Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Lipoproteins – e.g. – egg yolk proteins – cylomicrons – etc.
Patent
1995-01-23
1998-02-10
Tsang, Cecilia J.
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Lipoproteins, e.g., egg yolk proteins, cylomicrons, etc.
530324, 530412, 530417, 530418, 530427, 530824, 435242, 4352541, 4352543, 4352545, 4352548, 4352551, 435261, 435911, 435913, 435939, 424 7803, 424450, 514 7, 514 12, C07K 100, C12N 100, A61K 3800
Patent
active
057170703
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
The present invention relates to a family of lipid binding and transporting proteins, to the method for preparing them from fungi, as well as to their applications in cosmetology, in the agri-foodstuffs industry and in the pharmaceutical field.
The present invention also relates to the crude fungal extracts containing such proteins and to their applications in the pharmaceutical and cosmetological fields and in the agri-foodstuffs field.
Phospholipid transfer proteins (PLTP) have been isolated from fungi such as Mucor mucedo, Aspergillus ochraceus or Neurospora crassa (Intern. J. Biochem., 1990, 22, 1, 93-98; Develop. Plant Biol., 1984, 9, 303-306; BBA, 1992, 1126, 3, 286-290), from fungal cultures produced on synthetic media using glucose as carbon source. However, the culture of fungi on such synthetic media makes it possible to obtain only small quantities of PLTP (very low yields); consequently, such cultural methods are not industrially applicable.
The need for a method for preparing, in large quantities, lipid binding and transporting proteins capable of ensuring the intermembrane transport of lipids and the preparation of proteins permitting a selective transport of (PI)! is critically felt, especially for improving the efficacy of action of liposomes, whose applications are numerous both in the drug field and in cosmetology and generally for producing modified biomembranes.
SUMMARY OF THE INVENTION
Consequently, the present invention set itself the aim of providing phospholipid transfer proteins (PLTP) capable of being produced in large quantities by filamentous fungi and capable of ensuring selective intermembrane transport of lipids (phospholipids and sterols) and active ingredients; such proteins are more suitable for the requirements of practical use, especially in that they make it possible to improve the biological and surfactant activities of the membranes.
Indeed, membranes are the seat of numerous functions in the development of living organisms (cellular recognition and exchange, respiration, excretion and the like) and constitute a barrier which is difficult to modify; yet, biomembrane engineering involves the modification of their composition and their organization.
The subject of the present invention is a method for preparing phospholipid transfer proteins (PLTP) from non-toxic filamentous fungi of the type comprising the preparation a crude extract, the separation of the proteins from the said extract and the purification of the said proteins, which method is characterized in that the said fungi are cultured in a phospholipid-rich medium.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the steps in the preparation of crude extract.
FIG. 2 illustrates the method of phospholipid transfer.
FIG. 3 illustrates the specific activity (nmol of phosphatidylchloine transferred/min/mg) of various strains.
FIG. 4 illustrates the growth A. oryzae obtained from various inocula.
FIG. 5 illustrates the phospholipid transfer activity of A. oryzae obtained from various inocula.
FIG. 6 shows the steps in the purification of crude extract.
FIGS. 7(A, B1 and C1) shows the elution profiles of purified protein.
FIGS. 8(A, B2 and C2) shows the elution profiles of purified protein.
FIG. 9 illustrates the activity of the endoplaemic reticulum of a fungus cultured in a glucose-based liquid medium or in a phospholipid-rich liquid medium.
FIG. 10 illustrates phospholipid transfer activity obtained after culturing in a glucose-based liquid medium or in a phospholipid-rich liquid medium.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
According to an advantageous embodiment of the said method, the said phospholipid-rich culture medium comprises between 3 and 20 g/l of phospholipids.
According to another advantageous embodiment of the said method, it comprises: sufficient quantity of mycelium is obtained, proteins from the said ground product, by centrifugation) and recovery of the supernatant S1, speed above 20,000 g (second centrifugation) and recovery of the supernatant S2,
REFERENCES:
De Scheemaeker et al, Developments in Plant Biology, vol. 9, pp. 303-306, 1984.
Groudin et al, Int. J. Biochem., vol. 22, No. 1, pp. 93-98, 1990.
Basu et al, Biochimica et Biophysica Acta, vol. 1126, No. 3, pp. 286-290, 1992.
Tai et al., J. Biol. Chem., vol. 259, No. 19, pp. 12178-12183, Oct. 10, 1984.
Record et al, Biochemica et Biophysica Acta, vol. 1256, No. 1, pp. 18-24, Apr. 28, 1995.
Basu et al., "Purification of a Phosphatidylinositol/Phosphatidylcholine Transfer Protein From Neurospora Crassa", Biochimica et Biophysica Acta, vol. 1126, No. 3, (1992) Amsterdam NL, pp. 286-290.
Grondin et al., "Purification and Characterization of a Novel Phospholipid Transfer Protein From Filamentous Fungi", International Journal of Biochemistry, vol. 22, No. 1, 1990 Oxford GB, pp. 93-98.
De Scheemaeker et al., "Comparison Between Phospholipid Transfer Proteins in Two Filamentous Fungi", Developments in Plant Biology, vol. 9, (1984) Amsterdam NL pp. 303-306.
Yamada, "Lipid transfer proteins in palnts and microorganisms", vol. 33, No. 1, Jan. 1992, Tokyo, Japan, pp. 1-6.
Asther Marcel
Cahagnier Bernard
Lesage Laurence
Marion Didier
Record Eric
Institut National de la Recherche Agronomique-INRA
Mohamed Abdel A.
Tsang Cecilia J.
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