e. at a pH where the oxalate oxidases have a suitable activity.

The invention also relates to the use of the novel oxalate oxidase variants for the treatment or prophylaxis of oxalate-related diseases and to compositions comprising the oxalate oxidase variants. Patients with severe oxalate situations. Urinary and plasma oxalate originates from two sources: liver synthesis and dietary absorption and is removed mainly by kidney. Under certain disease conditions, including primary hyperoxaluria (PH) ( 1-4), secondary hyperoxaluria (SH) (5-7), Zellweger spectrum disorders (ZSD) (8), and chronic re- nal failure (CRF) or end-stage renal failure (ESRF) (9- 11), urinary oxalate and plasma oxalate concentrations can increase to very high concentrations, which cause severe oxalate related diseases.

PH, including type I, I I and I I I, are rare genetic disorders of glyoxylate metabolism in which specific hepatic enzyme deficiencies result in oxalate overproduction in the liver. Type I pertains to lack of hepatic alanine: glyoxylate aminotransferase (AGT) and type I I to lack of glyoxylate/hydroxypyruvate reductase (GR/HPR) (2). PH type II I results from defects in the liver-specific mitochondrial enzyme 4-hydroxy-2-oxo-glutarate aldolase (HOGA). There is another type of PH, referred to as non-l or non-l l, which shows a similar phenotype as PH, but which has the above mentioned enzymes represented in the liver ( 1). PH is the most severe form of hyperoxaluria and patients suffering from PH can produce plasma oxalate concentrations greater than 100 μηιοΙ/Ι_ if chronic or end-stage renal failure has been developed (3, 9). CaOx supersaturation in the blood of PH patients will lead to systemic oxalosis: CaOx crystals depositing in mul- tiple organs including kidneys, thyroid, myocardium, bone, skin, vessels and eyes. Deposition of CaOx crystals and high concentrations of oxalate in the kidneys will ultimately lead to ESRF and death if untreated ( 12).

ZSD patients have high incidence rates (83%) of hyperoxaluria (8). ZSD is character- ized by a general loss of peroxisomal functions caused by deficient peroxisomal assembly. Although the mechanism of oxalate synthesis in ZSD patients is unclear, the levels of urinary oxalate in some ZSD patients are comparable to PH patients.

Secondary hyperoxaluria (SH) is caused by over-absorption of dietary oxalate or oxa- late precursors either due to gastrointestinal (Gl) diseases, such as inflammatory bowel diseases (IBD), cystic fibrosis (CF), short bowel syndrome and bariatric surgery, or ingestion of a diets rich in oxalate or oxalate precursors (6, 13, 14). SH is very common and generally less severe than PH, but high urinary and plasma oxalate concentrations can occur in these cases due to consumption of rich oxalate diets, large ingested dos- es of vitamin C (an oxalate precursor), or in patients with chronic or end-stage renal failure {6, 13, 14). CRF and ESRF patients under chronic hemodialysis are likely to develop hyperoxaluria (9-11). The kidneys of these patients in combination with hemodialysis are unable to eliminate oxalate sufficiently, due to complications of CRF or ESRF. In addition, vitamin C is often injected intravenously as an antioxidant during hemodialysis, which is later metabolized to oxalate in the human body ( 15). Plasma oxalate concentrations in these patients can be found between 30-90 μηιοΙ/Ι_. In 2006, there were 345,000 patients under hemodialysis in the United States ( 16) and this number is on the rise. Hence, the number of patients with the risk of oxalosis is significant.

Toxic effects of high concentrations of oxalate and CaOx. Supersaturation of CaOx in blood can lead to oxalosis. CaOx crystals deposited in multiple organs in PH patients have been widely observed (2, 12) and CaOx crystals deposited in organs in SH patients ( 15, 17-20) and patients who undergo chronic hemodialysis have also been re- ported in many cases ( 11, 21-23). Deposition of CaOx crystals in these organs can result in organ dysfunction. Primary and secondary oxalosis are often one of the critical factors affecting the outcome of transplanted organs ( 11, 24-26). Many cases of transplant dysfunction are caused by primary or secondary hyperoxaluria ( 18, 27). Patients who undergo chronic hemodialysis experience accelerated atherosclerosis and premature death (28). Plasma oxalate is proposed as an atherogenic toxin due to its contribution to elevated intracellular calcium levels, exclusively in endothelial cells, and hence prevention of re-endothelialization. More significant toxic effects of oxalate and CaOx are its roles in developing CRF and ESRF ( 13, 14, 29-31). CaOx crystals deposit in the kidneys more frequent than in any other organ. It is proposed that CaOx crystals and/or high concentrations of oxalate evoke an inflammatory response and induce tubulointerstitial damage, which leads to fibrosis, loss of nephrons, and eventually to CRF and ESRF. Deposition of CaOx in the kidneys may accelerate kidney stone formation and growth. Due to the ability of CaOx stones to obstruct and cause physical damage to the kidneys, kidney stones are also a known risk for the development of renal failure (32).

Current treatments for severe oxalate situations. Since there is currently no cure for PH, ESRF will develop and kidney transplantation or combined liver/kidney transplantation is the eventual choice (33). For severe SH patients, limiting the dietary intake of oxalate and its precursors is beneficial. However, if ESRF has developed, chronic dialysis and renal transplantation will be the ensuing choices (27).

For patients suffering with CRF or ESRF who undergo chronic dialysis, blood oxalate accumulation has been widely observed due to the limited ability of oxalate to be removed by current dialysis techniques. Furthermore, the situation is often worsened by the administration of large doses of vitamin C, a commonly used antioxidant for hemo- dialysis. Deposition of CaOx crystals in organs has been found in many cases ( 11, 21- 23) and hyperoxaluria or formation of kidney stones is very common among these patients ( 10).

Oxalate degradation enzymes. There are two major oxalate degradation enzymes: oxalate decarboxylase and oxalate oxidase (www.brenda-enzymes.org/). However, no oxalate decarboxylase with activity at physiological pH (pH 7.4) has been found (www.brenda-enzymes.org/). Bougainvillea spectabilis leaves (Biochem. J. ,1962, 85, 33) with some oxalate oxidase activity at pH 7.4 has been reported. However, no oxalate oxidase activity in solution was detected.

The present invention is based on screening of more than 1000 plant species for oxalate oxidase activity. The plant materials were collected in significantly different growth conditions and classification categories The plant material was analyzed for oxalate oxidase activity at pH 7.4 as described in Example 1 herein.

In those cases where the plant material contained oxalate oxidase with an activity of pH 7.4, it was tried to obtain small amounts of the enzymes. These oxalate oxidases are usually associated with cell wall and are quite challenging to dissolve. The purification process includes homogenization of plant tissue into particles smaller than 100 microns, and suspension of the particles in a containing surfactant and high concentration of salts or sugar to extract for 2-5 days. The crude extract is then loaded into Q- sepharose column after dialysis to remove surfactant and salts. The partly purified en- zyme is then loaded into SDS-PAGE or native SDS-PAGE. In the native SDS-PAGE, the enzyme is still active and can be stained with activity assay solution. The band in SDS-PAGE can be cut out and used for amino acid sequence analysis of peptide segments of the protein by mass-spectrometry or used for N-terminal amino acid analysis. Only oxalate oxidases from banana and sweet beet have been purified. Especially, OxOx from Musa acuminate (banana) peel, Beta vulgaris (beet) stem, Bou- gainvillea spectabilis leaves, Mirabilis jalapa young leaf, Telosma cordatum (Brum, f.) /We/7 leaf, Jatropha gossypiifolia Linn. var. elegans Mueller leaf and Sauropus an- drogynus(L.)Merr leaf shows significant activity at pH 7.5. Thus, the present invention relates to oxalate oxidases derived from plant material and which has a sufficient activity (as defined herein) at pH 7.5. More specifically, the invention relates to a recombinant oxalate oxidase having a sufficient activity at pH 7.5 (as defined herein) and having amino acid sequences with an identity of at least 60% to the oxalate oxidases described herein, i.e. SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO:8. In particular the identity is at least 70% at least 75% at least 80% or at least 85%. Specifically, the identity is at least 90%, at least 95%, at least 98%, at least 99% or at least 100%.

The use of recombinant oxalate oxidases with amino acid sequences identical to those oxalate oxidase isolated or isolable from Musa acuminate (banana) peel, Beta vulgaris (beet) stem, Bougainvillea spectabilis leaves, Mirabilis jalapa young leaf, Telosma cordatum (Brum, f.) /Werr leaf, Jatropha gossypiifolia Linn. var. elegans Mueller leaf and Sauropus androgynus(L.)Merr \eai for the treatment or prophylaxis of oxalate-related disease is subject of the present invention.

OxOx from banana peels (Clinical Chemistry, 1985; 31 (4): 649) and beet stems (Clinical Chemistry, 1983; 29(10):1815-1819) have been reported in literature, but no activity around pH 7.4. It is suggested that the OxOx reported in literature may be different from those 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 sequences and DNA sequences, and show different properties as well. The 5100 and 5102 show very high activity (greater than 80% of the maximum activity) at pH 7.5, but the 5601 shows only 22% of the maximum activity at pH 7.5. Thus, it seems likely that none these OxO is the same as those reported in literature. Although only one OxOx gene has been cloned from banana in this work, there are dozens of related 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). The presence of an oxalate oxidase from Bougainvillea spectabilis leaves (Biochem. J. ,1962, 85, 33) has been reported in literature. OxOx from the other four plants are first discovered in this work. Thus, the present invention relates to oxalate oxidase or fragments or variants thereof isolated from banana, sweet beet, Mirabilis jalapa young leaf, Telosma cordatum (Brum, f.) /We/7 leaf, Jatropha gossypiifolia Linn. var. elegans Mueller leaf and Sauro- pus androgynus(L.)Merr leaf. The protein sequences of 5100, 5102, 5601 and 30640 are given in Example 2. 5100 and 5102 have 6 amino acids differently. 5102 shares 69% of identities of amino acids with 5601 . All four proteins share only 37% identities. As shown in example 6, the activity profiles of the OxOx 5100 and 5102 in the pH range of 4.5 to 8.0 are very similar, showing high activity at pH 7.0-8.0. However, the activity profile of the OxOx 5601 in the pH range of 4.5 to 8.0 is different from the ones for 5100 and 5102, but similar to the one of 30640, showing a little activity at neutral pH.

Polynucleotides

The present invention also relates to isolated polynucleotides encoding the oxalate oxi- dases according to the invention, i.e. oxalate oxidases having a suitable (as defined herein) activity at pH 7-8

Thus, in another aspect the invention relates to a nucleotide having a nucleic acid sequence with an identity of at least 60% with SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7. In particular the identity is at least 70% at least 75% at least 80% or at least 85%. Specifically, the identity is at least 90%, at least 95%, at least 98%, at least 99% or at least 100%.

As shown in example 2, Genes 5100 and 5102 have 8 nucleotides differently. 5102 shares 69% of identities with 5601 . All 4 genes share only 43% identities.

Nucleic acid constructs

The present invention relates to nucleic acid constructs comprising a polynucleotide encoding an oxalate oxidase of the present invention operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host under conditions compatible with the control sequence.

The polynucleotide may be manipulated in a variety of ways to provide for expression of an oxalate oxidase. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA are well known to a person skilled in the art. The control sequence may be a promoter, a polynucleotide recognized by a host cell for expression of a polynucleotide encoding an oxalate oxidase of the present invention. The promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heter- ologous to the host cell.

Examples of suitable promotes for directing transcription of the nucleic acid constructs of the present invention in a bacterial cell are promoters obtained from bacteria, virus or others, such as Lac, Trp, Tac, T7, P _L , or P _R .

Examples of suitable promotes for directing transcription of the nucleic acid constructs of the present invention in a fungal host cell are promoters obtained from yeast such as AOX1 , GAP. Examples of suitable promotes for directing transcription of the nucleic acid constructs of the present invention in a yeast host cell are promoters obtained from yeast such as AOX1 , GAP.

The control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator is operably linked to the 3'-terminus of the polynucleotide encoding an oxalate oxidase. Any terminator that is functional in the host cell may be used such as TAA, TGA, TAG.

The control sequence may also be a leader a non-translated region of an mRNA that is important for the translation by the host cell. The leader is operably linked to the 5'- terminus of the polynucleotide encoding an oxalate oxidase of the invention. Any leader that is functional in the host cell may be used.

Suitable examples of leaders may be alpha-mating factor secretion signal, PH01 , PHA-E, alpha-amylase signal sequence (An-amyS), Glucoamylase signal sequence (Aa-GluS), Inulinase signal sequence (Km-lnuS), Invertase signal sequence (Sc-lnvS). Other examples are HFB1 , HFB2, Sew, Dse, Exg, XPR2pre ,or Lip2prepro.

Expression vectors

The present invention also relates to recombinant expression vectors comprising a polynucleotide encoding an oxalate oxidase of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the oxalate oxidase at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide in a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g. a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide. The choice of vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

The vector may contain one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for both biocide or viral resistance, resistance to heavy metals, and the like.

Suitable markers may be ampicillin, kanamycin, or neomycin.

For integration into the host cell genome, the vector may rely on the polynucleotide's sequence encoding the oxalate oxidase or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s). For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term "origin of replication" or "plasmid replicator" means a polynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of origins of replication suitable for use in the present context are

pUC, and p15A.

More than one copy of polynucleotide of the present invention may be inserted into a host cell to increase the production of oxalate oxidase. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well-known to a person skilled in the art.

Host cells

The present invention also relates to recombinant host cells, comprising a polynucleotide encoding an oxalate oxidase of the present invention operably linked to one or more control sequences. A construct or vector comprising a polynucleotide is intro- duced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of host cell will to a large extent depend upon the gene encoding the oxalate oxidase and its source. The host cell may be any cell useful in the recombinant production of an oxalate oxidase, e.g. a prokaryote or a eukaryote.

The prokaryotic host cell may be a bacterium such as E.coli, or Bacillus subtilis

The eukaryotic host cell may be a mammalian, insect, plant or fungal cell. The fungal cell may be a yeast cell. Suitable examples are Pichia pastoris X-33, GS115, Yar- rowia lipolytica, Lactuca sativa L, Pisum sativum, and Nicotiana benthamiana. Methods for producing oxalate oxidases

The present invention also relates to methods of producing recombinant oxalate oxidases of the present invention, the method comprising i) cultivating a recombinant host cell of the present invention under conditions conductive for production of the oxalate oxidase, and ii) optionally recovering the oxalate oxidase.

The host cells are cultivated in a nutrient medium suitable for production of the oxalate oxidase using method well known in the art. For example the cells may be cultivated in multi-well plates, shake flask cultivation or small-scale or large-scale fermentation (including continuous, batch, fed-batch or solid state fermentation) in laboratory or indus- trial fermenters in a suitable medium and under conditions allowing the oxalate oxidase to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are: Small scale cultivation media: LB+2mM MnCI2

Large scale fermentation media:

Base medium (per L): KH ₂ P0 ₄ 13.5 g, Citrate acid. H20 0.85 g, yeast powder 10 g, MgS0 ₄ .7H ₂ 0 1 g, trace metals solution 2.5 ml, Antifoam 204: 400 microliter, 100 mg/ml Amp 1 ml. Set pH to 6.85 after autoclaving. (Trace metals solution and Anti- foam 204 are added after autoclaving. )

Trace metals solution (per L):

H ₂ S0 ₄ 0.5 ml, CuS0 ₄ .5H ₂ 0 25 mg, MnS0 ₄ 2.28 g , Na ₂ Mo0 ₄ -2H ₂ 0 25 mg,

ZnS0 ₄ .7H ₂ 0 0.5 g, CoCI ₂ -6H ₂ 0 45 mg, H3B03 0.5 g, CaCI ₂ .2H ₂ 0 3.25 g, FeS0 ₄ -7H ₂ 0 5 g.

Feed medium (per L): glycerol 300 g, yeast powder 60 g.

The oxalate oxidase may be detected using methods known in the art that are specific for oxalate oxidases. These detection methods include, but are not limited to colorimet- ric assay, HPLC method.

The oxalate oxidase may be purified by a variety of procedures known in the art including, but not limited to, chromatography, dialysis, gel separation techniques etc. Uses

As mentioned herein before a recombinant enzyme or a suitable derivative thereof according to the invention can be used in the treatment of diseases associated with the presence of excess of oxalate (compared to normal values). Examples of such disease are given above.

Oxalate oxidase compositions

It is envisaged that the recombinant oxalate oxidases as such or, if necessary, in a form, which reduces the possibility of unwanted immunological reactions in a patient, or which enhances e.g. the half-life of the enzyme in a body fluid such as blood or plas- ma. A general method is to subject the enzyme to pegylation.

The present invention also relates to pharmaceutical compositions for use in treating or preventing diseases associated with excess oxalate, wherein the composition contains a recombinant oxalate oxidase or a derivative thereof (e.g. a pegylated oxalate oxi- dase) having suitable activity at pH 7-8 (as described above).

The compositions may be designed to oral, parenteral or mucosal administration. Thus the administration may be oral, sublingual, application to the oral mucosa, or it may be intraveneous, subcutaneous, intramuscular, intraperitoneal, intrahecal etc. or it may be applied to the skin or a mucosa surface including ocular, buccal, nasal, vaginal and rectal mucosa.

The composition may be in solid, semi-solid or liquid form. Suitable solid compositions include powders, granules, pellets, capsules, tablets (included coated tablets), sachets, implants etc. Suitable semi-solid compositions include gels, pastes, cremes, ointments, vagitories, suppositories etc. Suitable liquid or fluid compositions include solutions, dispersion, emulsions, suspension, lotions, sprays, aerosols

The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the enzyme with the carrier which constitutes one or more accessory ingredients. In general the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. As mentioned above the recombinant oxalate oxidase may be administered orally in the form of an enteric coated pharmaceutical formulation or a pharmaceutical composition which by other means are protected from degradation of the enzyme in the stomach. The pharmaceutical formulation comprises the recombinant enzyme in a pharmaceutically acceptable dosage form. Depending upon the disorder and patient to be treated, as well as the route of administration, the compositions may be administered at varying doses.

For example, the recombinant enzymes of the invention can be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed- or controlled-release applications. For peroral administration means must be taken to avoid release or degradation of the enzyme in the stomach.

Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscar- mellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC), sucrose, gelatine and acacia. Additionally, lubricating agents such as magnesium stea- rate, stearic acid, glyceryl behenate and talc may be included. Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the compounds of the invention may be combined with various sweetening or flavour- ing agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

A tablet may be made by compression or moulding, optionally with one or more acces- sory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g. povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g. sodium starch glycolate, cross- linked povidone, cross-linked sodium carboxymethyl cellulose), surface-active or dis- persing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated e.g. with an enteric coating and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hy- droxypropylmethylcellulose in varying proportions to provide desired release profile.

Compositions for use in accordance with the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil- in-water liquid emulsion or a water-in-oil liquid emulsion.

It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.

Advantageously, agents such as preservatives and buffering agents can be dissolved in the vehicle. To enhance the stability, the composition can be frozen after filling into the vial and the water removed under vacuum. The dry lyophilized powder is then sealed in the vial and an accompanying vial of water for injection may be supplied to reconstitute the liquid prior to use. The composition may also be designed for parenteral administration. Such compositions may contain excipients, which are pharmaceutically acceptable for a composition to be injected. These may be in particular isotonic, sterile saline solutions (phosphate, sodium chloride) or dry, especially freeze-dried compositions, which upon addition, depending on the case, of sterilized water or physiological saline, are reconstituted to a ready-to-use composition.

The pharmaceutical compositions suitable for injection include sterile aqueous solu- tions or dispersions. The compositions may include solvents like water or ethanol, sesame oil, peanut oil or the like, glycerol or propylene glycol. pH-adjusting agent may be added to adjust pH of the composition and preservatives may also be added.

A person skilled in the art will be able to prepare a composition that is suitable for use in accordance with the present invention based on the disclosure herein and/or with guidance from Remington's Pharmaceutical Sciences, 18 ^th Ed. Mack Publishing Company, 1990 or newer editions.

The dosage to be administered of an oxalate oxidase will vary according to the particu- lar disease involved, the subject, and the nature and severity of the disease and the physical condition of the subject, and the selected route of administration.

The appropriate dosage can be readily determined by a person skilled in the art. The compositions may contain from 0.1 % by weight, preferably from 5-90%, more preferably from 10-80% by weight, of an oxalate oxidase of invention, depending on the particular composition and the method of administration.

It will be recognized by one of skill in the art that the optimal quantity and spacing of in- dividual dosages of a compound of the invention will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the age and condition of the particular subject being treated, and that a physician will ultimately determine appropriate dosages to be used. This dosage may be repeated as often as appropriate. If side effects develop the amount and/or frequency of the dosage can be altered or reduced, in accordance with normal clinical practice Definitions

cDNA: The term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature, spliced mRNA molecule obtained from a cell. cDNA lacks in- tron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term "coding sequence" means a polynucleotide, which directly specifies the amino acid sequence of the enzyme or variant of the enzyme. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG or TTG and ends with a stop codon such as TAA, TAG or TGA. The coding sequence may be genomic DNA, cDNA, synthetic DNA, or a combination thereof. Control sequences: The term "control sequences" means nucleic acid sequences necessary for expression of a polynucleotide encoding an oxalate oxidase or variant of the present invention. Each control sequences must be native (i.e. from the same gene) or foreign (i.e. from a different gene) to the polynucleotide encoding the oxalate oxidase or variant thereof or native or foreign to each other. Such control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequence with the coding region of the polynucleotide encoding the oxalate oxidase or variant thereof. Expression: The term "expression" includes any step involved in the production of an oxalate oxidase or variant thereof including, but not limited to, transcription, post- transcriptional modification, translation, post-translational modification, and secretion.

Expression vector: The term "expression vector" means a linear or circular DNA mol- ecule that comprises a polynucleotide encoding an oxalate oxidase or variant thereof and is operably linked to control sequences that provide for its expression.

Fragment: The term "fragment" means a polypeptide having one or more (e.g. several) amino acids absent from the amino and/or carboxyl terminus of the polypeptide thereof, wherein the fragment has oxalate oxidase activity. The fragment may have at least 85% of the amino acid residues e.g. at least 90% or at least 95% of the amino acid residues of the mature polypeptide.

Host cell : The term "host cell" means any cell type that is susceptible to transfor- mation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide as described herein.

Isolated: The term "isolated" means a substance in the form or environment that does not occur in nature. Non-limiting examples of isolated substances include i) any non- naturally occurring substance, ii) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide or cofactor, that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature, iii) any substance modified by the hand of man relative to that substance found in nature, or iv) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated.

Mutant: The term "mutant" means a polynucleotide encoding a variant.

Nucleic acid construct: The term "nucleic acid construct" means a nucleic acid mole- cule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, which comprises one or more control sequences. Operably linked: The term "operably linked" means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide, such that the control sequence directs expression of the coding sequence.

Optimum pH: The term "optimum pH" means the pH value where the enzymatic activ- ity is at its maximum level or at least 80% e.g. 85%, 90%, 95% or 98% of its maximum level.

Oxalate-related disease: The term "oxalate-related disease" means and disease or condition caused by an excess of oxalate in the body compared to normal level. Such disease or conditions include, but are not limited to, primary hyperoxaluria (PH), secondary hyperoxaluria (SH), Zellweger spectrum disorders (ZSD), and chronic renal fail- ure or end-stage renal failure (ESRF), autism, vulvodynia, oxalosis associated with end-stage renal disease, cardiac conductance disorders, Crohn' s disease, inflammatory bowel disease, colitis, urolithiasis, oxalosis associated with end-stange renal disease, sarcoidosis, asthma, COPD, fibromyalgia, Zellweger syndrome, bariatric surgery and other enteric disease states.

Sequence identity: The term "sequence identity" means the relatedness between two amino acid sequences or between two nucleotide sequences. Blast algoritm was used. It is provided by pubmed.gov, i.e., national center for biotechnology information.

Specific activity: The term "specific activity refers to the enzyme activities per mg of protein.

Unit: One unit of activity is defined as the enzyme amount required to produce 1 μηηοΐβ of formate from oxalate under the conditions described in Example 6 herein.

Variant: The term "variant" means a polypeptide having oxalate oxidase activity comprising an alteration, i.e. a substitution, insertion, and/or deletion, at one or more (e.g. several) positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of an amino acid occu- pying a position; and an insertion means adding an amino acid adjacent to and immediately following the amino acid occupying the position. The variants of the present invention have at least 20%, e.g. at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% of the oxalate oxidase activity of the parent oxalate oxidase.

Legends to figures

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).

Fig. 2 Schematic diagram of the Pichia expression plasmid Za-5102.

Fig. 3 Schematic diagram of the Pichia expression plasmid GAPZa-51 02.

Fig. 4 Schematic diagram of the Pichia expression plasmid Za-5601 . Fig. 5 Schematic diagram of the Pichia expression plasmid GAPZa-5601 . Fig. 6 Schematic diagram of the Pichia expression plasmid Za-30640.

Fig. 7 Schematic diagram of the Pichia expression plasmid GAPZa-5102.

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 (ZaA-5102).

Fig. 9 SDS-PAGE analysis of the expression of beet OxO in P. pastoris. M: protein marker; lanes 1 -9, X-33(Za-5601 ).

Fig. 10 SDS-PAGE analysis of the expression of banana OxO in P. pastoris. M: protein marker; lanes 1 -10, X-33(ZaA-30640).

Fig. 11 SDS-PAGE analysis of the expression of beet OxO in P. pastoris. M: protein marker; lanes 1 -9, X-33(GAPZa-5601 ). Fig. 12 PCR amplification of the OxO genes from cDNA library of sugar beet.

M. DNA Maker; Lanel -4, 5601 ; Lane5-8, 5102

Fig. 13 PCR amplification of the OxO gene from cDNA library of banana.

M. DNA Maker; Lanel -3, 30640

Fig.14 Schematic diagram of the plant expression vector

Fig.15 Histochemical assay of oxalate oxidase activity after overnight incubation in OxOx histological buffer

Fig.16 Detection of OxO activity of the purified cell wall fraction from pea plants by col- orimetric assay, pH 7.4. The left tube containing 2 mM oxalic acid; The right tube containing no oxalic acid. Fig. 17 Detection of OxOx activity of the purified 5102 fractions from pea plants. 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#. Fig. 19 Relative activity of recombinant oxalate oxidases.

Fig.21 Serum oxalate concentration curve from Example 12.

Fig.22 24h total urine oxalate curve from Example 12.

Fig 23 OxOx-B5102 antibody titer detection of different PEG-OxOx-B5102s from Example 12.

References

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Examples

Strains and plants

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

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

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

ligustrum japonicum

Pisum sativum Linn Thunb.var rvtundifolium B1

Solanum tuberosum L Pittosporum tobira

Solanum tuberosum L Acer palmatum

Colocasia esculenta E.pungens Thunb

Lactuca sativa L. var. capitata L. Prunus ceraifera

Bamboo Shoot Taiwania cryptomerioides Hayata

Brassica parachinensis Chaenomeles sinensis Koehne

Toona sinensis. A.Juss. Taxus wallichiana Zucc Pisum sativum Linn Ormosia henryi Prain Allium sativum L. Pyracantha fortuneana

Apium graveolens L. Mallotus repandus (Willd.) Muell. Arg Hordeum vulgare L. Distylium racemosum Sieb.et Zucc Hordeum vulgare L. Viburnum setigerum Hance

Hordeum vulgare L. Jasminum mesnyi

Cruciferae Brassica Chimonanthus praecox(L.)Link Allium epa L. Hovenia acerba Lindl.

Alliaceae Allium A. tuberosum Mahonia fortunei (Lindl. )Fedde Dioscorea opposita Cercis chinensis Bge

Vigna radiata Chionanthus retusus

Momordica charantia Fraxinus hupehensis

Cucumis sativus Linn Alpinia zerumbet

Benincasa hispida Asparagus myriocladus

Ananas comosus Cerasus conradinae (Koehne) Yu et Li Ananas comosus Juniperus chinensis cv. kaizuka Prunus ceraifera cv. Pissardii Brunfelsia acuminata(Pohl.)Benth Alliaceae Allium A. tuberosum Bambusa ventricosa McClure

Pisum sativum Linn Hedera nepalensis

Benincasa hispida Dendrobeaathamia capitata(WalL)

Hutoh.var.emeiensis(Fang et Hsi-

Benincasa hispida eh)Fang et.W.K.Hu

Vigna radiata Cyperus papyrus L.

Cruciferae Brassica Pilea nummulariifollia(Sw.)Wedd.

Momordica charantia Syngonium podophyllum

Ananas comosus :lresine herbstii Hook.f

Alliaceae Allium A. tuberosum Simlax China L

Alliaceae Allium A. tuberosum Gleditsia japonica

Benincasa hispida Euonymus grandiflorus Wall.

Vigna radiata Planch. cv.Fenghongpeier

Glycine max(L)Merrill Ilex cornuta Lindl. et Paxt.

Eleocharis dulcis Berchemia sinica Scheid

Symplocos sununtia Buch.-

Alliaceae Allium A. tuberosum Ham.ex. D.Don

Sorghum bicolor Dianthus chinensis L. Brassic campestris Olea europaea L

Galium aparine Begonia masoniana

Geranium carolinianumL. Marsilea quadrifolia

Euphorbia helioscopia Hibiscus rosa-sinensis L.

Portulaca oleracea L. Dracaena cochinchinen

Oxalis corniculata Common Rush

Vicia sepium linn. Manilkara zapota van Royen

Eugenia javanica Lam [ leaves ] Ligustrum lucidum Ait

Eugenia javanica Lam [fruit] Begonia thurstonin

Reineckea carnea Nephrolepis biserrata (Sw.) Schott Bougainvillea spectabilis wind Quisqualis indica

Bougainvillea spectabilis wind Asystasia gangetica

Sinojackta huanamelensis Podocarpus macrophyllus

Neolitsea sericea Artabotrys hexapetalus (L.f.) Bhandari

Heptacodium Miconiodes rehd Telosma cordatum ( Brum, f. ) Merr banana Forsythia viridissima

Populus bonatii Levi Osmanthus matsumuranus Hayata Vitis vinifera Phoebe zhennan S.Lee et F.N.Wei Prunus armeniaca L. Davidia involucrata Baill

Mirabilis jalapa Linn Neolepisorus ovatus

Mirabilis jalapa Linn Setcreasea pallida

Clerodendron japonicum (Thunb. )

Gordonia axillaris Sweet

Lindera aggregata (Sims) Ko- sterm Hydrangea macrophylla (Thunb.)Ser

Lllicium henryi Psidium littorale Raddi

PhoebechekiangensisC.B.Shang Savia miltiorrhiza Bunge

Fokienia hodginsii (Dunn) Henry

et Thomas Fagraea ceilanica

Viburnum macrocephalum

f.keteleeri Paeonia suffruticosa""

Zanthoxylum simullans Hance Sycopsis sinensis Oliver

Cinnamomum camphora Aesculus wilsonii Rehd

Acer albopurpurascens Hayata Edyeworthia chrysantha Lindl Daphniphyllum macropodum Miq 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 Vani- ot.) Rhizoma Alpiniae Officinarum

Podocarpus fleuryi Asarum forbesii Maxim

Tetracentron sinense Sarcandra glabra

Reinwardtia trigyna Peristrophe baphica

Valeriana hardwickii Peperomia tetraphylla

Fagopyrum dibotrys ( D.Don )

Hara Erythrina crista-galli L

Thalia dealbata Rumex japonicus Houtt

Muscari botryoides Mill. Melodinus hemsleyanus Diels. Fagus longipetiolata Seem Tibouchina aspera

Jussiaea reppens L. Rumex hastatus

Podocarpus fleuryi Hibiscus Sabdariffa Linn Ardisia crenata Sim Catharanthus roseus

Torreya fargesii Franch Tragopogon porrifolius Linn Aesculus wangii Hu ex Fang Mussaenda pubescens Ait. f. Nerium oleander Sedum sarmentosum Bunge Strobilanthes cusia Pilea cavaleriei Levi

Drynaria fortunei Sauropus rostrata Miq

Belamcanda chinensis Acorus gramineus

Kalimeris indica (Linn.) Sch. ) Euphorbia neriifolia L.

Rhoeo discolor Euphorbia tirucalli Linn

Fiveleaf Gynostemma Herb Dianthus serotinus

Sterculia nobililis Codariocalyx motorius

Choerospondias axillaris Acalypha hispida

Citrus medica L.var.sarcodactylis Acalypha hispida

Gynostemma compressum Garcinia oblongifolia

Chrysanthemum indicum Cynanchum stauntonii

SpilsheS acmella ( Linn ) Murr Aloe vera var. chinensis

Ilex kudmcha C. J. Tseng Polygonum capitatum

Cinnamomum cassia Iris japonica Rumex crispus L. var. japonicus Acanthopanax gracilistylus Clerodendranthus spicatus

(Thunb.) Dicranostigma leptopodum

Glechoma longituba Iris wilsonii

HymenocallisSpciosa Solanum mammosum Linn. Agrimonia pilosa Ledeb Rheum officinale

Dimocarpus longgana Lour. Rabdosia nervosa (Hemsl.) Lxora chinensis Plumbago zeylanica L Rhapis excelsa (Thunb.) Desmodium styracifolium Eranthemum pulchellum Andrews Salvia substolonifera Stib. Hypericum perforatum L Aspidistra elatior Bl.

Hippeastrum rutilum(Ker- Gawl.)Herb. Achyranthes bidentata Bl

Pogonatherum crinitum (Thunb.)

Kunth Achyranthes bidentata Bl

Echinacea purpurea Moench Pandanus tectorius

Pseudocalymma al- liaceum(Lam.)Sandwith Lygodium scandens (L. ) Sw. Sanguisorba officinalis L. Asparagus cochinchinensis desmodium triquetrum Lonicera dasystyla

Rabdosia japonica (Burm. f.) Hara Zanthoxylum nitidum

Tadehagi pseudotriquetrum (DC.) Billbergia pyramidalis Houttuynia cordata Thunb. Ficus sarmentosa

Gynura procumbens (Lour.) Merr. Pyrrosia drakeana

Vinca major Linn Ardisia pusilla A. DC.

Zephyranthes carinata Herb Boehmeria nivea

Platycodon grandiflorus Artemisia japonica Thunb Aquilaria sinensis Forsythia suspensa

Salvia cavaleriei Ardisia gigantifolia stapf Datura innoxia Mill. Tacca chantrieri Andre Codariocalyx gyroides Morinda officinalis How. Aerva sanguinolenta Ardisia villosa

Aster novi-belgii Hemerocallis citrina Baroni Colocasia antiquorum Schott Goodyera procera

Heteropanax fragrans Fallopia multiflora Thunbergia laurifolia Lindl. Campsis radicans (L.) Seem.

Iris germanica Piper longum Linn.

Alocasia macrorrhiza Passionfora edulis

Gelsemium elegans Prunella vulgaris

Cocculus laurifolius DC. Jasminum nudiflorum

Podocarous macrophyllus Kadsura longipedunculata

Syzygium jambos (L.) Alston Mentha haplocalyx

Buddleja asiatica Lour. Evodia lepta (Spreng.) Merr

Buddleja davidii Acacia confusa Merr

Caesalpinia decapetala Stephania kwangsiensis

Jatropha gossypiifolia Tetrastigma hemsleyanum

Peucedanum praeruptorum Dunn Herba Cayratica Jeponicae

Carpesium abrotanoides Linn. Asarum maximum Hemsl.

Euphorbia heberophylla L Argyreia acuta Lour.

Coleus amboinicus Lour Solallum nigrum L .ver

Lycoris aurea Curculigo capitulata

Sauropus androgynus(L.)Merr Fructus Amomi

Houttuynia emeiensis Polygonum chinense L.

Sophora flavescens Rosa chinensis

Salvia coccinea Linn selaginella moellendorfii hieron a plant in Tadehagi Herba Aristolochiae

Rhinacanthus nasutus (L.) Kurz Rubus cochinchinensis Tratt

Ardisia japonica (Thunb) Blume Rosa multiflora Thunb.

Trachelospermum jasminoides Rhododendron pulchrum Sweet Wisteria sinensis Dichotomanthus tristaniaecarpa Kurz Duchesnea indica Cyclobalanopsis glaucoides Schotky

Rhododendron anthopogonoides Ma¬

Ajuga decumbens Thunb xim

Potentilla kleiniana Malus halliana (Voss.) Koehne Pteris semipinnata L. Pistacia weinmannifolia

Senecio cannabifolius Less Ginkgo biloba

Ophitopogin japonicum Melaleuca bracteata

Polygonum hydropiper L. Rhododendron fuyuanense Z. H. Yang Pueraria lobata Ligustrumx Vicaryi

Mentha spicata Linn. Euonymus Japonicus Lonicera fulvotomentosa Cerasus cerasoides

Acacia confusa Merr Rhododendron 'nanhai'

Erythropalum scandens Bl. Rhododendron 'suimohua'

Caryota mitis Lour. Rhododendron delavayi Franch

Cirsium segetum Exochorda racemosa (Lindl.) Rehd

Curcuma kwangsiensis Rosmarinus officinalis

Artemisia argyi Cotoneaster franchetii

Limnophila sessiliflora Prunus serrulata

Alpinia katsumadai Hayata Pinus armandii Franch

Blumea balsamifera DC Pittosporum brevicalyx (Oliv.) Gagnep

Ficus pumila L Osyris wightiana Wall, ex Wight

Bryophyllum pinnatum Rhododendron 'mayingtao'

Agastache rugosa Rhododendron hancockii Hemsl.

Polygonum chinense L Rhododendron ciliicalyx Franch.

Jasminum amplexicaule Amygdalus persica

Gnetum montanum Markgr Abelia parvifolia Hemsl.

Rhododendron pachypodum Lonicera nitida 'Baggesens

Parakmeria yunnanensis Rhodedenron schlippenbachii

Sinocalycanthus chinensis Amygdalus persica f. atropurpurea

Cerasus campanulata Pyracarltha fortuneana

Quercus variabilis Blume Trigonobalanus verticillata

Rhododendron 'Wanxia' Rhododendron 'kunming'

Spiraea japonica L. f. Acer cappadocicum

Eucommia ulmoides Oliver Rhododendron mucronatum

Hovenia acerba Rhododendron 'fenpao'

Rhododendron fortunei Dianthus plumarius

Rhododendron leptothrium Musella lasiocarpa

magnolia grandiflora linn Rhododendron irroratum Franch

Rhododendron pachypodum Pistacia chinensis

Barleria cristata Hovenia acerba Lindl.

Vitex negundo Linn Michelia lacei W. W. Smith

Viburnum opulus L Distylium pingpienense (Hu) Walk.

Spiraea japonica L. f. Sedum Nicaeense All.

Cotoneaster salicifolius Aglaia odorata

Rhododendron albersenianum Ilex crenata cv. Convexa Makino Eriobotrya bengalensis Rhododendron 'wanxia'

Cerasus clarofolia Spiraea cantoniensis

Rhododendron vialii Lorpetalum chindensevar.rubrum Amygdalus persica Linn Photinia serrulata

Ailanthus altissima Rhododendron rigidum Franch Rhododendron taronense Rhododendron annae Franch. Rhododendrin davidii Rhododendron sinogrande

Sapiumsebiferum ( L. ) Roxb Cotoneaster horizontalis Dene Michelia maudiae Dunn Rhododendron aberconwayi Cowan Rhododendron spinuliferum Podocarpus macrophyllus var. maki Machilus yunnanensis Rhododendron decorum Fr Liquidambar formosana Hance Itea yunnanensis Franch.

Davidia involucrata Baill Lonicera nitida'Maigrun'

Prunus salicina Lindl. Michelia figo

Liriodendron chinensis ( Hemsl. )

Sinomanglietia glauca Sarg

Liquidambar formosana Hance Olea ferruginea Royle

Acer negundo L. Eriobotrya japonica

Magnolia soulangeana Soul. Luculia intermedia Hutch

Vinca minor Linn. Rhododendron 'jinzhizhu'

Rhododendron ciliatum Hook. f. Sect.Dacrycarpus Endl

magnolia grandiflora linn Ophiopogon japonicus cv . 'Nanus' Trigonobalanus doichangensis Ligustrum japonicum'Howardii' Hedera nepalensis Rhododendron pulchrum

Quercus variabilis Pvracantha angustifolia

Rhododendron simsii Chamaecyparis lawsoniana

Ilex cornuta Cedrus deodara

Fatsia japonica Thujopsis dolabrata

Lindera communis Ehretia dicksonii Hance

Pistacia weinmannifolia Acer cappadocicum

Alstonia yunnanensis Diels Camellia sasanqua

Machilus thunbergii Araucaria araucana

Bischofia polycarpa Ficus curtipes Corner

Cercidiphyllum japonicum Manglietia duclouxii

Viburnum odoratissimum Stranvaesia davidiana Lamium galeobdolon Rhamnus gilgiana Heppl.

Chaenomele japonica Agrimonia pilosa Ledeb.

Spiraea blumei Caragna fruten (L) koch

Agapanthus africanus Euonymus fortunei

Musella lasiocarpa Pieris formosa

Cryptomeria fortunei Fraxinus retusifoliolata

Weigela florida Sapindus delavayi

Paeonia lactiflora Elaeocarpus sylvestris

Parthenocissus tricuspidata Carayaillinoensis

Acer palmatum'Dissectum' Micheliamacclurel

Bougainvillea spectabilis Eurya loquaiana Dunn

Aesculus wangii Ficus virens

Mahonia fortunei Buxus microphylla

Osmanthus fragrans Lithocarpus henryi

Exbucklandia populnea Ficus religiosa Linn

Cinnamomum bodinieri Ligustrum lucidum

Machilus longipedicellata Disporopsis pernyi

Ficus virens Ait. Manglietiainsignis

Duranta repens Linn. Olea cuspidata

Manglietia grandis Hypoestes sanguinolenta

Olea europaea L. Cornus officinalis

Parakmeria yunnanensis Daphniphyllum longeracemosum

Tamarix chinensis Reineckia carnea

pyrus pyrifolia Acorus gramineus

Prunus serrulata Cerasus cerasoides

Iris confusa Dipteronia dyeriana Henry

Iris wilsonii Lindera megaphylla

Bambusa multiplex Liquidambar formosana Hance

Senecio scandens Podocarpus fleuryi

Neolitsea sericea Morus alba L

Fokienia hodginsii Zanthoxylum acanthopodium

Viburnum odoratissimum Ficus altissima Bl.

Cerasus pseudocerasus Iresine herbstii

Ilex cornuta Cedrus deodara

Fatsia japonica Thujopsis dolabrata Lindera communis Ehretia dicksonii Hance

Pistacia weinmannifolia Acer cappadocicum

Alstonia yunnanensis Diels Camellia sasanqua

Machilus thunbergii Araucaria araucana

Bischofia polycarpa Ficus curtipes Corner

Cercidiphyllum japonicum Manglietia duclouxii

Viburnum odoratissimum Stranvaesia davidiana

Lamium galeobdolon Rhamnus gilgiana Heppl.

Chaenomele japonica Agrimonia pilosa Ledeb.

Spiraea blumei Caragna fruten (L) koch

Agapanthus africanus Euonymus fortunei

Musella lasiocarpa Pieris formosa

Cryptomeria fortunei Fraxinus retusifoliolata

Weigela florida Sapindus delavayi

Paeonia lactiflora Elaeocarpus sylvestris

Parthenocissus tricuspidata Carayaillinoensis

Acer palmatum'Dissectum' Micheliamacclurel

Hypoestes sanguinolenta Eurya loquaiana Dunn

Aesculus wangii Ficus virens

Mahonia fortunei Buxus microphylla

Osmanthus fragrans Lithocarpus henryi

Exbucklandia populnea Ficus religiosa Linn

Cinnamomum bodinieri Ligustrum lucidum

Machilus longipedicellata Disporopsis pernyi

Ficus virens Ait. Manglietiainsignis

Duranta repens Linn. Olea cuspidata

Manglietia grandis Rhododendren simsii

Grape Strawberry

Loquat Pineapple leaves

Pineapple sarcocarp White gourd

Cucumber Young garlic shoot

Chives flower Purple cabbage

Tender leaf of Chinese toon Purple Chinese cabbage flower

Bamboo shoots Cabbage lettuce

Potato Potato sprout Chinese lettuce Horse bean

Pea shoots Taro

Pakchoi cabbage flower Pakchoi cabbage

Crowndaisy chrysanthemum Coriander herb

OxOx Activity Assay (HPLC method): T e 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 Dl water for OxOx activity analysis. 40 μΙ of solution or suspension was incubated with 360 μΙ 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 μΙ_ 1 .5 N H ₂ S0 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 su- pernatant was analyzed by an HPLC method to detect oxalate. One unit of activity is defined as the amount of enzyme required to degrade 1 μηιοΐθ of oxalate in one minute under the above conditions.

OxOx activity assay (colorimetric method): 20 μΙ_ solution sample is mixed into 580 μΙ_ 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 μηιοΐθ of oxalate in one minute under the above conditions.

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. Results

OxOx from banana peel, beet stem, Bougainvillea spectabilis leaves, Mirabilis jalapa young leaf, Telosma cordatum (Brum, f.) Merr leaf, Jatropha gossypiifolia Linn. var. el- egans 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. 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

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 genel 013, SEQ ID. 15) and sweet beet (the genel 08, 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 pro- tein 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. Results

DNA and protein sequences of beet OxOx 5100, 5102, 5601 and banana OxOx 30640. SEQ ID 1 - 5100 DNA:

ATGGTCTTTGCAATGAGCTTTACTTCTCATATTTAC- GTGGCTTCGGCCTCTGATCCTGGTCTCCTACAGGATTTTTGTGTGGGTG-

TAAATGACCCTGATTCAGCAGTGTTTGTAAATGGAAAATTCTGCAAGAACCCAAAA- GACGTGACAATCGACGATTTCTTATACAAAGGGTTTAATATTCCCTCAGA- CACAAACAACACTCAAAGAGCAGAAGCCACACTAGTAGATGTCAATCGAT- TTCCAGCACTTAACACATTAGGTGTAGCCATGGCTCGTGTAGACTTT- GCGTCCTTTGGCCTAAACACACCTCATTTGCACCCTCGTGGTTCTGAGA- TATTCGCGGTCCTAGAGGGGACTTTATATGCCGGCATTGTCACCACCGATAATAA- G CTTTTCG AC ACGGTGTTG A-

GAAAGGGTGACATGATTGTTTTCCCTCAAGGCTTAATCCACTTCCAGCTTAATCTT- GGCAAGACAGATGCTCTTGCTATTGCCTCTTTTGGGAGCCAATTTCCTGGACGAG- TTAATGTTGCTAATGGTGTCTTTGGAACTACGCCACAAATTTT- GGATGATGTACTTACCCAAGCGTTTCAGGTAGATAAGATGGTGATTGAG- CAACTTCGATCTCAGTTTTCAGGTCCAAACACATCAATCAACACTGGAA- GATCTATTCTTAAACTCTTAACTGATGTTGCT

SEQ ID 2 - 5100 protein:

MVFAMSFTSHIYVASASDPGLLQDFCVGVNDPD-

SAVFVNGKFCKNPKDVTIDDFLYKGFNIPSDTNNTQRAEATLVDVNRFPAL-

NTLGVAMARVDFASFGLNTPHLHPRGSEIFAVLEGTLYAGIVTT-

DNKLFDTVLRKGDMIVFPQGLIHFQLNLGKT-

DALAIASFGSQFPGRVNVANGVFGTTPQILDDVLTQAFQVDKMVIEQLRSQFSGPN- TS I NTG RS I LKLLTD VA

SEQ ID 3 - 5102 DNA Sequence (w/o N-terminal signal peptide sequence, 648 bp):

TCTGATCCTGGTCTCCTACAGGATTTTTGTGTGGGTGTAAATGACCCTGATTCAG-

CAGTGTTTGTAAATGGAAAATTCTGCAAGAACCCAAAAGACGTGACAATCGAC- GATTTCTTATACAAAGGGTTTAATATTCCCTCAGACACAAACAACACTCAAAGAG- CAGAAGCCACACTAGTAGATGTCAATCGATTTCCAGCACTTAACACATTAGGTG- TAGCCATGGCTCGTGTAGACTTTGCGTCCTTTGGCCTAAACACACCTCATTT- GCACCCTCGTGGTTCTGAGATATTCGCGGTGCTAGAGGGGACTTTATATGCCGG- CATTGTCACCACCGATTACAAGCTTTTCGACACGGTGTTGA- GAAAGGGTGACATGATTGTTTTCCCTCAAGGCTTAATCCACTTCCAGCTTAATCTT- GGCAAGACAGATGCTCTTGCTATTGCCTCTTTTGGGAGCCAATTTCCTGGACGAG- TTAATGTTGCTAATGGTGTCTTTGGAACTACGCCACAAATTTT- GGATGATGTACTTACCCAAGCGTTTCAGGTAGATGAGATGGTGATTCAG- CAACTTCGATCTCAGTTTTCAGGTCAAAACATATCAATCAACACTGGAA- GATCTATTCTTAAACTCTTAACTGATGTTGCT SEQ ID 4 - 5102 Protein Sequence (w/o N-terminal signal peptide sequence, 216 aa): SDPGLLQDFCVGVNDPDSAVFVNGKFCKNPKDVTIDDFIYKGF- NIPSDTNNTQRAEATLVDVNRFPALNTLGVAMARVDFASFGLNTPHLHPRGSEI- FAVLEGTLYAGIVTTDNKLFDTVLRKGDMIVFPQGLIHFQLNLGKT- DALAIASFGSQFPGRVNVANGVFGTTPQILDDVLTQAFQVDEMVIQQLRSQFSGPN- TS I NTG RS I LKLLTD VA

SEQ ID 5 - 5601 DNA Sequence (w/o N-terminal signal peptide sequence, 648 bp): TCCGATCCTGCACCCCTTCAAGATTTTTGTATTGCTGTAAATGATCCCAATTCTG- CAGTGCTTGTGAATGGAAAGCTTTGTAAGAACCCAAAAGAAGTGACAA- TAGATGATTTCTTGTACAAAGGGTTTAATATACCTGCAGACACAAACAACAC- TCAAGGAGCAAGTGCCACACTAGTGGACATTACTCTATTCCCTGCAG- TTAACACACAAGGAGTCTCCATGGCTCGTGTGGACTTTGCGCCATTT- GGACTAAACACCCCTCATTTACATCCTCGTGGCTCAGAGGTTTTCGCAG-

TGATGGAAGGGATTATGTATGCTGGTTTTGTGACCACTGATTATAAGCTCTATGATA - C AATTATA AA AA AG G GTG A-

TATTATTGTGTTTCCACAAGGTCTAATTCATTTCCAACTTAATCTTGGGAAGA- CAGATGCTTTAGCAATTGCCTCATTTGGGAGCCAAAATCCAGGGAGAATTAA- TATCGCTGACAGTGTGTTTGGTACTACTCCGCGTGTTCTAGATGATGTGCTTAC- CAAAGGATTTCAAATCGATGAGTTGTTGGTCAAGCAACTTCGTTCTCAGTTTTC- TACTGATAATATATCAACAAGCACTGGAAGGTCATTTTTGAAATTGC- TATCTGAAACTTAT SEQ ID 6 - 5601 Protein Sequence (w/o N-terminal signal peptide sequence, 216 aa): SDPAPLQDFCIAVNDPNSAVLVNGKLCKNPKEVTIDDFLYKGFNIPADTNNTQGA- SATLVDITLFPAVNTQGVSMARVDFAPYGLNTPHLHPRGSEVFAVMEGIMYAGFVTT- DYKLYDTIIKKGDIIVFPQGLIHFQLNLGKTDALAIASFGSQNPGRINI- ADSVFGTTPRVLDDVLTKGFQIDELLVKQLRSQFSTDNISTSTGRSFLKLLSETY

SEQ ID 7 - 30640 DNA Sequence (w/o N-terminal signal peptide sequence, 642 bp): TTTGATCCGAGTCCTCTCCAAGACTTTTGCGTTGCTGACTACGACTCCAAC- GTGTTTGTGAACGGATTCGCCTGCAAGAAAGCTAAGGATGTCACGG- CAGATGACTTCTACTTCACCGGCTTAGACAAGCCCGCGAGCACCGCCAAC- GAGCTTGGCGCAAACATCACTCTCGTCAACGTGGAAC-

GACTCCCAGGCCTCAACTCCCTTGGCGTCGCCATGTCTCGCATCGACTAC- GCGCCCTTCGGTCTCAACCCTCCTCACTCGCATCCACGATCGTCGGAGATACTG- CACGTGGCGGAAGGAACGCTCTACGCCGGCTTCGTCACCTCCAACAC- GGAAAACGGCAACCTTCTCTTCGCTAAGAAGCTGAAGAAGGGCGACGCGTTT- GTGTTCCCCAGGGGCCTCATACACTTCCAGTTCAACATCGGGGACAC- CGATGCGGTGGCGTTCGCTACCTTCGGCAGCCAGAGCCCGGGTCTCGTCAC- CACCGCCAACGCACTGTTCGGATCGAAGCCGCCCATCGCTGATTACATTCTT- GCCCAGGCCGTGCAGCTTAGCAAGACGACCGTGGGCTGGCTTCAGCAGCAG- CAGTGGTTGGACATCGCTCAAGAATATGGACAACGCTTAGTTCAAGCTAAT

SEQ ID 8 - 30640 Protein Sequence (w/o N-terminal signal peptide sequence, 213 aa): FDPSPLQDFCVADYDSNVFVNGFACKKAKDVTADDFYFTGLDKPASTANELGA- NITLVNVERLPGLNSLGVAMSRIDYAPFGLNPPHSHPRSSEILHVAEGTLYAGFV- TSNTENGNLLFAKKLKKGDAFVFPRGLIHFQFNIGDTDAVAFATFGSQSPGLVT- TANALFGSKPPIADYILAQ AVQ LS KTT VG W LQQQQW LD I AQ E YGQ R LVQ AN

DNA and protein sequences of beet germin-like proteins 108, 122, 303 and banana germin-like protein 1013. SEQ ID NO: 9 108 DNA sequence

ATGGCTCCCCTACTCTACCTTGTAGTATTCTTGCTT-

GCTCCTTTTCTCTCCCATGCTGCGGATCCCGATCCTTTGCTAGATTTTTGTG- TAGCGGACCTTAATGCCTCTCCCTCATTTGCTAATTTCCCTTGCAAACAAAC- CTCAAATGTGACCTCTGAAGATTTCTTCTTT- GATGGGTTTATGAATGAGGGAAACACATCAAACTCGTTT-

GGATCAAGGGTCACACCCGGAAACGTCCTCACATTTCCTGCCCTTAA-

TATGCTCGGGATTTCAATGAATCGGGTTGATCTTGCTGTGGATGG-

GATGAACCCGCCCCATTCCCACCCACGAGCAAGTGA-

GAGCGGTGTGGTGATGAAGGGGAGAGTTCTAGTAGGGTTCGTAACCACGGG- GAATGTGTACTATTCAAAGGTGTTGGTTCCAGGACAGATGTTT-

GTAATCCCAAGGGGGTTGGTTCATTTTCAAAAGAATGTTGGACAAAATAAGGCAC- TCATCATTACAGCTTTCAATAGTCAGAATCCAGGAGTAG-

TGTTATTATCCTCAACCCTGTTTGGTACAAACCCTTCAATTCCAGATGATGTTTTAA - GCCAAACTTTCCTAGTGGACCAGAGCATTGTCGAAGGAATAAAATCCAACTTTTGA

SEQ ID NO: 10 108 protein sequence MAPLLYLVVFLLAPFLSHAADPDPLLDFCVADLNASPSFANFPCKQTSNV- TSEDFFFDGFMNEGNTSNSFGSRVTPGNVLTFPALNMLGISMNRVDLAVDGMNP- PHSHPRASESGVVMKGRVLVGFVTTGNVYYSKVLVPGQMFVIPR- GLVHFQKNVGQNKALIITAFNS- QNPGVVLLSSTLFGTNPSIPDDVLSQTFLVDQSIVEGIKSNF ^*

SEQ ID N0: 11 122 DNA sequence:

Atggaagtcgtcgcagctgtatcttttctggccgtgttattggctctggtttcccctgcc ctcgccaatgatcctga- tatcttttctggccgtgttattggctctggtttcccctgccctcgccaatgatcctga- tatgctccaagatgtttgtgtcgctgattccacctctggagtgaaattgaatggattt- gcatgcaaggatgcagcaagcattacaccagaagacttcttctttgctggaa- tatccaaacccgg aatg acaaacaatacaatg aaatccctagtaaccgg agctaac- gtcgaaaagataccgggtttaaacacactcggagtgtccatgggtcg- tatcgacttcggcccaggtggtcttaacccacctcacactcacccac- gagccacagaaatggtctttgtgttatatggagaattggacgttggtttcctaac- tacttctaataagctcatttctaagcatattaaaactggtgaaacttttgtttttccta- gagggttagtccactttcagaaaaataatggggataaacctgctgctttagtcac- tgcttttaatagtcagttgcctggcacccaatcaatagctgccacgttgtttac- gtcgaccccacctgttccagataatgttttaactatgactttccaagtcggtacta- aacaagtccagaagatcaaggctaggctcgctcctaagaagtaa

SEQ ID N0: 12 122 protein sequence:

mevvaavsflavllalvspalandpdmlqdvcvadstsgvklngfackdaasitped - fffagiskpgmtnntmkslvtganvekipglntlgvsmgridfgpgglnp- phthpratemvfvlygeldvgflttsnkliskhiktgetfvfprglvhfqknngdk- paalvtafnsqlpgtqsiaatlftstppvpdnvltmtfqvgtkqvqkikarlapkk

SEQ ID NO: 13 303 DNA sequence: Atggcggctgtttgggtagtcttggtggtgctagcggcggcttttgctgttggggtcttt - gccagcgatcctgatatgcttcaagatgtttgtgttgctgatcgtacatctggaatatta g- tg aatgg attcacatgtaaaaatatg accatg ataacccctg aag acttcttcttcaccg - gaatttcacaaccaggccaaatcacaaataaaatccttggttctcgagtcaccg- gagcgaatgtgcaggacatccctggtctcaacaccttgggag- tctcgatggctcgtgtcgactttactccctacggtctaaacccacctcacattcacccta - gaatcgtccaccctcgtgccactgaaatgatctatgttcttaagggtgaattgtacgtt- ggttttataacgaccgacaataagctcatttccaaggttgttaaagctggagaagtattt - gttttccctagaggtttggctcactttcagaaaaacatgttgaaatatccagctgctgca tt- agctgccttcaacagccaacttccaggcactcaacaattt- gcagctgctctctttacttccaatcctcctgtgtctaatgatgtgtt- ggctcaggcttttaacattgacgaacacaatgtcaaaaagattagggctggccttactcc atag

SEQ ID NO: 14 303 protein sequence:

MAAVWVVLVVLAAAFAVGVFASDPDMLQDVCVADRTSGILVNGFTCKNMTMITPED- FFFTGISQPGQITNKILGSRVTGANVQDIPGLNTLGVSMARVDFTPYGLNP- PHIHPRATEMIYVLKGELYVGFITTDNKLISKVVKAGEVFVFPRGLAH- FQKNMLKYPAAALAAFNSQLPGTQQFAAALFTSNPPVSNDVLAQAFNIDEHNVK- KIRAGLTP

SEQ ID NO:15 1013 DNA sequenc

ATGGAGTCGCACTACACGAAGAGACCATTCCTCCTCTTTCTCTCCTTCAC-

CGTCCTCCTCGTGTTGATCCGCGCTGACCCTGATCCTCTCCAG-

GACTTCTGCGTCGCCGACCTCGGAGCTACTGTGGTCGTCAATGGGTTCCCGTG- CAAGCCCGCGTCCGGAGTCACGTCCGACGACTTCTTCTTCGCCG-

GACTGTCCAGGGAGGGCAACACCAGCAATATCTTCGGGTCCAACGTGACCAAC- GCCAACATGCTCAGCTTCCCGGGGCTCAACACCCTCGGCGTCTCCATGAAC- CGCGTCGACGTCGCCCCCGGCGGCAC- CAACCCGCCCCACAGCCACCCGAGGGCTAC- CGAGCTCATCATCCTCCTCAAGGGCCGGCTGCTGGTGGGGTTCATCAGCACCAG- TAACCAGTTCTTCTCCAAGGTCTTGAACCCCGGCGA-

GATGTTCGTGGTGCCCAAGGGGCTCATCCACTTCCAGTACAACGTCGGCAAGGA- GAAGGCGCTCGCCATCACCACCTTCGACAGCCAGCTCCCCGGAGTAG- TGATCGCCTCCACCACCCTGTTCGCATCGAATCCGGCGATTCCCGAC- GATGTGCTGGCCAAAGCTTTTCAGGTGGAC-

GCGAAGGTCGTCGCTCTCATCAAGTCCAAGTTTGAGAGATAA SEQ ID NO: 16 1013 protein sequence

MESHYTKRPFLLFLSFTVLLVLIRADPDPLQDFCVADLGATVVVNGFPCKPASGV- TSDDFFFAGLSREGNTSNIFGSNVTNANMLSFPGLNTLGVSMNRVDVAPGGTNP- PHSHPRATELIILLKGRLLVGFISTSNQFF-

SKVLNPGEMFVVPKGLIHFQYNVGKEKALAITTFDSQLPGVVIASTTL- FASNPAIPDDVLAKAFQVDAKVVALIKSKFER ^*

Example 3

Recombinant expression of oxalate oxidase by E. coli

Part 1 . Plasmid construction and protein expression

Expression plasmid construction:

The pAT plasmid was produced by deleting the DNA sequence between the 212 ^th bp and the 729 ^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 .

Protein production in small scale:

Cells were grown at 37°C in 200 ml_ LB medium (10 g tryptone, 5 g yeast extract, and 10 g NaCI in 950 mL deionized water.) supplemented with 100 μg mL ampicillin. When OD ₆₀₀ reached 0.6-0.8, expression was induced for 4 h at 37°C after addition of IPTG and MnCI 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.

Protein production in large scale:

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 μg/mL ampicillin and 5 mM MnCI2. 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 ₆₀₀ of 20 was induced with 0.8 mM IPTG, cultivated for another 20 h, and then harvested.

Part 2. Protein refolding

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.

Proteins were refolded by rapid dilution:

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.

The refolding buffers we have used:

I . 20 mM Tris, 300 mM NaCI, 1 mM MnCI ₂ , 1 mM GSH/0.2mM GSSG, pH8.0 2. 20 mM Tris, 300 mM NaCI, 1 mM MnCI ₂ , 1 mM GSH/0.2mM GSSG, 400 mM ar- ginine, pH8.0

3. 20 mM Tris, 300 mM NaCI, 1 mM MnCI ₂ , 1 mM GSH/0.2mM GSSG, 100 mM a- cyclodextrin, pH8.0

4. 20 mM Tris, 300 mM NaCI, 1 mM MnCI ₂ , 1 mM GSH/0.2mM GSSG, 2% b- cyclodextrin, pH8.0

5. 20 mM Tris, 300 mM NaCI, 1 mM MnCI ₂ , 1 mM GSH/0.2mM GSSG, 40% sucrose, pH8.0

6. 20 mM Tris, 300 mM NaCI, 1 mM MnCI ₂ , 1 mM GSH/0.2mM GSSG, 40% glucose, pH 8.0

7. 20 mM Tris, 1 mM MnCI ₂ , 1 mM GSH/0.2mM GSSG, pH8.0

8. 20 mM Tris, 1 mM MnCI ₂ , 1 mM GSH/0.2mM GSSG, 400 mM arginine, pH8.0

9. 20 mM Tris, 1 mM MnCI ₂ , 1 mM GSH/0.2mM GSSG, 50 mM betaine, pH8.0

10. 20 mM Tris, 1 mM MnCI ₂ , 50 mM betaine, pH8.0

I I . 20 mM Tris, 0.5-5 mM MnCI ₂ , 1 mM GSH/0.2mM GSSG, 50 mM betaine, pH8.0 12. 20 mM Tris, 1 mM MnCI ₂ , 1 mM GSH/0.2mM GSSG, 50 mM betaine, pH4.0-10.0 13. 20 mM Tris, 1 mM MnCI ₂ , 50 mM betaine, 0.05mM 1 -hexanol, 50mM acetamide, 300 mM KCI, pH8.0

Results:

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 Phenyl- sepharose column.

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.

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 in- elusion 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.

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 fer- mentor. 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.

5. The refolding conditions have not been optimized, but it has been observed that re- folding buffer pH at 8.0, stabilizers or solubility enhancers including betaine, NaCI or

KCI, acetamide, and 1 -hexanol, and the concentration of MnCI ₂ around 1 mM. are important for effective refolding.

Example 4

Recombinant expression of oxalate oxidase by P. pastoris Expression vector construction

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 pPICZaB and pGAPZaA vectors digested with the same restriction enzymes, resulting in the recombinant plasmids of Za-5102, GAPZa-5102, Za-5601 , GAPZa-5601 , Za-30640 and GAPZa-30640 (Figures 2-7). The recombinant plasmids of Za-5102, Za- 5601 and Za-30640 were linearized with Pme I (Mss I) and GAPZa-5102, GAPZa-5601 and GAPZa-30640 were linearized with Bin 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 1. Primers used to construct expression vectors

SEQ Oligonucleotides Sequence (5' to 3')

ID NO:

17 5102F CCGCTCGAGAAAAGATCTGATCCTGGTCTCCTACAG

18 5102R AAATATGCGGCCGCTCAAGCAACATCAGTTAAGAGTT

19 5601 F CCGCTCGAGAAAAGATCCGATCCTGCACCCCTT

20 5601 R AAATATGCGGCCGCTCAATAAGTTTCAGATAGCAATTTC

21 30640 F CCGCTCGAGAAAAGATTTGATCCGAGTCCTCTCCA

22 30640 R AAATATGCGGCCGCTCAATTAGCTTGAACTAAGCGTTG

Protein Expression

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 μg/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 expres- sion by shaking flask fermentation. The growth and induction media in shaking flask were BMGY (yeast extract 1 % (w/v), peptone 2% (w/v), 1 00 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 1 8-20 h to reach an OD ₆₀₀ of 3.0-6.0, then harvested by centrifugation and re-suspended in 50 mL BMMY medium containing 5 mM MnCI ₂ for methanol induction. The induction temperature was set at 28 °C, and 1 00% 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 sam- pies were concentrated by TCA precipitation. All the samples used for SDS-PAGE analysis are 30 μΙ of concentrated supernatant. For the constructive expression of GAP promoter, the growth was BYPD medium (1 0 g/l yeast extract, 20 g/l peptone, 20 g/l glucose, biotin 400 g/l, MnCI ₂ 5 mM and 1 00 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

Fed-batch fermentation: Inoculum (any recombinant yeast culture described above) was produced at 30 °C in a 2 I 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 I fermentor containing 2.8 I FM22 medium (KH ₂ P0 ₄ , 42.9 g/l; (NH ₄ ) ₂ S0 ₄ , 5 g/l; CaS0 ₄ ^» 2H ₂ 0, 1 .0 g/l;

K ₂ S0 ₄ , 14.3 g/l; MgS0 ₄ ^» 7H ₂ 0, 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):

CuS0 ₄ '5H ₂ 0, 2.0; Nal, 0.08; MnS0 ₄ ^» H ₂ 0, 3.0; Na ₂ Mo0 ₄ ^» 2H ₂ 0, 0.2; H ₃ B0 ₃ , 0.02; CaS0 ₄ '2H ₂ 0, 0.5; CoCI ₂ , 0.5; ZnCI ₂ , 7; FeS0 ₄ ^» 7H ₂ 0, 22; biotin, 0.2 and 1 ml/l concentrated H ₂ S0 ₄ [1 8]. The glycerol fed-batch solution contained ( ¹ ): 500 g glycerol (1 00 %) and 4 ml PTM4 stock solution. The methanol fed-batch solution consisted of 4 ml PTM4 stock solution and 1 I 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 0.1 6 h ^"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) am- monium 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.

Purification of recombinant protein

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. Results

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 (Figures 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

Construction of expression vectors

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 (Figures 12-13) were cloned into pHTE to produce pHTE-5601 , pHTE-5102 and pHTE-30640 (Figure 14), respectively. All positive recombinant plasmids were selected by colony PCR or by enzyme diges- tion and separately introduced into the A. tumefaciens strain GV3101 by the freeze/thaw method 5.1 Transient expression in tobacco leaves

Plant Materials

Wild-type N. benthamiana plants were grown in a greenhouse with a 1 (y8 hr light/dark cycle at 25 °C for 5 to 8 weeks.

5.1 .1 Bacterial Culture and Suspension Preparation

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 NaCI in 950 mL deionized water.) con- taining 25 mg/L rifampicin and 50 mg/L kanamycin with isolated colonies of Agrobacte- rium strains harbouring expression plasmids, overnight at 28°C under constant agitation at 220 rpm to grow preferentially to an OD (600 nm) >1 .2.

2. Inoculate fresh LB medium containing 25 mg/L rifampicin, 50 mg/L kanamycin and 20 μΜ 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).

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

4. Resuspend the bacterial pellets in 5 volume of bacteria resuspension solution(10 mM 2-N-morpholinoethanesulfonic acid (MES)) pH 5.5, 10 mM MgS0 ₄ , and 100 mM acetosyringone), and incubate for 4 h at room temperature before use. 5.1 .2 Syringe Infiltration and Plant Incubation

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 surround- ing the infiltration point is observed as the suspension enters the tissue in the apo- plastic space. For each point of infiltration, a surface of ~7 cm ² should be filled. Several points of infiltration may be necessary to completely inoculate each leaf.

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. 3. Incubate infiltrated plants in the greenhouse for 7 days, watering the plants as needed and continuing nitrogen fertilization. 5.1 .3 Leaf disk OxOx activity assay

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, 0.6mg/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.

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

5.2 Transient expression in pea

5.2.1 Plant Materials

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.

5.2.2 Bacterial Culture, Suspension Preparation and Vacuum-infiltration

Agrobacterium GV3101 cultures, bearing binary vectors, were grown in modified YEB media (6 g/Lyeast extract, 5 g/L peptone, 5 g/L sucrose, 2 mM MgS0 ₄ ) with antibiotics (100 mg/mL of kanamycin, 50mg/mL of rifampicin,) for 2 days at 28°C. For final scaled- up growth, initial 2-day cultures were diluted 1 :100 in the same YEB medium supplemented with antibiotics, 10 mM MES, pH 5.6, 20 μΜ of acetosyringone and allowed to grow 18-24h to an OD ₅₉₅ nm of about 2.4. Bacterial cells were supplemented with 55 g/L of sucrose and 200 μΜ 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 Agro- bacterium 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.

5.2.3 Enzyme assay

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 7000g. The supernatant was centrifuged for 15 min at 14,000g 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 1000g 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 50ml buffer A (50m M citric acid-phosphate, pH 6.0, 2M NaCI), and the resulting suspension was centrifuged for 20 min at 14,000g. The resulting supernatant was loaded onto Phenyl Sepharose HP column previously equilibrated with buffer A. The protein was eluted with a linear gradient of NaCI (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 NaCI (0-2 M) in buffer A. All the collection fractions were tested OxO activity by colorimetric meth- od.

Results

1 . The genes of OxOx 5102, 5601 and 30640 have been amplified (Figures 12-13)

2. The plant expression vector for OxOx 5102, 5601 and 30640 (Figure 14)

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

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

5. OxOx 5102 expressed by pea leaves has been purified and shows activity (Figures 17 and 18) 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 (Figure 16) Example 6

The activity of oxalate oxidases expressed by E. coli at different pH

Activity Assay: the enzyme solution 10 μΙ is added to 190 μΙ 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.

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

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).

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

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 figure 19 as relative activity, which is calculated by the activity divided by the maximum activity.

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 washing - OxOx-B5102

The harvested cell pellets were resuspended in a ratio of 30 g cell paste per 1 L wash buffer (50mM Tris-HCI, 2M urea, 50mM NaCI, 5mM EDTA, 5mM DTT, pH8.0). Benzo- nase nuclease (100 units per liter with 0.5mM MgCI ₂ ) was added to cell suspension to digest E. coli nucleic acid (including DNA and RNA), incubated at 37°C for 15min. 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 500ml 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

The washed inclusion body from Example 7 with purity >80% was dissolved in urea buffer (20 mM Tris-HCI, 8M urea, pH 8.0), and centrifuged at 13000g for 10min. 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, 30g/L NaCI, Tris-HCI, pH8.0) with 6 CV. The OxOx-B5102 protein was eluted by elution buffer C (8M urea, 160g/L NaCI, Tris-HCI, 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 concen- trated to 5mg/ml_ for protein refolding by 10K ultra-filtration tubes.

Example 9

Refolding of inclusion body

Refolding was performed as described herein before.

Example 10

Phenyl sepharose purification of OxOx-B5102 NaCI was added to the refolded OxOx-B5102 mixture to a final concentration of 500 mM, and then passed through 0.45μηι membrane. The clarified OxOx-B5102 solution was loaded into Phenyl sepharose column pre-balanced with 5 CV of balance buffer (10 mM Tris-HCI, 500 mM NaCI, pH 8.0). The column was further washed with 1 .5 CV of wash buffer (10 mM Tris-HCI, 250 mM NaCI, pH 8.0), and then OxOx-B5102 was eluted by elution buffer (10 mM Tris-HCI, 20% Isopropanol, pH 7.0). The target protein was precipitated by adding NaCI to the solution up to 50 mM and collected by centrifu- gation at 12000 g for 10 min, 4°C. The pellets were re-dissolved in borate buffer (10mM Na ₂ B ₄ 0 ₇ ~H ₃ B0 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

The concentration of OxOx-B5102 in borate buffer was adjusted to 2mg/ml_ for pegyla- tion. 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 2kD, 5kD, 10kD, 20kD, 4 armed-20kD, 30kD, 40kD and 4 armed-40kD 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 6h at 28°C, and then was stopped by adding glycine.

The pegylated OxOx-B5102 sample was loaded on size exclusion chromatography (GE HiLoad Superdex 16/600GL, USA) pre-balanced with phosphate buffer (10mM K ₂ HP0 ₄ ~KH ₂ P0 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 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 18~26°C, moisture 40-70%) and fed with distilled water and regular rat food every day. After 1 week of ac- climatization, 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 5000g for 5min at 4°C. Urine sample was collected from urine collection tube of metabolic cage every 12h (8:30 and 20:30) and acidified by using 2M HCI 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.

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 (1 ΟμΙ) was added to 96 well multi-plate for 6 wells. The reaction buffer contain- ing oxalate oxidase (1 OOmM citrate buffer, pH5.4, 10μΜ Amplex red, 1 U/mL HRP, 0.1 U/mL OxOx-B5102) were added into 3 wells and the background reaction buffer without OxOx (100mM citrate buffer, pH5.4, 10μΜ Amplex red, 1 U/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: 538nm; emission wavelength: 590nm). 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. 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. Following the acclimation period, OxOx-B5102 pegylated with 3 different molecular weights of PEG (20kD, 30kD, and 40kD 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. Serum oxalate and urine oxalate was monitored every day. The results showed that PEG-OxOx-B5102 could reduce 40-50% serum oxalate and 20-40% urine oxalate compared with placebo control (Fig.21 and Fig.22). Example 13

Immunogenicity evaluation of PEG-OxOx-B5102s

OxOx-B5102 and OxOx-B5102 pegylated with different molecular weights of PEG (2kD, 5kD, 10kD, 20kD, 30kD and 40kD) 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.

The ELISA method procedure: (1 ) rat serum samples were collected at different time- points (Od, 7d, 14d, 21 d, 28d, 45d, 60d) post injecting of PEG-OxOx-B5102 and stored at -20°C until use. (2) Dilute OxOx-B5102 to a final concentration of 1 C^g/ml in coating buffer (50mM carbonate/bicarbonate buffer, pH9.6) and transfer 100 μΙ 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 .5m M KH ₂ P0 ₄ ; 8.1 mM Na ₂ HP0 ₄ - 12H ₂ 0; 136mM NaCI; 2.7mM KCI; 0.05% Tween-20), and block non-specific binding sites by adding 300μΙ of blocking solution(1 .5mM KH ₂ P0 ₄ ; 8.1 mM Na ₂ HP0 ₄ - 12H ₂ 0; 136mM NaCI; 2.7mM KCI; 5% non-fat dry milk) to each well. (4) Seal plate and incubate at 37°C for 1 ~2 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 100μΙ per well to the ELISA plate. (7) Seal the plate and incubate at 37°C temperature for 1 hours or at 4°C overnight. Wash≥ 3 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 μΙ per well. (9) Seal the plate and incubate at room temperature for 1 hour. Wash≥ 5 times with PBS-T buffer. (10) For each plate, mix 6 ml of TMB Reagent A (0.5mM EDTA-Na; 5mM citric acid; 10% glycerol; 0.04% tetramethyl benzidine) with 6 ml TMB Reagent B (165mM sodium acetate; 8.3mM citric acid; 0.06% 30%-H ₂ O ₂ ) immediately prior to use. Transfer 100 μί into each well and incubate at room temperature for 30 min. To stop the reaction, add 100 μΙ of stopping solution (2M H ₂ S0 ₄ ). (11 ) Read the optical density (OD) for each well with a micro-plate reader set to 450 nm.

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 significant- ly 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.