WO2001029202A1 - Procede d'immobilisation d'une nouvelle penicilline v acylase de streptomyces lavendulae par l'obtention d'acide de 6-aminopenicilline - Google Patents

Procede d'immobilisation d'une nouvelle penicilline v acylase de streptomyces lavendulae par l'obtention d'acide de 6-aminopenicilline Download PDF

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Publication number
WO2001029202A1
WO2001029202A1 PCT/ES2000/000408 ES0000408W WO0129202A1 WO 2001029202 A1 WO2001029202 A1 WO 2001029202A1 ES 0000408 W ES0000408 W ES 0000408W WO 0129202 A1 WO0129202 A1 WO 0129202A1
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WO
WIPO (PCT)
Prior art keywords
penicillin
acylase
immobilization
apa
new
Prior art date
Application number
PCT/ES2000/000408
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English (en)
Spanish (es)
Inventor
Carmen Acebal Sarabia
Jesus Torres Bacete
Miguel Arroyo Sanchez
Raquel Torres Guzman
Isabel De La Mata Riesco
Maria Pilar Castillon Borreguero
Original Assignee
Universidad Complutense De Madrid Rectorado
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 Universidad Complutense De Madrid Rectorado filed Critical Universidad Complutense De Madrid Rectorado
Priority to AU10308/01A priority Critical patent/AU1030801A/en
Publication of WO2001029202A1 publication Critical patent/WO2001029202A1/fr

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Classifications

    • 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)
    • C12N9/84—Penicillin amidase (3.5.1.11)
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/06—Enzymes or microbial cells immobilised on or in an organic carrier attached to the carrier via a bridging agent
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • C12N11/087—Acrylic polymers
    • 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
    • C12P37/00—Preparation of compounds having a 4-thia-1-azabicyclo [3.2.0] heptane ring system, e.g. penicillin
    • C12P37/06—Preparation of compounds having a 4-thia-1-azabicyclo [3.2.0] heptane ring system, e.g. penicillin by desacylation of the substituent in the 6 position

Definitions

  • the present invention falls within the biotechnological field of obtaining 6-aminopenicillanic acid from natural penicillins, and more specifically, from penicillin V.
  • the invention relates to a new method of covalent immobilization on Eupergit C of penicillin. V acylase isolated from Streptomyces lavendulae cultures.
  • the present invention relates to a process for the immobilization of a new penicillin V acylase from Streptomyces lavendulae to obtain 6-aminopenicillanic acid (6-APA)
  • this invention allows the industrial production of 6- APA from penicillin V through the use of a new preparation of penicillin V acylase (penicillin amidohydrolase, EC 3.5.1.11) immobilized.
  • This biocatalyst has been prepared by covalent immobilization of Streptomyces lavendulae ATCC 13664 penicillin V acylase in the Eupergit C commercial support (Rhóm GmbH, Kirschenallee, Germany).
  • PVA is produced by the actinomycete Streptomyces lavendulae ATCC 13664 according to the protocol described by Torres et al, 1998 (Torres, R., de la Mata, I., Castillón, MP, Arroyo, M., Torres, J., Acebal, C. (1998) Purification and characterization of penicillin V acylase from Streptomyces lavendulae. Progress in Biotechnology 15: Stability and Stabilization of Biocatalysts, A. Ballesteros, FJ Plou, JL Iborra and PJ Halling Eds. Elsevier. Amsterdam, pp. 719-724 .), partially purified by precipitation with acetone and subsequently lyophilized.
  • Immobilization of PVA is performed on Eupergit C, a support consisting of a copolymer of methacrylamide, N, N '- methylene bis-methacrylamide and oxirane groups.
  • the enzyme covalently binds to the support through its epoxide groups (oxirane groups).
  • This new biocatalyst allows a remarkable improvement of the enzyme's stability against temperature and pH. At the same time, it allows its reuse, which implies an economic advantage when carrying out industrial reactions to obtain 6-APA.
  • a unit of activity has been considered as the ⁇ moles of 6-aminopenicillanic acid produced in one minute at 40 ° C and pH 9.5 and rated spectophotometrically by reaction with p-dimethyl-aminobenzaldehyde (PDAB method) according to the specifications of Balasingham, K., Warburton, D., Dunnill, P. and Lilly, HD (The isolation and kinetics of penicillin An ⁇ dase from Escherichia coli. Biochim. Biophys. Acta. 1972, 276, 250-256).
  • PDAB method p-dimethyl-aminobenzaldehyde
  • the immobilized preparation of PVA in Eupergit C has shown an important improvement in its kinetic characteristics after a treatment with bovine serum albumin (BSA), after the binding of the enzyme. This treatment has allowed to increase the activity of the immobilized derivative by 60% compared to that presented before the modification with BSA.
  • BSA bovine serum albumin
  • ECPVA penicillin V acylase
  • ECPVA Under the optimal test conditions established (pH 9.5 and 40 ° C), ECPVA has a half-life of 304 hours, while increasing its stability considerably to 40 ° C and pH 7, increasing its half-life to at least 1150 hours . Finally, ECPVA can be used in successive reactions of 1 hour for at least 50 cycles, keeping 100% of the performance exhibited in the first activity cycle.
  • This example refers to the method of immobilization of the
  • the enzyme solution is maintained for 5 minutes in the presence of phenoxyacetic acid, and subsequently 1 gram of dried Eupergit C is added.
  • the mixture is incubated, with reciprocating stirring and at room temperature, in a 25 ml erlenmeyer flask. After 24 hours, 3.6 grams of wet immobilized derivative of PVA are obtained in Eupergit C. Then, it is washed twice with 40 ml of distilled water and another 2 times with 40 ml of 0.1 M potassium phosphate buffer and pH 8.
  • the efficiency of the enzyme binding to the support is determined by assessing the presence of protein by the Bradford method (Bradford, MM A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principie of protein-dye binding, Anal. Biotech. 1916, 72, 248-245) and enzymatic activity (PDAB method) in each of the immobilized derivative washes. The purpose is to verify that all the enzyme that has come into contact with the support has been attached to it.
  • the result of the immobilization shows that 100% of the PVA is bound to Eupergit C.
  • the example shows the reaction conditions in the hydrolysis of penicillin V to 6-aminopenicillanic acid catalyzed by the immobilized derivative of penicillin V acylase in Eupergit C.
  • the conditions are as follows: 1. 0.36 grams of derivative per milliliter of reaction volume.
  • Buffer phosphate-citrate-borate (1: 1: 1) 0.1 M pH 9.5
  • penicillin V The hydrolysis of penicillin V is monitored by assessing the amount of 6-aminopenicillanic acid produced, for which 50 ⁇ l aliquots are taken and diluted with 0.25 ml of distilled water, to subsequently add 0.9 ml of 20% acetic acid
  • the 6-APA produced is evaluated by the PDAB method.
  • the immobilized derivative according to example 1 shows an activity of 0.245 Ul / g of immobilized derivative.
  • the yield of the hydrolysis reaction of penicillin V (5mM) catalyzed by this derivative is 76% after one hour of reaction.
  • the hydrolysis of penicillin V catalyzed by ECPVA is monitored in the same way as indicated in example 3.
  • the immobilized and modified derivative (ECPVA) shows an activity of 0.393 Ul / g of derivative.
  • the yield of the hydrolysis reaction of penicillin V (5 mM) catalyzed by ECPVA reaches 80% after one hour of reaction.

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

Abstract

L'immobilisation de la pénicilline V acylase de Streptomyces lavendulae ATCC 13664 sur le support commercial Eupergit C a permis d'obtenir le biocatalyseur ECPVA, une nouvelle pénicilline V acylase immobilisée en vue de l'obtention de 6-APA à partir de la pénicilline V. L'invention concerne le procédé permettant de réaliser cette immobilisation dans les conditions de réaction qui rendent possible l'hydrolyse de pénicilline V catalysée par ECPVA.
PCT/ES2000/000408 1999-10-22 2000-10-23 Procede d'immobilisation d'une nouvelle penicilline v acylase de streptomyces lavendulae par l'obtention d'acide de 6-aminopenicilline WO2001029202A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU10308/01A AU1030801A (en) 1999-10-22 2000-10-23 Method for immobilizing a novel penicillin v acylase of streptomyces lavendulae for obtaining 6-amino penicillanic acid

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ES009902332A ES2163997B2 (es) 1999-10-22 1999-10-22 Procedimiento para la inmovilizacion de una nueva penicilina v acilasa de "streptomyces lavendulae" para la obtencion de acido 6-aminopenicilanico.
ES9902332 1999-10-22

Publications (1)

Publication Number Publication Date
WO2001029202A1 true WO2001029202A1 (fr) 2001-04-26

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Application Number Title Priority Date Filing Date
PCT/ES2000/000408 WO2001029202A1 (fr) 1999-10-22 2000-10-23 Procede d'immobilisation d'une nouvelle penicilline v acylase de streptomyces lavendulae par l'obtention d'acide de 6-aminopenicilline

Country Status (3)

Country Link
AU (1) AU1030801A (fr)
ES (1) ES2163997B2 (fr)
WO (1) WO2001029202A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006008296A1 (fr) * 2004-07-19 2006-01-26 Fidia Farmaceutici S.P.A. Procede de preparation de catalyeurs enzymatiques, catalyseurs pouvant etre obtenu au moyen de ce procede et leur utilisation
CN100465271C (zh) * 2006-12-28 2009-03-04 浙江大学 一种在聚电解质离子的协同作用下对青霉素酰化酶的固定方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5500352A (en) * 1993-03-24 1996-03-19 Sepracor, Inc. Membrane filtration process for 6-aminopenicillanic acid
EP0730035A1 (fr) * 1995-02-28 1996-09-04 ACS DOBFAR S.p.A. Procédé enzymatique amélioré pour la production de pénicillines et céphalosporines

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5500352A (en) * 1993-03-24 1996-03-19 Sepracor, Inc. Membrane filtration process for 6-aminopenicillanic acid
EP0730035A1 (fr) * 1995-02-28 1996-09-04 ACS DOBFAR S.p.A. Procédé enzymatique amélioré pour la production de pénicillines et céphalosporines

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BALDARO E. ET AL.: "Pen G acylase catalized resolution of phenylacetate esters of secondary alcohols", TETRAHEDRON: ASYMMETRY, vol. 4, no. 5, 1993, pages 1031 - 1034 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006008296A1 (fr) * 2004-07-19 2006-01-26 Fidia Farmaceutici S.P.A. Procede de preparation de catalyeurs enzymatiques, catalyseurs pouvant etre obtenu au moyen de ce procede et leur utilisation
CN100465271C (zh) * 2006-12-28 2009-03-04 浙江大学 一种在聚电解质离子的协同作用下对青霉素酰化酶的固定方法

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Publication number Publication date
AU1030801A (en) 2001-04-30
ES2163997A1 (es) 2002-02-01
ES2163997B2 (es) 2004-07-01

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