WO1990013638A1 - Method of inactivating lipase - Google Patents
Method of inactivating lipase Download PDFInfo
- Publication number
- WO1990013638A1 WO1990013638A1 PCT/DK1990/000093 DK9000093W WO9013638A1 WO 1990013638 A1 WO1990013638 A1 WO 1990013638A1 DK 9000093 W DK9000093 W DK 9000093W WO 9013638 A1 WO9013638 A1 WO 9013638A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- protease
- inactivation
- phospholipase
- carried out
- range
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims description 21
- 230000000415 inactivating effect Effects 0.000 title claims description 6
- 102000004882 Lipase Human genes 0.000 title claims description 3
- 108090001060 Lipase Proteins 0.000 title claims description 3
- 239000004367 Lipase Substances 0.000 title claims description 3
- 235000019421 lipase Nutrition 0.000 title claims description 3
- 239000004365 Protease Substances 0.000 claims abstract description 45
- 108091005804 Peptidases Proteins 0.000 claims abstract description 44
- 230000002779 inactivation Effects 0.000 claims abstract description 27
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims abstract 9
- ZIIUUSVHCHPIQD-UHFFFAOYSA-N 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide Chemical compound CC1=CC(C)=CC(C)=C1S(=O)(=O)NC1=CC=CC(C(F)(F)F)=C1 ZIIUUSVHCHPIQD-UHFFFAOYSA-N 0.000 claims description 27
- 102000015439 Phospholipases Human genes 0.000 claims description 27
- 108010064785 Phospholipases Proteins 0.000 claims description 27
- 230000000694 effects Effects 0.000 claims description 12
- 150000003904 phospholipids Chemical class 0.000 claims description 8
- 238000010438 heat treatment Methods 0.000 claims description 2
- 239000000463 material Substances 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims 2
- -1 phospho Chemical class 0.000 claims 1
- 230000029087 digestion Effects 0.000 abstract description 3
- 102100037611 Lysophospholipase Human genes 0.000 abstract 1
- 108010058864 Phospholipases A2 Proteins 0.000 abstract 1
- 102000035195 Peptidases Human genes 0.000 description 35
- 235000019419 proteases Nutrition 0.000 description 25
- 108010056079 Subtilisins Proteins 0.000 description 8
- 102000005158 Subtilisins Human genes 0.000 description 8
- 108090000631 Trypsin Proteins 0.000 description 6
- 102000004142 Trypsin Human genes 0.000 description 6
- 239000012588 trypsin Substances 0.000 description 6
- 241000283690 Bos taurus Species 0.000 description 5
- 230000000813 microbial effect Effects 0.000 description 5
- 108010056587 rennilase Proteins 0.000 description 5
- 108010009355 microbial metalloproteinases Proteins 0.000 description 4
- 108090000145 Bacillolysin Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 108091005507 Neutral proteases Proteins 0.000 description 3
- 102000035092 Neutral proteases Human genes 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 229940108461 rennet Drugs 0.000 description 3
- 108010058314 rennet Proteins 0.000 description 3
- 241000194108 Bacillus licheniformis Species 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000235403 Rhizomucor miehei Species 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 230000003301 hydrolyzing effect Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 210000000496 pancreas Anatomy 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- DVLFYONBTKHTER-UHFFFAOYSA-N 3-(N-morpholino)propanesulfonic acid Chemical compound OS(=O)(=O)CCCN1CCOCC1 DVLFYONBTKHTER-UHFFFAOYSA-N 0.000 description 1
- 241000607534 Aeromonas Species 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000193744 Bacillus amyloliquefaciens Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 108091005658 Basic proteases Proteins 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 108090000317 Chymotrypsin Proteins 0.000 description 1
- 108010067372 Pancreatic elastase Proteins 0.000 description 1
- 102000016387 Pancreatic elastase Human genes 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- 241000228150 Penicillium chrysogenum Species 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 241000183024 Populus tremula Species 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 108700034501 Staphylococcus aureus auR Proteins 0.000 description 1
- 101710119637 Trypsin-7 Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 229960002376 chymotrypsin Drugs 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 230000003861 general physiology Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 239000003998 snake venom Substances 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
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
- C12N9/99—Enzyme inactivation by chemical treatment
-
- 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)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
Definitions
- This invention relates to a method of inactivating phospholipase and to a method of hydrolyzing phospholipid.
- Enzyme products containing phospholipase A 2 (E.C. 3.1.1.4) of porcine pancreatic origin are used industrially for hydrolysis of phospholipids for improvement of the emulsifying power.
- Many users of phospholipase A 2 face a problem of inactivation of the phospholipase after hydrolysis because the enzyme is very stable. For instance, the enzyme resists boiling for 5 minutes at pH 4.0 (G.H. de Haas et al.: Biochim. Biophys. Acta (159), 103-117 (1968)).
- inactivation at high temperatures can lead to degradation of the phospholipids.
- JP-A 63-233,750 discloses inactivation by digestion of the phospholipase by protease, followed by heat inactivation of the protease. Inactivation of the protease was carried out at 90°C or higher for 30 minutes or more, and it was stated that protease could be inactivated at 80-90°C for 5-30 minutes. However, these temperatures may still be too high to avoid degradation of the phospholipid. It is the object of the invention to provide a method of inactivating phospholipase, avoiding such high temperatures.
- the invention provides a method of inactivating phospholipase comprising inactivation of phospholipase with protease below 75 ⁇ C followed by inactiva ⁇ tion of protease below 75°C.
- the invention also provides a method of hydrolyzing phospholipid, comprising treatment of material containing phospholipid with phospholipase followed by inactivation of the phospholipase by said method.
- the phospholipase used in the invention may be phospholipase A 2 , e.g. from pancreas (e.g. porcine) or snake venom. These are known to have a high degree of homology (M.J.Spron et al.: Eur. J. Biochem. (137), 537-544 (1983)), and can therefore be inactivated in essentially the same way.
- the protease used in the invention should have a substrate specificity that makes it possible to inactivate the phospholipase, and its stability characteristics should be such that it can be largely inactivated at the conditions used. Many suitable proteases are known, of animal, plant and microbial origin. Some examples follow: Animal proteases: Trypsin (e.g. from porcine or bovine pancreas) , chymotrypsin, elastase and pepsin. Plant proteases: Papain.
- Microbial proteases alkaline Bacillus proteases (e.g. subtilisin Carlsberg from B. licheniformis. subtilisin Novo or subtilisin BPN'), neutral protease from Bacillus amylo- liquefaciens (also termed B. subtilis) , alkaline Asper illus proteases, Streptomvces ⁇ riseus protease, Aeromonas s . neutral protease, Penicillium notatum protease, Staphylococcal protease, acid microbial proteases, and microbial rennet from Mucor miehei.
- Bacillus proteases e.g. subtilisin Carlsberg from B. licheniformis. subtilisin Novo or subtilisin BPN'
- neutral protease from Bacillus amylo- liquefaciens also termed B. subtilis
- alkaline Asper illus proteases Streptomvces
- the temperature throughout the process of the invention is below 75 ⁇ C, preferably below 60°C, and most preferably below 55°C.
- the process of the invention comprises two sequential steps: (1) inactivation of phospholipase with protease and (2) inactivation of protease.
- the transition from step (1) to (2) may involve heating and/or changing the pH (especially lowering the pH) . It may be particularly convenient to heat at essentially unchanged pH, or to change the pH (e.g. lower the pH) at essentially unchanged temperature.
- the amount of protease is preferably sufficient to reduce the phospholipase activity to below 10%, most preferably below 1% of the activity before inactivation.
- the phospholipase inactivation is typically carried out at pH 4-9 (especially 6-8), 20-55°C for 10-120 minutes.
- the conditions for the subsequent inactivation of protease may be chosen according to the stability characteristics of the protease in question, but will generally be within the ranges pH 4-8, 20-75°C (especially 40-55 ⁇ C) for 10-120 minutes, and the remaining protease activity will usually be below 10%, e.g. below 2% and espe ⁇ cially below 1% of the activity originally present. Examples of suitable conditions for essentially complete inactivation (to less than 0.1%) of some proteases follow:
- Alcalase ® 2.4 L alkaline protease from B. licheniformis
- Neutrase ® 1.5 MG neutral protease from B ⁇ . amyloliquefaciens
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
Phospholipase A2 can be inactivated by digestion with protease followed by inactivation of the protease, using temperatures in both steps below 75°C.
Description
METHOD OF INACTIVATING LIPASE
TECHNICAL FIELD
This invention relates to a method of inactivating phospholipase and to a method of hydrolyzing phospholipid.
BACKGROUND OF THE INVENTION
Enzyme products containing phospholipase A2 (E.C. 3.1.1.4) of porcine pancreatic origin are used industrially for hydrolysis of phospholipids for improvement of the emulsifying power. Many users of phospholipase A2 face a problem of inactivation of the phospholipase after hydrolysis because the enzyme is very stable. For instance, the enzyme resists boiling for 5 minutes at pH 4.0 (G.H. de Haas et al.: Biochim. Biophys. Acta (159), 103-117 (1968)). However, inactivation at high temperatures can lead to degradation of the phospholipids.
In an attempt to avoid the use of excessive temperatures for inactivation, JP-A 63-233,750 (Q.P. Corporation) discloses inactivation by digestion of the phospholipase by protease, followed by heat inactivation of the protease. Inactivation of the protease was carried out at 90°C or higher for 30 minutes or more, and it was stated that protease could be inactivated at 80-90°C for 5-30 minutes. However, these temperatures may still be too high to avoid degradation of the phospholipid. It is the object of the invention to provide a method of inactivating phospholipase, avoiding such high temperatures.
STATEMENT OF THE INVENTION
Surprisingly, we have found that phospholipase can be inactivated by digestion with protease followed by inactivation of the protease, using temperatures in both steps below 75°C.
Accordingly, the invention provides a method of inactivating phospholipase comprising inactivation of phospholipase with protease below 75βC followed by inactiva¬ tion of protease below 75°C. The invention also provides a method of hydrolyzing phospholipid, comprising treatment of material containing phospholipid with phospholipase followed by inactivation of the phospholipase by said method.
DETAILED DESCRIPTION OF THE INVENTION
The phospholipase used in the invention may be phospholipase A2, e.g. from pancreas (e.g. porcine) or snake venom. These are known to have a high degree of homology (M.J. Dufton et al.: Eur. J. Biochem. (137), 537-544 (1983)), and can therefore be inactivated in essentially the same way. The protease used in the invention should have a substrate specificity that makes it possible to inactivate the phospholipase, and its stability characteristics should be such that it can be largely inactivated at the conditions used. Many suitable proteases are known, of animal, plant and microbial origin. Some examples follow: Animal proteases: Trypsin (e.g. from porcine or bovine pancreas) , chymotrypsin, elastase and pepsin. Plant proteases: Papain.
Microbial proteases: alkaline Bacillus proteases (e.g. subtilisin Carlsberg from B. licheniformis. subtilisin Novo or subtilisin BPN'), neutral protease from Bacillus amylo- liquefaciens (also termed B. subtilis) , alkaline Asper illus proteases, Streptomvces σriseus protease, Aeromonas s . neutral protease, Penicillium notatum protease, Staphylococcal protease, acid microbial proteases, and microbial rennet from Mucor miehei.
The temperature throughout the process of the invention is below 75βC, preferably below 60°C, and most preferably below 55°C.
The process of the invention comprises two sequential steps: (1) inactivation of phospholipase with
protease and (2) inactivation of protease. The transition from step (1) to (2) may involve heating and/or changing the pH (especially lowering the pH) . It may be particularly convenient to heat at essentially unchanged pH, or to change the pH (e.g. lower the pH) at essentially unchanged temperature.
The amount of protease is preferably sufficient to reduce the phospholipase activity to below 10%, most preferably below 1% of the activity before inactivation. A suitable amount will generally correspond to a protease activity of 0.001 to 0.2 AU/g in the mixture (AU = Anson unit, Journal of General Physiology, 22, 79-89 (1959)).
The phospholipase inactivation is typically carried out at pH 4-9 (especially 6-8), 20-55°C for 10-120 minutes. The conditions for the subsequent inactivation of protease may be chosen according to the stability characteristics of the protease in question, but will generally be within the ranges pH 4-8, 20-75°C (especially 40-55βC) for 10-120 minutes, and the remaining protease activity will usually be below 10%, e.g. below 2% and espe¬ cially below 1% of the activity originally present. Examples of suitable conditions for essentially complete inactivation (to less than 0.1%) of some proteases follow:
Protease PH Temperature Time Subtilisin Carlsberg 7 75βC 60 min. do. 5 70βC 30 min. do. 4 50°C 30 min.
Neutral
B.amyloliquef ciens 7 65°C 90 min. do. 7 70°C 20 min. do. 7 75βC 10 min. do. 4 50°C 30 min.
Porcine trypsin 7 75°C 180 min
EXAMPLE
Five commercial proteases from Novo Industri A/S were tried: Alcalase® 2.4 L (alkaline protease from B. licheniformis) . Neutrase® 1.5 MG (neutral protease from B^. amyloliquefaciens) Pancreatic Trypsin Novo (PTN, porcine) 6.0 S salt-free. Crystalline Bovine Trypsin 3000K, and Rennilase® 500 MG-TL (Ther olabile, modified microbial rennet from M. miehei) . Each protease was added in the amount indicated below (AU/ml or KRU/ml) to a solution of Lecitase® (trademark of Novo Nordisk A/S, porcine pancreatic phospholipase A2) at a concentration of 110 IU/ml (IU = International Unit, measured according to G.H. de Haas et al: Biochem. Biohphys. Acta (159), 103-117 (1968)) in a buffer containing 50 mM sodium acetate, 50 mM 3-(N-morpholino)propanesulfonic acid, and 10 mM CaCl2. Inactivation of phospholipase and protease, respectively, was carried out through two incubations of 2 hours each, at the conditions indicated below. The table below indicates the initial activities of phospholipase and protease the final activities measured after the treatment. It is seen from the table that Rennilase® inactivates the phospholipase to below 10%, and the four other proteases to below 1%.
It is also seen that of the PTN 90% has become inactivated, and of the Alcalase®, Neutrase®, Rennilase® and bovine trypsin more than 90% has become inactivated.
Incubation conditions Phospholipase activity (IU/ml) Protease activity*
Protease First Second 2 hours 2 hours Initial Final Initial Final
Rennilase® 500 MG-TL pH 6, 40°C pH 8, 55°C 110 8 5 < 0.03
10 PTN 6.OS Salt-free pH 8, 40°C pH 8, 55°C 110 < 1 0.062 0.0062
Crystalline Bovine Trypsin 3000 K pH 7, 30°C pH 8, 55°C 110 < 1 0.204 0.0019
15 Alcalase® 2.4 L pH 8, 50°C pH 4, 50°C 110 < 1 0.024 < 0.001
Neutrase® 1.5 MG pH 6, 40°C pH 8, 55°C 110 < 1 0.015 < 0.001
20 Protease activities of PTN, bovine trypsin, Alcalase®, and Neutrase® were measured in AU (Anson Units) per ml, and activity of Rennilase® was measured in KRU (Kilo Rennet Units) per ml. KRU is measured by NOVO Analysis Method AF 67/3-e, available on request.
Claims
1. A method of inactivating phospholipase, characterized by comprising inactivation of phospholipase with protease below 75°C, followed by inactivation of the protease below 75βC.
2. The method of Claim 1, wherein said steps are both carried out below 60°C, preferably below 55βC.
3. The method of Claim 1 or 2, wherein the phospholipase activity remaining after the inactivation is less than 10%, preferably less than 1%.
4. The method of any of Claims 1 - 3, wherein the inactivation of phospholipase is carried out at a pH in the range 4-9 (preferably 6-8) , a temperature in the range 20- 55°C and a reaction time in the range 10 - 120 minutes.
5. The method of any of Claims 1 - 4, wherein the protease activity remaining after inactivation is below 10%, preferably below 2%, and most preferably below 1%.
6. The method of any of Claims 1 - 5, wherein the inactivation of protease is carried out by heating, essentially without adjusting the pH.
7. The method of any of Claims 1 - 5, wherein the inactivation of protease is carried out by changing the pH, essentially without changing the temperature.
8. The method of any of Claims 1 - 5, wherein the inactivation of protease is carried out by changing the pH, and increasing the temperature.
9. The method of any of Claims 1 - 8, wherein the inactivation of protease is carried out at a temperature in the range 20-75°C (preferably 40-55βC) , a pH in the range 4- 8 and a reaction time in the range 10 - 120 minutes.
510. A method of preparing hydrolyzed phospholipid by treatment of material containing phospholipid with phospho¬ lipase, characterized by comprising subsequent inactivation of the phospholipase by the method of any of Claims 1 - 9.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DK206489A DK206489D0 (en) | 1989-04-28 | 1989-04-28 | METHOD OF INACTIVATING PHOSPHOLIPASE |
| DK2064/89 | 1989-04-28 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1990013638A1 true WO1990013638A1 (en) | 1990-11-15 |
Family
ID=8109819
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/DK1990/000093 WO1990013638A1 (en) | 1989-04-28 | 1990-04-11 | Method of inactivating lipase |
Country Status (3)
| Country | Link |
|---|---|
| AU (1) | AU5632590A (en) |
| DK (1) | DK206489D0 (en) |
| WO (1) | WO1990013638A1 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0567662A1 (en) * | 1992-04-25 | 1993-11-03 | Societe Des Produits Nestle S.A. | Process for aromatizing of milk chocolate |
| WO1997020921A1 (en) * | 1995-12-07 | 1997-06-12 | Novo Nordisk A/S | Selective inactivation of enzyme activities |
| EP0899331A1 (en) * | 1997-08-22 | 1999-03-03 | Societe Des Produits Nestle S.A. | Purified proteolytic enzyme and procedure for purification |
| EP3113625A1 (en) * | 2014-02-21 | 2017-01-11 | Clariant Produkte (Deutschland) GmbH | Composition for the enzymatic degumming of oil |
-
1989
- 1989-04-28 DK DK206489A patent/DK206489D0/en not_active Application Discontinuation
-
1990
- 1990-04-11 WO PCT/DK1990/000093 patent/WO1990013638A1/en unknown
- 1990-04-11 AU AU56325/90A patent/AU5632590A/en not_active Abandoned
Non-Patent Citations (2)
| Title |
|---|
| CHEMICAL ABSTRACTS, Volume 103, No. 5, 5 August 1985, (Columbus, Ohio, USA); CHRISTEN, G.L. et al.: "Effect of histidine on thermostability of lipase and protease of Pseudomonas fluorescens 27", see page 326, abstract 3532f & J. DIARY SCI. 1985, 68(3), 594-604. * |
| DIALOG INFORMATION SERVICES, File 351, World Patent Index 81-90, Dialog accession no. 88-318043/45, QP Corp: "Treated phospholipid prod. prepn. - by treating phospholipid with phospholipase, treating with protease and inactivating by heating"; & JP,A,63 233 750, 29-09-1988, 8845 (Basic). * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0567662A1 (en) * | 1992-04-25 | 1993-11-03 | Societe Des Produits Nestle S.A. | Process for aromatizing of milk chocolate |
| US5393538A (en) * | 1992-04-25 | 1995-02-28 | Nestec S.A. | Preparation of crumb-flavored milk chocolate |
| WO1997020921A1 (en) * | 1995-12-07 | 1997-06-12 | Novo Nordisk A/S | Selective inactivation of enzyme activities |
| US6080564A (en) * | 1995-12-07 | 2000-06-27 | Novo Nordisk A/S | Selective inactivation of Aspergillus proteases |
| EP0899331A1 (en) * | 1997-08-22 | 1999-03-03 | Societe Des Produits Nestle S.A. | Purified proteolytic enzyme and procedure for purification |
| US6420156B2 (en) | 1997-08-22 | 2002-07-16 | Nestec S.A. | Purified proteolytic enzyme and method of purification |
| EP3113625A1 (en) * | 2014-02-21 | 2017-01-11 | Clariant Produkte (Deutschland) GmbH | Composition for the enzymatic degumming of oil |
Also Published As
| Publication number | Publication date |
|---|---|
| DK206489D0 (en) | 1989-04-28 |
| AU5632590A (en) | 1990-11-29 |
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