ES2545639T3 - Pectato liasas, ácidos nucleicos que las codifican y métodos para su preparación y uso - Google Patents

Pectato liasas, ácidos nucleicos que las codifican y métodos para su preparación y uso Download PDF

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ES2545639T3
ES2545639T3 ES04758802.5T ES04758802T ES2545639T3 ES 2545639 T3 ES2545639 T3 ES 2545639T3 ES 04758802 T ES04758802 T ES 04758802T ES 2545639 T3 ES2545639 T3 ES 2545639T3
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nucleic acid
seq
sequence
encodes
pectate
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Inventor
Janne Kerovuo
Arne Solbak
Kevin Gray
Ryan Mccann
Shalaka Purohit
Joel Gerendash
Giselle Janssen
Samun Dahod
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BASF Enzymes LLC
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BASF Enzymes LLC
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01067—Galacturan 1,4-alpha-galacturonidase (3.2.1.67)
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
    • C12N15/8246—Non-starch polysaccharides, e.g. cellulose, fructans, levans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01015—Polygalacturonase (3.2.1.15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/02—Carbon-oxygen lyases (4.2) acting on polysaccharides (4.2.2)
    • C12Y402/02002—Pectate lyase (4.2.2.2)

Abstract

Un ácido nucleico aislado, sintético, o recombinante que comprende (a) una secuencia de ácidos nucleicos que tiene una identidad de secuencia de al menos un 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, o superior o que es idéntica en un 100 % a la SEC ID Nº: 77, sobre una región de al menos 950, 1000, 1050, 1100, 1150 o más restos, o la longitud completa de un gen o de un transcrito, en la que el ácido nucleico codifica al menos un polipéptido que tiene una actividad de pectato liasa; (b) una secuencia de ácidos nucleicos que comprende una secuencia como se establece en la SEC ID Nº: 77; (c) una secuencia de ácidos nucleicos que codifica un polipéptido que tiene una secuencia como se establece en las SEC ID Nº: 78, SEC ID Nº: 132 o SEC ID Nº: 134; (d) una secuencia que se hibrida en condiciones rigurosas con un ácido nucleico que comprende la SEC ID Nº: 77, SEC ID Nº: 131 o SEC ID Nº: 133, en la que el ácido nucleico codifica un polipéptido que tiene una actividad de pectato liasa, en la que las condiciones rigurosas incluyen una etapa de lavado que comprende un lavado en 0,2X SSC a una temperatura de aproximadamente 65 ºC durante aproximadamente 15 minutos; (e) el ácido nucleico de (a), (b), (c) o (d) que codifica un pectato liasa pero que carece de una secuencia señal, un dominio prepro, o una combinación de los mismos; (f) el ácido nucleico de (a), (b), (c), (d) o (e) que codifica un pectato liasa pero que también tiene una secuencia heteróloga; o (g) una secuencia complementaria con (a), (b), (c), (d), (e), o (f).

Description

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ejemplo, la formulación puede comprender una enzima de la invención en una cantidad entre aproximadamente 1000 u/ml y 10.000 u/ml.
Los estudios de solubilidad con producto liofilizado (SEC ID Nº: 134) resuspendido en agua indicaban que la
5 solubilidad de la enzima puede ser tan elevada como 25000 u/ml a 4 ºC. Por lo tanto, en un aspecto, la invención proporciona formulaciones que tienen un nivel tan elevado como aproximadamente 25000 u/ml, o superior. En un aspecto la invención proporciona formulaciones que comprenden una enzima de la invención en una cantidad entre aproximadamente 100 u/ml and 25000 u/ml, 30000 u/ml, 35000 u/ml o 40000 u/ml, o superior.
10 Diseño de la formulación:
La invención proporciona formulaciones que comprenden al menos una enzima de la invención, y, en aspectos alternativos, comprende adicionalmente cualquier aditivo o aditivos. Las formulaciones de la invención se pueden basar en aditivos conocidos en otras formulaciones enzimáticas, por ejemplo, análogas. Por ejemplo, las
15 formulaciones de la invención pueden comprender los aditivos y/o condiciones que se exponen en las Tablas 3, 4, 5 y 6, que siguen a continuación, o cualquier variación de las mismas. Por ejemplo, las formulaciones de la invención pueden comprender glicerol, sacarosa, cloruro sódico, dextrina, propilenglicol, sorbitol, sulfato sódico o TRIS, o un equivalente.
20 En un aspecto, una formulación de la invención puede ser una formulación basada en agua, o, una formulación basada en aceite.
Se realizaron dos fases de estudios de estabilidad de la formulación; estos estudios usaron la enzima a modo de ejemplo de la SEC ID Nº: 134.
25 Estudio de estabilidad acelerada a 37 ºC
Nota: se trata de formulaciones basadas en tampón.
30 • Identificar sistemáticamente diversos aditivos
•
Someter a ensayo diferentes valores de pH
•
Formulaciones a aproximadamente 2000 u/ml.
•
La SEC ID Nº: 134 fue la enzima a modo de ejemplo sometida a ensayo
35 Tabla 3 (SN = número de muestra)
SN
pH Glicerol Sacarosa Cloruro sódico Dextrina Propilen glicol sorbitol Sulfato sódico Conc. Eficaz de TRIS
1
5 40 mM
2
6 40 mM
3
7 40 mM
4
8 40 mM
5
7,5 35 % 20 mM
6
7,5 50 % 20 mM
7
7,5 35 % 20 mM
8
7,5 20 % 20 mM
9
7,5 10 % 20 mM
10
7,5 30 % 20 mM
11
7,5 100 mM 20 mM
12
7,5 35 % 20 mM
13
5,5 35 % 40 mM
14
5,5 50 % 40 mM
Formulaciones con el mayor rendimiento (usando la SEC ID Nº: 134 como una enzima a modo de ejemplo de la invención) basadas en el aspecto físico y en la retención de actividad superior a un 80 %:
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Claims (1)

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ES04758802.5T 2003-04-04 2004-04-02 Pectato liasas, ácidos nucleicos que las codifican y métodos para su preparación y uso Expired - Lifetime ES2545639T3 (es)

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Application Number Priority Date Filing Date Title
US46084203P 2003-04-04 2003-04-04
US460842P 2003-04-04
US48479803P 2003-07-03 2003-07-03
US484798P 2003-07-03
PCT/US2004/010229 WO2004090099A2 (en) 2003-04-04 2004-04-02 Pectate lyases, nucleic acids encoding them and methods for making and using them

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US (2) US7592434B2 (es)
EP (2) EP1613733B1 (es)
CA (1) CA2521402C (es)
DK (2) DK2341136T3 (es)
ES (2) ES2545639T3 (es)
HU (1) HUE030493T2 (es)
MX (1) MXPA05010681A (es)
PL (2) PL2341136T3 (es)
WO (1) WO2004090099A2 (es)

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