CA1202584A - Proteases fixed on a carrier and their use in biotechnology - Google Patents
Proteases fixed on a carrier and their use in biotechnologyInfo
- Publication number
- CA1202584A CA1202584A CA000415462A CA415462A CA1202584A CA 1202584 A CA1202584 A CA 1202584A CA 000415462 A CA000415462 A CA 000415462A CA 415462 A CA415462 A CA 415462A CA 1202584 A CA1202584 A CA 1202584A
- Authority
- CA
- Canada
- Prior art keywords
- gel
- protease
- carrier
- fixed
- proteases
- 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.)
- Expired
Links
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Preparation or treatment thereof
- A23L2/70—Clarifying or fining of non-alcoholic beverages; Removing unwanted matter
- A23L2/84—Clarifying or fining of non-alcoholic beverages; Removing unwanted matter using microorganisms or biological material, e.g. enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12H—PASTEURISATION, STERILISATION, PRESERVATION, PURIFICATION, CLARIFICATION OR AGEING OF ALCOHOLIC BEVERAGES; METHODS FOR ALTERING THE ALCOHOL CONTENT OF FERMENTED SOLUTIONS OR ALCOHOLIC BEVERAGES
- C12H1/00—Pasteurisation, sterilisation, preservation, purification, clarification, or ageing of alcoholic beverages
- C12H1/003—Pasteurisation, sterilisation, preservation, purification, clarification, or ageing of alcoholic beverages by a biochemical process
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Physiology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Nutrition Science (AREA)
- Polymers & Plastics (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Detergent Compositions (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
- Materials For Medical Uses (AREA)
Abstract
ABSTRACT
Proteases fixed to non-hardened gel-forming proteins by covalent cross-linking and their use for removing protein turbidity from drinks such as wine, beer or fruit juices.
.
Proteases fixed to non-hardened gel-forming proteins by covalent cross-linking and their use for removing protein turbidity from drinks such as wine, beer or fruit juices.
.
Description
S~
The invention relates to proteases covalently fixed on a carrier protein in a novel manner, the preparation thereof and the use thereof in biotechnology.
Proteases are of considerable importance in -the manufac-ture of drin~s since protein turbidity may occur in many clear drinks (such as wine, beer and fruit juices~ as a result of heating or cooling.
One of the present approaches to solving this problem is treatment with bentonite. This certainly achieves the required stability but at the same time there are a number of serious dis-advantages. Thus, the strong adsorptive effect also elimina~es a number of valuable substances such as aromatic substances, bouquet and/or dyestuffs, and moreover some undesirable metal ions may be introduced, and thirdly considerable quantities of sediment are produced which are ~aborious to process It has there~ore already been proposed that the proteins which cause cloudiness under the e~fect o~ heat or cold be decom-posed by means of proteases (vide Z~ fur Lebensmitteluntersuchung und forschung 134 (1967~, pages 87-97).
However, there are disadvantages with this method too.
First, the costs of enzyme enrichment and isolation are extremel~
high since the enzymes are unstable and cannot be recovered either.
Moreover, it is inadvisable or even illegal according to the laws on foodstuffs to have any soluble enzymes remaining in the drink.
One way of solving these problems is to make the enzymes used insoluble by fixing them on carrier substances which are in-soluble in water. This enables the enzymes to be recovered and ~' 5~
re-used several times.
A number of methods of fixing enzymes are already known.
A synopsis of the latest results in this field is provided, for example, by J.C. Johrson (Editor) in "Immobilized Enzymes, Prepara-tion and Engineering", Noyes Data Corp., Park Ridge, N~J., USA
(1979).
The fixing of the enzymes on carriers results in other advantages in addition to those mentioned above. These include, for example-a continuous method of operation increased stability a smaller reaction volume no contamination of the reaction mixtures no heat precipita-tion of the enzyme.
There is no standard process for bonding an enzyme to an insoluble carrier substance. In order to reach the objective of obtaining maximum biological acti~ity of the enzyme, the carrier substance and method of immobilization must be individually matched to the enzyme~
~0 Particularly preferred carrier substances for this purpose (on the grounds that they are easy ~o process and inexpen-sive) are gel-forming proteins such as gelatine or fresh egg white (see for example German Auslegeschrift 22 46 002). However, these carrier substances are not suitable for fixing enzymes with a proteolytic activity since they are hydrolysed by them.
It has therefore already been proposed ~see German ~uslegeschrift 26 36 206.1-411 that the gel-~orming proteins be denatured by chemical hardening (e.g. with formaldehyde, glutardi-aldehyde or diisocyanate) in order to make them safe from attack by the proteases fixed on them. In this way, rennin or even pepsin, for example, may be fixed on a carrier (vide Biotechnology Letters 1 (1979) pages 225-230).
Surprisingly, it has now been found that proteases can also be fixed to gel-forming proteins even if the proteins are not subjected to a complex preliminary chemical treatment.
The invention therefore relates to a process for immobil-izing proteases on gel~forming proteins which is characterised in that the protease is mixed with gel-forming protein (e.g. gelatine, egg white, soya protein and the like, preferably albumin), the mixture is treated with a water-miscible organic solvent (pre cipitating agent) and subsequently the resulting particle suspension is reacted with difunctional and/or polyfunctional cross-linking agents, The invention ~urther relates to the enzyme preparations fixed on a carrier ~hich are obtained in this way and their use in decomposing proteins.
The mixture of proteolytic enzyme and gel-forming protein may be prepared by dissolving or suspending the enzyme in an aqueous solution o~ the gel-forming protein~ The temperature is preferably adjusted so that the active mixture is obtained in liquid ~orm.
The mixture of proteolytic enzyme and gel-forming protein is preferably prepared by first dissolving the proteolytic enzyme (e~g. pepsin, papain, etc) in water and then mixing it with an aqueous solution o~ the gel~forming protein, This mixture is then ; combined with an organic solvent which has ~n enzyme-precipitating ~Zi3Z5~3~
activity and is compatible with the enzyme~ Examples o~ such a sol~ent include acetone, ethanol, isopropanol and butanol~ The particle suspension formed by the precipitating agent is allowed to react with difunctional or polyfunctional cross-linking agents in such a manner and for such a time that the mixture of proteolytic enzyme and gel-forming protein i5 cross-linked as comple-tely as possible. The particulate mixture may also be separated from the organic liquid beforehand and on~y then treated with the cross-linking agent~
For the cross-linking reactisn, difunctional and poly-functional cross-linking agents for proteins may be used. Prefer-ably, an aqueous solution of glutardialdehyde is used. It is also possible to dissolve the coupling reagent in one of the enzyme-precipitating liquids mentioned above and add it in this form to the aqueous mixture of proteolytic enzyme and gel-forming protein.
This reaction mixture consisting of proteolytic enzyme, gel-forming protein, enzyme-precipitating liquid and cross-linking agent is ~igorously stirred for some time, preferably fxom 0.5 to 4 hours. The coupling reaction is usually carried out at ambient ~0 ~emperature.
Ater the bonding reaction has ended~ the preparation is ~ashed several times, pre~erably wi,th water. The product thus obtained can then be dried to give the required water content (e.gO
by spray drying or drying with acetone).
The enzyme product~s prepared by this procedure may be used in batch processes for the proteolysis o~ protein (protein degradation), with the enzyme product being separated off and re-i~DZ5~3~
used, or in continuous processes in an enzyme reactor. They areparticularly suitable for treating protein-containing beverages (such as wine, beer and fruit juices) where the end product should not contain any residual enzymes The proteolytic enzyme used is preferably pepsin. The pepsin activity of both the soluble form and the immobilised form is determined by the Anson method (Rick,W & Fritsch, W.-P. in Bergmeyer (Editor): Methoden der enzymatischen Analyse, 3rd edition, volume 1, pages 1086-1090, Verlag Chemie, Weinheim, 1974).
The following Examples serve to illustrate the invention:
Example 1
The invention relates to proteases covalently fixed on a carrier protein in a novel manner, the preparation thereof and the use thereof in biotechnology.
Proteases are of considerable importance in -the manufac-ture of drin~s since protein turbidity may occur in many clear drinks (such as wine, beer and fruit juices~ as a result of heating or cooling.
One of the present approaches to solving this problem is treatment with bentonite. This certainly achieves the required stability but at the same time there are a number of serious dis-advantages. Thus, the strong adsorptive effect also elimina~es a number of valuable substances such as aromatic substances, bouquet and/or dyestuffs, and moreover some undesirable metal ions may be introduced, and thirdly considerable quantities of sediment are produced which are ~aborious to process It has there~ore already been proposed that the proteins which cause cloudiness under the e~fect o~ heat or cold be decom-posed by means of proteases (vide Z~ fur Lebensmitteluntersuchung und forschung 134 (1967~, pages 87-97).
However, there are disadvantages with this method too.
First, the costs of enzyme enrichment and isolation are extremel~
high since the enzymes are unstable and cannot be recovered either.
Moreover, it is inadvisable or even illegal according to the laws on foodstuffs to have any soluble enzymes remaining in the drink.
One way of solving these problems is to make the enzymes used insoluble by fixing them on carrier substances which are in-soluble in water. This enables the enzymes to be recovered and ~' 5~
re-used several times.
A number of methods of fixing enzymes are already known.
A synopsis of the latest results in this field is provided, for example, by J.C. Johrson (Editor) in "Immobilized Enzymes, Prepara-tion and Engineering", Noyes Data Corp., Park Ridge, N~J., USA
(1979).
The fixing of the enzymes on carriers results in other advantages in addition to those mentioned above. These include, for example-a continuous method of operation increased stability a smaller reaction volume no contamination of the reaction mixtures no heat precipita-tion of the enzyme.
There is no standard process for bonding an enzyme to an insoluble carrier substance. In order to reach the objective of obtaining maximum biological acti~ity of the enzyme, the carrier substance and method of immobilization must be individually matched to the enzyme~
~0 Particularly preferred carrier substances for this purpose (on the grounds that they are easy ~o process and inexpen-sive) are gel-forming proteins such as gelatine or fresh egg white (see for example German Auslegeschrift 22 46 002). However, these carrier substances are not suitable for fixing enzymes with a proteolytic activity since they are hydrolysed by them.
It has therefore already been proposed ~see German ~uslegeschrift 26 36 206.1-411 that the gel-~orming proteins be denatured by chemical hardening (e.g. with formaldehyde, glutardi-aldehyde or diisocyanate) in order to make them safe from attack by the proteases fixed on them. In this way, rennin or even pepsin, for example, may be fixed on a carrier (vide Biotechnology Letters 1 (1979) pages 225-230).
Surprisingly, it has now been found that proteases can also be fixed to gel-forming proteins even if the proteins are not subjected to a complex preliminary chemical treatment.
The invention therefore relates to a process for immobil-izing proteases on gel~forming proteins which is characterised in that the protease is mixed with gel-forming protein (e.g. gelatine, egg white, soya protein and the like, preferably albumin), the mixture is treated with a water-miscible organic solvent (pre cipitating agent) and subsequently the resulting particle suspension is reacted with difunctional and/or polyfunctional cross-linking agents, The invention ~urther relates to the enzyme preparations fixed on a carrier ~hich are obtained in this way and their use in decomposing proteins.
The mixture of proteolytic enzyme and gel-forming protein may be prepared by dissolving or suspending the enzyme in an aqueous solution o~ the gel-forming protein~ The temperature is preferably adjusted so that the active mixture is obtained in liquid ~orm.
The mixture of proteolytic enzyme and gel-forming protein is preferably prepared by first dissolving the proteolytic enzyme (e~g. pepsin, papain, etc) in water and then mixing it with an aqueous solution o~ the gel~forming protein, This mixture is then ; combined with an organic solvent which has ~n enzyme-precipitating ~Zi3Z5~3~
activity and is compatible with the enzyme~ Examples o~ such a sol~ent include acetone, ethanol, isopropanol and butanol~ The particle suspension formed by the precipitating agent is allowed to react with difunctional or polyfunctional cross-linking agents in such a manner and for such a time that the mixture of proteolytic enzyme and gel-forming protein i5 cross-linked as comple-tely as possible. The particulate mixture may also be separated from the organic liquid beforehand and on~y then treated with the cross-linking agent~
For the cross-linking reactisn, difunctional and poly-functional cross-linking agents for proteins may be used. Prefer-ably, an aqueous solution of glutardialdehyde is used. It is also possible to dissolve the coupling reagent in one of the enzyme-precipitating liquids mentioned above and add it in this form to the aqueous mixture of proteolytic enzyme and gel-forming protein.
This reaction mixture consisting of proteolytic enzyme, gel-forming protein, enzyme-precipitating liquid and cross-linking agent is ~igorously stirred for some time, preferably fxom 0.5 to 4 hours. The coupling reaction is usually carried out at ambient ~0 ~emperature.
Ater the bonding reaction has ended~ the preparation is ~ashed several times, pre~erably wi,th water. The product thus obtained can then be dried to give the required water content (e.gO
by spray drying or drying with acetone).
The enzyme product~s prepared by this procedure may be used in batch processes for the proteolysis o~ protein (protein degradation), with the enzyme product being separated off and re-i~DZ5~3~
used, or in continuous processes in an enzyme reactor. They areparticularly suitable for treating protein-containing beverages (such as wine, beer and fruit juices) where the end product should not contain any residual enzymes The proteolytic enzyme used is preferably pepsin. The pepsin activity of both the soluble form and the immobilised form is determined by the Anson method (Rick,W & Fritsch, W.-P. in Bergmeyer (Editor): Methoden der enzymatischen Analyse, 3rd edition, volume 1, pages 1086-1090, Verlag Chemie, Weinheim, 1974).
The following Examples serve to illustrate the invention:
Example 1
2 kg of pepsin were dissolved in 10 1 of distilled water at ambient temperature. This enzyme solution was mixed with an aqueous albumin solution (200 g of albumin in 5 1 of distilled water). The resulting suspension was added to 24 1 o~ isopropanol (80%) with stirring, at a temperature of 25C, and then 1 litre of glutardialdehyde ~25%) was added thereto. This reaction mix-ture was stirred at 25C for 2 hours~ At the end of -this time, the reaction product was separated by centrifuging and thoxoughly ~0 washed with 200 1 of water. The washed product was dried using a laboratory spray drier with a 2-component nozzle with an air inlet temperature of 155C, an air exit temperature of 75C and an air throughput of 420 Nm3/h. The pepsin used (20 kg) had a total start-ing activity of 6000 AE ~Anson units). The dry preparation ixed on a carrier amounted to 415 g and had a total activity of 3800 AE, corresponding to a recovery of 63.3% after immobilization and spray dxying.
~zs~
Example 2 20 g of pepsin (5 AE/g) obtained from pigs' stomachs were dissolved with 2 g of egg albumin in 150 ml of distilled water.
200 ml of ice-cold acetone were stirred into this solution. After some minutes, lO ml of a 25% glutardialdehyde solution were added to this suspension and th~ resulting mixture was stirred or shaken at ambient temperature. After a reaction period of about 2 hours, the reaction product was filtered off and thoroughly washed with distilled water. The flltrate was suspended in ice-cold acetone.
The immobilised enzyme product was separated from the acetone by ; filtration and then the filtrate was dried at 45C in a vacuum-type drying cupboard. 2.5 g of dry preparation fixed on a carrier were obtained, with an activity of ll AE/g,
~zs~
Example 2 20 g of pepsin (5 AE/g) obtained from pigs' stomachs were dissolved with 2 g of egg albumin in 150 ml of distilled water.
200 ml of ice-cold acetone were stirred into this solution. After some minutes, lO ml of a 25% glutardialdehyde solution were added to this suspension and th~ resulting mixture was stirred or shaken at ambient temperature. After a reaction period of about 2 hours, the reaction product was filtered off and thoroughly washed with distilled water. The flltrate was suspended in ice-cold acetone.
The immobilised enzyme product was separated from the acetone by ; filtration and then the filtrate was dried at 45C in a vacuum-type drying cupboard. 2.5 g of dry preparation fixed on a carrier were obtained, with an activity of ll AE/g,
Claims (11)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. Process for the preparation of proteases fixed on a carrier of gel-forming proteins, characterised in that the proteases are mixed with a gel-forming protein, the mixture is treated with an organic solvent having an enzyme-precipitating activity and subsequently the particle suspension formed is reacted with one or more cross-linking agents.
2. Process as claimed in claim 1, characterised in that albumin is used as the gel-forming protein.
3. Process as claimed in one of claims 1 or 2, characterised in that the protease is pepsin.
4. Process as claimed in claim 1, characterised in that glutardialdehyde is used as a cross-linking agent.
5. Process as claimed in claim 1,characterised in that the protease is pepsin, the gel-forming protein is albumin, and the cross-linking agent is glutardialdehyde.
6. Protease preparation fixed on a carrier, characterised in that the protease is covalently bonded, by means of a cross-linking agent, to a non-hardened gel-forming protein.
7. Protease preparation as claimed in claim 6, characterised in that pepsin is covalently bonded by means of glutardialdehyde to a gel-forming protein.
8. Protease preparation as claimed in claim 7, characterised in that the gel-forming protein is albumin.
9. A method of removing residual enzymes from protein-containing fluids which comprises treating the fluid with a protease fixed on a carrier according to claim 6.
10. Method according to claim 9 wherein the fluid is a beverage.
11. Method according to claim 10 wherein the fluid is wine or beer.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19813145684 DE3145684A1 (en) | 1981-11-19 | 1981-11-19 | CARRIER-FIXED PROTEASES AND THEIR APPLICATION IN BIOTECHNOLOGY |
| DEP3145684.7 | 1981-11-19 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1202584A true CA1202584A (en) | 1986-04-01 |
Family
ID=6146625
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000415462A Expired CA1202584A (en) | 1981-11-19 | 1982-11-12 | Proteases fixed on a carrier and their use in biotechnology |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0080077B1 (en) |
| AT (1) | ATE19652T1 (en) |
| AU (1) | AU9075682A (en) |
| CA (1) | CA1202584A (en) |
| DE (2) | DE3145684A1 (en) |
| ZA (1) | ZA828483B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0659217B2 (en) * | 1985-08-06 | 1994-08-10 | 三井東圧化学株式会社 | Method for producing immobilized enzyme and device for use in the method |
| DE3636735C2 (en) * | 1986-10-29 | 1995-07-06 | Akzo Gmbh | Biochemically active matrix and process for its production |
| EP1088887A1 (en) * | 1999-09-23 | 2001-04-04 | Dsm N.V. | Crosslinked enzyme aggregates |
| FR2810336B1 (en) * | 2000-06-19 | 2003-01-31 | Prod Oenologiques J Laffort & | PROCESS FOR THE MANUFACTURE OF AN ADJUSTED CLARIFICATION PRODUCT FROM A WINE AND CLARIFICATION PRODUCT OBTAINED |
| US20030165592A1 (en) * | 2002-02-28 | 2003-09-04 | Daqing Sun | Effective use of protease in winemaking |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CH538003A (en) * | 1968-03-29 | 1973-01-31 | Anvar | Process for obtaining textile articles carrying enzymes |
| US3597219A (en) * | 1968-09-27 | 1971-08-03 | Monsanto Co | Chillproofing of beverages using insoluble polymer-enzyme product |
-
1981
- 1981-11-19 DE DE19813145684 patent/DE3145684A1/en not_active Withdrawn
-
1982
- 1982-10-30 AT AT82110043T patent/ATE19652T1/en not_active IP Right Cessation
- 1982-10-30 DE DE8282110043T patent/DE3271040D1/en not_active Expired
- 1982-10-30 EP EP82110043A patent/EP0080077B1/en not_active Expired
- 1982-11-12 CA CA000415462A patent/CA1202584A/en not_active Expired
- 1982-11-18 ZA ZA828483A patent/ZA828483B/en unknown
- 1982-11-19 AU AU90756/82A patent/AU9075682A/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| DE3271040D1 (en) | 1986-06-12 |
| EP0080077A3 (en) | 1983-11-16 |
| AU9075682A (en) | 1983-05-26 |
| EP0080077B1 (en) | 1986-05-07 |
| DE3145684A1 (en) | 1983-05-26 |
| EP0080077A2 (en) | 1983-06-01 |
| ATE19652T1 (en) | 1986-05-15 |
| ZA828483B (en) | 1984-07-25 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKEX | Expiry |