WO2004018634A2 - Polynucleotide codant une oxalate decarboxylase issue de aspergillus niger et procedes d'utilisation - Google Patents

Polynucleotide codant une oxalate decarboxylase issue de aspergillus niger et procedes d'utilisation Download PDF

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WO2004018634A2
WO2004018634A2 PCT/US2003/026404 US0326404W WO2004018634A2 WO 2004018634 A2 WO2004018634 A2 WO 2004018634A2 US 0326404 W US0326404 W US 0326404W WO 2004018634 A2 WO2004018634 A2 WO 2004018634A2
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oxalate
oxalate decarboxylase
cell
plant
enzyme
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PCT/US2003/026404
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WO2004018634A3 (fr
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Nigel Gordon John Richards
Christopher Harry Chang
Ammon B. Peck
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University Of Florida
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)

Definitions

  • the subject invention was made with government support under a research project supported by National Institutes of Health Grant No. DK53556.
  • Oxalic acid a compound that is toxic to almost all organisms (Hodgkinson, 1977), plays several important roles in fungal growth and metabolism (Dutton et al, 1996), and in biological mechanisms underlying fungal pathogenesis.
  • Aspergillus niger which can colonize lung tissue in immunocompromised individuals, excretes enough oxalate to fo ⁇ n crystalline calcium salts as part of necrotizing otomycosis (Landry et al, 1993) and, in certain cases, can give rise to fatal pulmonary oxalosis (Kimmerling et al, 1992; Metzger et al, 1984).
  • Oxalate decarboxylase catalyzes a remarkable transformation in which the C-C bond in oxalate is cleaved to give carbon dioxide and formate:
  • Oxalate decarboxylase was first isolated from basidiomycete fungi (Shimazono, 1955), and has subsequently been identified in several species of filamentous fungi, including Myrothecium verrucaria (Lillehoj et al, 1965), certain strains of Aspergillus niger (Emiliani et al, 1964) and Flammulina velutipes (Mehta et al, 1991), and the common button mushroom Agaricus bisporus (Kathiara et al, 2000).
  • OxDC expression can also be induced in the white-rot fungus Coriolus versicolor (Shimazono et al, 1957), and very recent work has also shown that OxDC is present in Bacillus subtilis (Tanner et al, 2000), although this appears to be the only bacterium in which the presence of this enzyme has been unambiguously demonstrated. While it has been demonstrated that the bacterial OxDC is manganese-dependent (Tanner et al, 2001), the detailed catalytic mechanism by which oxalate is converted to formate and carbon dioxide has not yet been elucidated. Early experiments employing the Aspergillus niger OxDC showed that (i) enzymatic
  • CO 2 evolution requires oxalate to the exclusion of other biologically relevant carboxylic acids, (ii) oxygen is required for catalytic turnover, although high oxygen tensions inhibit the enzyme (Emiliani et al, 1968), and (iii) a sub-stoichiomerric quantity of oxygen is converted to hydrogen peroxide during the reaction.
  • Weak reductants such as phenylenediamines and diphenols activate the enzyme, whereas treatment with strong reductants such as dithionite and hydroxylamine eliminate OxDC activity. No evidence was found for the presence of exogenous cofactors in the native Aspergillus niger OxDC, and the enzyme was reported not to contain iron and copper ions as purified.
  • Mn(III) and Mn(IV) are the redox active forms of the metal during catalysis (Anand et al, 2002), there is no published evidence to support such a claim. Equally, the intermediacy of a protein-based radical cannot be ruled out on the basis of current biochemical and structural information on Bacillus subtilis OxDC. This proposal has the merit of rationalizing the observed correlation between the amounts of hydrogen peroxide formed under the assay conditions and the partial pressure of oxygen.
  • Kidney-urinary tract stone disease 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. Other disease states have also been associated with excess oxalate. These include, vulvodynia, oxalosis associated with end- stage renal disease, cardiac conductance disorders, Crohn's disease, and other enteric disease states.
  • Oxalic acid (and/or its salt-oxalate) is found in a wide diversity of foods, and is therefore, a component of many constituents in human and animal diets. Increased oxalate absorption may occur after foods containing elevated amounts of oxalic acid are eaten. Foods such as spinach and rhubarb are well known to contain high amounts of oxalate, but a multitude of other foods and beverages also contain oxalate. Because oxalate is found in such a wide variety of foods, diets that are low in oxalate and which are also palatable are hard to formulate. In addition, compliance with a low oxalate diet is often problematic. Normal tissue enzymes also produce endogenous oxalate metabolically. Oxalate
  • kidney fluids dietary oxalate that is absorbed as well as oxalate that is produced metabolically
  • tissue enzymes dietary oxalate that is absorbed as well as oxalate that is produced metabolically
  • This excretion occurs mainly via the kidneys.
  • concentration of oxalate in kidney fluids is critical, with increased oxalate concentrations causing increased risk for the formation of calcium oxalate crystals and thus the subsequent formation of kidney stones.
  • Kidney-urinary tract stone disease occurs in as much as 12% of the population in Western countries and about 70% of these stones are composed of calcium oxalate or of calcium oxalate plus calcium phosphate.
  • Some individuals e.g., patients with intestinal disease such as Crohn's disease, inflammatory bowel disease, or steatorrhea and also patients that have undergone jejunoileal bypass surgery
  • Oxalate is also a problem in patients with end-stage renal disease and there is recent evidence (Solomons et al, 1991) that elevated urinary oxalate is also involved in vulvar vestibulitis (vulvodynia). Bacteria that degrade oxalate have been isolated from human feces (Allison et al,
  • U.S. Patent No. 6,355,242 and published international patent application WO 98/52586 disclose delivery of bacteria and/or oxalate-degrading enzymes to intestinal tracts of persons or animals, thereby reducing oxalate in the intestinal tract of those persons or animals who are at risk for oxalate related disease.
  • OxDC of Aspergillus niger which converts oxalate directly to formate and carbon dioxide without the need for exogenous co-factors, can provide a therapeutic approach at a significant reduction in cost.
  • a second benefit of using Aspergillus niger OxDC is that the enzyme has a pH-optimum of 4.2, making it useful for oxalate degradation in the upper intestine. Since Aspergillus niger is also used in the production of citrate, which is then added to food products and dietary supplements, it is likely that no significant side effects will be observed when this form of OxDC is administered in the human gastrointestinal tract.
  • the subject invention pertains to polynucleotides encoding the enzyme oxalate decarboxylase from the filamentous fungus Aspergillus niger and methods of use.
  • the polynucleotides can be used to express oxalate Aspergillus niger decarboxylase that can be used to degrade oxalate for therapeutic and other purposes.
  • the subject invention also pertains to cells and microbes, such as bacteria, which are transformed with a polynucleotide of the present invention encoding an oxalate decarboxylase enzyme.
  • the subject invention also pertains to plants that are transformed with a polynucleotide of the present invention encoding an oxalate decarboxylase enzyme.
  • Transformed plants of the present invention expressing oxalate decarboxylase can be administered to a human or animal as a constituent of a meal, for example, as a salad or vegetable.
  • the transformed plant of the present invention can be administered to an animal as a constituent of feed or the plant can be grown in a pasture in which animals are allowed to graze and feed upon the plant.
  • the subject invention also concerns the use of Aspergillus niger oxalate decarboxylase, or a microbe transformed with a polynucleotide of the invention to express oxalate decarboxylase of the invention, to achieve therapeutic oxalate degradation in a human or animal.
  • the subject invention also pertains to use of oxalate decarboxylase of the invention to degrade oxalate present in fluids, such as blood and urine.
  • oxalate decarboxylase of the invention can be coated or attached to a surface, for example, that of a catheter or other medical device, that might come into contact with a fluid containing oxalate.
  • the attached enzyme can prevent oxalate accumulation or encrustation on those surfaces of a device that are in contact with the fluid.
  • Figure 1 shows an SDS-PAGE gel of oxalate decarboxylase purification fractions obtained from the procedures described in "Materials and Methods" section herein. Lanes from left to right are crude extract, methanol re-suspension, Q-Sepharose fractions and phenyl Sepharose fractions.
  • Figure 2 shows the deduced primary structure of Aspergillus niger oxalate decarboxylase protein from the cDNA sequence shown in Figure 3.
  • Amino acids that define the signal peptide of the protein are shown in italic font. Standard one letter code is used to represent amino acids.
  • Figure 3A-C shows the alignment of the nucleotide sequences of the gene encoding oxalate decarboxylase Aspergillus niger (genomic) OxDC and the cDNA obtained from mRNA isolated from the fungus (cDNA). Underlined residues in the genomic sequence indicate the location of the two introns deduced to be present in the gene by comparison of the sequences. These are both flanked by canonical sequences shown in bold typeface. The TAG sequence at the 3'-end of the gene, also shown in bold typeface, indicates the end of the region coding for the protein product.
  • Figure 4 shows the DNA sequence encoding oxalate decarboxylase as cloned from genomic DNA of Aspergillus niger.
  • Figure 5 shows the deduced primary structure of Bacillus subtilis yvrk protein. Standard one letter code is used to represent amino acids.
  • SEQ ID NO. 1 is a genomic polynucleotide of Aspergillus niger encoding an oxalate decarboxylase enzyme that can be used according to the present invention.
  • SEQ ID NO. 2 is a cDNA sequence of Aspergillus niger encoding an oxalate decarboxylase enzyme that can be used according to the present invention.
  • SEQ ID NO. 3 is the amino acid sequence of an oxalate decarboxylase enzyme of Aspergillus niger encoded by SEQ ID NO. 1.
  • SEQ ID NO. 4 is an amino acid sequence of an oxalate decarboxylase enzyme of the invention with the amino acid leader sequence removed.
  • SEQ ID NO. 5 is a sequence of a PCR primer that can be used according to the present invention.
  • SEQ ID NO. 6 is a sequence of a PCR primer that can be used according to the present invention.
  • SEQ ID NO. 7 is a partial sequence of the oxalate decarboxylase enzyme of the present invention.
  • SEQ ID NO. 8 is a predicted partial sequence of the oxalate decarboxylase enzyme of the present invention.
  • SEQ ID NO. 9 is the deduced primary structure of Bacillus subtilis yvrk protein according to the present invention.
  • the subject invention concerns polynucleotides encoding the enzyme oxalate decarboxylase from the filamentous fungus Aspergillus niger.
  • the amino acid sequence of the oxalate decarboxylase enzyme from Aspergillus niger is shown in Figure 2 (SEQ ID NO.
  • the subject invention pertains to the enzyme having the sequence shown in Figure 2 (SEQ ID NO. 3), as well as the enzyme lacking the leader sequence (shown in italics in Figure 2), i.e., a polypeptide of SEQ ID NO 4.
  • a cDNA sequence that encodes the oxalate decarboxylase enzyme from Aspergillus niger is shown in the bottom row of nucleotide sequence in Figure 3 (SEQ ID NO. 2).
  • the genomic sequence from Aspergillus niger encoding oxalate decarboxylase is shown in the top row of nucleotide sequence in Figure 3 (SEQ ID NO. 1).
  • the subject invention also concerns the polypeptides of the invention complexed with a metal.
  • the metal is manganese, iron, or copper. More preferably, the metal is manganese.
  • the subject invention also concerns pharmaceutical and nutraceutical compositions for the introduction of the oxalate decarboxylase of the present invention and/or bacteria or other cells that have been transformed with a polynucleotide of the present invention into the intestine of a human or animal.
  • the transformed bacteria or cell or enzyme has been lyophilized or frozen.
  • a liquid or paste form can be encapsulated in a gel capsule or provided with other forms of enteric protection.
  • the gel capsule material or the material providing enteric protection is resistant to degradation by the acidity and enzymes of the stomach but can be degraded, with concomitant release of the enzyme and or transformed bacteria or cell of the invention, by the higher pH and bile acid contents present in the human or animal intestinal tract.
  • the subject invention also concerns transgenic animals in which a polynucleotide encoding an oxalate decarboxylase of the invention has been incorporated into the animal's genome.
  • Methods for preparing transgenic animals are well known in the art.
  • the subject invention also concerns an enzyme delivery system comprising a plant which has been transformed with a polynucleotide of the subject invention encoding oxalate decarboxylase, which when expressed can degrade oxalate.
  • Transformed plants of the present invention expressing oxalate decarboxylase can be administered to a human or animal as a constituent of a meal, for example, as a salad or vegetable.
  • the transformed plant of the present invention can be administered to an animal as a constituent of feed or the plant can be grown in a pasture in which animals are allowed to graze and feed upon the plant.
  • the subject application also concerns plants transformed with polynucleotides of the present invention that encode oxalate decarboxylase from Aspergillus niger.
  • Plants that can be transformed with the subject polynucleotide include both monocotyledonous and dicotyledonous plants.
  • Plants within the scope of the present invention include monocotyledonous plants, such as rice, wheat, barley, oats, sorghum, maize, lilies, and millet, and dicotyledonous plants, such as peas, alfalfa, chickpea, chicory, clover, kale, lentil, prairie grass, soybean, tobacco, potato, sweet potato, radish, cabbage, rape, apple trees, and lettuce.
  • the plant is a cereal. Cereals to which this invention applies include, for example, maize, wheat, rice, barley, oats, rye, and millet.
  • the subject invention also concerns methods for degrading oxalate present in fluids, such as blood and urine, using oxalate decarboxylase of the present invention.
  • the subject invention can be used to prevent or minimize encrustation of oxalate crystals on any device, such as a catheter, that comes into contact with oxalate-containing fluids.
  • An oxalate decarboxylase enzyme of the invention can be provided on or in any devices that come into contact with fluids that contain or may contain oxalate.
  • the enzyme can be coated to or attached on the inside of a medical catheter or stent.
  • the enzyme could also be provided in dialysis cartridges to degrade oxalate present in a patient's biological fluid.
  • the subject invention also concerns methods and compositions for assaying for the presence of oxalate.
  • the method comprises contacting a sample to be assayed with an oxalate decarboxylase of the present invention and then determining the presence of either carbon dioxide or formate generated from the reaction of the enzyme with oxalate.
  • the subject invention also concerns pharmaceutical and nutraceutical compositions for the introduction of the oxalate decarboxylase of Bacillus subtilis and/or bacteria or other cells that have been transformed with a polynucleotide encoding the oxalate decarboxylase of Bacillus subtilis into the intestine of a human or animal.
  • the transformed bacteria or cell or enzyme has been lyophilized or frozen.
  • a liquid or paste form can be encapsulated in a gel capsule or provided with other forms of enteric protection.
  • the gel capsule material or the material providing enteric protection is resistant to degradation by the acidity and enzymes of the stomach but can be degraded, with concomitant release of the enzyme and/or transformed bacteria or cell of the invention, by the higher pH and bile acid contents present in the human or animal intestinal tract.
  • the subject invention also concerns transgenic animals in which a polynucleotide encoding oxalate decarboxylase o ⁇ Bacillus subtilis has been incorporated into the animal's genome. Methods for preparing transgenic animals are well known in the art.
  • the subject invention also concerns an enzyme delivery system comprising a plant which has been transformed with a polynucleotide encoding oxalate decarboxylase of Bacillus subtilis, which when expressed can degrade oxalate.
  • Transformed plants of the present invention expressing oxalate decarboxylase of Bacillus subtilis can be administered to a human or animal as a constituent of a meal, for example, as a salad or vegetable.
  • the transformed plant of the present invention can be administered to an animal as a constituent of feed or the plant can be grown in a pasture in which animals are allowed to graze and feed upon the plant.
  • the subject application also concerns plants transformed with polynucleotides encoding the oxalate decarboxylase of Bacillus subtilis.
  • Plants that can be transformed with the subject polynucleotide include both monocotyledonous and dicotyledonous plants.
  • Plants within the scope of the present invention include monocotyledonous plants, such as rice, wheat, barley, oats, sorghum, maize, lilies, and millet, and dicotyledonous plants, such as peas, alfalfa, chickpea, chicory, clover, kale, lentil, prairie grass, soybean, tobacco, potato, sweet potato, radish, cabbage, rape, apple trees, and lettuce.
  • the plant is a cereal. Cereals to which this invention applies include, for example, maize, wheat, rice, barley, oats, rye, and millet.
  • the subject invention also concerns methods for degrading oxalate present in fluids, such as blood and urine, using oxalate decarboxylase of Bacillus subtilis.
  • the subject invention can be used to prevent or minimize encrustation of oxalate crystals on any device, such as a catheter, that comes into contact with biological fluids.
  • Oxalate decarboxylase of Bacillus subtilis can be provided on or in any devices that come into contact with fluids that contain or may contain oxalate.
  • the enzyme can be coated to or attached on the inside of a medical catheter or stent.
  • the enzyme could also be provided in dialysis cartridges to degrade oxalate present in a patient's biological fluid.
  • the methods and compositions of the present invention can be used with humans and other animals.
  • the other animals contemplated within the scope of the invention include domesticated, agricultural, or zoo- or circus-maintained animals.
  • domesticated animals include, for example, dogs, cats, rabbits, ferrets, guinea pigs, hamsters, pigs, monkeys or other primates, and gerbils.
  • Agricultural animals include, for example, horses, mules, donkeys, burros, cattle, cows, pigs, sheep, and alligators.
  • Zoo- or circus-maintained animals include, for example, lions, tigers, bears, camels, giraffes, hippopotamuses, and rhinoceroses.
  • Polynucleotides of the present invention can be composed of either RNA or DNA.
  • the polynucleotides are composed of DNA.
  • the subject invention also encompasses those polynucleotides that are complementary in sequence to the polynucleotides disclosed herein.
  • polynucleotide sequences can encode oxalate decarboxylase enzymes disclosed herein.
  • references to "essentially the same" sequence refers to sequences that encode amino acid substitutions, deletions, additions, or insertions, which do not materially alter the functional activity of the polypeptide, encoded by the polynucleotides of the present invention.
  • amino acids other than those specifically exemplified in the sequence of oxalate decarboxylase disclosed herein is also contemplated within the scope of the present invention.
  • Amino acids can be placed in the following classes: non-polar, uncharged polar, basic, and acidic.
  • Conservative substitutions whereby an oxalate decarboxylase polypeptide having an amino acid of one class is replaced with another amino acid of the same class fall within the scope of the subject invention so long as the oxalate decarboxylase having the substitution still retains substantially the same activity as wild type polypeptide.
  • Table 1 below provides a listing of examples of amino acids belonging to each class.
  • non-natural amino acids include, but are not limited to, ornithine, citrulline, hydroxyproline, homoserine, phenylglycine, taurine, iodotyrosine, 2,4-diaminobutyric acid, ⁇ -amino isobutyric acid, 4- aminobutyric acid, 2-amino butyric acid, ⁇ -amino butyric acid, ⁇ -amino hexanoic acid, 6- amino hexanoic acid, 2-amino isobutyric acid, 3-amino propionic acid, norleucine, norvaline, sarcosine, homocitrulline, cysteic acid, ⁇ -butylglycine, ⁇ -butylalanine, phenylglycine, cyclohexylalanine, ⁇ -alanine
  • Non-natural amino acids also include amino acids having derivatized side groups.
  • any of the amino acids in the protein can be of the D (dextrorotary) form or L (levorotary) form.
  • the scope of the invention also includes amino acid substitutions in the sequence of the polypeptide that change the pH optimum at which the polypeptide exhibits the highest level of enzymatic activity. Techniques for making such amino acid substitutions and assaying the polypeptide for pH optimum are well known in the art (Neves-Peterson et al, 2001; Nielson et al, 1999; Shaw et al, 1999).
  • Polynucleotides and proteins of the subject invention can also be defined in terms of more particular identity and/or similarity ranges with those exemplified herein.
  • the sequence identity will typically be greater than 60%, preferably greater than 75%, more preferably greater than 80%, even more preferably greater than 90%, and can be greater than 95%.
  • the identity and/or similarity of a sequence can be 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, ' 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% as compared to a sequence exemplified herein.
  • the subject invention also concerns polynucleotides, which encode fragments of a full-length oxalate decarboxylase enzyme of the invention, so long as those fragments retain substantially the same functional activity as full-length polypeptide.
  • the fragments of an oxalate decarboxylase polypeptide encoded by these polynucleotides are also within the scope of the present invention. Fragments of the full-length sequence can be prepared using standard techniques known in the art.
  • the subject invention also contemplates those polynucleotide molecules encoding oxalate decarboxylase enzymes having sequences that are sufficiently homologous with the wild type sequence of Aspergillus niger so as to permit hybridization with that sequence under standard stringent conditions and standard methods (Maniatis et al, 1982).
  • stringent conditions for hybridization refers to conditions wherein hybridization is typically carried out overnight at 20-25 C below the melting temperature (Tm) of the DNA hybrid in 6x SSPE, 5x Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA.
  • Tm melting temperature
  • Tm 81.5 C+16.6 Log[Na+]+0.41(%G+C)-0.61(% formamide)-600/length of duplex in base pairs.
  • Washes are typically carried out as follows: (1) Twice at room temperature for 15 minutes in lx SSPE, 0.1% SDS (low stringency wash).
  • Aspergillus niger ATCC 26550 was maintained on potato dextrose agar plates at 4 °C. Inoculating a starter culture of ATCC medium 950 with a loopful of Aspergillus niger spores produced mycelium for OxDC purification.
  • the literature procedures for inducing OxDC production were modified by the substitution of sucrose for glucose (by weight), NH 4 C1 for NaNO 3 (by molarity) and the addition of 10 mM sodium oxalate (Sigma). After growth at 30 °C for several days, this starter culture was used to inoculate larger cultures.
  • Fungus for DNA isolation was grown on yeast extract-peptone- dextrose medium (ATCC medium 1005), with cultures being shaken at 37 °C until the mycelium was confluent. Mycelium was harvested by vacuum filtration, washed with de- ionized water, frozen in powdered dry ice, lyophilized, and stored at -80 °C until used in subsequent experiments.
  • yeast extract-peptone- dextrose medium ATCC medium 1005
  • Enzyme Assay consisted of 50 mM NaOAc pH 5.2, 0.2% Tween 20, 2 mM o- phenylenediamine, 30 mM potassium oxalate, and enzyme in a volume of 100 ⁇ L. Turnover was initiated by addition of substrate. Mixtures were incubated at ambient temperature (21- 22 °C), and then the reaction was quenched by addition of 10 ⁇ L 1 N NaOH. The amount of formate produced in the enzyme-catalyzed reaction was measured using a formate dehydrogenase (FDH) assay consisting of 100 mM potassium phosphate pH 7.8, 1.5 mM NAT , and 0.1 LU. FDH (1 mL final volume). Absorbance at 340 nm was measured after incubation at 37 °C for 30 minutes. Formate was quantitated by comparison to a standard curve generated by spiking protein-free OxDC assays with known amounts of sodium formate.
  • FDH formate dehydrogenase
  • OxDC treated in this manner were then divided into two aliquots for EPR and inductively coupled plasma- atomic emission (ICP-AE) spectroscopy.
  • Metal content was determined by ICP-AE spectroscopy using protein samples made by diluting 100 ⁇ L of OxDC (0.5 mg) with 9.9 mL deionized water. All analyses were performed in the Department of Chemistry at the University of Florida. Calculations of the metal content in native Aspergillus niger OxDC employed standard procedures (see supplementary material).
  • GTCCTCGAGAAAAGATACCAG-3' (SEQ ID NO. 5) was employed to introduce a Xliol site and a proteolytic cleavage site, for use in future expression experiments, immediately upstream of the codon of Tyr-24 in the putative Aspergillus niger gene sequence.
  • This primer was combined with a reverse primer (5'-TCATCTACTCACTTGGGCTCCGAATTG -3') (SEQ ID NO. 6) matching the 3'-end of the gene in Aspergillus phoenices.
  • Competent JM109 cells >10 8 CFU/ ⁇ g were transformed, and white colonies screened by Xhol digestion of alkaline lysis/miniprepped plasmid DNA. Plasmid that produced two bands upon Xliol digestion was purified, and submitted for nucleotide sequencing at the Interdisciplinary Center for Biotechnology Research (ICBR) at the University of Florida.
  • ICBR Interdisciplinary Center for Biotechnology Research
  • Example 1 Isolation. Purification, and Assay of Native Oxalate Decarboxylase from Aspergillus niger.
  • OxDC expression is induced so as to reduce oxalate concentrations in the mycelium to a non- toxic level.
  • OxDC as a single band on SDS-PAGE ( Figure 1), with a molecular weight in the range expected based on studies of the enzyme isolated from Flammulina velutipes (Kathiara et al, 2000). Purified Aspergillus niger OxDC exhibited a specific activity of 10 LU./mg, as determined from steady-state formate production under initial velocity conditions.
  • Example 2 Deduced Primary Structure o ⁇ Aspergillus niger OxDC.
  • the protein product encoded by the yvrk gene in Bacillus subtilis shows some homology to Aspergillus niger OxDC with 197 (52%) residues in the bacterial OxDC being identical to those in the fungal enzyme.
  • MALDI-TOF measurements on the purified fungal OxDC indicated that the mass of a single subunit of the enzyme is 48,700-48,800 Da, which is consistent with that calculated for the deduced amino acid sequence with the observed N- terminal residue, assuming that the protein contains metal ions and is glycosylated. While the bacterial OxDC has been shown to exist as a hexamer consisting of a hypothetical dimer of trimers (Tanner et al, 2001), the quaternary structure of the fungal enzyme remains to be unambiguously established.
  • OxDC contains two metal-binding sites per polypeptide.
  • ICP-AE analysis of the purified enzyme from Aspergillus niger showed the sample to contain approximately 0.75 and 0.25 subunit-equivalents Mn and Cu, respectively. If bound Cu and Mn ions were both required for catalysis, it is anticipated that the maximum activity of the purified fungal enzyme would correspond to 3/16 of the theoretical V ma .
  • Example 4 Cloning, Expression and Purification o ⁇ Bacillus subtilis Oxalate Decarboxylase.
  • the upstream and downstream PCR primers were designed based on the published sequence of the gene in the GenBank database, in order to clone the bacterial gene, and express and characterize its encoded protein. These primers were such that the yvrK coding sequence would be in-frame with the T7 control elements that are part of the pET-9a expression vector (Stratagene). An Ndel site was included at the N-terminal methionine, and a BamHl site after the termination codon o ⁇ yvrK. B. subtilis 168 genomic DNA was purified from an overnight 5 L culture using a Genomic DNA Miniprep kit (Qiagen).
  • the yvrK sequence was amplified for 31 cycles (95 °C denaturation, 30s; 45 °C annealing, 30 s; 74 °C extension, 2 min).
  • the resulting DNA was digested with Ndel and BamHl, then ligated into pET-9a digested similarly.
  • Competent JM109 cells were transformed with the ligation mixture and with pET-9a as a control, and transformants selected on Luria-Bertani broth (LB) containing 30 ⁇ g/mL kanamycin (LBK).
  • LB Luria-Bertani broth
  • LBK Luria-Bertani broth
  • LBK Luria-Bertani broth
  • LBK Luria-Bertani broth
  • a plasmid produced from pET-9a/ vrrv:JM109 by standard alkaline lysis miniprep was used to transform the expression strain BL21(DE3), and the expression of the 7vrA;-encoded protein was tested by inoculating 0.5 L of LBK supplemented with pET-9a/ vrrv:BL21(DE3).
  • the cells were grown at 37 °C and shaken at 200 r.p.m. When the cultures reached A ⁇ oo of 2 they were heat shocked in water bath at 42 °C for 18 min before the addition of isopropyl thiogalactoside (IPTG) and MnCl 2 to final concentrations 1 and 5 mM respectively.
  • the cells were harvested after 4 h of shaking by centrifugation (5,000 x g, 15 min, 4 °C). Pellets were resuspended in 50 mL lysis buffer (50 mM Tris/HCl pH 7; 10 ⁇ M MnCl 2 ) and sonicated for 30 s at 80 % power. After sonication, lysis pellets were separated from the crude extract by centrifugation (8000 rpm, 20 min, 4°C) and resuspended in 50 mL of extraction buffer containing 1 M sodium chloride, 0.1% Triton X-100, and 10 mM 2-mercaptoethanol. The mixture was stirred overnight at room temperature.
  • the fractions were pooled as for the DEAE column and diluted 15-fold before they were loaded onto a Q-Sepharose Hi- Performance (Amersham Pharmacia Biotech) column.
  • the protein was eluted with an imidazole hydrochloride buffer (50 mM, pH 7.0, containing 10 M MnCl 2 ) and a 500 L NaCl gradient (0 to 1 M) as for the DEAE column.
  • Protein precipitated with 70 % ammonium sulfate was centrifuged and redissolved in 10 ml 20 mM hexamethylenetetramine-HCl pH 7. Ammonium sulfate was dialyzed out against 1L of the same amine buffer for 5 h at 4 °C.
  • Kesarwani M., Azam, M., Natarajan, K., Mehta, A., and Datta, A. (2000) "Oxalate Decarboxylase from Collybia Velutipes. Molecular Cloning and its Overexpression to Confer Resistance to Fungal Infection in Transgenic Tobacco and Tomato" J. Biol Chem. 275:7230-7238.

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Abstract

La présente invention concerne des polynucléotides codant l'enzyme oxalate décarboxylase issue du champignon filamenteux Aspergillus niger et des procédés d'utilisation des polynucléotides. L'invention se rapporte également à des procédés d'utilisation de l'enzyme oxalate décarboxylase issue de Bacillus subtilis.
PCT/US2003/026404 2002-08-20 2003-08-20 Polynucleotide codant une oxalate decarboxylase issue de aspergillus niger et procedes d'utilisation WO2004018634A2 (fr)

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Cited By (8)

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EP1755655A2 (fr) * 2004-05-07 2007-02-28 OxThera, Inc., Procedes et compositions de reduction des concentrations d'oxalate
US8142775B2 (en) 2006-08-02 2012-03-27 Althea Technologies, Inc. Crystallized oxalate decarboxylase and methods of use
CN102597225A (zh) * 2009-07-02 2012-07-18 奥克斯泰拉知识产权公司 重组草酸盐降解酶的纯化和分离以及含有草酸盐降解酶的喷雾干燥粒子
US8486389B2 (en) 1997-05-23 2013-07-16 Oxthera, Inc. Compositions and methods for treating or preventing oxalate-related disease
WO2016161455A2 (fr) 2015-04-02 2016-10-06 Captozyme, Llc Enzymes de dégradation des oxalates à haute efficacité pour la dégradation d'oxalates insolubles et solubles
US9714456B2 (en) 2013-01-18 2017-07-25 Allena Pharmaceuticals, Inc. Crystallized oxalate decarboxylase and methods of use
WO2018054132A1 (fr) * 2016-09-23 2018-03-29 武汉康复得生物科技股份有限公司 Oxalate décarboxylase glycosylée, sa préparation et son utilisation
US11077238B2 (en) 2013-06-07 2021-08-03 Allena Pharmaceuticals, Inc. Compositions, methods, and devices for dialysis

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WO1998042827A2 (fr) * 1997-03-21 1998-10-01 Pioneer Hi-Bred International, Inc. Gene codant pour un oxalate decarboxylase isole a partir d'aspergillus phoenices

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WO1998042827A2 (fr) * 1997-03-21 1998-10-01 Pioneer Hi-Bred International, Inc. Gene codant pour un oxalate decarboxylase isole a partir d'aspergillus phoenices

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SANDERSON K.E. ET AL.: 'Storing, shipping and maintaining records on bacterial strains.' METHODS ENZYMOL. vol. 204, 1991, pages 248 - 264, XP008050095 *
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Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8486389B2 (en) 1997-05-23 2013-07-16 Oxthera, Inc. Compositions and methods for treating or preventing oxalate-related disease
US10149866B2 (en) 2000-02-09 2018-12-11 Oxthera Intellectual Property Ab Compositions and methods for treating or preventing oxalate-related disease
EP1755655A4 (fr) * 2004-05-07 2011-03-30 Oxthera Inc Procedes et compositions de reduction des concentrations d'oxalate
EP1755655A2 (fr) * 2004-05-07 2007-02-28 OxThera, Inc., Procedes et compositions de reduction des concentrations d'oxalate
EP2803366B1 (fr) 2004-06-15 2018-08-01 OxThera Intellectual Property AB Compositions pour le traitement ou la prévention de maladie liée à l'oxalate
US10653726B2 (en) 2004-06-15 2020-05-19 Oxthera Intellectual Property Ab Compositions and methods for treating or preventing oxalate-related disease
US10369203B2 (en) 2006-08-02 2019-08-06 Allena Pharmaceuticals, Inc. Crystallized oxalate decarboxylase and methods of use
CN103272225A (zh) * 2006-08-02 2013-09-04 阿尔西亚技术股份有限公司 结晶化的草酸脱羧酶和使用方法
US9155785B2 (en) 2006-08-02 2015-10-13 Ajinomoto Althea, Inc. Crystallized oxalate decarboxylase and methods of use
US8142775B2 (en) 2006-08-02 2012-03-27 Althea Technologies, Inc. Crystallized oxalate decarboxylase and methods of use
US9821041B2 (en) 2006-08-02 2017-11-21 Allena Pharmaceuticals, Inc. Crystallized oxalate decarboxylase and methods of use
CN103272225B (zh) * 2006-08-02 2018-05-22 味之素阿尔西亚有限公司 结晶化的草酸脱羧酶和使用方法
US8741284B2 (en) 2006-08-02 2014-06-03 Althea Technologies, Inc. Crystallized oxalate decarboxylase and methods of use
CN104805067A (zh) * 2009-07-02 2015-07-29 奥克斯泰拉知识产权公司 重组草酸盐降解酶的纯化和分离以及含有草酸盐降解酶的喷雾干燥粒子
CN102597225A (zh) * 2009-07-02 2012-07-18 奥克斯泰拉知识产权公司 重组草酸盐降解酶的纯化和分离以及含有草酸盐降解酶的喷雾干燥粒子
US10570468B2 (en) 2013-01-18 2020-02-25 Allena Pharmaceuticals, Inc. Crystallized oxalate decarboxylase and methods of use
US10246755B2 (en) 2013-01-18 2019-04-02 Allena Pharmaceuticals, Inc. Crystallized oxalate decarboxylase and methods of use
US9714456B2 (en) 2013-01-18 2017-07-25 Allena Pharmaceuticals, Inc. Crystallized oxalate decarboxylase and methods of use
US11208700B2 (en) 2013-01-18 2021-12-28 Allena Pharmaceuticals, Inc. Crystallized oxalate decarboxylase and methods of use
US11077238B2 (en) 2013-06-07 2021-08-03 Allena Pharmaceuticals, Inc. Compositions, methods, and devices for dialysis
EP3277099A4 (fr) * 2015-04-02 2018-10-24 Captozyme, LLC Enzymes de dégradation des oxalates à haute efficacité pour la dégradation d'oxalates insolubles et solubles
WO2016161455A2 (fr) 2015-04-02 2016-10-06 Captozyme, Llc Enzymes de dégradation des oxalates à haute efficacité pour la dégradation d'oxalates insolubles et solubles
US11603524B2 (en) 2015-04-02 2023-03-14 Oxidien Pharmaceuticals, Llc High efficiency oxalate-degrading enzymes for degradation of insoluble and soluble oxalate
WO2018054132A1 (fr) * 2016-09-23 2018-03-29 武汉康复得生物科技股份有限公司 Oxalate décarboxylase glycosylée, sa préparation et son utilisation
US11085033B2 (en) 2016-09-23 2021-08-10 Wuhan Kangfude Biotechnology Co., Ltd. Glycosylated oxalate decarboxylase and preparation and application thereof

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