WO1993010224A1 - A cholesterol esterase acting lipase from pseudomonas cepacia - Google Patents
A cholesterol esterase acting lipase from pseudomonas cepacia Download PDFInfo
- Publication number
- WO1993010224A1 WO1993010224A1 PCT/DK1992/000324 DK9200324W WO9310224A1 WO 1993010224 A1 WO1993010224 A1 WO 1993010224A1 DK 9200324 W DK9200324 W DK 9200324W WO 9310224 A1 WO9310224 A1 WO 9310224A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cholesterol esterase
- lipase
- cholesterol
- ala
- cepacia
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L15/00—Egg products; Preparation or treatment thereof
- A23L15/25—Addition or treatment with microorganisms or enzymes
-
- 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
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01013—Sterol esterase (3.1.1.13)
-
- D—TEXTILES; PAPER
- D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
- D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
- D21C5/00—Other processes for obtaining cellulose, e.g. cooking cotton linters ; Processes characterised by the choice of cellulose-containing starting materials
- D21C5/005—Treatment of cellulose-containing material with microorganisms or enzymes
Definitions
- the present invention relates to novel cholesterol esterases. More specifically, the invention relates to novel cholesterol esterases derived from strains of Pseudomonas cepacia. In a more specific embodiment, the invention relates to a cholesterol esterase acting lipase.
- maltophilia (3.4% of the studied cultures) had cholesterol esterase activity. Lipolytic activity was found in P. aureofaciens (in 100% of the studied strains), P. cepacia (in 75%), P. maltophilia (in 60%), P. fluorescens (in 13.3%), and P. aeruginosa (in 60%).
- At least one strain of Pseudomonas cepacia is able to produce extracellular cholesterol esterase.
- the component responsible for the cholesterol esterase activity is also the component responsible for the lipase activity.
- the invention provides cholesterol esterases obtainable from a strain of Pseudomonas cepacia.
- the invention provides a process for the preparation of cholesterol esterases of the invention, which process comprises cultivation of a cholesterol esterase producing strain of Pseudomonas cepacia in a suitable nutrient medium, containing carbon and nitrogen sources and inorganic salts, followed by recovery of the desired enzyme.
- the invention relates to the use of a lipase obtainable from the strain P. cepacia DSM 3401 as a cholesterol esterase.
- the present invention provides a cholesterol esterase obtainable from a strain of Pseudomonas cepacia. Moreover, it has surprisingly been found that in this case the enzyme responsible for the cholesterol esterase activity is identical to the enzyme responsible for the lipase activity, i.e. the very same protein possesses lipase activity as well as cholesterol esterase activity. Accordingly, the invention provides an enzyme having cholesterol esterase activity as well as lipase activity, i.e. being a cholesterol esterase acting lipase or vice versa.
- the invention provides novel cholesterol esterases obtainable from a strain of P. cepacia, preferably the strain P. cepacia DSM 3401 , or a mutant or a variant thereof.
- the cholesterol esterase of the invention can be further described by the following physical-chemical characteristics.
- the enzyme possesses cholesterol esterase activity at pH values of from below pH 4 to above pH 10, determined at 37°C with cholesterol oleate as substrate.
- the enzyme has a pH optimum in the range of from pH 5 to pH 7, more specifically around pH 6.
- the enzyme has a molecular weight of approximately 30 kD, as determined by SDS-PAGE, and a pi of approximately 4.0. Maximum activity, among the substrates tested, on cholesterolpalmitate.
- the cholesterol esterase of the invention has the amino acid sequence identified by the sequence listing attached to this specification.
- the cholesterol esterase of the invention can be obtained by cultivation of a cholesterol esterase producing strain of P. cepacia, preferably the strain DSM 3401 , in a suitable nutrient medium, containing carbon and nitrogen sources and inorganic salts, followed by recovery of the desired enzyme.
- a highly purified lipase preparation designated Pseudomonas cepacia 751 OA lipase, was obtained from the strain DSM 3401 by the methods described in Examples 1-2 of this specification.
- amino acid degradation the amino acid sequence of the enzyme was found to be the one identified in the sequence listing attached to this specification. By the method described in Example 3 of this specification it was demonstrated that this enzyme also possesses cholesterol esterase activity.
- the cholesterol esterase of the invention may also be obtained by recombinant DNA-technology. By expression of only one protein it is possible to achieve two enzymatic activities, lipase activity and cholesterol esterase activity.
- the invention also relates to the use of a cholesterol esterase acting lipase for hydrolysis of eggs,
- the invention relates to use of a cholesterol esterase of the invention in processes for hydrolysis of resin in pulp.
- Mechanical pulping alone or combined with a gentle chemical treatment, is widely used in the manufacture of pulps. These processes occur at pH in the range 4-9, and the com- ponents of the wood undergo relatively small chemical changes.
- the pulp therefore has a considerable content of triglycerides, esters, and waxes from resin.
- Residual resin may cause problems during the subsequent use of the pulp.
- agglomerated resin may cause paper breakage during paper manufacture or during printing as well as lowering of the paper quality. It is known that the hydrophobic part of resin contains considerable amounts of triglycerides and other esters. It is, therefore, desirable to hydrolyze these, as the hydrolysis products are more easily removed in aqueous systems.
- a culture of Pseudomonas cepacia, DSM 3401 , on an agar slant was transferred to five 500 ml shaking flasks, each with 100 ml of Bullion-3 medium, and shaken at 30°C for 1 day (200 rpm, amplitude 2,5 cm).
- composition of Bullion-3 medium was as follows:
- the medium was autoclaved at 121°C for 40 minutes, The culture broth of these Bullion-3 shake flasks was used as a seed culture for inoculating two hundred 500 ml shake flasks, each with 200 ml of PL-1 medium.
- composition of the PL-1 medium was as follows:
- the medium was autoclaved at 121°C for 40 minutes.
- Each PL-1 shake flask was inoculated with 0.5-2 ml of Bullion-3 culture broth, and shaken with 200 rpm (amplitude 2.5 cm) at 30°C for 5 days.
- the culture broth from the shake flasks was pooled at harvest, totalling 39.5 I with an enzyme yield of 53 LU/ml.
- the culture broth was centrifuged for 35 minutes at 4100 xg by means of a Beckman Model J-6 centrifuge.
- the supernatant was concentrated by filtration (washed with approximately 1 volume of water) to 1.4 I by a Pellicon ultrafiltration apparatus from Millipore with a 10.000 MW cut off filter sheet.
- the concentrate was freeze dried, and the yield was 56.2 g of powder with an enzyme activity of 21.500 LU/g.
- One Lipase Unit is the amount of enzyme which, under standard conditions (i.e. at30.0°C; pH 7.0; and tributyrine substrate) liberates 1 ⁇ mol titratable butyric acid per minute.
- a folder AF 95/5 describing this analytical method is available upon request to Novo Nordisk A/S, Denmark.
- the ultrafiltration concentrate obtained according to Example 1 was suspended in water and applied on a hydrophobic XAD-8 resin matrix, and eluted with 60% (v/v) 96% ethanol.
- ammonium acetate was added to a final concentration of 0.5 M.
- the enzyme was applied on a ToyopearlTM-Butyl column and eluted with 0.05 M glycine buffer, pH 9.3.
- Buffer 0.4 M KH 2 PO 4 ; pH 7.0; 37 °C (54.4 mg/ml)
- Substrate 0.0086 M cholesterol oleate (5.6 mg/ml).
- One cholesterol esterase unit is defined as the amount of enzyme that will hydrolyse 1 ⁇ mole of cholesterol oleate per minute at pH 7.0 and 37°C. Enzymes Examined
- AH enzymes were scanned for approximately 600 seconds. The spectrophotometric determinations are presented in Fig. 1. Next the gradient dA/dt, and ultimately the cholesterol esterase activity was calculated using the expression given above. The results are presented in Table 1 , below.
- Example 3 further characterization of the cholesterol esterase acting lipase obtained according to Example 1-2 is presented.
- the assay for cholesterol esterase activity set forth in Example 3 has been accomplished.
- the substrate specificity was examined at pH 7.0, 37°C, with a series of cholesterol esters having hydrocarbon chains from 2 to 18.
- the pH profile was determined at 37°C between pH 4 and pH 10, and with cholesterol oleate as substrate.
- the cholesterol esterase activity was measured as a function of the temperature. Cholesterol oleate was used as substrate, and the measurements were made at pH 7.0.
- ORGANISM Pseudomonas cepacia
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Enzymes And Modification Thereof (AREA)
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DK1867/91 | 1991-11-15 | ||
DK911867A DK186791D0 (da) | 1991-11-15 | 1991-11-15 | Nye enzymer |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1993010224A1 true WO1993010224A1 (en) | 1993-05-27 |
Family
ID=8108650
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/DK1992/000324 WO1993010224A1 (en) | 1991-11-15 | 1992-11-09 | A cholesterol esterase acting lipase from pseudomonas cepacia |
Country Status (3)
Country | Link |
---|---|
AU (1) | AU2942292A (da) |
DK (1) | DK186791D0 (da) |
WO (1) | WO1993010224A1 (da) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1994023052A1 (en) * | 1993-04-02 | 1994-10-13 | Novo Nordisk A/S | A method of hydrolysing cholesterol esters by using a pseudomonas fragi cholesterol esterase |
WO1998028394A1 (en) * | 1996-12-20 | 1998-07-02 | The Procter & Gamble Company | Detergent compositions comprising cholesterol esterase |
WO2002075045A1 (es) * | 2001-03-16 | 2002-09-26 | Consejo Superior De Investigaciones Científicas | Procedimiento para el control enzimático de los depósitos de brea (pitch) formados durante la fabricación de pasta de papel utilizando una esterasa que hidroliza tanto triglicéridos como ésteres de esteroles |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4283494A (en) * | 1978-04-26 | 1981-08-11 | Meito Sangyo Kabushiki Kaisha | Microbial lipase, process for its preparation and microbiologically pure culture therefor |
US4677068A (en) * | 1984-12-24 | 1987-06-30 | Boehringer Mannheim Gmbh | Process for obtaining cholesterol esterase |
WO1991000908A1 (en) * | 1989-07-07 | 1991-01-24 | Novo Nordisk A/S | Dna encoding a lipase and a lipase modulator |
-
1991
- 1991-11-15 DK DK911867A patent/DK186791D0/da not_active Application Discontinuation
-
1992
- 1992-11-09 AU AU29422/92A patent/AU2942292A/en not_active Abandoned
- 1992-11-09 WO PCT/DK1992/000324 patent/WO1993010224A1/en active Application Filing
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4283494A (en) * | 1978-04-26 | 1981-08-11 | Meito Sangyo Kabushiki Kaisha | Microbial lipase, process for its preparation and microbiologically pure culture therefor |
US4677068A (en) * | 1984-12-24 | 1987-06-30 | Boehringer Mannheim Gmbh | Process for obtaining cholesterol esterase |
WO1991000908A1 (en) * | 1989-07-07 | 1991-01-24 | Novo Nordisk A/S | Dna encoding a lipase and a lipase modulator |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1994023052A1 (en) * | 1993-04-02 | 1994-10-13 | Novo Nordisk A/S | A method of hydrolysing cholesterol esters by using a pseudomonas fragi cholesterol esterase |
US6066486A (en) * | 1993-04-02 | 2000-05-23 | Novo Nordisk A/S | Method of hydrolyzing cholesterol esters by using a Pseudomonas fragi cholesterol esterase |
WO1998028394A1 (en) * | 1996-12-20 | 1998-07-02 | The Procter & Gamble Company | Detergent compositions comprising cholesterol esterase |
WO2002075045A1 (es) * | 2001-03-16 | 2002-09-26 | Consejo Superior De Investigaciones Científicas | Procedimiento para el control enzimático de los depósitos de brea (pitch) formados durante la fabricación de pasta de papel utilizando una esterasa que hidroliza tanto triglicéridos como ésteres de esteroles |
ES2179768A1 (es) * | 2001-03-16 | 2003-01-16 | Consejo Superior Investigacion | Procedimiento para el control enzimatico de los depositos de brea (pitch) formados durante la fabricacion de pasta de papel utilizando una esterasa que hidroliza tanto trigliceridos como esteres de esteroles. |
Also Published As
Publication number | Publication date |
---|---|
DK186791D0 (da) | 1991-11-15 |
AU2942292A (en) | 1993-06-15 |
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