ES2521940A1 - Variantes de beta-glucosidasa con actividad de transglicosilación reducida - Google Patents

Variantes de beta-glucosidasa con actividad de transglicosilación reducida Download PDF

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Publication number
ES2521940A1
ES2521940A1 ES201330678A ES201330678A ES2521940A1 ES 2521940 A1 ES2521940 A1 ES 2521940A1 ES 201330678 A ES201330678 A ES 201330678A ES 201330678 A ES201330678 A ES 201330678A ES 2521940 A1 ES2521940 A1 ES 2521940A1
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Spain
Prior art keywords
beta
variants
transglycosylation activity
glucosidase variants
glucosidase
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ES201330678A
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English (en)
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ES2521940B1 (es
Inventor
Bruno DÍEZ GARCÍA
Ana GÓMEZ RODRÍGUEZ
Jorge GIL MARTÍNEZ
Noelia VALBUENA CRESPO
Antonio Javier MORENO PÉREZ
Rafael DUEÑAS SÁNCHEZ
Ana María MUÑOZ GONZÁLEZ
Dolores PÉREZ GÓMEZ
Sandra GAVALDÁ MARTÍN
Laura SÁNCHEZ ZAMORANO
Consolación ÁLVAREZ NÚÑEZ
María De Los Ángeles BERMÚDEZ ALCANTARA
Pablo GUTIÉRREZ GÓMEZ
Ricardo Arjona Antolín
Lucía MARTÍN PÉREZ
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Abengoa Bioenergia Nuevas Technologias SA
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Abengoa Bioenergia Nuevas Technologias SA
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Priority to ES201330678A priority Critical patent/ES2521940B1/es
Priority to PCT/ES2014/070391 priority patent/WO2014181023A1/es
Publication of ES2521940A1 publication Critical patent/ES2521940A1/es
Application granted granted Critical
Publication of ES2521940B1 publication Critical patent/ES2521940B1/es
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Classifications

    • C—CHEMISTRY; METALLURGY
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • 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)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2445—Beta-glucosidase (3.2.1.21)
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • 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)
    • C12Y302/01021—Beta-glucosidase (3.2.1.21)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Variantes de beta-glucosidasa con actividad de transglicosilación reducida. La presente invención se refiere a variantes de beta-glucosidasa con actividad de transglicosilación reducida. La invención también se refiere a una construcción génica, a una célula huésped y a una composición enzimática que comprende dichas variantes. También se proporciona un procedimiento para producir dichas variantes, un procedimiento para producir azúcar fermentable y un procedimiento para producir un bioproducto, tal como bioetanol, a partir de material celulósico con las variantes de beta-glucosidasa, la célula huésped o la composición enzimática que comprende dichas variantes.

Description

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beta-glucosidasa de la cepa 31L2D seleccionada y otros transformantes analizados en
presencia y en ausencia de celobiosa se muestra en la Figura 3.
Para determinar la secuencia del gen bgl1 expresado en 31L2D, se amplificó un fragmento de ADN que contenía Pcbh1-bgl1 a partir del ADN genómico usando los oligonucleótidos 3 y
4.
Oligonucleótido 3 (SEQ ID NO: 9): El sitio de restricción SacI está subrayado. 5´-CGAGGAGCTCCTTACAAAAAAAAGGTATCC-3´
Oligonucleótido 4 (SEQ ID NO: 10): Los sitios de restricción BamHI, SmaI y PstI están subrayados. El codón de terminación está en recuadro.
5´TTCCTGCAGCCCGGGGGATCCTCAAGGAAGCTCAATCTTGAGATCCAACTTCC-3´
El oligonucleótido 3 incluye el sitio de restricción SacI e hibrida en el extremo 3' de Pcbh1. El oligonucleótido 4 hibrida en el extremo 3' de bgl1 e incluye el codón de terminación de bgl1 y los sitios de restricción BamHI, SmaI y PstI para clonar en el plásmido de expresión pBASE1. Los oligonucleótidos 3 y 4 se usaron para amplificar el fragmento Pcbh1-bgl1 usando ADN genómico de la cepa 31L2D como diana (obtenido usando el kit DNeasy Plant Mini Kit de Qiagen) con la ADN polimerasa iProof High-Fidelity (BioRad) y se programaron durante un ciclo a 98 ºC durante 2 minutos y 30 ciclos de 98 ºC durante 10 segundos, 72 ºC durante 90 segundos y 72 ºC durante 10 minutos. El fragmento de ADN amplificado se digirió con las enzimas de restricción SacI y BamHI y se clonó en pABC344 digerido previamente con las mismas enzimas de restricción. La mezcla de ligación se transformo en
células
electrocompetentes de Escherichia coli XL1Blue MRF siguiendo el protocolo
proporcionado por el fabricante (Stratagene). El plásmido recombinante
se denominó
pABC410 y se muestra en la Figura 4.
El gen bgl1 de pABC410 se secuenció. El bgl1 mutado mostró una mutación: la guanina de la posición 633 de la secuencia de nucleótidos de bgl1 nativo SEQ ID NO: 3 se había mutado a timina, dando, por tanto, una secuencia de nucleótidos de SEQ ID NO: 6, que codificaba una preproteína (SEQ ID NO: 5, preproteína Bgl1Q211H), en la que la glutamina
(Q) en el residuo 211 de la preproteína nativa SEQ ID NO: 2 se había intercambiado por histidina (H), de modo que 31L2D expresaba una versión madura mutada (SEQ ID NO: 4)
26
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Claims (1)

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ES201330678A 2013-05-10 2013-05-10 Variantes de beta-glucosidasa con actividad de transglicosilación reducida Expired - Fee Related ES2521940B1 (es)

Priority Applications (2)

Application Number Priority Date Filing Date Title
ES201330678A ES2521940B1 (es) 2013-05-10 2013-05-10 Variantes de beta-glucosidasa con actividad de transglicosilación reducida
PCT/ES2014/070391 WO2014181023A1 (es) 2013-05-10 2014-05-09 Variantes de beta-glucosidasa con actividad de transglicosilación reducida

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
ES201330678A ES2521940B1 (es) 2013-05-10 2013-05-10 Variantes de beta-glucosidasa con actividad de transglicosilación reducida

Publications (2)

Publication Number Publication Date
ES2521940A1 true ES2521940A1 (es) 2014-11-13
ES2521940B1 ES2521940B1 (es) 2015-09-09

Family

ID=50928133

Family Applications (1)

Application Number Title Priority Date Filing Date
ES201330678A Expired - Fee Related ES2521940B1 (es) 2013-05-10 2013-05-10 Variantes de beta-glucosidasa con actividad de transglicosilación reducida

Country Status (2)

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ES (1) ES2521940B1 (es)
WO (1) WO2014181023A1 (es)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011041594A1 (en) * 2009-09-30 2011-04-07 Codexis, Inc. Recombinant c1 b-glucosidase for production of sugars from cellulosic biomass
WO2011066457A2 (en) * 2009-11-25 2011-06-03 Codexis, Inc. Recombinant beta-glucosidase variants for production of soluble sugars from cellulosic biomass

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1117808B1 (en) 1998-10-06 2004-12-29 Mark Aaron Emalfarb Transformation system in the field of filamentous fungal hosts: in chrysosporium
US7122330B2 (en) 2000-04-13 2006-10-17 Mark Aaron Emalfarb High-throughput screening of expressed DNA libraries in filamentous fungi
EP2599863B1 (en) 2009-11-20 2017-08-02 Danisco US Inc. Beta-glucosidase variants with improved properties
BR112014004171A2 (pt) * 2011-08-22 2018-11-06 Codexis Inc variantes da proteína glicósideo hidrolase gh61 e cofatores que aumentam a atividade de gh61

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011041594A1 (en) * 2009-09-30 2011-04-07 Codexis, Inc. Recombinant c1 b-glucosidase for production of sugars from cellulosic biomass
WO2011066457A2 (en) * 2009-11-25 2011-06-03 Codexis, Inc. Recombinant beta-glucosidase variants for production of soluble sugars from cellulosic biomass

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BHAT, K.M. et al. "Purification and characterization of a n extracellular beta-glucosidase from the thermophilic fungus Sporotrichum thermophile and its influence on cellulase activity". JOURNAL OF GENERAL MICROBIOLOGY. 01.11.1993. Vol. 139, N°. 11, páginas 2825 - 2832; todo el documento. *
KIM, T.J. et al. "Role of the glutamate 332 residue in the transglycosilation activity of Thermus maltogenic amylase". BIOCHEMISTRY. 13.06.2000. Vol. 39, N°. 23, páginas 6773 - 6780; resumen. *

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WO2014181023A1 (es) 2014-11-13
ES2521940B1 (es) 2015-09-09

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