WO1990013638A1 - Method of inactivating lipase - Google Patents

Method of inactivating lipase Download PDF

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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
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WIPO (PCT)
Prior art keywords
protease
inactivation
phospholipase
carried out
range
Prior art date
Application number
PCT/DK1990/000093
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French (fr)
Inventor
Kim Brint Pedersen
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Novo Nordisk A/S
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Filing date
Publication date
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Publication of WO1990013638A1 publication Critical patent/WO1990013638A1/en

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    • 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
Figure imgf000007_0001
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.
PCT/DK1990/000093 1989-04-28 1990-04-11 Method of inactivating lipase WO1990013638A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DK2064/89 1989-04-28
DK206489A DK206489D0 (en) 1989-04-28 1989-04-28 METHOD OF INACTIVATING PHOSPHOLIPASE

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WO1990013638A1 true WO1990013638A1 (en) 1990-11-15

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Cited By (4)

* Cited by examiner, † Cited by third party
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

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
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)

* Cited by examiner, † Cited by third party
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

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DK206489D0 (en) 1989-04-28

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