WO1993002180A1

WO1993002180A1 - Method for selective lipolysis, and a lipase mixture and microorganism suitable for use in the method

Info

Publication number: WO1993002180A1
Application number: PCT/EP1992/001589
Authority: WO
Inventors: Albrecht Weiss; Günter LAZAR; Karin Grundel
Original assignee: Cognis Gesellschaft Für Bio- Und Umwelttechnologie Mbh
Priority date: 1991-07-22
Filing date: 1992-07-13
Publication date: 1993-02-04
Also published as: DE4124248A1

Abstract

The aim of the invention is to use strains of the microorganism species Geotrichum to increase the selectivity of raw-lipase mixtures. This has been achieved by the discovery of a strain which produces a raw-lipase mixture capable of selectively splitting fatty-acid triglycerides so that oleic acid is the favoured product.

Description

"Process for selective fat cleavage, suitable lipase mixture and microorganism"

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.

It is known that fungi of the Geotrichum species and in particular the Geotrichum candidum species are capable of producing lipases. Corresponding strains and their lipases have been described by various authors, the lipases differing greatly from strain to strain.

This is how Sugihara et al in J. Bioche describe it. 107, 426-430 (1990) the type separation and characterization of two molecular forms of a Geotrichum candidum lipase. Lipase 1 has a molecular weight of 64 kDa and an isoelectric point of 4.3, whereas Lipase 2 has a molecular weight of 66 kDa and an isoelectric point of 4.3. T. Jacobsen et al describe in Enzyme Microb. Techno !. 1989, vol. 11, 90 lipases from Geotrichum candidum with a molecular weight of 53 kDa and 37 kDa, respectively. A similar molecular weight (53 kDa to 55 kDa with isoelectric points of 4.33) is described by Tsujisaka et al Agr. Biol. Chem. 37 (6) 1457, (1973). Finally, J. Shimada et al in J. Biochem. 106 (3) 383 (1989) and 107, 703 (1990) two lipases from Geotrichum candidum, the enzyme designated Lipase 2 having been cloned and found to contain 544 amino acids.

There has also been repeated evidence in the scientific literature that some of the lipases from Geotrichum candidum are capable of selectively cleaving fatty acid triglycerides, i.e. from such fatty acid

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 .

Reference is also made to the following work: T. A. Marks in Lipids, Vol. 3 (2) 143, (1967), to M. Iwai et al in Agr. Organic !. Chem., 37 (4), 929, (1973), on J. Kroll, Pharmazie 28, (4), 263 (1973) and on J.A. Alford in J. Lipid Research Vol. 5, 390 (1964). Some of these references describe the isolation of special G. candidum lipases and the selectivity of corresponding isolates.

In summary, it can be said that fungi of the Geotrichum candidum species produce extracellular lipases and that some of these lipases are capable of selective fat cleavage.

These findings are of scientific importance for the practice of the oleochemist, but are not technically usable. If a microorganism strain produces a lipase mixture which contains only a selective lipase in a mixture with a non or not very selective lipase, this strain or its enzyme products can only be used for the selective cleavage of fatty acid triglycerides if they are selective Lipase is cleaned of other lipases.

However, the separation of lipases from a lipase mixture is very difficult and, because of the similarity of the enzymes, is at best possible on a laboratory scale using complex, mostly chromatographic, methods.

For the production of high-purity oleic acid, for which there is technical need, it is of interest to have a lipase preparation which can be prepared by conventional fermentation methods and without complex separation processes and which is capable of selectively cleaving triglycerides.

It is therefore an object of the invention to provide a microorganism which delivers such a lipase preparation during fermentation and a Show methods for the selective cleavage of triglycerides with the help of this lipase preparation.

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.

Another object of the invention is a lipase mixture from Geotrichum sp. 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.

To obtain the strain according to the invention, 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. On the basis of the damage caused by the mutation method, it can be assumed that 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.

To produce the lipase mixture from the strain according to the invention, this is grown under conventional fermentation conditions. 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.

To obtain 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.

If so desired, the lipase mixture of Geotrichum sp. However, 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.

For triglyceride cleavage, 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.

When esters are cleaved by the lipase mixture according to the invention, the aqueous phase can be adjusted to pH values between 4 and 8 with the aid of a buffer. However, this is not a mandatory requirement, since the lipase mixtures according to the invention and the individual lipases are not very pH-dependent. According to a preferred embodiment, 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.

From the liquid phase, 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.

As demonstrated in the examples, the lipase mixture shows the advantage of high selectivity. Thus, when using 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.

B e i s p i e l e

Example 1: Isolation of the wild trunk

Smears with sterile and moist cotton swabs from substrates from the Henkel oil and fat companies were spread on corn-malt agar plates.

Corn-malt aqar:

- Corn flour extract 1000 ml

- Malt extract 30 g

- 10% KOH 2 ml

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).

Olive oil aqar plates:

- Autoclave 1/15 M Sörensen phosphate buffer pH 7.0 and 1.5% agar

- Add 0.1% olive oil, triolein or trilinolein at approx. 80 ° C

- Homogenize for 3 - 5 minutes with ultrasound (400 W)

- Pour 15 ml each into Petri dishes

When a microorganism secretes 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.

Medium:

Yeast extract (5 g / 1)

Olive oil (10 g / 1)

Peptone (6 g / 1)

NaCl (1.4 g / 1) pH 6.3

Incubation conditions:

The olive oil used serves as an inducer of lipase production.

150 ml of medium were inoculated with the mold in 500 ml Erlenmeyer and incubated at 26 ° C. and 160 rpm with shaking. Maximum lipase activity was measured after 72-96 hours.

Example 2: Einspormycelien / Mutationsverfahren

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.

Medium 1:

Yeast extract (10 g / 1) olive oil (10 g / 1) pH 6.3

One isolate showed a 50% increase in lipase activity (2 U / ml).

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.

After 4 mutation cycles, the isolated leaky mutant DSM 6590 showed a lipase activity increased by a factor of 50 compared to the wild type.

Example 3: Schütte cultures / fermentations

A fermentation medium was prepared containing:

Olive oil 21 g / 1 corn steep liquor 10 g / 1 yeast extract KAT 20 g / 1 (obtained from Ohly, Hamburg)

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.

Example 4: Enzyme processing

The culture supernatants obtained from the fermentation were separated from cells and solid media components using a continuous centrifuge.

The mixture was then concentrated using an ultrafiltration unit and thin-film evaporation. The filtrate thus obtained was freeze-dried. A raw preparation was obtained. 24 grams of crude preparation with a specific lipase activity of 3000 U / g resulted from the fermentation, corresponding to a 63-fold drying. Example 5: Enzyme purification

Raw preparation:

spec. Activity: 3000 U / g protein content: 5%

The crude preparation was dissolved in water and dialyzed overnight against Tris buffer (A buffer). The lipase was then bound via an anion exchanger.

Conditions:

Anion exchanger: Q-Sepharose / Pharmacia

A buffer: Tris 30 mM pH 7.6

B buffer: Tris 30 mM pH 7.6 + 1 M NaCl

The lipase was eluted in a linear salt gradient (0-1 M NaCl), which was built up with the buffers A and B.

In a second step, the lipase solution was applied to a gel filtration column and eluted with ammonium bicarbonate buffer.

- Gel filtration: Sephadex G 25 / Pharmacia (column: 100 cm high, 11.3 cm diameter)

- Ammonium bicarbonate buffer - 50 M

- Amount of lipase solution added: 1200 ml

- spec. Activity: 50 U / ml

The lipase-containing fraction was lyophilized (freeze-dried). Lyophilisate:

spec. Activity: 60000 U / g protein housing: 60% electrophoresis native: 2 lipase bands Electrophoresis SDS: 1 band, molecular weight 67 kDa Isoelectric points: pl 4.3 and pl 4.5

Example 6: Separation of lipases

The two electrophoretically proven lipases of the purified lyophilisate were separated by HPLC.

Conditions:

TSK OEAE 3 SW column / Supelco

A buffer: Na phosphate 50 mM pH = 5.8

B buffer: Na phosphate 50 mM pH = 5.8 + 1 M NaCl

Gradient: from 0 to 20% B buffer in 1 hour

Flow rate: 1 ml / min

Inject: 100 μl of a 10 mg / ml stock solution in A buffer

Two fractions, each with a lipase, were obtained, which were used for selectivity studies.

Example 7: Selectivity studies

Both the two separate isoenzymes and the purified lyophilisate and the crude preparation originating from the fermentation were used for selectivity studies. Conditions:

Reaction approach:

Phosphate buffer pH 7.0 - 0.05 M

Palm oil as a substrate

Buffer / substrate ratio 4/1 (v / v)

30 ° C / shake

Total volume of reaction mixture 1 ml

Amount of lipase crude preparation 2 mg / batch

Amount of lipase lyophilisate 0.1 mg / batch with lipase replenishment: same amounts of lipases dissolved in 100 μl

The following approaches were examined:

Follow up with fresh enzyme solution

Subsequent dosing of substrate

Reaction in the unbuffered system

Reaction at different buffer / substrate ratios

Variation of the substrate and thus the fatty acid composition:

Palm oil, fancy tallow, soybean oil

Selectivity of the lipase values after a reaction time of 24 hours

according to the invention according to the invention comparative example

Claims

Patent claims

1. Microorganism strain of the species Geotrichum generated by a wild strain with mutation and selection with the deposit name DSM 6590 and the ability to generate a lipase mixture which selectively cleaves oleic acid from triglycerides of fatty acid mixtures, as well as mutants and variants of this strain.

2. Lipase mixture from Geotrichum sp. DSM 6590 can be produced by culturing the microorganism 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 them by evaporation and / or precipitation and / or freeze-drying.

3. Geotrichum sp. Lipase DSM 6590 with a molecular weight of 67 kDa and an isoelectric point pl 4.3.

4. Geotrichum sp. Lipase DSM 6590 with a molecular weight 67 kDa and an isoelectric point pl 4.5.

5. Use of lipases according to claims 2 to 4 for the selective cleavage of oleic acid from fatty acid triglycerides.

6. Embodiment according to claim 5, characterized in that the ratio of lipid phase to aqueous phase between 10 to 90 and 90 to 10, preferably between 60 to 40 and 90 to 10 is set.

7. Embodiment according to one of claims 5 or 6, characterized in that the lipid phase additionally contains emulsifiers, in particular partial glycerides.

8. Embodiment according to one of claims 5 to 7, characterized in that the aqueous phase contains buffering substances and is adjusted to a pH between 4 and 8.

9. Embodiment according to one of claims 5 to 8, characterized in that the pH is not readjusted during the fat splitting reaction.

10. Embodiment according to one of claims 1 to 9, characterized in that the fat cleavage reaction is carried out at temperatures between 10 and 60 ° C, in particular 30 ° C and 40 ° C.

11. Embodiment according to one of claims 5 to 10, characterized in that the lipases are preferably used in an amount of 0.5 to 50 U / g fat or oil in an amount of 2 to 15 U / g.