WO1993002180A1 - Procede pour la lipolyse selective, melange de lipase adequat, ainsi que micro-organisme - Google Patents

Procede pour la lipolyse selective, melange de lipase adequat, ainsi que micro-organisme Download PDF

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Publication number
WO1993002180A1
WO1993002180A1 PCT/EP1992/001589 EP9201589W WO9302180A1 WO 1993002180 A1 WO1993002180 A1 WO 1993002180A1 EP 9201589 W EP9201589 W EP 9201589W WO 9302180 A1 WO9302180 A1 WO 9302180A1
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WO
WIPO (PCT)
Prior art keywords
lipase
embodiment according
lipases
geotrichum
dsm
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Application number
PCT/EP1992/001589
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German (de)
English (en)
Inventor
Albrecht Weiss
Günter LAZAR
Karin Grundel
Original Assignee
Cognis Gesellschaft Für Bio- Und Umwelttechnologie Mbh
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Application filed by Cognis Gesellschaft Für Bio- Und Umwelttechnologie Mbh filed Critical Cognis Gesellschaft Für Bio- Und Umwelttechnologie Mbh
Publication of WO1993002180A1 publication Critical patent/WO1993002180A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6418—Fatty acids by hydrolysis of fatty acid esters
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C1/00—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids
    • C11C1/02—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids from fats or fatty oils
    • C11C1/04—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids from fats or fatty oils by hydrolysis
    • C11C1/045—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids from fats or fatty oils by hydrolysis using enzymes or microorganisms, living or dead
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

Definitions

  • the invention relates to a process for the selective elimination of oleic acid from triglycerides, suitable lipases or lipase mixtures and a microorganism or its mutants or variants which is capable of delivering this lipase mixture.
  • Triglycerides which are made up of mixtures of fatty acids, prefer to release oleic acid, described E. Charton et al in a lecture to the CEC-GBF International Workshop 1990 on September 13, 1990 that two extracellular lipases were extracted from the strains Geotrichum candidum ATCC 34614 and CMICC 335426, and that one of these lipases is able to split off cis-9 fatty acids from the glycerides .
  • a lipase preparation which can be prepared by conventional fermentation methods and without complex separation processes and which is capable of selectively cleaving triglycerides.
  • the main subject of the invention is therefore a microorganism strain of the species Geotrichum produced by mutation and selection from a wild strain with the deposit name DSM 6590 and the ability to produce a lipase mixture which selectively releases oleic acid from triglycerides of fatty acid mixtures, as well as mutants and variants of this tribe.
  • DSM 6590 can be produced by cultivating the microorganism under customary fermentation conditions in the presence of esters of oleic acid, separating the cells and solid media components and, if desired, removing low molecular weight components by ultrafiltration, concentrating by evaporation and / or precipitation and / or freeze-drying.
  • a third object of the invention is the use of lipases according to claims 2 to 4 for the selective elimination of oleic acid from fatty acid triglycerides.
  • the microorganism Geotrichum sp. was developed by mutation and selection from a wild strain which was found in the vicinity of the applicant's oleochemical plants.
  • the wild trunk is likely to be assigned to the species Geotrichum candidum.
  • Macroscopic and microscopic studies show that the fungus forms a well-developed mycelium. End parts of the hyphae disintegrate into short arthrospores, which can be characterized as cube-like or short cylindrical with flat ends. Cytological studies (Giemsa staining) showed that the hyphae are septate and that the hypha segments contain 5 - 10 nuclei.
  • the oidiospores predominantly contain 1 or 2 nuclei in a ratio of 1: 1; only a few oidiospores have 3 or more nuclei.
  • oidia of an isolate of the wild strain were mutagenized with N-methyl-N-nitroso-N'-nitroguanidine in such a way that that about 3.6% of the oidia remained viable.
  • the mutated oidia were plated out in different dilution stages so that individual colonies grew well separated from one another. Mutants were isolated using the Lederberg's stamp method. Mutants with increased lipase activity were again subjected to treatment with N-methyl-N-nitroso-N'-nitroguanidine. After four cycles of mutation, the strain of the invention was obtained. It is a leaky mutant, that is to say a mutant which can be recognized as damaged due to its external shape.
  • Lipase activity is increased by a factor of 50 compared to the wild strain.
  • the mutant generated differs very greatly from the wild strain due to the — accidental intervention — of the mutating agent.
  • the mutant according to the invention is in the German collection for microorganisms as Geotrichum sp. DSM 6590 deposited according to the Budapest contract.
  • the fermentation medium should be an N source, such as. B. yeast extract, a C source, an inductor and, if desired, trace elements. It has been found that liquid fatty acid triglycerides are advantageously used, and that they can simultaneously take on the function of a nutrient (C source) and that of an inducer for lipase formation.
  • An advantageous amount of fatty acid triglycerides in the fermentation is between 5 and 50 g / l fermentation medium. It is' doing a particular advantage of the invention claimed tribe that the use of the inductor does not adversely affect the desired selectivity of the lipase, but nevertheless allows the economic yield of lipase. This is surprising and is in contrast to the findings of the specialist literature.
  • a lipase mixture from the fermentation medium, cells and solid media components are separated.
  • the separation can take place in the usual way, for example by centrifugation or by filtering devices. It is then expedient to concentrate the fermentation medium containing the lipase, for example by ultrafiltration or Thin film evaporation can happen. It is also possible to isolate the lipases in a suitable manner by precipitation steps.
  • the crude lipase mixture obtained with the help of the strain according to the invention essentially contains two lipases.
  • the raw mixture can advantageously be used for industrial processes without separating these lipases, since both lipases selectively split off monounsaturated fatty acids with a double bond in the cis-9 position.
  • DSM 6590 can also be separated into two individual ipases, namely into a lipase with a molecular weight of approx. 67 kDa and an isoelectric point pl 4.3 and into a lipase with the same molecular weight and an isoelectric point pl 4.5.
  • the two lipases can be separated analytically by electrophoresis. On a preparative scale, it is possible using column chromatographic methods, in particular HPLC.
  • the lipases according to the invention can generally be used for esterification and for cleaving esters, in particular for cleaving tri-glycerides. Because of the selectivity of the lipase mixture and the individual lipases, it is preferably used to split off monounsaturated acids, in particular oleic acid, from triglycerides rich in oleic acid. So with the aid of the lipase mixture according to the invention from oleic acid-rich triglycerides, such as. B. from sunflower oil or sunflower oil new varieties, but also from palm oil or animal oils and fats oleic acid with a very high degree of purity.
  • the lipase is used as a concentrated fermentation solution, which may have been purified by ultrafiltration, as a precipitate or in freeze-dried form and is added to oil-water mixtures. It is advantageous to set the ratio of lipid phase (oil phase) to aqueous phase between 10:90 and 90:10, preferably between 60:40 and 90:10. In many types of lipase use, it can be advantageous to increase the phase interface between oil and water. This can be done mechanically by stirring. However, emulsifiers can also be added. Partial glycerides in particular have proven themselves as emulsifiers. B. glycerol monooleate or stearate.
  • the aqueous phase can be adjusted to pH values between 4 and 8 with the aid of a buffer.
  • the pH is therefore not readjusted when fatty acid triglycerides are cleaved.
  • Suitable temperatures for the cleavage reaction are between 10 ° C and 60 ° C, in particular between 30 ° C and 40 ° C.
  • the amounts of lipases used should be between 0.5 and 50 U / g fat or oil. Amounts of 2 to 15 U / g are preferred.
  • (U) corresponds to the release of 1 mol of fatty acid per minute.
  • the lipase mixtures according to the invention or else the individual lipases can also be immobilized in a conventional manner or used in a carrier-bound manner.
  • the lipase mixture shows the advantage of high selectivity.
  • an unpurified and non-separated enzyme preparation for example in the case of palm oil, only a maximum of 2 to 3% of the saturated fatty acids are released enzymatically, whereas oleic acid is largely eliminated.
  • the maize Ma! Z agar plates were incubated with the smears at 25 ° C. for 72 hours.
  • the microorganisms were separated on corn-malt agar plates and the pure cultures obtained in this way were transferred to fat / oil agar plates (olive oil).
  • a lipase extracellular lipase
  • a clear zone forms around the cell colony.
  • the most active lipase formers can be identified with the help of the "lysis zones”.
  • the selectivity of the lipases formed can also be roughly estimated by the selection of the triglycerides.
  • a wild-type lipase generator obtained in this way showed a particularly good lipolytic activity against triolein and trilinolein.
  • the wild type obtained was fermented in shake flask cultures and the excretion of extracellular lipase examined.
  • the cell-free culture broths contained lipase activities of 0.7-1.0 U / ml culture filtrate.
  • the olive oil used serves as an inducer of lipase production.
  • One-cycle mycelia which were obtained by plating out suitable dilution stages of an oidiene wash-off of the wild type, were examined for lipase activity in the following medium.
  • Oidia of this isolate (spores which the fungus forms for propagation) were mutagenized with N-methyl-N-nitroso-N'-nitroguanidine in such a way that about 3.6% remained capable of germination.
  • the mutated oidia were plated out in different dilution stages in such a way that individual colonies grew well separated from one another (conditions see below).
  • Hard (“normal”) and leaky mutants (mutants which are recognizable as damaged due to their external shape) were isolated by means of the Lederberg stamp method. Mutants with increased lipase activity were again subjected to the N-methyl-N-nitroso-N'-nitroguanidine treatment.
  • the isolated leaky mutant DSM 6590 showed a lipase activity increased by a factor of 50 compared to the wild type.
  • a fermentation medium was prepared containing:
  • the pH was adjusted to 7.0.
  • the pH was kept constant for 120 hours. It was fermented with stirring at 26 ° C.
  • the reactor volume of air was blown into the fermenter every minute.
  • the culture supernatants obtained from the fermentation were separated from cells and solid media components using a continuous centrifuge.
  • the crude preparation was dissolved in water and dialyzed overnight against Tris buffer (A buffer). The lipase was then bound via an anion exchanger.
  • a buffer Tris 30 mM pH 7.6
  • the lipase was eluted in a linear salt gradient (0-1 M NaCl), which was built up with the buffers A and B.
  • the lipase solution was applied to a gel filtration column and eluted with ammonium bicarbonate buffer.
  • the lipase-containing fraction was lyophilized (freeze-dried). Lyophilisate:
  • the two electrophoretically proven lipases of the purified lyophilisate were separated by HPLC.
  • Amount of lipase lyophilisate 0.1 mg / batch with lipase replenishment same amounts of lipases dissolved in 100 ⁇ l

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
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  • Tropical Medicine & Parasitology (AREA)
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  • Botany (AREA)
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  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Dans des souches de micro-organismes de l'espèce Geotrichum, il s'agissait d'augmenter la sélectivité des mélanges de lipase brute. On y est parvenu en découvrant une souche fournissant un mélange de lipase brute, lequel permet un clivage sélectif de triglycérides d'acide gras, avec clivage préféré de l'acide oléique.
PCT/EP1992/001589 1991-07-22 1992-07-13 Procede pour la lipolyse selective, melange de lipase adequat, ainsi que micro-organisme WO1993002180A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DEP4124248.3 1991-07-22
DE4124248A DE4124248A1 (de) 1991-07-22 1991-07-22 Verfahren zur selektiven fettspaltung, dazu geeignete lipasemischung und mikroorganismus

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WO1993002180A1 true WO1993002180A1 (fr) 1993-02-04

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7381560B2 (en) 1992-11-13 2008-06-03 Biogen Idec Inc. Expression and use of anti-CD20 antibodies
US9228212B2 (en) 2009-03-02 2016-01-05 Arkema France Method for producing ricinoleic acid ester by selective enzymatic transesterification

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5470741A (en) * 1992-07-22 1995-11-28 Henkel Corporation Mutant of Geotrichum candidum which produces novel enzyme system to selectively hydrolyze triglycerides

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0243338A2 (fr) * 1986-04-25 1987-10-28 Fina Research S.A. Segment d'ADN codant pour une lipase spécifique, vecteurs pour l'expression de celui-ci, micro-organismes transformés par ces vecteurs et utilisation de ces micro-organismes pour la production de lipase
WO1989003419A1 (fr) * 1987-10-13 1989-04-20 The Lubrizol Corporation Procede de production de preparations a base d'acide cis-9-octadecenoique

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0243338A2 (fr) * 1986-04-25 1987-10-28 Fina Research S.A. Segment d'ADN codant pour une lipase spécifique, vecteurs pour l'expression de celui-ci, micro-organismes transformés par ces vecteurs et utilisation de ces micro-organismes pour la production de lipase
WO1989003419A1 (fr) * 1987-10-13 1989-04-20 The Lubrizol Corporation Procede de production de preparations a base d'acide cis-9-octadecenoique

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, vol. 116, no. 5, 3. März 1992, Columbus, Ohio, US; abstract no. 36667a, BAILLARGEON, MARY WELCH ET AL. 'Geotrichum candidum NRRL Y-553 lipase: purification,characterization and fatty acid specificity.' Seite 309 ; *
CHEMICAL ABSTRACTS, vol. 70, no. 13, 31. März 1969, Columbus, Ohio, US; abstract no. 54422c, SAMPUGNA, J. ET AL. 'Suitability of Geotrichum candidum lipase for the stereospecific analysis of some triglycerides.' Seite 53 ; *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7381560B2 (en) 1992-11-13 2008-06-03 Biogen Idec Inc. Expression and use of anti-CD20 antibodies
US9228212B2 (en) 2009-03-02 2016-01-05 Arkema France Method for producing ricinoleic acid ester by selective enzymatic transesterification

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