Classifications

• • 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
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/18—Carboxylic ester hydrolases (3.1.1)
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
  • A23J7/00—Phosphatide compositions for foodstuffs, e.g. lecithin
• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
  • C11B3/00—Refining fats or fatty oils
  • C11B3/003—Refining fats or fatty oils by enzymes or microorganisms, living or dead

Definitions

• This invention relates to a novel phospholipase, DNA encoding it and to its production and use.
• Phospholipids such as lecithin or phosphatidylcholine, consist of glycerol esterified with two fatty acids in an outer (sn-1) and the middle (sn-2) positions and esterified with phosphoric acid in the third position; the phosphoric acid, in turn, may be esterified to an amino-alcohol.
• Phospholipases are enzymes which participate in
• phospholipase activity can be distinguished, including phospholipase A1 and A2 which hydrolyze one fatty acyl group (in the sn-1 and sn-2 position, respectively) to form lysophospholipid; and lysophospholipase (or phospholipase B) which can hydrolyze the remaining fatty acyl group in lysophospholipid.
• This invention relates to a phospholipase that has the ability
• Enzymes with phospholipase B activity have been reported from various fungal sources, including Penicillium notatum (also known as P. chrysogenum; N. Kawasaki, J. Biochem., 77, 1233-44, 1975; N. Masuda et al., Eur. J. Biochem., 202,
• Saccharomyces cerevisiae M. Ichimasa et al., Agric. Biol. Chem., 49 (4), 1083-89, 1985; F. Paultauf et al., J. Biol. Chem., 269, 19725-30, 1994), Torulaspora delbrueckii (old name Saccharomyces rosei; Y. Kuwabara, Agric. Biol. Chem., 52 (10), 2451-58, 1988; FEMS, Microbiol. Letters, 124, 29-34), Schizosaccharomyces pombe (H. Oishi et al., Biosci. Biotech. Biochem., 60 (7), 1087-
• an acidic phospholipase can be obtained from a strain of the genus Hyphozyma. It is able to hydrolyze both fatty acyl groups in intact phospholipid. Advantageously, it has no lipase activity and is active at very low pH; these properties make it very suitable for use in oil degumming, as enzymatic and alkaline hydrolysis (saponification) of the oil can both be suppressed.
• the phospholipase is not membrane bound, making it suitable for commercial production and purification.
• WO 93/24619 discloses a lipase from Hyphozyma sp. LF-132 (CBS 648.91), but the production of phospholipase by this genus has never been reported.
• the phospholipase of this invention can be obtained from the same strain as the known lipase, and that the two enzymes can be separated.
• a first aspect of the invention provides an isolated phospholipase which is able to hydrolyze both fatty acyl groups in a phospholipid, is derivable from a strain of Hyphozyma, and has optimum phospholipase activity at about 50°C and pH 3 measured at the conditions described in Example 3.
• the invention also provides an isolated phospholipase which is able to hydrolyze both fatty acyl groups in a phospholipid, and is a polypeptide comprising at its N-terminal a partial amino acid sequence which is the sequence shown in positions 1-497 of SEQ ID NO: 11 , or is at least 50 % identical therewith
• the invention provides an isolated phospholipase which is able to hydrolyze both fatty acyl groups in a phospholipid, and is a polypeptide containing amino acid sequences which are at least 50 % identical with the amino acid sequences shown in SEQ ID NO: 1-8, disregarding Xaa.
• the invention further provides an isolated DNA sequence which encodes said phospholipase.
• Yet another aspect of the invention provides a method of producing a phospholipase, comprising cultivation of a phospholipase-producing strain of Hyphozyma in a suitable nutrient medium, followed by recovery of the phospholipase
• a further aspect of the invention provides a method for producing a phospholipase, comprising isolating a DNA sequence encoding the phospholipase from a phospholipase-producing strain of Hyphozyma, combining the DNA fragment with appropriate expression signal(s) in an appropriate vector, transforming a suitable heterologous host organism with the vector, cultivating the transformed host organism under conditions leading to expression of the phospholipase, and recovering the phospholipase from the culture medium
• the invention also provides use of said phospholipase in a process comprising treatment of a phospholipid or lysophospholipid with the phospholipase so as to hydrolyze fatty acyl groups.
• the invention provides a process for reducing the content of phospholipid in a vegetable oil, comprising treating the oil with an aqueous dispersion of an acidic phospholipase at pH 1.5-3 so as to hydrolyze a major part of the phospholipid, and separating an aqueous phase containing the hydrolyzed phospholipid from the oil.
• Figs. 1 , 2 and 3 show the temperature profile, pH profile and thermostability, respectively, of phospholipase from Hyphozyma sp. CBS 648.91. Further details are given in Example 3.
• Fig. 4a-d gives a comparison of SEQ ID NO: 11 with 3 prior-art sequences.
• the phospholipase of the invention is able to hydrolyze both acyl groups in a phospholipid molecule (such as phosphatidyl choline or lecithin) without intermediate accumulation of lysophospholipid and is also able to hydrolyze the fatty acyl group of a lysophospholipid (such as lysophosphatidyl choline or lyso-lecithin).
• a phospholipase of the invention is not membrane bound.
• a preferred enzyme is derived from Hyphozyma sp. strain CBS 648.91. Its molecular weight is about 94 kDa by SDS, about 87 kDa by gel filtration, and 92 kDa by mass spectrometry. It is believed to be glycosylated. It has an iso-electric point of about 5.6. It has no lipase activity, i.e. it does not hydrolyze triglycerides. The influence of pH and temperature on the activity of this phospholipase is shown in Fig. 1 and 2. As shown in these figures, the enzyme has optimum activity at about pH 3 and 50°C.
• Fig. 3 shows the thermostability of this enzyme, expressed as the residual activity after 10 minutes at pH 7 at various temperatures. It is seen that the enzyme retains more than 90 % activity at temperatures up to 50°C, more than 75% up to 60°C and more than 50% up to 70°C.
• 1 unit phospholipase activity unit
• DPPC dipalmitoyi phosphatidylcholine
• the amount of released fatty acid is determined by NEFA-C test Wako.
• 1 International Unit (IU) is the amount of phospholipase that releases one ⁇ (micro)-equivalent of free fatty acid per minute from egg yolk in the presence of calcium and deoxycholate at pH 8.0 and 40°C in a pH-stat.
• the released fatty acids are titrated with 0.1 N sodium hydroxide and the base volume is monitored as a function of time.
• the following test is used to identify if a given enzyme has the ability to hydrolyze both fatty acyl groups of a phospholipid without the accumulation of lysophospholipid.
• a substrate solution is prepared containing 2% L- ⁇ (alpha)- phosphatidylcholine, dipalmitoyi (product of Wako Pure Chemical Industries Ltd.) and
• Triton X-100 A buffer solution is prepared containing 0.4 M citrate buffer (pH 5).
• Enzyme solutions are prepared containing various amounts of the sample to be analyzed.
• 0.5 ml of the substrate solution, 0.25 ml of the buffer solution and 0.05 ml of 0.1 N CaCI 2 are mixed and incubated at 40°C.
• 0.1 ml of the enzyme solution is added and incubated for 1 hour.
• the reaction is terminated by adding 0.1 ml of 1 N HCI.
• Partial sequences SEQ ID NO: 1-8 were determined by sequencing of phospholipase from Hyphozyma sp. CBS 648.91 after enzymatic hydrolysis. In these sequences, Xaa represents an amino acid that could not be determined. SEQ ID NO: 1 is an N-terminal sequence, and the others are internal sequences. Xaa in SEQ ID NO: 1 is believed to be a Pro residue. Xaa in SEQ ID NO: 3, 7 and 8 and both Xaa in SEQ ID NO: 5 are believed to be glycosylated Asn residues.
• a nearly complete DNA sequence (SEQ ID NO: 9) was determined for the gene encoding the phospholipase from Hyphozyma sp. CBS 648.91. This sequence was determined from the genomic locus and includes an open reading frame of 552 amino acids and 213 base pairs of sequence upstream of the putative translation initiation codon. The methods used for sequence isolation and determination are well known in the art. Details are given in the examples.
• the long, uninterrupted open reading frame identified in this sequence was translated and compared to the partial peptide sequences SEQ ID NO: 1-8. The translated sequence was identical to seven of the partial peptide sequences at all positions, SEQ ID NO: 1-7, and overlapped the most distal partial peptide sequence, SEQ ID NO: 8 by 10 amino acids.
• SEQ ID NO: 11 a sequence of 573 amino acid residues (shown as SEQ ID NO: 11) has been determined.
• the amino terminus of the mature peptide is determined by comparison with SEQ ID NO: 1.
• the sequenced open reading frame extends upstream an additional 115 amino acids. There is only one Met codon in this region, 76 amino acids from the start of the mature peptide (position -76).
• the 14 amino acids immediately following this methionine residue appear to constitute a secretion signal sequence (G. von Heijne, Nucleic Acids Res, 14, 4683-4690, 1986), indicating both that this is the translation initiation codon and that the encoded protein is secreted.
• the intervening 61 amino acids must constitute a propeptide.
• the peptide sequence from Hyphozyma was aligned with the phospholipase B sequences from three other fungi, Penicillium notatum (Genbank X60348), Saccharomyces cerevisiae (Genbank L23089) and Torulaspora delbrueckii (Genbank D32134), as shown in Fig. 4a-d.
• Penicillium notatum Genbank X60348
• Saccharomyces cerevisiae Genbank L23089
• Torulaspora delbrueckii Genebank D32134
• the portion of the Hyphozyma phospholipase sequence we have determined is 38% identical to the phospholipase from Penicillium notatum, 37% identical to the phospholipase from Saccharomyces cerevisiae, and 38% identical to the phospholipase from Torulaspora delbrueckii.
• the full length Penicillium, Saccharomyces, and Torulaspora sequences extend from 112 to 145 residues further than the partial Hyphozyma sequence, suggesting that the full length for the translated Hyphozyma peptide is approximately 700 amino acid residues.
• the phospholipase of the invention may contain an N-terminal sequence as shown at positions 1-497 of SEQ ID NO: 11 or a sequence derived therefrom by substitution, deletion or insertion of one or more amino acids.
• the derived sequence may be at least 50 % identical, e.g. at least 60%, preferably at least 70%, especially at least 80 or at least 90% identical with said partial sequence.
• the phospholipase of the invention may contain a further 150-250 (e.g. 180-220) amino acid residues at the C- terminal
• the phospholipase of this invention may be derived from a fungal strain of the genus Hyphozyma, a genus of yeast-like Hyphomycetes described in de Hoog, G.S & Smith, M.Th., Antonie van Leeuwenhoek, 47, 339-352 (1981).
• the strain belongs to the species defined by the strain Hyphozyma sp. LF132, CBS 648.91 , which is described in WO 93/24619.
• This strain was classified in the genus Hyphozyma, but it did not match any of the previously described species of Hyphozyma, so it is believed to define a new species. It is particularly preferred to use said strain or a mutant or variant thereof having the ability to produce phospholipase.
• Hyphozyma sp. strain designated LF132 by the inventors
• CBS Centraal Bureau voor Schimmelcultures
• the phospholipase of the invention may be produced by cultivation of the microorganism described above in a suitable nutrient medium, containing carbon and nitrogen sources and inorganic salts, followed by recovery of the enzyme.
• the nutrient medium may be formulated according to principles well known in the art.
• the phospholipase may be recovered from the culture broth and purified to remove lipase activity, e.g. as described in the examples of this specification.
• An alternative method of producing the phospholipase of the invention comprises transforming a suitable host cell with a DNA sequence encoding the phospholipase, cultivating the transformed organism under conditions permitting the production of the enzyme, and recovering the enzyme from the culture.
• the host organism is preferably a eukaryotic cell, in particular a fungal cell, such as a yeast cell or a filamentous fungal cell, preferably a strain of Aspergillus, Fusarium, Trichoderma or Saccharomyces, most preferably A. niger, A. oryzae, F. graminearum, F. sambucinum, F. cerealis or S. cerevisiae.
• the production of the phospholipase in such host organisms may be done by the general methods described in EP 238,023 (Novo Nordisk), WO 96/00787 (Novo Nordisk) or EP 244,234 (Alko).
• the DNA sequence can be isolated from a phospholipase-producing Hyphozyma strain by extraction of DNA by methods known in the art, e.g. as described by Sambrook et al., (1989), Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Lab.; Cold Spring Harbor, NY.
• DNA sequence of the invention can also be isolated by any general method involving
• DNA encoding a phospholipase of the invention may, in accordance with well-known procedures, conveniently be isolated from a phospholipase- producing Hyphozyma strain, by use of synthetic oligonucleotide probes prepared on the basis of a peptide sequence disclosed herein.
• the phospholipase of the invention can be used in any application where it is desired to hydrolyze the fatty acyl group(s) of a phospholipid or lyso-phospholipid, such as lecithin or lyso-lecithin.
• the phospholipase is preferably used at pH 1.5-5 (e.g.
• the phospholipase may be inactivated after the reaction by a heat treatment, e.g. at pH 7,
• the phospholipase of the invention can be used in the preparation of dough, bread and cakes, e.g. to improve the elasticity of the bread or cake.
• the phospholipase can be used in a process for making bread, comprising adding the phospholipase to the ingredients of a dough, kneading the dough and baking the dough to make the bread. This can be done in analogy with US 4,567,046 (Kyowa Hakko), JP-A 60-78529 (QP Corp.), JP-A 62-111629 (QP Corp.), JP-A 63- 258528 (QP Corp.) or EP 426211 (Unilever).
• the phospholipase of the invention can also be used to improve the filterability of an aqueous solution or slurry of carbohydrate origin by treating it with the phospholipase.
• This is particularly applicable to a solution or slurry containing a starch hydrolysate, especially a wheat starch hydrolysate since this tends to be difficult to filter and to give cloudy filtrates.
• the treatment can be done in analogy with EP 219,269 (CPC International).
• the phospholipase of the invention can be used in a process for reducing the content of phospholipid in an edible oil, comprising treating the oil with the phospholipase so as to hydrolyze a major part of the phospholipid, and separating an aqueous phase containing the hydrolyzed phospholipid from the oil.
• This process is applicable to the purification of any edible oil which contains phospholipid, e.g. vegetable oil such as soy bean oil, rape seed oil and sunflower oil.
• the vegetable oil Prior to the enzymatic treatment, the vegetable oil is preferably pretreated to remove slime (mucilage), e.g. by wet refining.
• the oil will contain 50-250 ppm of phosphorus as phospholipid at the start of the treatment with phospholipase, and the process of the invention can reduce this value to below 5-10 ppm.
• the enzymatic treatment is conducted by dispersing an aqueous solution of the phospholipase, preferably as droplets with an average diameter below 10 ⁇ (micro)m.
• the amount of water is preferably 0.5-5% by weight in relation to the oil.
• An emulsifier may optionally be added. Mechanical agitation may be applied to maintain the emulsion.
• the enzymatic treatment can be conducted at a pH in the range 1.5-5.
• the process pH may be in the range 3.5-5 in order to maximize the enzyme performance, or a pH in the range 1.5-3 (e.g. 2-3) may be used in order to suppress the alkaline hydrolysis of triglycerides (saponification).
• the pH may be adjusted by adding citric acid, a citrate buffer or HCI.
• a suitable temperature is generally 30-70°C (particularly 30-45°C, e.g. 35- 40°C).
• the reaction time will typically be 1-12 hours (e.g. 2-6 hours), and a suitable enzyme dosage will usually be 100-5000 IU per liter of oil (e.g. 200-2000 IU/I) or 0.1 - 10 mg/l (e.g. 0.5-5 mg/l).
• the enzymatic treatment may be conducted batchwise, e.g. in a tank with stirring, or it may be continuous, e.g. a series of stirred tank reactors.
• the enzymatic treatment is followed by separation of an aqueous phase and an oil phase. This separation may be performed by conventional means, e.g. 5 centrifugation.
• the aqueous phase will contain phospholipase, and the enzyme may be re-used to improve the process economy.
• the process can be conducted according to principles known in the art, e.g. in analogy with US 5,264,367 (Metallgesellschaft, Rohm); K. Dahlke &
• the strain was cultivated at 27-30 C for 3-4 days.
• the culture broth was subjected to liquid/solid separation by centrifugation. After centrifugation, a 25 phospholipase activity of 1 unit/g culture broth was obtained (unit defined above).
• the supernatant was desalted and freeze-dried resulting in a crude powder preparation.
• MW molecular weight
• the temperature profile was determined at pH 3.0 and 4.0 in a range of 40 to 70°C.
• the phospholipase was incubated for 10 minutes, and the activity was determined by the method described above.
• the temperature profile is presented in Fig. 1 as relative activity (taking the maximum activity as 100%). It is seen from this figure that both at pH 3 and 4, the phospholipase has high activity (more than 50% of optimum) at temperatures of 40 to 60°C with a temperature optimum around 50°C.
• the pH profile was determined at 40°C using glycine-HCI buffer at pH 2, 2,5 and 3, and citrate buffer at pH 3, 4, 5 and 6.
• the results are presented in Fig. 2 as relative activity (taking the maximum activity as 100%). Due to a change of buffer system (glycine-HCI, citrate), the figure is made up of two curves, one representing the interval of pH 2.0 to 3.0 and the other representing the interval of pH 3.0 to 6.0. From the figure it appears that the phospholipase is active at pH values of 2 to 5, and the pH optimum is around 3.
• thermostability was determined by incubating in 0.1 M phosphate buffer (pH 7) for 10 minutes at temperatures of 40-80°C and determining the residual activity after the incubation. The results were 100% at 40°C, 95% at 50°C, 82% at 60°C, 55% at 70°C and 9% at 80°C. These results are also shown in Fig. 3.
• a substrate solution was prepared by dissolving 2% of crude soy bean lecithin
• Lyso-phosphatidylcholine (LPC) was treated for 10 minutes at 40°C, other conditions being the same as described in Example 4.
• the chromatogram showed that about two thirds of the LPC was hydrolyzed, and that fatty acid was formed together with a small amount of phosphatidylcholine.
• Hyphozyma as follows. The enzyme dosage, the reaction pH and temperature were varied, and the resulting content of phospholipid was measured.
• the equipment consisted of a 1 I jacketed steel reactor fitted with a steel lid, a propeller (600 rpm), baffles, a temperature sensor, an inlet tube at the top, a reflux condenser (4 °C) at the top, and an outlet tube at the bottom.
• the reactor jacket was connected to a thermostat bath.
• the outlet tube was connected via silicone tubing to an in-line mixer head equipped with a high shear screen (8500 rpm, flow ca. 1.1 l/minute).
• the mixer head was fitted with a cooling coil (5-10 °C) and an outlet tube, which was connected to the inlet tube of the reactor via silicone tubing.
• a temperature sensor was inserted in the silicone tubing just after the mixer head. The only connection from the reactor/mixer head system to the atmosphere was through the reflux condenser.
• the initial performance was calculated from the initial rate of phosphorus removal from the oil, taking the optimum as 100 %.
• DNA encoding the phospholipase of Hyphozyma was isolated by two different methods. The 5' end of the gene was isolated by cloning. A genomic library of Hyphozyma DNA partially digested with Sau3A was screened at high stringency using standard methods (Sambrook et al., (1989), Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Lab.; Cold Spring Harbor, NY) with a probe specific to the phospholipase sequence. This probe was amplified from total Hyphozyma DNA with degenerate primers designed using the previously determined partial peptide sequences with SEQ ID NO: 1 and 5.
• Standard PCR conditions were used for amplification (Saiki el al., Science, 239, 487-491 , 1988), including 0.5mM MgCI 2 , a 45°C annealing temperature, and primers PLMStrl (SEQ ID NO: 12) and PLMStr ⁇ (SEQ ID NO: 13).
• the clone pMStr16 hybridized to the probe, and therefore was isolated and a portion of the insert was sequenced.
• An additional internal portion of the phospholipase-encoding gene was isolated using PCR with Hyphozyma DNA and the primers PLHaW2 (SEQ ID NO: 14) and PLMStr7 (SEQ ID NO: 15).
• PLHaW2 was designed using the sequence determined from pMStr16, and PLMStr7 was designed from the sequence of the partial peptide with SEQ ID NO: 8. Standard conditions were used for the PCR reactions, with 1.5 mM MgCI 2 , and a 46°C annealing temperature. The resulting amplified fragment was isolated and sequenced.
• ORGANISM Hyphozyma sp .
• ORGANISM Hyphozyma sp.
• ORGANISM Hyphozyma sp.
• ORGANISM Hyphozyma sp.
• Tyr Ala Ser lie Leu Ala Ala Val Ala Gly Lys Arg Asn Glu Gly Tyr 20 25 30
• ORGANISM Hyphozyma sp .
• Trp Glu Phe Asn Pro Tyr Glu Phe Gly Ser Trp Asn Pro Xaa Val Ser 20 25 30
• ORGANISM Hyphozyma sp .
• ORGANISM Hyphozyma sp.
• ORGANISM Hyphozyma sp.
• ORGANISM Hyphozyma sp.
• ATC TGG GAT CTC GAG TCC AAC CTC ATC GTC CCC GAG GAC GGC AAG GTC 954 lie Trp Asp Leu Glu Ser Asn Leu lie Val Pro Glu Asp Gly Lys Val 160 165 170
• CBS Centraal Bureau voor Schimmelcultures

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