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WO2003070928B1 - Hydrolysis of lactose with immobilised thermoresistant lactase and the production method thereof - Google Patents

Hydrolysis of lactose with immobilised thermoresistant lactase and the production method thereof

Info

Publication number
WO2003070928B1
WO2003070928B1 PCT/ES2003/000081 ES0300081W WO03070928B1 WO 2003070928 B1 WO2003070928 B1 WO 2003070928B1 ES 0300081 W ES0300081 W ES 0300081W WO 03070928 B1 WO03070928 B1 WO 03070928B1
Authority
WO
WIPO (PCT)
Prior art keywords
galactosidase
enzyme
lactose
thermus
chimeric
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.)
Ceased
Application number
PCT/ES2003/000081
Other languages
Spanish (es)
French (fr)
Other versions
WO2003070928A1 (en
Inventor
Benevides Costa Chitun Pessela
Herrero Alejandro Vian
Seijas Jose Manuel Guisan
Santiago Alfonso Vi Carrascosa
Lafuente Roberto Fernandez
Ruiz Cesar Mateo
Lopez Jose Luis Garcia
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Consejo Superior de Investigaciones Cientificas CSIC
Original Assignee
Consejo Superior de Investigaciones Cientificas CSIC
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Consejo Superior de Investigaciones Cientificas CSIC filed Critical Consejo Superior de Investigaciones Cientificas CSIC
Priority to AU2003209775A priority Critical patent/AU2003209775A1/en
Publication of WO2003070928A1 publication Critical patent/WO2003070928A1/en
Publication of WO2003070928B1 publication Critical patent/WO2003070928B1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • C12P19/14—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
    • 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)
    • C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
    • C12N9/2471—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
    • 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)
    • C12Y302/01023—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase

Landscapes

  • 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)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)

Abstract

The invention relates to a method for the hydrolysis of lactose catalysed by immobilised/stabilised recombinant derivatives of β-galactosidase from Thermus sp. (strain t2) fused with polyhistidine tails (imac/?-galactosidase) and cloned in E. coli CECT 5970. The inventive method is suitable for use in the dairy industry and in biotechnology where it is applied to different milk derivatives, such as milk, whey or any other compound comprising lactose, in order to prevent intolerance problems and improve the organoleptic properties thereof. With respect to the environment, the method can be used to eliminate highly-contaminating lactose waste (highly-reducing sugar), by transforming said waste into a product with added value (fructose and glucose syrup). The enzyme is thermoresistant and contains no impurities and, moreover, the preparation method thereof involves single-step purification which is carried out with a mesophilic microorganism.

Claims

REIVINDICACIONES MODIFICADAS[Recibidas por la Ofícina Internacional el 05 de agosto de 2003 (06.08.03): reivindicaciones 1 a 17 remplazadas por las reivindicaciones 1 a 15] MODIFIED CLAIMS [Received by the International Of fi ce on August 05, 2003 (06.08.03): claims 1 to 17 replaced by claims 1 to 15] 1.- Derivado de la enzima β-galactosidasa quimérica recombinante de Thermus sp. T2, caracterizado porqué:1.- Derived from the recombinant chimeric β-galactosidase enzyme from Thermus sp. T2, characterized by: (a) presenta la secuencia de aminoácidos descrita en SEQ (D NO:2, con un 5 módulo (IMAC) con una cola de histidiπas (IMAC/β-galactosidasa),(a) presents the amino acid sequence described in SEQ (D NO: 2, with a 5-module (IMAC) with a tail of histidiπas (IMAC / β-galactosidase), (b) está purificado por cromatografía de afinidad a quelatos metálicos en soportes que contengan metales de transición,(b) is purified by affinity chromatography to metal chelates in supports containing transition metals, (c) esta estabilizada e inmovilizada por uniones covaíeπtes o adsorción física sobre soportes adecuados (en un solo paso o en dos) y 0 (d) presenta capacidad para hidrolizar y transglicosilar lactosa(c) is stabilized and immobilized by covalent unions or physical adsorption on suitable supports (in a single step or in two) and 0 (d) has the ability to hydrolyze and transglycosylate lactose 2.- Derivado de la enzima β-galactosidasa quimérica recombiπaπte de Thermus sp. T2 según la reivindicación 1, caracterizado porque el procedimiento de obtención comprende los siguientes pasos: 52.- Derivative of the recombinant β-galactosidase enzyme recombiπaπte from Thermus sp. T2 according to claim 1, characterized in that the obtaining process comprises the following steps: (a) aislamiento del fragmento de ADN que contiene la secuencia de nucleótidos que codifica para la β-galactosidasa de Thermus sp. T2 por digestión del plásmido pBGT1 con enzimas de restricción,(a) isolation of the DNA fragment containing the nucleotide sequence coding for β-galactosidase from Thermus sp. T2 by digestion of the plasmid pBGT1 with restriction enzymes, (b) digestión del plásmtdo pTrcHisB que contiene la secuencia de 0 nucleótidos que codifica para un módulo con una cola de 6 histidinas. que permite la purificación por cromatografía de afinidad con quelatos metálicos,(b) digestion of the plasmid pTrcHisB containing the sequence of 0 nucleotides coding for a module with a tail of 6 histidines . which allows purification by affinity chromatography with metal chelates, (c) fusión de fragmento de ADN de (a) al resto de plásmido pTrcHisB de (b) obteniéndose un nuevo plásmido, el pBGT3, que contiene una 5 secuencia nucleotidica SEQ ID NO:1 nueva, que codifica para una proteína β-galactosidasa quimérica con cola de histidínas: IMAC/β- galactosidasa,(c) fusion of DNA fragment from (a) to the rest of plasmid pTrcHisB of (b) obtaining a new plasmid, pBGT3, which contains a new nucleotide sequence SEQ ID NO: 1, which codes for a protein β-galactosidase chimeric with histidine tail: IMAC / β-galactosidase, (d) transformación de una célula hospedante con dicho plásmido recombinante; 0 (e) cultivo de las células hospedantes transformadas en un medio de cultivo adecuado y en condiciones que permitan la producción de β- galactosidasa quimérica IMAC/β-galactosidasa, (f) purificación por adsorción selectiva de la enzima β-galactosidasa quimérica con la cola de polihistidina diana medíante cromatografía de afinidad a quelatos metálicos (p.e.- Co o Ni) a concentración inferior a 5μM para asegurar una única interacción con el quelato del soporte, (g) inmovilización-estabilización en soportes rígidos (resinas epoxiacrilicas muy entrecruzadas en presencia de agentes porogénicos, vidrio poroso, otros silicatos, azarosa, celulosa, etc.), conteniendo como grupos para la unión covaleπte grupos aldehidos o epóxido, y que a la vez pueden tener grupos para adsorber físicamente las enzimas (p.e., derivados de boronato, grupos con carga positiva, grupos con carga negativa, quelatos,...) y (h) estabilización adicional si procede de la estructura cuaternaria por entrecruzamiento posterior con polímeros polifuncionales, covalente con dextrano-aldehído o físico con polímeros tipo polietilenimina.(d) transformation of a host cell with said recombinant plasmid; 0 (e) culture of the transformed host cells in a suitable culture medium and under conditions that allow the production of chimeric β-galactosidase IMAC / β-galactosidase, (f) purification by selective adsorption of the chimeric β-galactosidase enzyme with the target polyhistidine tail by means of metal chelate affinity chromatography (peCo or Ni) at a concentration below 5μM to ensure a single interaction with the chelate of the support, (g) immobilization-stabilization in rigid supports (highly cross-linked epoxyacrylic resins in the presence of porogenic agents, porous glass, other silicates, azarose, cellulose, etc.), containing as groups for the covalent union of aldehyde or epoxide groups, and they may have groups to physically adsorb the enzymes (eg boronate derivatives, positively charged groups, negatively charged groups, chelates, ...) and (h) additional stabilization if it comes from the quaternary structure by subsequent crosslinking with polyfunctional polymers , covalent with dextran-aldehyde or physical with polyethyleneimine polymers. 3.- Derivado de la enzima β-galactosidasa quimérica recombinaπte de Thermus sp. T2, según las reivindicaciones 1 y 2 caracterizado porqué la inmovilización- estabilización se realiza a pH alcalino para forzar la unión covalente.3.- Derivative of the recombinant chimeric β-galactosidase enzyme from Thermus sp. T2, according to claims 1 and 2 characterized in that the immobilization-stabilization is carried out at alkaline pH to force the covalent binding. 4.- Derivado de la enzima β-galactosidasa quimérica recombinante de Thermus sp. T2, según las reivindicaciones 1 y 2 caracterizado porqué la inmovilización- estabilización utiliza soportes con grupos epóxidos y que tras la reacción se bloquean con grupos hidrofilicos (aminoácidos, aminoalcoholes, azúcares, tioaminos, etc).4.- Derivative of the recombinant chimeric β-galactosidase enzyme from Thermus sp. T2, according to claims 1 and 2, characterized in that the immobilization-stabilization uses supports with epoxide groups and that after the reaction are blocked with hydrophilic groups (amino acids, aminoalcohols, sugars, thioamines, etc.). 5.- Derivado de la enzima β-galactosidasa quimérica recombinante de Thermus sp. T2, según las reivindicaciones 1 y 2 caracterizado porque la inmovilización, estabilización se realiza utilizando soportes epóxido o aldehido -IDA-Co, Ni o Zn.5.- Derivative of the recombinant chimeric β-galactosidase enzyme from Thermus sp. T2, according to claims 1 and 2 characterized in that the immobilization, stabilization is carried out using epoxide or aldehyde-AD-Co, Ni or Zn supports. 6.- Derivado de la enzima β-galactosidasa quimérica recombinante de Thermus sp. T2, según las reivindicaciones 1 y 2 caracterizado porqué la inmovilización se realiza porqué se adsorbe físicamente sobre soportes recubiertos con polímeros policatiónicos del tipo polietilenimina, polialilamina, dextrano aminado u otros polisacáridos aminados.6.- Derivative of the recombinant chimeric β-galactosidase enzyme from Thermus sp. T2, according to claims 1 and 2, characterized in that the immobilization is carried out because it is physically adsorbed on polymer-coated supports. polycationic of the polyethylenimine, polyallylamine, aminated dextran or other amino polysaccharides type. 7.- Derivado de la enzima β-galactosidasa quimérica recombiπaπte de Thermus sp. T2, según la reivindicación 2 caracterizado porqué la estabilización de la estructura cuaternaria utiliza dextrano aldehido para conseguir el entrecruzamiento químico de las subunidades, realizado en presencia de agentes protectores (p.e., sustratos) para evitar la distorsión del centro activo de la enzima.7.- Derivative of the recombinant β-galactosidase enzyme recombiπaπte from Thermus sp. T2, according to claim 2, characterized in that the stabilization of the quaternary structure uses dextran aldehyde to achieve the chemical crosslinking of the subunits, carried out in the presence of protective agents (e.g., substrates) to avoid distortion of the active center of the enzyme. 8.- Enzima β-galactosidasa quimérica recombinante IMAC/β-galactosidasa de Thermus sp. T2 caracterizada SEQ ID NO:28.- Recombinant chimeric β-galactosidase enzyme IMAC / β-galactosidase from Thermus sp. T2 characterized SEQ ID NO: 2 9.- Secuencia nucleotídica caracterizada por la secuencia SEQ ID NO:1 que codifica para una proteína β-galactosidasa quimérica según 89. Nucleotide sequence characterized by the SEQ ID NO: 1 sequence coding for a chimeric β-galactosidase protein according to 10- Células hospedantes transformadas caracterizadas por contener el plásmido pBGT3 según la reivindicación 2, que contiene SEQ ID NO:1, preferentemente la cepa pura de Escherichia coli CECT 5970, sus mutantes o sus derivados transformados.10- Transformed host cells characterized by containing the plasmid pBGT3 according to claim 2, which contains SEQ ID NO: 1, preferably the pure strain of Escherichia coli CECT 5970, its mutants or its transformed derivatives. 11.- Procedimiento de tratamiento enzimático de lactosa, azúcares o análogos estructurales, caracterizado porque11.- Procedure for the enzymatic treatment of lactose, sugars or structural analogs, characterized in that (a) se utiliza el biocatalizador (enzima) el derivado de la enzima β- galactosidasa quimérica recombinante de Thermus sp. T2 purificado, inmovilizado-estabilizado según (as reivindicaciones 1-7,(a) the biocatalyst (enzyme) is the derivative of the recombinant chimeric β-galactosidase enzyme from Thermus sp. T2 purified, immobilized-stabilized according to claims 1-7, (b) con utilización preferente entre 25 y 75 ° C y(b) with preferred use between 25 and 75 ° C and 12.- Procedimiento de tratamiento eπzirpático de lactosa, azúcares o análogos estructurales según la reivindicación 110, caracterizado porque se realiza la hidrólisis de concentrados de lactosa (desde 1 hasta 40%) hasta 75 ° C desde pH 4 hasta pH 8 mediante el biocatalizador (enzima) purificado, inmovilizado- estabilizado según las reivindicaciones 1-712. Process for the eπzirpathic treatment of lactose, sugars or structural analogs according to claim 110, characterized in that the hydrolysis of lactose concentrates (from 1 to 40%) is carried out up to 75 ° C from pH 4 to pH 8 by the stabilized immobilized-stabilized biocatalyst (enzyme) according to claims 1-7 13.- Procedimiento de tratamiento enzimático de lactosa, azúcares o análogos estructurales según la reivindicación 10, caracterizado porque se realiza la hidrólisis de los mismos en leche entera, desnatada o semidesnatada hasta 75 ° C mediante el biocatalizador (enzima) purificado, ¡nmovilizado-estabil izado según las reivindicaciones 1-7.13. Process for the enzymatic treatment of lactose, sugars or structural analogs according to claim 10, characterized in that the hydrolysis thereof is carried out in whole, skimmed or semi-skimmed milk up to 75 ° C by the purified biocatalyst (enzyme), immobilized- stabilized according to claims 1-7. 14.- Procedimiento de tratamiento enzimático de lactosa, azúcares o análogos estructurales según la reivindicación 10, caracterizado porque se realiza la hidrólisis de los mismos en suero lácteo, concentrado o no, o de permeados de suero lácteo de diferentes orígenes (pH 4-8) y hasta 75 ° C mediante el biocatalizador (enzima) purificado, inmovilízado-estabilizado según las reivindicaciones 1-7.14. Procedure for the enzymatic treatment of lactose, sugars or structural analogs according to claim 10, characterized in that the hydrolysis thereof is carried out in milk whey, concentrated or not, or of permeate of milk whey from different origins (pH 4-8). ) and up to 75 ° C by the immobilized-stabilized purified biocatalyst (enzyme) according to claims 1-7. 15.- Procedimiento de tratamiento enzimático de lactosa , azúcares o análogos estructurales según la reivindicación 10, caracterizado porque se realiza en cualquier liquido que los contenga, a pH de 4 a 8 hasta 75 ° C, caracterizado porque se utiliza el biocatalizador (enzima) purificado, inmovilizado-estabiiizado según las reivindicaciones 1-7. 15. Process for the enzymatic treatment of lactose, sugars or structural analogs according to claim 10, characterized in that it is made in any liquid that contains them, at a pH of 4 to 8 up to 75 ° C, characterized in that the biocatalyst (enzyme) is used. purified, immobilized-stabilized according to claims 1-7.
PCT/ES2003/000081 2002-02-21 2003-02-14 Hydrolysis of lactose with immobilised thermoresistant lactase and the production method thereof Ceased WO2003070928A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003209775A AU2003209775A1 (en) 2002-02-21 2003-02-14 Hydrolysis of lactose with immobilised thermoresistant lactase and the production method thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ESP200200419 2002-02-21
ES200200419A ES2214933B1 (en) 2002-02-21 2002-02-21 HYDROLYSIS OF LACTOSE WITH IMMOBILIZED THERMORESISTENT LACTASE AND ITS METHOD OF OBTAINING.

Publications (2)

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WO2003070928A1 WO2003070928A1 (en) 2003-08-28
WO2003070928B1 true WO2003070928B1 (en) 2003-12-18

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ES (1) ES2214933B1 (en)
WO (1) WO2003070928A1 (en)

Also Published As

Publication number Publication date
ES2214933B1 (en) 2005-12-01
WO2003070928A1 (en) 2003-08-28
ES2214933A1 (en) 2004-09-16
AU2003209775A1 (en) 2003-09-09

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