ES2533469T3 - Método de preparación de un producto tratado con calor - Google Patents
Método de preparación de un producto tratado con calor Download PDFInfo
- Publication number
- ES2533469T3 ES2533469T3 ES07115541.0T ES07115541T ES2533469T3 ES 2533469 T3 ES2533469 T3 ES 2533469T3 ES 07115541 T ES07115541 T ES 07115541T ES 2533469 T3 ES2533469 T3 ES 2533469T3
- Authority
- ES
- Spain
- Prior art keywords
- preparing
- treated product
- heat treated
- aspergillus
- asparaginase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 238000000034 method Methods 0.000 title abstract 2
- 102000015790 Asparaginase Human genes 0.000 abstract description 8
- 108010024976 Asparaginase Proteins 0.000 abstract description 8
- 229960003272 asparaginase Drugs 0.000 abstract description 6
- DCXYFEDJOCDNAF-UHFFFAOYSA-M asparaginate Chemical compound [O-]C(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-M 0.000 abstract description 6
- 108090000623 proteins and genes Proteins 0.000 abstract description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 4
- 238000010438 heat treatment Methods 0.000 abstract 2
- 239000002994 raw material Substances 0.000 abstract 2
- GXCLVBGFBYZDAG-UHFFFAOYSA-N N-[2-(1H-indol-3-yl)ethyl]-N-methylprop-2-en-1-amine Chemical compound CN(CCC1=CNC2=C1C=CC=C2)CC=C GXCLVBGFBYZDAG-UHFFFAOYSA-N 0.000 abstract 1
- 150000001720 carbohydrates Chemical class 0.000 abstract 1
- 238000001035 drying Methods 0.000 abstract 1
- 102000004169 proteins and genes Human genes 0.000 abstract 1
- 241000228212 Aspergillus Species 0.000 description 6
- 240000006439 Aspergillus oryzae Species 0.000 description 5
- 235000002247 Aspergillus oryzae Nutrition 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- DLFVBJFMPXGRIB-UHFFFAOYSA-N Acetamide Chemical compound CC(N)=O DLFVBJFMPXGRIB-UHFFFAOYSA-N 0.000 description 2
- 241000351920 Aspergillus nidulans Species 0.000 description 2
- 241000228245 Aspergillus niger Species 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 101100163949 Caenorhabditis elegans asp-3 gene Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 239000004606 Fillers/Extenders Substances 0.000 description 1
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 description 1
- 102000005924 Triose-Phosphate Isomerase Human genes 0.000 description 1
- 108700015934 Triose-phosphate isomerases Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 101150069003 amdS gene Proteins 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A21—BAKING; EDIBLE DOUGHS
- A21D—TREATMENT OF FLOUR OR DOUGH FOR BAKING, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS
- A21D8/00—Methods for preparing or baking dough
- A21D8/02—Methods for preparing dough; Treating dough prior to baking
- A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
- A21D8/042—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
- A23L19/10—Products from fruits or vegetables; Preparation or treatment thereof of tuberous or like starch containing root crops
- A23L19/12—Products from fruits or vegetables; Preparation or treatment thereof of tuberous or like starch containing root crops of potatoes
- A23L19/18—Roasted or fried products, e.g. snacks or chips
- A23L19/19—Roasted or fried products, e.g. snacks or chips from powdered or mashed potato products
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
- A23L7/10—Cereal-derived products
- A23L7/117—Flakes or other shapes of ready-to-eat type; Semi-finished or partly-finished products therefor
-
- 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)
- C12N9/82—Asparaginase (3.5.1.1)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01118—Glucose 1-dehydrogenase (NAD+) (1.1.1.118)
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- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01119—Glucose 1-dehydrogenase (NADP+) (1.1.1.119)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01124—Fructose 5-dehydrogenase (NADP+) (1.1.1.124)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01147—16-Alpha-hydroxysteroid dehydrogenase (1.1.1.147)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01148—Estradiol 17alpha-dehydrogenase (1.1.1.148)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
- C12Y101/03009—Galactose oxidase (1.1.3.9)
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- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/99—Oxidoreductases acting on the CH-OH group of donors (1.1) with other acceptors (1.1.99)
- C12Y101/9901—Glucose dehydrogenase (acceptor) (1.1.99.10)
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- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/99—Oxidoreductases acting on the CH-OH group of donors (1.1) with other acceptors (1.1.99)
- C12Y101/99011—Fructose 5-dehydrogenase (1.1.99.11)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/99—Oxidoreductases acting on the CH-OH group of donors (1.1) with other acceptors (1.1.99)
- C12Y101/99018—Cellobiose oxidase (1.1.99.18)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/99—Oxidoreductases acting on the CH-OH group of donors (1.1) with other acceptors (1.1.99)
- C12Y101/99028—Glucose-fructose oxidoreductase (1.1.99.28)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
- C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
- C12Y102/01003—Aldehyde dehydrogenase (NAD+) (1.2.1.3)
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- C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
- C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
- C12Y102/01004—Aldehyde dehydrogenase (NADP+) (1.2.1.4)
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- C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
- C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
- C12Y102/01005—Aldehyde dehydrogenase [NAD(P)+] (1.2.1.5)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
- C12Y104/03—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
- C12Y104/03002—L-Amino-acid oxidase (1.4.3.2)
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- C12Y—ENZYMES
- C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
- C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
- C12Y110/03002—Laccase (1.10.3.2)
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- C12Y—ENZYMES
- C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
- C12Y111/01—Peroxidases (1.11.1)
- C12Y111/01007—Peroxidase (1.11.1.7), i.e. horseradish-peroxidase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
- C12Y301/03004—Phosphatidate phosphatase (3.1.3.4)
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- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
- C12Y301/03005—5'-Nucleotidase (3.1.3.5)
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- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/13—Dipeptidases (3.4.13)
- C12Y304/13018—Cytosol nonspecific dipeptidase (3.4.13.18), i.e. glycyl-leucine dipeptidase
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- C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
- C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
- C12Y305/01001—Asparaginase (3.5.1.1)
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- C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
- C12Y305/01002—Glutaminase (3.5.1.2)
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- C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
- C12Y305/01026—N4-(Beta-N-acetylglucosaminyl)-L-asparaginase (3.5.1.26)
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- C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
- C12Y305/01043—Peptidyl-glutaminase (3.5.1.43)
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- C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
- C12Y305/01052—Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase (3.5.1.52), i.e. glycopeptidase
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- Enzymes And Modification Thereof (AREA)
- General Preparation And Processing Of Foods (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Polymers With Sulfur, Phosphorus Or Metals In The Main Chain (AREA)
- Heating, Cooling, Or Curing Plastics Or The Like In General (AREA)
- Processing Of Solid Wastes (AREA)
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Abstract
Método para preparar un producto tratado con calor, que comprende las fases secuenciales de: a) proveer una materia prima que comprende hidrato de carbono, proteína y agua b) tratar la materia prima con una asparaginasa y c) tratar con calor hasta alcanzar un contenido de agua final por debajo del 35% en peso, donde el tratamiento con calor implica fritura a temperaturas de 150-180ºC, horneado en aire caliente a 160-310ºC durante 2-10 minutos, calentamiento en placa caliente y/o secado de la malta verde.
Description
5
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E07115541
23-03-2015
Soluciones concentradas
[0032] 50 mM tampón Tris, pH 8.6
189mM Solución de L-Asparraguina
1,5 M Ácido tricloroacético (ATC)
Reactivo de Nessler, Aldrich Stock No. 34,514-8 (Sigma-Aldrich, St. Louis, Mo. USA)
Asparaginasa, Sigma Stock No. A4887 (Sigma-Aldrich, St. Louis, Mo. USA)
Ensayo
Reacción enzimática:
[0033]
500 micro-I tampón
100 micro-I Solución de L-asparraguina
350 micro-I agua
son mezclados y equilibrados a 37ºC.
se añaden 100 micro-I de solución enzimática y las reacciones son incubadas a 37ºC durante 30 minutos.
[0034] Las reacciones son detenidas colocándolas en hielo y añadiendo 50 micro-I de 1.5M ATC.
[0035] Las muestras son mezcladas y centrifugadas durante 2 minutos a 20.000 g
Medición de amonio libre:
[0036] Se mezclan 50 micro-I de la reacción enzimática con 100 micro-I de agua y 50 micro-I de reactivo de Nessler. La reacción es mezclada y la absorbencia es medida a 436nm después de 1 minuto.
Estándar:
[0037] El concentrado de asparaginasa (Sigma A4887) es diluido 0,2, 0,5, 1, 1,5, 2, y 2,5 U/ml.
Ejemplo 1: expresión de una asparaginasa de Aspergillus oryzae en Aspergillus oryzae
[0038] Se generaron bibliotecas de ADNc de ARNm de Aspergillus oryzae, se ordenaron y almacenaron en una base de datos de ordenador como se describe en WO 00/56762.
[0039] Se comparó la secuencia péptida de asparaginasa de Saccharomyces cerevisiae (Kim,K.-W.; Kamerud, J.Q.; Livingston,D.M.; Roon,R.J., (1988) Asparaginase II of Saccharomyces cerevisiae. Characterization of the ASP3 gene. J. Biol. Chem. 263:11948), con traducciones de las secuencias de ADNc parcial de Aspergillus oryzae usando el programa TFASTXY, versión 3.2t07 (Pearson et al. Genomics (1997) 46:24-36). Se identificó una secuencia de A. oryzae traducida como teniendo una identidad del 52% para asparaginasa II de levadura a través de un recubrimiento de 165 aminoácidos. Se determinó la secuencia completa del inserto de ADNc del clon correspondiente (depositado como DSM 15960) y se presentó como SEC ID NO: 1, y el péptido traducido de esta secuencia, AoASP, es presentado como SEC ID NO: 2. Esta secuencia fue usada para designar cebadores para la amplificación PCR del gen de codificación AoASP de DSM 15960, con sitios de restricción apropiados añadidos a las extremidades del cebador para facilitar la subclonación del producto PCR (cebadores AoASP7 y AoASP8, SEC ID Nos: 14 y 15). La amplificación PCR fue realizada usando Extensor Hi-Fidelity PCR Master Mix (ABgene, Surrey, U.K.) siguiendo las instrucciones del fabricante y usando una temperatura de hibridación de 55ºC para los primeros 5 ciclos y 65ºC para un ciclo adicional y un tiempo de extensión de 1,5 minutos.
[0040] El fragmento de PCR fue restringido con BamHl y Hindlll y clonado en el vector de expresión Aspergillus pMStr57 usando técnicas estándares. El vector de expresión pMStr57 contiene los mismos elementos que pCaHj483 (WO 98/00529), con modificaciones menores hechas en el promotor NA2 de Aspergillus como se describe para el vector pMT2188 en WO 01/12794, y tiene secuencias para la selección y propagación en E. coli, y selección y expresión en Aspergillus. Específicamente, la selección en Aspergillus es facilitada por el gen amdS de Aspergillus nidulans, que permite el uso de acetamida como única fuente de nitrógeno. La expresión en Aspergillus es mediada por un promotor (NA2) de amilasa II neutra modificada de Aspergillus niger que es fusionado en la secuencia líder 5' del gen de codificación de triosa fosfato isomerasa (tpi) de Aspergillus nidulans, y el terminador del gen de codificación de amiloglucosidasa de Aspergillus niger. El gen de codificación de asparaginasa del constructo de expresión de Aspergillus resultante, pMStr90, fue secuenciado y la secuencia coincidía completamente con aquella determinada previamente para el inserto de DSM 15960.
5
Claims (1)
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2007
- 2007-12-07 US US11/952,593 patent/US20080096260A1/en not_active Abandoned
- 2007-12-07 US US11/952,640 patent/US8124396B2/en not_active Expired - Fee Related
-
2012
- 2012-01-18 US US13/352,511 patent/US8507246B2/en not_active Expired - Fee Related
-
2013
- 2013-05-10 US US13/891,859 patent/US8859023B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| AU2003271552B2 (en) | 2009-04-23 |
| EP1553848B1 (en) | 2007-09-05 |
| DE60316168D1 (de) | 2007-10-18 |
| US20060275879A1 (en) | 2006-12-07 |
| EP1886582A2 (en) | 2008-02-13 |
| ES2293012T3 (es) | 2008-03-16 |
| WO2004032648A1 (en) | 2004-04-22 |
| EP2294928A1 (en) | 2011-03-16 |
| CA2501741C (en) | 2012-04-17 |
| US8507246B2 (en) | 2013-08-13 |
| DK1886582T3 (en) | 2015-04-20 |
| EP1886582A3 (en) | 2010-07-14 |
| DE60316168T2 (de) | 2008-05-29 |
| US20130236598A1 (en) | 2013-09-12 |
| US7396670B2 (en) | 2008-07-08 |
| EP1553848A1 (en) | 2005-07-20 |
| CA2501741A1 (en) | 2004-04-22 |
| DK1553848T3 (da) | 2008-01-21 |
| US8859023B2 (en) | 2014-10-14 |
| AU2003271552A1 (en) | 2004-05-04 |
| EP2294928B1 (en) | 2014-08-06 |
| US20080096260A1 (en) | 2008-04-24 |
| EP1886582B1 (en) | 2015-01-14 |
| US20120114793A1 (en) | 2012-05-10 |
| US20080095883A1 (en) | 2008-04-24 |
| DK2294928T3 (da) | 2014-11-10 |
| ES2511292T3 (es) | 2014-10-22 |
| ATE372059T1 (de) | 2007-09-15 |
| US8124396B2 (en) | 2012-02-28 |
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