WO2006040345A2 - Amidases from aspergillus niger and their use in a food production process - Google Patents
Amidases from aspergillus niger and their use in a food production process Download PDFInfo
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- WO2006040345A2 WO2006040345A2 PCT/EP2005/055242 EP2005055242W WO2006040345A2 WO 2006040345 A2 WO2006040345 A2 WO 2006040345A2 EP 2005055242 W EP2005055242 W EP 2005055242W WO 2006040345 A2 WO2006040345 A2 WO 2006040345A2
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- Prior art keywords
- acrylamide
- seq
- coffee
- amidase
- food
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23F—COFFEE; TEA; THEIR SUBSTITUTES; MANUFACTURE, PREPARATION, OR INFUSION THEREOF
- A23F5/00—Coffee; Coffee substitutes; Preparations thereof
- A23F5/16—Removing unwanted substances
- A23F5/163—Removing unwanted substances using enzymes or microorganisms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23F—COFFEE; TEA; THEIR SUBSTITUTES; MANUFACTURE, PREPARATION, OR INFUSION THEREOF
- A23F5/00—Coffee; Coffee substitutes; Preparations thereof
- A23F5/24—Extraction of coffee; Coffee extracts; Making instant coffee
- A23F5/243—Liquid, semi-liquid or non-dried semi-solid coffee extract preparations; Coffee gels; Liquid coffee in solid capsules
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
- A23L5/25—Removal of unwanted matter, e.g. deodorisation or detoxification using enzymes
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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
- C12N9/14—Hydrolases (3)
- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
Definitions
- the present invention relates to processes for the production of a food, pet food, or feed product involving at least one heating step, and to food, pet food, or feed products obtained by such a process. Furthermore, the present invention relates to a novel enzymes suitable for the process according to the invention.
- Acrylamide has been produced commercially for a number of years. Hence, its toxicological status is well evaluated. Acrylamide is mainly used for the production of poly-acrylamide, and the latter compound is used for various applications, such as the production of drinking water, soil stabilization, industrial wastewater treatment, the winning of oil, and laboratory applications.
- Patent applications US 2004/0058046 and US 2004/0058054 provide methods to prevent acrylamide formation by treatment of an intermediate form of a food product with an enzyme that breaks down amino acids involved in the formation of acrylamide, in particular asparagine.
- the modification of amino acids may be undesirable in view of the product's quality, for instance when the relevant amino acids represent a major fraction of the product's mass. This is for example the case for potatoes wherein about 0.1 wt% asparagine is present with respect to the potatoes dry weight.
- the acrylamide formed on potato chips is about 100 ppb, indicating that only a small fraction of the asparagine present in the potato is formed into acrylamide. Applying the methods according to the above-mentioned patent applications, however, would result in the need to modify all or at least the majority of the nutritional relevant amino acid asparagine present.
- this method is difficult to apply, for instance when none of the intermediate forms of the food product, that occur prior to the heating step, contains sufficient moisture to allow an externally supplied enzyme to act. Finally, it may be that acrylamide is formed in spite of all measures taken to avoid this.
- the wording "food” is here and hereafter defined to include both foodstuffs for human consumption and foodstuffs for animal consumption, including pet food and feed, unless explicitly specified differently in the description.
- Food includes beverages.
- the wording "food product” is defined to cover raw material, food intermediates and food products being ready for consumption, unless explicitly specified differently in the description.
- the present invention provides a process for the production of a food product involving at least one heating step, comprising adding an enzyme capable to modify acrylamide after said heating step.
- the form of the food product to which the enzyme is applied does not have to be the final product - additional processing steps may take place after the addition of the enzyme.
- the preferred food products are food products that are heat treated in dried condition and therefore contain an amount of acryl amide to be reduced to lower levels. By dried condition is meant comprising less than 20wt%, preferably less than 15 wt%.
- Coffee beans, coffee powder, coffee drinks, or coffee containing drink is the most preferred food to be treated. Coffee beans are -A
- WO 2004/037007 discloses such a pre treatment.
- acrylamide forming was tried to be solved by bringing an exogenously added enzyme in contact with a compound inside a more-or-less solid matrix.
- heating steps are those in which a part of the intermediate food product is exposed to temperatures at which the formation of acrylamide is promoted, e.g. 105 0 C or higher, 12O 0 C or higher.
- the temperature of the heating step in a food production process is maximally 25O 0 C, more generally up to 22O 0 C or 200 0 C.
- the heating step in the process according to the invention may be carried out in ovens, for instance at a temperature between 150-250 0 C, such as for the baking of bread and other baked products; in oil such as the frying of
- French fries, potato crisps, or tofu for example at 150-200 0 C; on a hotplate or on or under a heated grill.
- Preferred heating steps are roasting or grilling.
- the food product may be made from at least one raw material that is of plant origin, for example tubers such as potato, sweet potato, or cassava; legumes, such as peas or soy beans; aromatic plants, such as tobacco, coffee or cocoa; nuts; or cereals, such as wheat, rye, corn, maize, barley, groats, buckwheat, rice, or oats. Also food products made from more than one raw material are included in the scope of this invention, for example food products comprising both corn and potato.
- the food product may also comprise at least one raw material that is of animal origin.
- the food product may also comprise at least one raw material that is of fungal origin, such as raw materials derived from mushrooms or fungal protein.
- An example of food products in which the process according to the invention can be suitable are roasted products, and/or powders, mixtures, and/or extracts made from roasted products.
- roasted products are: coffee or cocoa beans; nuts, such as peanuts, almonds, walnuts, pecan nuts, hazelnuts; cereals, such as roasted corn; roots, such as chicory; isolated substances, such as sugar used for caramel.
- the process according to the invention is particularly suitable for coffee or cocoa beans.
- the present invention also relates to products derived from roasted materials.
- products derived from roasted materials are brewed coffee, coffee extract, coffee concentrate instant coffee, liquid coffee drinks, ground coffee, the decaf version of these coffee products, cocoa powder, chocolate, instant chocolate drink, nut paste, beer, whisky, malt, malt extract, soy sauce.
- the present invention is extremely suitable for roasted food products, since in the production of these products generally no step is included comprising sufficient moisture to allow enzyme action on the product intermediates prior to the heating step.
- Particularly preferred processes are those that involve a liquid or moist environment after the heating step, such as the liquid extraction of roasted or heated materials or mixing of ground roasted or heated materials with another, moist material or with moisture. Examples of products made with such processes are coffee, cocoa drink, chocolate, almond spice, soy sauce and caramel.
- the process according to the invention can also be used to other food products comprising acrylamide.
- Acrylamide is formed in food by the reaction of asparagine and glucose when the food is heated at temperatures of 100 - 120 degrees Celcius and higher.
- the acrylamide formation will increase with increasing temperatures.
- Several foods are treated at temperatures whereby acrylamide will be formed such as baked bread in an oven of 225 degrees Celcius, French fries when fried in oil of about 160 to 180 degrees Celcius or coffee beans roasted at temperatures of 180°C and higher.
- An example hereof is the enzymatic treatment of asparagines using asparaginases. This treatment was shown to be very successful. For example in bread, French fries and chips the amount of acrylamide formed was substantially reduced. In these cases the enzyme was added to the food before heating.
- French fries or chips could be sprayed with an enzyme solution.
- an enzyme solution for French fries and bread, only the outer surface of the food will be exposed to a temperature above 100 degrees Celcius. In bread the inner part will not be heated above 80 or 90 degrees Celcius because of the presence of water. Also the inner part of French fries will not be heated above the boiling temperature of water and in the inner part substantially no acrylamide will be formed and thus only the enzymatic treatment of the outer surface of the French fries is enough to prevent acrylamide formation.
- this asparinases is less effective in food products which will be heat treated in such away that the whole food product will have a temperature of above 120 degrees Celcius and whereby there are no methods to incorporate the enzyme in the whole food product in a commercially attractive way.
- these food products are solid and not kneadable (thus not like a bread dough), have a thickness of at least 3 mm, and contain only minor amounts of water, in general less than 20 wt%, preferably less than 12wt% of water.
- a good example hereof are coffee beans. These beans are roasted to produce coffee beans suitable for making coffee. During roasting the whole bean will have a temperature of more than 150 degrees Celcius.
- Acrylamide will not be only formed in the outer layer, like in case of French fries or bread, but acrylamide is also formed throughout the whole bean.
- coffee berries are picked, dehulled, matured (“fermented”), washed, and dried (either to the open air or by forced hot air). These steps take at the coffee producer (in the producing country).
- the resulting product is the green coffee bean, with a moisture content between 9.8 and 12.5 % (the recommendation is 1 1 -12). In this stage the bean is well-keepable.
- the present invention therefore is preferably used in the process of producing food which is a coffee product preferably coffee beans, ground coffee, instant coffee, liquid coffee drinks, coffee extracts and the decaf version of coffee products.
- the process of the present invention is most advantageously applied in making regular coffee products.
- the present invention discloses a process to reduce the level of acrylamide in food by treating food which has been heat treated at temperatures above 100 degrees Celcius, preferably above 120 degrees Celcius and most preferably above 150 degrees Celcius and subsequently is contacted under aqueous conditions with an amidase enzyme.
- the aqueous conditions are obtained by adding a liquid or paste containing water.
- the liquid or paste may contain the enzyme but the liquid or paste may also be added separately from the enzyme.
- the food product may be in aqueous state (for example coffee extract or chocolate or cocoa drink), in an paste state (for example peanut butter or almond spice), in an emulsion form (for example peanut butter) as long as the enzymatically treated food contains at least 15 wt%, preferably at least 25 wt%, more preferably at least 40 wt% and most preferably at least 60 wt% of water.
- the food product of the present invention is preferably a brewed coffee, liquid coffee drinks, coffee extracts, cocoa drinks or chocolate drinks. After the enzymatical treatment, the food product may be dried or partially dried. So also concentrated coffee extract, instant coffee, instant chocolate drink or instant cocoa drinks with were enzymatically treated with an amidase are part of the present invention.
- amidase can be inactivated. This can be done by for example a short heat treatment at a suitable temperature for example at 90O.
- a short heat treatment at a suitable temperature for example at 90O.
- the present invention provides roasted coffee beans, coffee extracts, concentrated coffee drinks, coffee drinks, brewed coffee containing less than 50 ppb, preferably less than 30 ppb and most preferably less than 10 ppb of acrylamide based on dry matter coffee.
- the present invention also comprises packages such as sealed foil packages, optionally vacuum sealed, jars or pots made of metal, glass or polymers and the like which comprise at least 50 grams, preferably at least 100 grams of roasted coffee beans, coffee extracts, concentrated coffee drinks, coffee drinks, brewed coffee containing less than 50 ppb, preferably less than 30 ppb and most preferably less than 10 ppb of acrylamide based on dry matter coffee.st preferably less than 10 ppb of acrylamide based on dry matter coffee. In general these packages will contain less than 1000 kg, preferably less than
- the enzyme used in the process of the invention is an enzyme capable of modifying acrylamide.
- enzyme is meant “one enzyme” as well as "a combination of more than one enzyme”.
- the enzyme is capable of modifying short-chain non- cyclic amides.
- short chain non-cyclic amides comprise at most 6 C-atoms in their aliphatic chain, more preferably at most 5, even more preferably at most 4 and most preferably at most 3.
- the aliphatic chain can be linear or branched.
- the enzyme is capable of modifying acrylamide, acetamide and/or propionamide.
- a preferred modification of acrylamide is through the action of an amidase.
- the enzyme preparation used in the process of the invention is derived from a micro-organism and obtained by fermentation processes known in the art.
- the micro-organism may be a bacterium, a fungus or a yeast.
- the enzyme can be obtained from various sources, such as for example from plants, animals and microorganisms, such as for example Alcaligenes, Arthrobacter, Brevibacterium, Escherichia, Klebsiella, Mycobacterium, Streptomyces, Saccharomyces, Pseudomonas, Rhodococcus, Xanthomonas, Aspergillus and Bacillus species, preferably Aspergillus, Baccillus or Saccharomyces, more preferably Aspergillus.
- An example of a suitable Escherichia strain is Escherichia coli.
- An example of a suitable Rhodococcus strain is Rhodococcus rhodochrous.
- suitable Pseudomonas strains are P. aeruginosa, P. cepacia, and P. chloroapis.
- An example of suitable Streptomyces strains is Streptomyces lividans.
- suitable Saccharomyces strains are for example Saccharomyces cerevisae, Saccharomyces uvarum, Saccharomyces bayanus, Saccharomyces pastorianus or Saccharomyces paradoxus.
- suitable Aspergillus strains are Aspergillus oryzae, Aspergillus nidulans (Emericella nidulans) or Aspergillus niger.
- Bacillus strains are Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus lichen if ormis, Bacillus megateruim, Bacillus stearothemophilus, Bacillus subtilis or Bacillus thuringiensis.
- the enzyme is obtained from food-grade organisms, for example
- Aspergillus niger Saccharomyces cerevisiae, or Bacillus subtilis, most preferably Aspergillus niger.
- the enzyme is provided in a stabile form, usually a liquid, a powder, a granulate, or an encapsulated form.
- a stabile form usually a liquid, a powder, a granulate, or an encapsulated form.
- any additives and stabilizers known to be useful in the art to improve and/or maintain the enzyme's activity may be applied.
- the enzyme When the enzyme is contained in a liquid form, it may be applied to the product by any conceivable method, for instance by soaking, spraying or mixing.
- the enzyme can for example also be added to a liquid extract, an emulsion or a suspension of an intermediate of the food.
- the enzyme can be produced in situ by a micro-organism capable of producing said enzyme.
- the enzyme can be added in an amount of about at between 100-0.1 U/L, more preferably lower than 50 U/L, even more preferably at most 10 U/L or between 5 and 1 U/L (wherein 1 U is defined as hydrolysing 1 ⁇ mol amide per minute), see Example 8.
- Amidases are enzymes that hydrolyse the C-N chemical bond of an amide, thereby producing ammonia and the corresponding acid. For instance an acetamidase converts acetamide to ammonia and acetic acid, and an acrylamidase converts acrylamide to ammonia and acrylic acid (propenoic acid).
- Amidases are a well-researched class of enzymes, classified as “Enzymes hydrolysing carbon-nitrogen bonds, other than peptide bonds” (EC 3.5). Of particular interest is the group acting on linear amides (3.5.1 ). Another group of enzymes that catalyse a similar reaction are the beta-lactam acylases (EC 3.5.1 .1 1 ), where the C and the N are also both substituted, for instance glutaryl acylase, where the C is part of glutaric acid, and the N part of a beta-lactam ring.
- Additional enzymes that act in a similar fashion are asparaginase (3.5.1 .1 ), glutaminase (3.5.1 .2), 6-aminohexanoate-cyclic- dimer hydrolase (EC 3.5.2.12), fatty acid amide hydrolase, chitin deacetylase (3.5.1 .41 ), glutamyl-tRNA (Gln)-amidotransferase.
- the hydrolytic reaction that they catalyse is similar to that for peptidases (EC 3.4), but in the case of peptidases both the C and the N of the hydrolysed bond are contained in amino acids.
- amidases according to the invention being to modify acrylamide once it is formed, is essentially different from the use of for example asparaginase to break down amino acids in order to prevent the formation of acrylamide as is described in the prior art, for example US 2004/0058046 or US 2004/0058054. Many, but not all, of these enzymes share a common structural motif: a conserved region rich in glycine, serine, and alanine residues. It has been reported that there are three major families of amidases: the amidase signature group (examples: A. oryzae, S. typhimurium); the nitrilase group (examples: P. aeruginosa, R.
- Substrates for amidases include acetamide, indoleacetamide, phenylacetamide, para-nitro-phenylacetamide, para-nitro-acetanilide, chloracetamide, propionamide, butyramide, isobutyramide, succinamide, acrylamide, methacrylamide, benzamide, and nicotinamide.
- amidases from various sources to hydrolyse acrylamide has been reported in the prior art for removal of monomeric acrylamide from polyacrylamide products, to allow for a safer application on the polymer.
- US patent 4,925,797 describes the use of an amidase from Methylophilus methylotrophus for this purpose.
- the authors of this patent are concerned with the presence of acrylamide in polymers that may come into contact with food, and they provide a method to remove the acrylamide from the polymer to avoid contamination of the food.
- amidase from Rhodococcus sp. is used to decrease acrylamide levels in polyacrylamide gels.
- US patent 6,248,551 describes acrylamidase from Helicobacter pylori. Other authors have used immobilized cells to achieve the breakdown of acrylamide, for instance in US patent 6,146,861 where Rhodococcus rhodochrous cells are used for this purpose.
- acrylamide hydrolysing activities are also known from fungi.
- the Emericella acetamidase is applied as a selection marker for genetically transformed fungal cells in the field of molecular genetics.
- fungal amidases have not been employed for the effective removal of acrylamide.
- successful application of acrylamide- hydrolysing enzymes in food successful in the sense that acrylamide is reduced to acceptable levels, has not yet been achieved.
- the levels to be achieved - and hence the concentration of the enzyme substrate are extremely low, being in the ppb range, such as lower than 200 ppb, more preferably lower than 100 ppb, even more preferably lower than 50 ppb and most preferably lower than 20 ppb.
- the filamentous fungus Aspergillus niger harbours multiple genes coding for enzymes that are able to hydrolyse acrylamide. Moreover we have found some of these enzymes to be secreted into the culture liquid, which improves the ease of producing and isolating these. Moreover, we have found that some of these enzymes are capable of effectively removing acrylamide from foodstuffs, in particular from foodstuffs where acrylamide has been formed during a heating step.
- the invention provides newly identified polynucleotide sequences comprising genes that encode novel amidases which for example can be yielded from Aspergillus niger.
- novel amidases can be used in the process for food production of the present invention, for example in production of coffee extract.
- the invention also provides for novel polynucleotides encoding novel amidase enzymes.
- the present invention provides novel polynucleotides encoding an amidase, tentatively called AMID01 , AMID02, AMIDOG, AMID04, AMID05, AMID06, AMID07, AMID08, AMID09, AMID10 and AMID11 (hereinafter referred to AMID01 -1 1 ), having an amino acid sequence chosen from the group respectively consisting of SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32 and 33 (hereinafter ieferred to SEQ ID NO: 23-33) or functional equivalents of any of them.
- the sequence of the genes encoding AMID01 -1 1 was determined by sequencing a genomic clone obtained from Aspergillus niger.
- the invention provides polynucleotide sequences comprising the gene encoding the AMID01 - 1 1 amidase as well as its complete cDNA sequence and its coding sequence.
- the invention relates to an isolated polynucleotide comprising the nucleotide sequence chosen from the group consisting of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or 1 1 (hereinafter referred to as SEQ ID NO: 1-1 1 ) or chosen from the group consisting of SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 (hereinafter referred to as SEQ ID NO: 12-22) or functional equivalents of any of them.
- the invention relates to an isolated polynucleotide hybridisable, preferably under stringent conditions, more preferably under highly stringent conditions, to a polynucleotide chosen from the group consisting of SEQ ID NO: 1 -1 1 or chosen from the group consisting of SEQ ID NO: 12-22.
- polynucleotides may be obtained from filamentous fungi, in particular from Aspergillus niger.
- the invention relates to an isolated polynucleotide having a nucleotide sequence chosen from the group consisting of SEQ ID NO: 1-1 1 or chosen from the group consisting of SEQ ID NO: 12-22.
- the invention also relates to an isolated polynucleotide encoding at least one functional domain of a polypeptide chosen from the group consisting of SEQ ID NO: 23- 33 or functional equivalents of any of them.
- gene and “recombinant gene” refer to nucleic acid molecules which may be isolated from chromosomal DNA, which include an open reading frame encoding a protein, e.g. an A. niger asparaginase.
- a gene may include coding sequences, non-coding sequences, introns and regulatory sequences.
- a gene refers to an isolated nucleic acid molecule as defined herein.
- a nucleic acid molecule of the present invention such as a nucleic acid molecule having the nucleotide sequence chosen from the group consisting of SEQ ID NO: 1 -1 1 or chosen from the group consisting of SEQ ID NO: 12-22 or a functional equivalent of any of them, can be isolated using standard molecular biology techniques and the sequence information provided herein. For example, using all or portion of the nucleic acid sequence chosen from the group consisting of SEQ ID NO: 1 -1 1 or chosen from the group consisting of SEQ ID NO: 12-22 as a hybridization probe, nucleic acid molecules according to the invention can be isolated using standard hybridization and cloning techniques (e. g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
- nucleic acid molecule encompassing all or a portion of a nucleic acid sequence chosen from the group consisting of SEQ ID NO: 1 -1 1 or chosen from the group consisting of SEQ ID NO: 12-22 can be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence information contained in a sequence chosen from the group consisting of SEQ ID NO: 1 -1 1 or chosen from the group consisting of SEQ ID NO: 12-22.
- PCR polymerase chain reaction
- a nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
- the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
- an isolated nucleic acid molecule of the invention comprises the nucleotide sequence chosen from the group consisting of SEQ ID NO: 12- 22.
- the sequence chosen from the group consisting of SEQ ID NO: 12-22 correspond respectively to the coding region of the A. niger AMID01-1 1 cDNA.
- This cDNA comprises sequences encoding the A. niger AMID01-1 1 polypeptide respectively according to SEQ ID NO: 23-33.
- an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence chosen from the group consisting of SEQ ID NO: 1 -1 1 or chosen from the group consisting of SEQ ID NO: 12-22 or a functional equivalent of any of these nucleotide sequences.
- a nucleic acid molecule which is complementary to another nucleotide sequence is one which is sufficiently complementary to the other nucleotide sequence such that it can hybridize to the other nucleotide sequence thereby forming a stable duplex.
- One aspect of the invention pertains to isolated nucleic acid molecules that encode a polypeptide of the invention or a functional equivalent thereof such as a biologically active fragment or domain, as well as nucleic acid molecules sufficient for use as hybridisation probes to identify nucleic acid molecules encoding a polypeptide of the invention and fragments of such nucleic acid molecules suitable for use as PCR primers for the amplification or mutation of nucleic acid molecules.
- an "isolated polynucleotide” or “isolated nucleic acid” is a DNA or RNA that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived.
- an isolated nucleic acid includes some or all of the 5' non-coding (e.g., promotor) sequences that are immediately contiguous to the coding sequence.
- the term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene encoding an additional polypeptide that is substantially free of cellular material, viral material, or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized). Moreover, an "isolated nucleic acid fragment" is a nucleic acid fragment that is not naturally occurring as a fragment and would not be found in the natural state.
- nucleic acid molecule As used herein, the terms “polynucleotide” or “nucleic acid molecule” are intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.
- the nucleic acid molecule can be single-stranded or double-stranded, but preferably is double- stranded DNA.
- the nucleic acid may be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.
- Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to at least any one of the nucleic acid molecule chosen from the group consisting of AMID01 -1 1 , e.g. its coding strand. Also included within the scope of the invention are the complement strands of the nucleic acid molecules described herein.
- sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases.
- the specific sequences disclosed herein can be readily used to isolate the complete gene from filamentous fungi, in particular A. n/gerwhich in turn can easily be subjected to further sequence analyses thereby identifying sequencing errors.
- nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art.
- a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.
- a nucleic acid molecule according to the invention may comprise only a portion or a fragment of the nucleic acid sequence chosen from the group consisting of SEQ ID NO: 1 -11 or chosen from the group consisting of SEQ ID NO: 12-22, for example a fragment which can be used as a probe or primer or a fragment encoding a portion of an AMID01-11 protein.
- the nucleotide sequence determined from the cloning of the AMID01- 11 gene and cDNA allows for the generation of probes and primers designed for use in identifying and/or cloning other AMID01-11 family members, as well as AMID01 -11 homologues from other species.
- the probe/primer typically comprises substantially purified oligonucleotide which typically comprises a region of nucleotide sequence that hybridizes preferably under highly stringent conditions to at least about 12 or 15, preferably about 18 or 20, preferably about 22 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 or more consecutive nucleotides of a nucleotide sequence chosen from the group consisting of SEQ ID NO: 1-11 or chosen from the group consisting of SEQ ID NO: 12-22 or of a functional equivalent of any of them.
- Probes based on the AMID01 -11 nucleotide sequences can be used to detect transcripts or genomic AMID01 -11 sequences encoding the same or homologous proteins for instance in other organisms.
- the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor.
- the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor.
- Such probes can also be used as part of a diagnostic test kit for identifying cells which express an AMID01 -11 protein.
- the two sequences are the same length.
- a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
- the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. MoI. Biol.
- the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1 , 2, 3, 4, 5, or 6.
- the percent identity two amino acid or nucleotide sequence is determined using the algorithm of E. Meyers and W.
- nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. MoI. Biol. 215:403 — 10.
- Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402.
- the default parameters of the respective programs e.g., XBLAST and NBLAST
- the default parameters of the respective programs e.g., XBLAST and NBLAST
- hybridizing is intended to describe conditions for hybridization and washing under which nucleotide sequences at least about 50%, at least about 60%, at least about 70%, more preferably at least about 80%, even more preferably at least about 85% to 90%, more preferably at least 95% homologous to each other typically remain hybridized to each other.
- hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45 0 C, followed by one or more washes in 1 X SSC, 0.1 % SDS at 50 0 C, preferably at 55 0 C, preferably at 60 0 C and even more preferably at 65 0 C.
- SSC sodium chloride/sodium citrate
- Highly stringent conditions include, for example, hybridizing at 68 0 C in 5x SSC/5x Denhardt's solution / 1 .0% SDS and washing in 0.2x SSC/0.1 % SDS at room temperature. Alternatively, washing may be performed at 42 0 C.
- a polynucleotide which hybridizes only to a poly A sequence such as the 3' terminal poly(A) tract of mRNAs), or to a complementary stretch of T (or U) resides, would not be included in a polynucleotide of the invention used to specifically hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-standed cDNA clone).
- cDNA libraries constructed from other organisms e.g. filamentous fungi, in particular from the species Aspergillus can be screened.
- Aspergillus strains can be screened for homologous AMID01 -1 1 polynucleotides by Northern blot analysis.
- cDNA libraries can be constructed from RNA isolated from the appropriate strain, utilizing standard techniques well known to those of skill in the art.
- a total genomic DNA library can be screened using a probe hybridisable to an AMID01-1 1 polynucleotide according to the invention.
- Homologous gene sequences can be isolated, for example, by performing PCR using two degenerate oligonucleotide primer pools designed on the basis of nucleotide sequences as taught herein.
- the template for the reaction can be cDNA obtained by reverse transcription of mRNA prepared from strains known or suspected to express a polynucleotide according to the invention.
- the PCR product can be subcloned and sequenced to ensure that the amplified sequences represent the sequences of a new AMID01 -1 1 nucleic acid sequence, or a functional equivalent thereof.
- the PCR fragment can then be used to isolate a full length cDNA clone by a variety of known methods.
- the amplified fragment can be labeled and used to screen a bacteriophage or cosmid cDNA library.
- the labeled fragment can be used to screen a genomic library.
- RNA can be isolated, following standard procedures, from an appropriate cellular or tissue source.
- a reverse transcription reaction can be performed on the RNA using an oligonucleotide primer specific for the most 5' end of the amplified fragment for the priming of first strand synthesis.
- RNA/DNA hybrid can then be "tailed" (e.g., with guanines) using a standard terminal transferase reaction, the hybrid can be digested with RNase H, and second strand synthesis can then be primed (e.g., with a poly-C primer).
- second strand synthesis can then be primed (e.g., with a poly-C primer).
- Whether or not a homologous DNA fragment encodes a functional AMID01-1 1 protein may easily be tested by methods known in the art.
- Vectors Another aspect of the invention pertains to vectors, preferably expression vectors, containing a polynucleotide sequence as described above, encoding an AMID01-11 protein or a functional equivalent thereof.
- vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
- plasmid which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
- viral vector Another type of vector, wherein additional DNA segments can be ligated into the viral genome.
- vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
- Other vectors e.g., non-episomal mammalian vectors
- certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors”.
- expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
- plasmid and "vector” can be used interchangeably herein as the plasmid is the most commonly used form of vector.
- the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
- viral vectors e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses
- the recombinant expression vectors of the invention can comprise a nucleic acid according to invention in a form suitable for expression of the nucleic acid in a host cell.
- the recombinant expression vector can for example include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed.
- the vector according tot eh invention preferably comprises the polynucleotide sequence according to the invention operatively linked with regulatory sequences suitable for expression of said polynucleotide sequence in a suitable host cell.
- "operatively linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
- regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signal).
- Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cells and those which direct expression of the nucleotide sequence only in a certain host cell (e.g. tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
- the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, encoded by nucleic acids as described herein (e.g. AMID01 -1 1 proteins, mutant forms of AMID01-1 1 proteins, fragments, variants or functional equivalents thereof, etc.).
- the recombinant expression vectors of the invention can be designed for expression of AMID01-1 1 proteins in prokaryotic or eukaryotic cells.
- AMID01-1 1 proteins can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells, fungal cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
- the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
- Expression vectors useful in the present invention include chromosomal-, episomal- and virus-derived vectors e.g., vectors derived from bacterial plasmids, bacteriophage, filamentous fungi, yeast episome, yeast chromosomal elements, viruses such as baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
- vectors derived from bacterial plasmids, bacteriophage, filamentous fungi, yeast episome, yeast chromosomal elements, viruses such as baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses and vectors derived from combinations thereof, such as those
- the DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
- an appropriate promoter such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
- Other suitable promoters will be known to the skilled person.
- promoters are preferred that are capable of directing a high expression level of asparaginases in filamentous fungi. Such promoters are known in the art.
- the expression constructs may contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation.
- the coding portion of the mature transcripts expressed by the constructs will include a translation initiating A
- Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
- transformation and “transfection” are intended to refer to a variety of art- recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-percipitation, DEAE-dextran-mediated transfection, transduction, infection, lipofection, cationic lipidmediated transfection or electroporation.
- Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2 d ,ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Davis et al., Basic Methods in Molecular Biology (1986) and other laboratory manuals.
- a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
- selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methatrexate.
- Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding an AMID01 -1 1 protein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g. cells that have incorporated the selectable marker gene will survive, while the other cells die).
- the expression vectors will preferably contain selectable markers.
- markers include dihydrofolate reductase or neomycin resistance for eukarotic cell culture and tetracyline or ampicilling resistance for culturing in E. coli and other bacteria.
- Representative examples of appropriate host include bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS and Bowes melanoma; and plant cells. Appropriate culture media and conditions for the above- described host cells are known in the art.
- vectors preferred for use in bacteria are pQE70, pQE60 and PQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16A, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233- 3, pDR540, pRIT5 available from Pharmacia.
- preferred eukaryotic vectors are PWLNEO, pSV2CAT, pOG44, pZT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.
- Other suitable vectors will be readily apparent to the skilled artisan.
- bacterial promotors for use in the present invention include E. coli lad and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR, PL promoters and the trp promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus ("RSV”), and metallothionein promoters, such as the mouse metallothionein-l promoter.
- retroviral LTRs such as those of the Rous sarcoma virus ("RSV")
- metallothionein promoters such as the mouse metallothionein-l promoter.
- Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type.
- enhancers include the SV40 enhancer, which is located on the late side of the replication origin at bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
- appropriate secretation signal may be incorporated into the expressed polypeptide.
- the signals may be endogenous to the polypeptide or they may be heterologous signals.
- the polypeptide may be expressed in a modified form and may include not only secretion signals but also additional heterologous functional regions. Thus, for instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification.
- Polypeptides according to the invention provides an isolated polypeptide having the amino acid sequence chosen from the group consisting of amino acid sequences according to SEQ ID NO: 23- 33, an amino acid sequence obtainable by expressing respectively the polynucleotide of SEQ ID NO: 1-11 , in an appropriate host or obtainable by expressing respectively the polynucleotide of SEQ ID NO: 12-22 in an appropriate host. Also, a peptide or polypeptide comprising a functional equivalent of the above polypeptides is comprised within the present invention. The above polypeptides are collectively comprised in the term "polypeptides according to the invention"
- peptide and oligopeptide are considered synonymous (as is commonly recognized) and each term can be used interchangeably as the context requires to indicate a chain of at least two amino acids coupled by peptidyl linkages.
- polypeptide is used herein for chains containing more than seven amino acid residues. All oligopeptide and polypeptide formulas or sequences herein are written from left to right and in the direction from amino terminus to carboxy terminus. The one-letter code of amino acids used herein is commonly known in the art and can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2 d ,ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989)
- isolated polypeptide or protein is intended a polypeptide or protein removed from its native environment.
- recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purpose of the invention as are native or recombinant polypeptides which have been substantially purified by any suitable technique such as, for example, the single-step purification method disclosed in Smith and Johnson, Gene 67:31-40 (1988).
- the AMID01-11 amidase according to the invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification.
- Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells.
- polypeptides of the present invention may be glycosylated or may be non-glycosylated.
- polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. Protein fragments
- the invention also features biologically active fragments of the polypeptides according to the invention.
- Biologically active fragments of a polypeptide of the invention include polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the AMID01 -1 1 protein (e.g., the amino acid sequence respectively chosen from the group consisting of SEQ ID NO: 23-33), which include fewer amino acids than the full length protein, and exhibit at least one biological activity of the corresponding full- length protein.
- biologically active fragments comprise a domain or motif with at least one activity of the AMID01-1 1 protein.
- a biologically active fragment of a protein of the invention can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length.
- biologically active portions in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the biological activities of the native form of a polypeptide of the invention.
- the invention also features nucleic acid fragments which encode the above biologically active fragments of the AMID01 -1 1 protein.
- Functional equivalents and “functional variants” are used interchangeably herein.
- Functional equivalents of AMID01 -1 1 DNA are isolated DNA fragments that encode a polypeptide that exhibits a particular function of the AMID01 -1 1 A. n/geramidase as defined herein.
- a functional equivalent of an AMID01 -1 1 polypeptide according to the invention is a polypeptide that exhibits at least one function of an A. niger amidase as defined herein. Functional equivalents therefore also encompass biologically active fragments.
- Functional protein or polypeptide equivalents may contain only conservative substitutions of one or more amino acids chosen from the group consisting of SEQ ID NO: 23-33 or substitutions, insertions or deletions of non-essential amino acids.
- a non-essential amino acid is a residue that can be altered in an amino acid chosen from the group consisting of SEQ ID NO: 23-33 without substantially altering the biological function.
- amino acid residues that are conserved among the AMID01-1 1 proteins of the present invention are predicted to be particularly unamenable to alteration.
- amino acids conserved among the AMID01 -1 1 proteins according to the present invention and other amidases are not likely to be amenable to alteration.
- substitution is intended to mean that a substitution in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
- These families are known in the art and include amino acids with basic side chains (e.g. lysine, arginine and hystidine), acidic side chains (e.g.
- uncharged polar side chains e.g., glycine, asparagines, cjutamine, serine, threonine, tyrosine, cysteine
- non-polar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
- beta-branched side chains e.g., threonine, valine, isoleucine
- aromatic side chains e.g., tyrosine, phenylalanine tryptophan, histidine.
- nucleic acid equivalents may typically contain silent mutations or mutations that do not alter the biological function of encoded polypeptide. Accordingly, the invention provides nucleic acid molecules encoding AMID01 -1 1 proteins that contain changes in amino acid residues that are not essential for a particular biological activity. Such AMID01-1 1 proteins differ in amino acid sequence chosen from the group consisting of SEQ ID NO: 23-33 yet retain at least one biological activity.
- the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises a substantially homologous amino acid sequence of at least about 40%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homologous to the amino acid sequence shown any amino acid sequence chosen from the group consisting of SEQ ID NO: 23-33.
- An isolated nucleic acid molecule encoding an AMID01-11 protein homologous to the protein according to any protein chosen from the group consisting of SEQ ID NO: 23- 33 can be created by introducing one or more nucleotide substitutions, additions or deletions into the coding nucleotide sequences chosen from the group consisting of SEQ ID NO: 1-11 or chosen from the group consisting of SEQ ID NO: 12-22 such that one or more amino acid substitutions, deletions or insertions are introduced into the encoded protein.
- Such mutations may be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
- orthologues of the A. niger AMID01-11 protein are proteins that can be isolated from other strains or species and possess a similar or identical biological activity. Such orthologues can readily be identified as comprising an amino acid sequence that is substantially homologous to an amino acid sequence chosen from the group consisting of SEQ ID NO: 23-33.
- substantially homologous refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., with similar side chain) amino acids or nucleotides to a second amino acid or nucleotide sequence such that the first and the second amino acid or nucleotide sequences have a common domain.
- amino acid or nucleotide sequences which contain a common domain having about 40%, preferably 65%, more preferably 70%, even more preferably 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity or more are defined herein as sufficiently identical.
- nucleic acids encoding other AMID01 -11 family members which thus have a nucleotide sequence that differs from the group consisting of SEQ ID NO: 1-11 or from the group consisting of SEQ ID NO: 12-22, are within the scope of the invention.
- nucleic acids encoding AMID01-11 proteins from different species which thus have a nucleotide sequence which differs from the group consisting of SEQ ID NO: 1 -1 1 or from the group consisting of SEQ ID NO: 12-22 are within the scope of the invention.
- Nucleic acid molecules corresponding to variants (e.g. natural allelic variants) and homologues of the AMID01 -1 1 DNA of the invention can be isolated based on their homology to the AMID01 -1 1 nucleic acids disclosed herein using the cDNAs disclosed herein or a suitable fragment thereof, as a hybridisation probe according to standard hybridisation techniques preferably under highly stringent hybridisation conditions.
- allelic variants of the AMID01 -1 1 sequence the skilled person will recognise that changes can be introduced by mutation into the nucleotide sequences chosen from the group consisting of SEQ ID NO: 1 -1 1 or from the group consisting of SEQ ID NO: 12-22 thereby leading to changes in the amino acid sequence of the AMID01 -1 1 protein without substantially altering the function of the AMID01-1 1 protein.
- improved AMID01 -1 1 proteins are provided.
- Improved AMID01-1 1 proteins are proteins wherein at least one biological activity is improved. Such proteins may be obtained by randomly introducing mutations along all or part of the AMID01-1 1 coding sequence, such as by saturation mutagenesis, and the resulting mutants can be expressed recombinantly and screened for biological activity. For instance, the art provides for standard assays for measuring the enzymatic activity of amidases and thus improved proteins may easily be selected.
- the AMID01 -1 1 protein has an amino acid sequence according to SEQ ID NO: 3.
- the AMID01 -1 1 polypeptide is substantially homologous to the amino acid sequence chosen from the group consisting of SEQ ID NO: 23-33 and retains at least one biological activity of a polypeptide chosen from the group consisting of SEQ ID NO: 23-33, yet differs in amino acid sequence due to natural variation or mutagenesis as described above.
- the AMID01 -1 1 protein has an amino acid sequence encoded by an isolated nucleic acid fragment capable of hybridising to a nucleic acid chosen respectively from the group consisting of SEQ ID NO: 1-1 1 or from the group consisting of SEQ ID NO: 12-22, preferably under highly stringent hybridisation conditions.
- the AMID01 -1 1 protein is a protein which comprises an amino acid sequence at least about 40%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homologous to the amino acid sequence as respectively chosen from the group consisting of SEQ ID NO: 23-33 and retains at least one functional activity of the polypeptide as chosen from the group consisting of SEQ ID NO: 23-33.
- Functional equivalents of a protein according to the invention can also be identified e.g. by screening combinatorial libraries of mutants, e.g. truncation mutants, of the protein of the invention for asparaginase activity.
- a variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level.
- a variegated library of variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential protein sequences is expressible as individual polypeptides.
- libraries of fragments of the coding sequence of a polypeptide of the invention can be used to generate a variegated population of polypeptides for screening a subsequent selection of variants.
- a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of the coding sequence of interest with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector.
- an expression library can be derived which encodes Nterminal and internal fragments of various sizes of the protein of interest.
- REM Recursive ensemble mutagenesis
- DNA sequence polymorphisms that may lead to changes in the amino acid sequence of the AMID01 -1 1 protein may exist within a given population. Such genetic polymorphisms may exist in cells from different populations or within a population due to natural allelic variation. Allelic variants may also include functional equivalents.
- Fragments of a polynucleotide according to the invention may also comprise polynucleotides not encoding functional polypeptides. Such polynucleotides may function as probes or primers for a PCR reaction. Nucleic acids according to the invention irrespective of whether they encode functional or non-functional polypeptides, can be used as hybridization probes or polymerase chain reaction (PCR) primers. Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having an AMID01 -1 1 activity include, inter alia, (1 ) isolating the gene encoding the AMID01 -1 1 protein, or allelic variants thereof from a cDNA library e.g. from other organisms than A.
- in situ hybridization e.g. FISH
- metaphase chromosomal spreads to provide precise chromosomal location of the AMID01-1 1 gene as described in Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988);
- Northern blot analysis for detecting expression of AMID01 -1 1 mRNA in specific tissues and/or cells and 4) probes and primers that can be used as a diagnostic tool to analyse the presence of a nucleic acid hybridisable to the AMID01 -1 1 probe in a given biological (e.g. tissue) sample.
- Also encompassed by the invention is a method of obtaining a functional equivalent of an AMID01-1 1 gene or cDNA.
- a method entails obtaining a labelled probe that includes an isolated nucleic acid which encodes all or a portion of the sequence chosen from the group consisting of SEQ ID NO: 23-33 or a variant thereof; screening a nucleic acid fragment library with the labelled probe under conditions that allow hybridisation of the probe to nucleic acid fragments in the library, thereby forming nucleic acid duplexes, and preparing a full-length gene sequence from the nucleic acid fragments in any labelled duplex to obtain a gene related to the AMID01 -1 1 gene.
- an AMID01 -1 1 nucleic acid of the invention is at least 40%
- ID NO: 1-1 1 or from the group consisting of SEQ ID NO: 12-22 or the complement of any of them.
- an AMID01 -1 1 polypeptide of the invention is at least 40%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homologous to the amino acid sequence shown in a peptide sequence chosen from the group consisting of SEQ ID NO: 23-33.
- the invention features cells, e.g., transformed host cells or recombinant host cells that contain a nucleic acid encompassed by the invention.
- a "transformed cell” or “recombinant cell” is a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a nucleic acid according to the invention.
- Both prokaryotic and eukaryotic cells are included, e.g., bacteria, fungi, yeast, and the like, especially preferred are cells from filamentous fungi, in particular Aspergillus niger.
- a host cell can be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in a specific, desired fashion. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may facilitate optimal functioning of the protein.
- Various host cells have characteristic and specific mechanisms for post- translational processing and modification of proteins and gene products. Appropriate cell lines or host systems familiar to those of skill in the art of molecular biology and/or microbiology can be chosen to ensure the desired and correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used.
- Host cells are well known in the art. Host cells also include, but are not limited to, mammalian cell lines such as CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and choroid plexus cell lines.
- mammalian cell lines such as CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and choroid plexus cell lines.
- the polypeptides according to the invention can be produced by a stably-transfected cell line.
- a number of vectors suitable for stable transfection of mammalian cells are available to the public, methods for constructing such cell lines are also publicly known, e.g., in Ausubel et al. (supra).
- the invention provides food products obtainable by the process of the invention as described hereinbefore or by the use of the novel asparaginase as described hereinbefore to produce food products.
- These food products are characterized by significantly reduced acrylamide levels in comparison with the food products obtainable by production processes that do not comprise adding one or more enzymes in an amount that is effective in reducing the level of amino acids which are involved in the formation of acrylamide during said heating step.
- the process according to the invention can be used to obtain a decrease of the acrylamide content of the produced food product preferably more than 50%, more preferably more than 70%, even more preferably 80% and most preferably more than 90% compared to a food product obtained with the conventional process.
- the first type contains the "standard" amdS selection marker, which allows a direct selection of transformed strains on the selective substrate acetamide. This is quite suitable for secreted amidases, but for intracellular enzymes it has the drawback that there will be a background activity of the marker acetamidase. Therefore, for a number of amidases that were predicted to be intracellular, a second type of construct was used, employing a marker-less construct, introduced by co-transformation with a second vector harbouring the phleomycin resistance marker.
- Oligonucleotide primers were designed to amplify the gene encoding the gene of interest from the genome of A. niger CBS513.88. As said before the primers suitable to pick up the genes according to the invention can be made as known to the person skilled in the art. Table 2 shows the nucleotide sequence of the oligonucleotide primers that were used for amplification of some of the amidase genes according to the invention.
- a Pad restriction site is located for facilitation of the cloning procedure.
- a linker to the start codon was added.
- the 3'-end of the upstream primer is complementary to the 5'-end of the gene of interest, including the ATG start codon.
- the structure of all upstream primers was: 5'- CCCTTAATTAACTCATAGGCATCATG-gene specific sequence-3', where the ATG start codon is underlined.
- an Ascl restriction site is located for facilitation of the cloning procedure.
- the 3'-end of the upstream primer is complementary to the reverse of the 3'-end of the gene of interest, approximately 100 base pairs downstream of the stop codon.
- the structure of all downstream primers was: 5'-
- the length of the gene specific sequence in the primers was 20-25 nucleotides, depending on the GC content of the complementary part of the primer.
- Genomic DNA was isolated from A. niger CBS513.88 according to standard procedure, and used in a PCR reaction with both the upstream and downstream primers to amplify the gene encoding the amidase of interest. A person skilled in the art will know how to optimize PCR conditions for each gene and primer combination.
- PCR fragments were cloned in the pCR2.1 (Invitrogen) vector and amplified in E. coli. After digestion of the plasmid with Pad and Ascl, the amidase genes were subcloned in an Aspergillus expression vector.
- the expression vector was pGBFIN5 (WO 9932617).
- the expression vector was also digested with Pad and Ascl. Correct orientation of the insert in the resulting plasmids was checked by digestion with suitable restriction enzymes and sequence analysis of the inserted gene. This cloning procedure positions the amidase gene downstream of the Aspergillus nigerg ⁇ aA promoter, and upstream of the glaA terminator. Additionally the Aspergillus nidulans amdS gene is present on this plasmid for convenient selection in A. niger (WO 9846772). The structure of these expression plasmids is depicted in Figure 1.
- the expression vector was pGBFINGFP-2 ( Figure 2).
- the expression vector was also digested with Pad and Ascl. Correct orientation of the insert in the resulting plasmids was checked by digestion with suitable restriction enzymes and sequence analysis of the inserted gene.
- This cloning procedure replaces the GFP gene in the pGBFINGFP-2 vector with the gene of interest, and positions the amidase gene downstream of the Aspergillus n/gerglaA promoter, and upstream of the glaA terminator, resulting in pGBFINAAA-1 ( Figure 3).
- a phleomycin resistance gene is present on this plasmid, for convenient selection in A. niger.
- A. niger strains containing multiple copies of the expression cassette were used for generation of sample material by cultivation of the strains in shake flask cultures.
- a useful method for cultivation of A. niger strains and separation of the mycelium from the culture broth is described in WO 9846772. The culture broth was subsequently used for purification of the amidase and measurement of amidase activity.
- amidase enzymes were obtained by growing the transformed A. niger strains in the following way.
- Fresh spores (106-107) of A. niger strains were inoculated in 20 ml CSL-medium (100 ml flask, baffle) and grown for 20-24 hours at 34 O and 170 rpm. After inoculation of 5-10 ml CSL pre-culture in 100 ml of the following medium: 150 g/l maltose, 60 g/l bacto soytone, 1 g/l NaH 2 PO 4 , 15 g/l (NH 4 ) 2 SO 4 , 1 g/l MgSO4.4H2O, 0.08 g/l tween-80, 0.02 g/l Basildon, 20 g/l Morpholino Ethane Sulfonicacid (MES), 1 g/l L-arginine, in 500 ml baffled shake flasks, at 30O and 250 rpm for 5 days.
- MES Morpholino Ethane Sulfonicacid
- cell-free extracts were prepared by grinding the biomass in the pellet fraction to powder in liquid nitrogen.
- the supernatant fractions were stored at 4 O (or -20 °C).
- 1 H NMR spectra were recorded on a Bruker DRX-600 operating at a proton frequency of 600 MHz at a probe temperature of 300 K. A 5mm triple resonance probe with self-shielded gradients was used. 1 H NMR spectra of all reference compounds were acquired in order to show that all the compounds involved have unique NMR signals, based on which they can be identified and quantified (not shown). In order to create perfect reference spectra of every relevant compound, a stock solution of each compound was prepared in D 2 O (Cambridge Isotope Laboratories). Stock solutions were prepared in concentrations of 10 mg/ml by weighing the substrate or the reference compound and adding D 2 O.
- Acetamide (catalog number 12,263-7, lot16813BA-453, Aldrich, Wl USA): 1 .99 (s, CH 3 ) ppm.
- Acetic acid (catalog number 1 .00063, lot K31668363, Merck NJ USA): 1 .90 (s, CH 3 ) ppm.
- Propionamide (catalog number 14,393-6, lot 25009JB-413, Aldrich, Wl USA): 1 .10 (t, CH 3 ), 2.27 (q, CH 2 ) ppm.
- Propionic acid (catalog number P- 1386, lot 083 K3404, Sigma, St Louis, Mo USA): 1 .09 (t, CH 3 ), 2.37 (q, CH 2 ) ppm.
- the activity determination was essentially performed according to Skouloubris et al. [MoI. Microbiol. 40: 596-609, 2001 ].
- a 100 mM solution of the amide substrates was prepared in PEB. 200 ml of this substrate solution was mixed with 50 ml cell extract, and the mixture was incubated for 30 minutes at 30 - 37O.
- the activity of the enzyme was quantified by determining the amount of ammonia that had been released.
- the colorimetric assay was influenced by the amount of cell extract used in the incubation. If increasing amounts of A. niger cell extract were added to the highest concentration of the calibration curve (25 ml 5 mM (NH 4 ) 2 SO 4 ), the light absorption decreased from 0.44 all the way to 0 ( Figure 4). Therefore, the calibration lines must be made in the presence of the appropriate amount of cell extract. Moreover, it was found that the nature of the cell extract was of influence. If calibration lines were made with either 20 ml A. niger extract or 20 ml K. lactis extract, the first line had a significantly lower slope (Figure 5).
- the extracellular amidases ZDK and ZDO were partially purified by a sequence of chromatographic steps from culture supernatant, prepared as described previously.
- the supernatant was first concentrated 5 to 7-fold by ultrafiltration, using a Biomax-10 membrane, and the pH was adjusted to the value of the buffer of the first chromatographic step.
- the collected fractions were analysed for the presence of the desired protein by SDS-PAGE, the fractions containing the desired protein were pooled, again concentrated by ultrafiltration, and their pH adjusted to the pH of the subsequent chromatographic step.
- Step 3 was omitted for the ZDK purification
- Cell-free extracts were prepared as described previously. These extracts were used to determine the activity of the intracellular amidases expressed in A. niger by the NMR method of Example 3. First, the assay conditions were checked using the known amdS amidases from E. nidulans and its homologue from A. niger (Seq ID NO: 1 1 ).
- amidase ZDO has a preference for propionamide as a substrate.
- the relative activity towards acrylamide is comparable to the amdS amidases.
- the specific activity can be estimated, using an NH3 calibration curve, and the estimated protein content. Specific activities of 0.7 and 0.2 mmol.min-1 .mg-1 were calculated for ZDK and ZDO, respectively .
- Example 9 Amidases in Aspergillus niger and other micro-organisms The known sequence of these new amidases were used as a probe to find additional amidases in the genomes of other micro-organisms. A number of additional homologous enzymes were identified in fungi and bacteria.
- Detection was performed using on-line chemical ionisation mass spectrometry in positive ion mode, with methane as ionisation gas.
- the characteristic ions m/z 72 (acrylamide) and m/z 75 (13C3 acrylamide) were monitored for quantification.
- MSD mass selective detector
- Concentrated enzyme solutions of ZDK and ZDO were prepared by ultrafiltration, and subsequently stabilized by addition of 50% w/v (final volume) glycerol, 0.02% (w/v)
- Freshly brewed coffee was divided into 6 ml portions, and incubated at 30 0 C.
- the samples were freeze-dried and analyzed for acrylamide according to the method described above.
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EA200700852A EA014853B1 (en) | 2004-10-15 | 2005-10-13 | Amidases from aspergillus niger and their use in a food production process with lowered acrylamide level |
BRPI0516523-7A BRPI0516523A (en) | 2004-10-15 | 2005-10-13 | process for food production |
EP05810939A EP1799822A2 (en) | 2004-10-15 | 2005-10-13 | Amidases from aspergillus niger and their use in a food production process |
US11/664,419 US20080008780A1 (en) | 2004-10-15 | 2005-10-13 | Amidases from Aspergillus Niger and Their Use in a Food Production Process |
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EP04105077 | 2004-10-15 | ||
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EP04107053 | 2004-12-29 | ||
EP05106200.8 | 2005-07-07 | ||
EP05106200A EP1741721A1 (en) | 2005-07-07 | 2005-07-07 | Method for production of a compound in a eukaryotic cell |
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Cited By (4)
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WO2012032472A3 (en) * | 2010-09-06 | 2012-05-03 | Danisco A/S | Food additive comprising an amidase for detoxifying ochratoxin |
EP2437625B1 (en) * | 2009-06-02 | 2016-10-05 | R.J.Reynolds Tobacco Company | Thermal treatment process for tobacco materials |
WO2021148509A1 (en) | 2020-01-21 | 2021-07-29 | Anka Angewandte Kaffeetechnologie Gmbh | Enzyme for decomposing acrylamide |
WO2023017147A1 (en) | 2021-08-12 | 2023-02-16 | Anka Angewandte Kaffeetechnologie Gmbh | Method for the removal of acrylamide from foods and stimulants |
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US7811618B2 (en) | 2002-09-19 | 2010-10-12 | Frito-Lay North America, Inc. | Method for reducing asparagine in food products |
US7393550B2 (en) | 2003-02-21 | 2008-07-01 | Frito-Lay North America, Inv. | Method for reducing acrylamide formation in thermally processed foods |
US8110240B2 (en) | 2003-02-21 | 2012-02-07 | Frito-Lay North America, Inc. | Method for reducing acrylamide formation in thermally processed foods |
US8486684B2 (en) | 2007-08-13 | 2013-07-16 | Frito-Lay North America, Inc. | Method for increasing asparaginase activity in a solution |
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US20110135803A1 (en) * | 2008-07-09 | 2011-06-09 | Starbucks Corporation D/B/A Starbucks Coffee Company | Dairy containing beverages with enhanced flavors and method of making same |
US20110135802A1 (en) * | 2008-07-09 | 2011-06-09 | Starbucks Corporation D/B/A Starbucks Coffee Company | Dairy containing beverages with enhanced flavors and method of making same |
US8284248B2 (en) | 2009-08-25 | 2012-10-09 | Frito-Lay North America, Inc. | Method for real time detection of defects in a food product |
US8158175B2 (en) | 2008-08-28 | 2012-04-17 | Frito-Lay North America, Inc. | Method for real time measurement of acrylamide in a food product |
US9095145B2 (en) | 2008-09-05 | 2015-08-04 | Frito-Lay North America, Inc. | Method and system for the direct injection of asparaginase into a food process |
US9215886B2 (en) * | 2008-12-05 | 2015-12-22 | Frito-Lay North America, Inc. | Method for making a low-acrylamide content snack with desired organoleptical properties |
US20150250200A1 (en) * | 2012-09-05 | 2015-09-10 | Vural Gokmen | Instant coffee or coffee substitute with reduced acrylamide and hydroxymethyl furfural content and production method thereof |
CN116987750B (en) * | 2023-08-08 | 2024-01-26 | 吉林省雁鸣湖大豆生物科技有限责任公司 | Plant active protein powder and preparation method thereof |
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US7504490B1 (en) * | 1998-10-16 | 2009-03-17 | Oscient Pharmaceuticals Corporation | Nucleic acid and amino acid sequences relating to Apergillus fumigatus for diagnostics and therapeutics |
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2005
- 2005-10-13 WO PCT/EP2005/055242 patent/WO2006040345A2/en active Application Filing
- 2005-10-13 US US11/664,419 patent/US20080008780A1/en not_active Abandoned
- 2005-10-13 EA EA200700852A patent/EA014853B1/en not_active IP Right Cessation
- 2005-10-13 EP EP05810939A patent/EP1799822A2/en not_active Ceased
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2437625B1 (en) * | 2009-06-02 | 2016-10-05 | R.J.Reynolds Tobacco Company | Thermal treatment process for tobacco materials |
WO2012032472A3 (en) * | 2010-09-06 | 2012-05-03 | Danisco A/S | Food additive comprising an amidase for detoxifying ochratoxin |
WO2021148509A1 (en) | 2020-01-21 | 2021-07-29 | Anka Angewandte Kaffeetechnologie Gmbh | Enzyme for decomposing acrylamide |
WO2021148508A1 (en) | 2020-01-21 | 2021-07-29 | Anka Angewandte Kaffeetechnologie Gmbh | Enzymes for the removal of acrylamide |
WO2023017147A1 (en) | 2021-08-12 | 2023-02-16 | Anka Angewandte Kaffeetechnologie Gmbh | Method for the removal of acrylamide from foods and stimulants |
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US20080008780A1 (en) | 2008-01-10 |
BRPI0516523A (en) | 2008-09-09 |
EA014853B1 (en) | 2011-02-28 |
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