WO1998026055A1 - Esterases immobilisees obtenues a partir d'extraits bruts et leur utilisation - Google Patents

Esterases immobilisees obtenues a partir d'extraits bruts et leur utilisation Download PDF

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Publication number
WO1998026055A1
WO1998026055A1 PCT/EP1997/006894 EP9706894W WO9826055A1 WO 1998026055 A1 WO1998026055 A1 WO 1998026055A1 EP 9706894 W EP9706894 W EP 9706894W WO 9826055 A1 WO9826055 A1 WO 9826055A1
Authority
WO
WIPO (PCT)
Prior art keywords
esterase
esterases
immobilized
crude extract
activity
Prior art date
Application number
PCT/EP1997/006894
Other languages
German (de)
English (en)
Other versions
WO1998026055A9 (fr
Inventor
Heidi Hummel
Mathias Reymann
Heiko Karels
Original Assignee
Schering Aktiengesellschaft
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 Schering Aktiengesellschaft filed Critical Schering Aktiengesellschaft
Priority to EP97952909A priority Critical patent/EP0948607A1/fr
Priority to AU56610/98A priority patent/AU729928B2/en
Priority to JP52621498A priority patent/JP2001505772A/ja
Publication of WO1998026055A1 publication Critical patent/WO1998026055A1/fr
Publication of WO1998026055A9 publication Critical patent/WO1998026055A9/fr

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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
    • C12P33/00—Preparation of steroids
    • 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)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • 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
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/38—Nucleosides
    • C12P19/385—Pyrimidine nucleosides
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters

Definitions

  • the present invention relates to immobilized proteins from crude extract and their use for fermentation in bioreactors.
  • Important enzymes that are used in biochemical or biotechnological conversions in fermenters or bioreactors are esterases.
  • Carboxylesterases are very common in nature. They catalyze the hydrolysis of carboxylic acid esters to free acid anions and alcohols.
  • esterases can be immobilized directly from the crude extract of tissues and cells of animals or plants without prior isolation and purification by binding to resins.
  • Suitable resins for this are, for example, epoxy resins.
  • the present invention therefore relates to esterases covalently bound and thus immobilized on resins from crude extracts of animal or plant cells and animal or plant tissue.
  • the present invention thus relates in particular to resin-bound esterase from the crude extract of pork liver.
  • a suitable resin for this is, for example, the epoxy resin Eupergit C.
  • the resin-bound pork liver crude extract esterase can be used to cleave carboxylic acid esters to free acid anions and alcohols in bioreactors with high yield and high activity.
  • the immobilized esterase according to the invention can preferably be used for the cleavage of nitroisophthalic acid dimethyl ester (NIPA-DME) to nitroisophthalic acid monomethyl ester (NIPA-MME), the undesired reaction to nitroisophthalic acid (NIPA) not taking place.
  • NIPA-DME nitroisophthalic acid dimethyl ester
  • NIPA-MME nitroisophthalic acid monomethyl ester
  • NIPA nitroisophthalic acid
  • the immobilized esterase according to the invention can preferably be used for the saponification of 2-fluoro-ara-adenine triacetate to 2-F-ara-adenine (F-araA), a precursor of Fludara.
  • a further preferred use of the immobilized esterase according to the invention is the use in partial saponification from the ReichsteinS-17.21 diacetate to the ReichsteinS-17 monoacetate
  • the immobilized esterase according to the invention made it possible to achieve a high yield of the end product in the fermentation. This was not predictable, since it is generally known that isolated and purified enzymes are used in enzymatic reactions in order to achieve the greatest possible conversion or high yield. Contamination of the enzyme is generally considered to inhibit activity. Furthermore, it can generally be assumed that further enzymes present in the crude extract interfere with the reaction or even catalyze further enzymatic reactions.
  • Fig. 2 shows the structure required for the fermentation
  • Fig. 3a shows the specific loss of activity of the immobilized at
  • 3b shows the normalized loss of activity of the immobilizate during long-term use.
  • Fresh pork liver is digested with potassium phosphate buffer at pH 7.5 in a mass ratio of 1: 4 at room temperature for 3 minutes in a homogenizer. The homogenate is centrifuged. The supernatant is decanted off. The sediment is discarded. The amount of supernatant corresponds to the buffer used. The supernatant obtained in this way is the raw pig liver extract, which has a high catalytic activity. 66 mg of total protein / ml of extract were obtained.
  • the volume activity of the esterase is approximately 6.5 U / ml, the specific activity is approximately 0.1 U / mg.
  • the quaternary structure of the pig liver esterase is in most cases a trimeric protein with three identical subunits and each with an active center with a molecular weight of approx. 60 kd. Therefore, only one band at 60 kd may occur in gel electrophoresis.
  • a pig liver extract which was prepared as described in Example 1, was therefore applied in various dilutions to an SDS gel in addition to commercially available pig liver esterase and marker proteins. The result of the gel electrophoresis is shown in FIG. 1.
  • isolated and purified pig liver esterase is treated for comparison.
  • the immobilisates of the crude extracts from Example 3 are placed in the reactor.
  • the product can be suctioned off with a filter without completely emptying the reactor.
  • the reactor can then be reloaded with substrates. This process can be repeated until the enzyme has lost its activity. It has been shown in practice that the process can be repeated more than 100 times without loss of activity.
  • the stability of the immobilized enzyme when used repeatedly is decisive for the use of the immobilized product on an industrial scale.
  • the liver was disrupted 1: 4 with 1 M phosphate buffer (pH 7.5).
  • the activity of the immobilizate for the swollen state is 8.94 U / g, based on the wet weight.
  • a long-term study was carried out with this immobilizate, the use of NIPA-DME per batch being 10 g / l.
  • the reaction was carried out at 38 ° C and pH 7.5 for 24 hours. As a result, the immobilizate was separated from the solution and renewed
  • the immobilisate Use of the immobilisate.
  • the decrease in time of the specific activity and the normalized activity of the enzyme is shown in Fig. 3a and Fig. 3b.
  • the immobilized esterase from the raw pig liver extract still has 65% residual activity.
  • the half-life of the immobilized product is approximately 250 hours, making it ideal for technical use in the fermenter. If the immobilizate is loaded with 10 g / l NIPA-DME for 10 hours, at least 100 batches can be run with an average reaction time of 5 hours.
  • the controlled fermentation was carried out in a Biostat ED fermenter in accordance with the arrangement shown in FIG. 2.

Abstract

L'invention concerne des protéines immobilisées obtenues à partir d'extraits bruts, ainsi que leur utilisation pour la fermentation dans des bioréacteurs.
PCT/EP1997/006894 1996-12-11 1997-12-10 Esterases immobilisees obtenues a partir d'extraits bruts et leur utilisation WO1998026055A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP97952909A EP0948607A1 (fr) 1996-12-11 1997-12-10 Esterases immobilisees obtenues a partir d'extraits bruts et leur utilisation
AU56610/98A AU729928B2 (en) 1996-12-11 1997-12-10 Immobilized esterases from crude extract and their use
JP52621498A JP2001505772A (ja) 1996-12-11 1997-12-10 粗製抽出物からの固定化されたエステラーゼおよびその使用

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19653730A DE19653730C2 (de) 1996-12-11 1996-12-11 Immobilisierte Proteine aus Rohextrakt und deren Verwendung zur Umsetzung von Estern
DE19653730.4 1996-12-11

Publications (2)

Publication Number Publication Date
WO1998026055A1 true WO1998026055A1 (fr) 1998-06-18
WO1998026055A9 WO1998026055A9 (fr) 1998-12-17

Family

ID=7815820

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1997/006894 WO1998026055A1 (fr) 1996-12-11 1997-12-10 Esterases immobilisees obtenues a partir d'extraits bruts et leur utilisation

Country Status (7)

Country Link
EP (1) EP0948607A1 (fr)
JP (1) JP2001505772A (fr)
AU (1) AU729928B2 (fr)
CZ (1) CZ208799A3 (fr)
DE (1) DE19653730C2 (fr)
HU (1) HUP0000599A2 (fr)
WO (1) WO1998026055A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104861023B (zh) * 2007-08-03 2017-04-12 卡斯欧皮亚公司 用于获得11‑脱氧皮醇的17α‑单酯和/或其9,11‑脱氢衍生物的酶法

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100371971B1 (ko) * 1999-09-30 2003-02-14 주식회사 펩트론 천연물 유래 화합물 라이브러리의 제조방법
ITMI20011762A1 (it) 2001-08-10 2003-02-10 Cosmo Spa Esteri di 17alfa,21-diidrossipregnene, loro uso come agenti anti-androgenetici e procedimenti per la loro preparazione
EP3108879A1 (fr) 2015-06-25 2016-12-28 Cassiopea S.p.A. Formulation à concentration élevée

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0302284A2 (fr) * 1987-08-06 1989-02-08 RAMOT UNIVERSITY, AUTHORITY FOR APPLIED RESEARCH & INDUSTRIAL DEVELOPMENT LTD. Enzymes stabilisées
EP0345658A2 (fr) * 1988-06-08 1989-12-13 BASF Aktiengesellschaft Procédé de préparation d'un biocatalyseur et son application pour la séparation de mélanges racémiques
EP0346865A2 (fr) * 1988-06-16 1989-12-20 The Du Pont Merck Pharmaceutical Company Phosphorylare polynucléotide immobilisée dans des particules époxy-actives
EP0562373A2 (fr) * 1992-03-23 1993-09-29 Siemens Aktiengesellschaft Immobilisation de substances biochimiques
US5262313A (en) * 1991-06-14 1993-11-16 Andcare, Inc. Carrageeman-immobilized esterase

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4897352A (en) * 1988-01-15 1990-01-30 The Dow Chemical Company Acrylate based adsorbent resin for the immobilization of enzymes

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0302284A2 (fr) * 1987-08-06 1989-02-08 RAMOT UNIVERSITY, AUTHORITY FOR APPLIED RESEARCH & INDUSTRIAL DEVELOPMENT LTD. Enzymes stabilisées
EP0345658A2 (fr) * 1988-06-08 1989-12-13 BASF Aktiengesellschaft Procédé de préparation d'un biocatalyseur et son application pour la séparation de mélanges racémiques
EP0346865A2 (fr) * 1988-06-16 1989-12-20 The Du Pont Merck Pharmaceutical Company Phosphorylare polynucléotide immobilisée dans des particules époxy-actives
US5262313A (en) * 1991-06-14 1993-11-16 Andcare, Inc. Carrageeman-immobilized esterase
EP0562373A2 (fr) * 1992-03-23 1993-09-29 Siemens Aktiengesellschaft Immobilisation de substances biochimiques

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
G. SPAGNA ET AL.,: "Pectinlyase immobilization on epoxy supports for application in the food processing industry", JOURNAL OF CHEMICAL TECHNOLOGY UND BIOTECHNOLOGY, vol. 57, no. 4, 1993, BARKING, ESSEX, GB, pages 379 - 385, XP000378237 *
K. BURG ET AL.,: "Neue synthetische Träger zur Fixierung von Enzymen", DIE ANGEWANDTE MAKROMOLECULARE CHEMIE, vol. 157, 1988, BASEL, CH, pages 105 - 121, XP002064486 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104861023B (zh) * 2007-08-03 2017-04-12 卡斯欧皮亚公司 用于获得11‑脱氧皮醇的17α‑单酯和/或其9,11‑脱氢衍生物的酶法

Also Published As

Publication number Publication date
HUP0000599A2 (en) 2000-07-28
AU729928B2 (en) 2001-02-15
JP2001505772A (ja) 2001-05-08
DE19653730C2 (de) 1999-06-24
AU5661098A (en) 1998-07-03
DE19653730A1 (de) 1998-06-18
EP0948607A1 (fr) 1999-10-13
CZ208799A3 (cs) 1999-09-15

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