WO1998026055A9 - 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
- WO1998026055A9 WO1998026055A9 PCT/EP1997/006894 EP9706894W WO9826055A9 WO 1998026055 A9 WO1998026055 A9 WO 1998026055A9 EP 9706894 W EP9706894 W EP 9706894W WO 9826055 A9 WO9826055 A9 WO 9826055A9
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- esterase
- esterases
- crude extract
- immobilized
- activity
- Prior art date
Links
- 239000000287 crude extract Substances 0.000 title claims abstract description 27
- 238000000855 fermentation Methods 0.000 claims abstract description 10
- 230000004151 fermentation Effects 0.000 claims abstract description 10
- 210000004185 Liver Anatomy 0.000 claims description 27
- 241000282898 Sus scrofa Species 0.000 claims description 13
- 229920005989 resin Polymers 0.000 claims description 10
- 239000011347 resin Substances 0.000 claims description 10
- 239000002253 acid Substances 0.000 claims description 5
- 150000001298 alcohols Chemical class 0.000 claims description 4
- 150000001450 anions Chemical class 0.000 claims description 4
- 150000001733 carboxylic acid esters Chemical class 0.000 claims description 4
- 238000003776 cleavage reaction Methods 0.000 claims description 4
- 239000003822 epoxy resin Substances 0.000 claims description 3
- 229920000647 polyepoxide Polymers 0.000 claims description 3
- 238000007127 saponification reaction Methods 0.000 claims description 3
- 210000001519 tissues Anatomy 0.000 claims description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-M acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 2
- UZMFVOACDBUXRK-UHFFFAOYSA-N dimethyl 2-nitrobenzene-1,3-dicarboxylate Chemical compound COC(=O)C1=CC=CC(C(=O)OC)=C1[N+]([O-])=O UZMFVOACDBUXRK-UHFFFAOYSA-N 0.000 claims description 2
- 235000013311 vegetables Nutrition 0.000 claims description 2
- NUULGBPUCNUUQD-UHFFFAOYSA-N 2-Nitroisophthalic Acid Monomethyl Ester Chemical compound COC(=O)C1=CC=CC(C(O)=O)=C1[N+]([O-])=O NUULGBPUCNUUQD-UHFFFAOYSA-N 0.000 claims 1
- 108010058683 Immobilized Proteins Proteins 0.000 abstract description 2
- 230000000694 effects Effects 0.000 description 25
- 108090000790 Enzymes Proteins 0.000 description 12
- 102000004190 Enzymes Human genes 0.000 description 12
- 235000015277 pork Nutrition 0.000 description 12
- 238000006243 chemical reaction Methods 0.000 description 10
- 239000000284 extract Substances 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 239000000758 substrate Substances 0.000 description 4
- 230000036462 Unbound Effects 0.000 description 3
- 238000001502 gel electrophoresis Methods 0.000 description 3
- 239000008057 potassium phosphate buffer Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 229940040511 Liver Extract Drugs 0.000 description 2
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- CAHWDGJDQYAFHM-UHFFFAOYSA-N 2-Nitroisophthalic acid Chemical compound OC(=O)C1=CC=CC(C(O)=O)=C1[N+]([O-])=O CAHWDGJDQYAFHM-UHFFFAOYSA-N 0.000 description 1
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonium chloride Substances [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 1
- 210000004369 Blood Anatomy 0.000 description 1
- 102000013392 Carboxylesterase Human genes 0.000 description 1
- 108010051152 Carboxylesterase Proteins 0.000 description 1
- 102000004308 Carboxylic Ester Hydrolases Human genes 0.000 description 1
- 108090000863 Carboxylic Ester Hydrolases Proteins 0.000 description 1
- 235000019750 Crude protein Nutrition 0.000 description 1
- 102000016901 Glutamate dehydrogenases Human genes 0.000 description 1
- 108091000037 Glutamate dehydrogenases Proteins 0.000 description 1
- 230000036499 Half live Effects 0.000 description 1
- 108010093096 Immobilized Enzymes Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 210000003097 Mucus Anatomy 0.000 description 1
- 102000005924 Triose-phosphate isomerases Human genes 0.000 description 1
- 108020003073 Triose-phosphate isomerases Proteins 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 238000011138 biotechnological process Methods 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M buffer Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- 230000003197 catalytic Effects 0.000 description 1
- 230000024881 catalytic activity Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000002255 enzymatic Effects 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- GIUYCYHIANZCFB-FJFJXFQQSA-N fludarabine phosphate Chemical compound C1=NC=2C(N)=NC(F)=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@@H]1O GIUYCYHIANZCFB-FJFJXFQQSA-N 0.000 description 1
- 229960005304 fludarabine phosphate Drugs 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000011068 load Methods 0.000 description 1
- -1 methyl nitroisophthalate Chemical compound 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- OZAIFHULBGXAKX-UHFFFAOYSA-N precursor Substances N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000002123 temporal effect Effects 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- ILJSQTXMGCGYMG-UHFFFAOYSA-M triacetate(1-) Chemical compound CC(=O)CC(=O)CC([O-])=O ILJSQTXMGCGYMG-UHFFFAOYSA-M 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
Definitions
- the present invention relates to immobilized proteins from crude extract and their use for fermentation in bioreactors.
- Important enzymes which are used in biochemical or biotechnological reactions 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 attachment to resins.
- Suitable resins for this purpose are, for example, epoxy resins.
- the present invention therefore relates to resins covalently bonded and thus immobilized esterases from crude extracts of animal or plant cells and animal or vegetable tissue.
- the present invention thus relates in particular to resins bound esterase from the crude extract of pork liver.
- a resin suitable for this purpose is, for example, the epoxy resin Eupergit C.
- the ester-bound pork liver crude extract esterase can be used to cleave carboxylic acid esters to free acid anions and alcohols in high yield, high activity bioreactors.
- the immobilized esterase according to the invention can preferably be used for the cleavage of dimethyl nitroisophthalate (NIPA-DME) to methyl nitroisophthalate (NIPA-MME), the undesired reaction to nitroisophthalic acid (NIPA) not taking place.
- NIPA-DME dimethyl nitroisophthalate
- NIPA-MME methyl nitroisophthalate
- NIPA nitroisophthalic acid
- the immobilized esterase of the present invention may preferably be used for saponifying 2-fluoro-ara-adenine triacetate to 2-F-ara-adenine (F-araA), a precursor of Fludara.
- Another preferred use of the immobilized esterase according to the invention is the use in the partial saponification of the Reichstein S-17,21 diacetate to the Reichstein S-17 monoacetate
- Fig. 1 shows the SDS gel electrophoresis (12.5% acrylamide gel) with purified esterase from pig liver and pig liver esterase from the crude extract.
- Fig. 2 shows the structure required for the fermentation
- Fig. 3a shows the specific loss of activity of Imobilisats in
- FIG. 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 thus obtained is the pork liver crude extract which has a high catalytic activity. There were obtained 66 mg total protein / ml extract.
- the volume activity of the esterase is about 6.5 U / ml, the specific activity is about 0.1 U / mg.
- porcine liver esterase The quaternary structure of porcine liver esterase is in most cases a trimeric protein with three identical subunits and one active site each with a molecular weight of about 60 kd. In gel electrophoresis, therefore, only one band may occur at 60 kd.
- a pig liver extract which was prepared as described in Example 1, was applied at various dilutions to an SDS gel in addition to commercially available pig liver esterase and marker proteins. The result of gel electrophoresis is shown in FIG.
- porcine liver esterase is treated for comparison.
- the immobilizates of the crude extracts from Example 3 are initially charged in the reactor.
- the product can be exhausted without the reactor to be completely exhausted with a filter.
- the reactor can be re-charged 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 immobilizate For the immobilizate, kinetic studies were carried out with 10 g / l and 50 g / l NIPA-DME initial concentration. Kinetic studies indicate that the kinetics of substrate affinity and the inhibition constant for free esterase methanol are consistent with those of immobilized esterase. Thus, the bound enzyme behaves in its kinetic properties as the free enzyme.
- the specific activity of the moist immobilized esterase is about 8.5 U / g or 0.51 mol substrate / hour and kg of immobilizate. The total activity of the immobilizate compared to that of the pork liver extract used is reduced by the immobilization process to about 50%.
- 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, with the use of NIPA-DME per batch being 10 g / l. The reaction was carried out at 38 ° C and a pH of 7.5 over a period of 24 hours. This resulted in the separation of the immobilizate from the solution and the renewed
- the controlled fermentation was carried out in a Biostat ED fermenter according to the arrangement shown in FIG.
- immobilized crude extract corresponds to about 0.75 g of immobilized purified esterase.
Abstract
L'invention concerne des protéines immobilisées obtenues à partir d'extraits bruts, ainsi que leur utilisation pour la fermentation dans des bioréacteurs.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| 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 |
| 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 |
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 WO1998026055A1 (fr) | 1998-06-18 |
| WO1998026055A9 true 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) |
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
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