EP0948607A1 - Esterases immobilisees obtenues a partir d'extraits bruts et leur utilisation - Google Patents
Esterases immobilisees obtenues a partir d'extraits bruts et leur utilisationInfo
- Publication number
- EP0948607A1 EP0948607A1 EP97952909A EP97952909A EP0948607A1 EP 0948607 A1 EP0948607 A1 EP 0948607A1 EP 97952909 A EP97952909 A EP 97952909A EP 97952909 A EP97952909 A EP 97952909A EP 0948607 A1 EP0948607 A1 EP 0948607A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- esterase
- esterases
- immobilized
- crude extract
- activity
- 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.)
- Withdrawn
Links
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.
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
L'invention concerne des protéines immobilisées obtenues à partir d'extraits bruts, ainsi que leur utilisation pour la fermentation dans des bioréacteurs.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE19653730 | 1996-12-11 | ||
DE19653730A DE19653730C2 (de) | 1996-12-11 | 1996-12-11 | Immobilisierte Proteine aus Rohextrakt und deren Verwendung zur Umsetzung von Estern |
PCT/EP1997/006894 WO1998026055A1 (fr) | 1996-12-11 | 1997-12-10 | Esterases immobilisees obtenues a partir d'extraits bruts et leur utilisation |
Publications (1)
Publication Number | Publication Date |
---|---|
EP0948607A1 true EP0948607A1 (fr) | 1999-10-13 |
Family
ID=7815820
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP97952909A Withdrawn EP0948607A1 (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) |
Families Citing this family (4)
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 |
ITMI20071616A1 (it) | 2007-08-03 | 2009-02-04 | Cosmo Spa | Processo enzimatico per l'ottenimento di 17-alfa monoesteri del cortexolone e/o suoi 9,11-deidroderivati. |
EP3108879A1 (fr) | 2015-06-25 | 2016-12-28 | Cassiopea S.p.A. | Formulation à concentration élevée |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
IL83451A (en) * | 1987-08-06 | 1991-06-10 | Univ Ramot | Stabilized water soluble enzymes and a method for their preparation |
US4897352A (en) * | 1988-01-15 | 1990-01-30 | The Dow Chemical Company | Acrylate based adsorbent resin for the immobilization of enzymes |
DE3819467A1 (de) * | 1988-06-08 | 1989-12-14 | Basf Ag | Verfahren zur herstellung eines biokatalysators und dessen verwendung zur razematspaltung |
IL90600A0 (en) * | 1988-06-16 | 1990-01-18 | Du Pont | Polynucleotide phosphorylase immobilized on epoxy-activated beads |
US5262313A (en) * | 1991-06-14 | 1993-11-16 | Andcare, Inc. | Carrageeman-immobilized esterase |
EP0562373A3 (en) * | 1992-03-23 | 1994-05-25 | Siemens Ag | Immobilisation of biochemical substances |
-
1996
- 1996-12-11 DE DE19653730A patent/DE19653730C2/de not_active Expired - Lifetime
-
1997
- 1997-12-10 CZ CZ992087A patent/CZ208799A3/cs unknown
- 1997-12-10 HU HU0000599A patent/HUP0000599A2/hu unknown
- 1997-12-10 WO PCT/EP1997/006894 patent/WO1998026055A1/fr not_active Application Discontinuation
- 1997-12-10 JP JP52621498A patent/JP2001505772A/ja active Pending
- 1997-12-10 EP EP97952909A patent/EP0948607A1/fr not_active Withdrawn
- 1997-12-10 AU AU56610/98A patent/AU729928B2/en not_active Ceased
Non-Patent Citations (1)
Title |
---|
See references of WO9826055A1 * |
Also Published As
Publication number | Publication date |
---|---|
DE19653730C2 (de) | 1999-06-24 |
HUP0000599A2 (en) | 2000-07-28 |
CZ208799A3 (cs) | 1999-09-15 |
WO1998026055A1 (fr) | 1998-06-18 |
AU729928B2 (en) | 2001-02-15 |
DE19653730A1 (de) | 1998-06-18 |
JP2001505772A (ja) | 2001-05-08 |
AU5661098A (en) | 1998-07-03 |
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Legal Events
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PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
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Effective date: 19990429 |
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RBV | Designated contracting states (corrected) |
Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LI LU MC NL PT SE |
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STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN |
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18W | Application withdrawn |
Withdrawal date: 20010517 |