Classifications

• • 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)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
  • C12N9/62—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi from Aspergillus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
  • A61K36/06—Fungi, e.g. yeasts
  • A61K36/062—Ascomycota
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/814—Enzyme separation or purification
  • Y10S435/815—Enzyme separation or purification by sorption

Definitions

• fibrinolytic material produced up to the present has been found to have the above-referred-to beneficial lytic action on fibrin clots, it has also been found to contain blood pressure lowering agents and it will be appreciated the value of such material would be materially enhanced if such blood pressure lowering agents were removed.
• Another important object is to provide not only a fibrinolytic material free from such blood pressure reducing material but a material having high fibrinolytic activity.
• Another important object is to enable the removal of such blood pressure lowering agents with a relatively simple procedure so that such enhanced fibrinolytic material may be produced in substantial quantities.
• the principal feature of the invention resides in filtering the fibrinolytic material through a cross-linked dextran known in the trade as Sephadex G50, which has a cross linked dextran having a water regain value of 5.7 as defined in the Journal Acta histochemica Bd. 11, 1961, page 306.
• the starting fibrinolytic material employed is a relatively crude preparation of a substance produced by growing strains of the mould Aspergillus oryzae, strain B1273, for example, by the deep culture techniques as set out in said co-pending application Serial No. 156,142.
• This strain B1273 of Aspergillus oryzae is available from the Quartermaster Research and Development Center, Dept. of Army, Natick, Mass. (see Proc. Soc. Exper. Biol. & Med. 99, 505, 1958 and 102, 203, 1959).
• the starting material may be a more purified material such as, for example, the purified fibrinolytic materials disclosed in said co-pending application Serial No. 156,144.
• such fibrinolytic material in either the crude or more purified form is filtered through a column of cross-linked dextran described un- 3,140,984 Patented July 14, 1964 Ice der the trademark Sephadex G50.
• the filter is then developed with an eluting agent and the eluate collected in fractions and the fractions of high fibrinolytic activity may then be pooled and dried in vacuo from the frozen state to produce fibrinolytic product which not only has a high potency but also is substantially free from blood pressure lowering agents.
• the filtering of the starting fibrinolytic material through the cross-linked dextran, Sephadex G50 is carried out at low temperatures, for example, at temperatures slightly above the freezing point of water to provide increased yields of the desired blood-pressure-lowering-agent-free fibrinolytic material.
• Example I 1.0 gram of fibrinolytic material having an activity of approximately 5000 units per mg. (as determined by a fibrin plate method of assay as is well understood in the art) (see proceedings of the Society for Experimental Biology and Medicine, volume 102, 1959, pages 201 to 203) but also having the characteristic of reducing the blood pressure when administered was dissolved in 15 ml. of water and the acidity adjusted to pH 4.8 with hydrochloric acid. This solution was filtered through crosslinked dextran (medium Sephadex G50) in a column approximately 2" in diameter and 22" in height and having a temperature slightly above the freezing point of water, about 3 C. to 6 C.
• crosslinked dextran medium Sephadex G50
• the filter was developed with an eluting agent consisting of distilled water adjusted to pH 4.8 by the careful addition of hydrochloric acid.
• the eluate was collected in 15-ml fractions.
• the fractions with high activity (fractions 33 to 41) were pooled and dried in vacuo from the frozen state.
• the yield was mg. of material having a potency of approximately 12,000 units per mg. This product was tested and found to be substantially completely free of any blood pressure lowering agents.
• the eluting agent was adjusted to pH 4.8, in similar examples the pH varied from about ph 3.5 to about pH 5.5 and similar yields obtained.
• Example II Although the material obtained from Example I has remarkably high activity in addition to being free from blood pressure lowering agents and is suitable for clinical use, its activity may be further increased as follows according to these examples.
• Example I A quantity of the material of Example I having a potency of 12,000 units per mg. was dialysed against running tap water yielding a fibrinolytic product containing about 18,000 units per mg.
• Example III 50 mg. of a relatively cruder fibrinolytic material having a potency of between 3,000 and 4,000 units per mg. were dissolved in 10 ml. of 8 molar urea solution and the solution filtered through a column of cross-linked dextran, Sephadex G50, as in Example I. Water was then run through the column and approximately thirty 3 ml. fractions collected. Four of the fractions (fractions 13-16 inclusive) containing most of the activity were freeze-dried to give 11 mg. of a product having fibrinolytic activity of approximately 8,000 units per mg. Again, the use of the cross-linked dextran, Sephadex G50, was found to not only materially increase the potency of the fibrinolytic material but also to remove the blood pressure lowering agents present in the starting material.
• the filtering the material through the cross-linked dextran is best accomplished by using the column in a refrigerator as it appears increased yields are obtained when work is carried out at low temperature, that is, a temperature slightly above the freezing point of water.
• a process for purifying fibrinolytic material produced from Aspergillus oryzae B127 3 and removing blood pressure lowering agents therefrom consisting in filtering an aqueous solution of said fibrinolytic material through cross-linked dextran known as Sephadex G50, developing the filter with an eluting agent and collecting the eluate which contains the desired resulting fibrinolytic material.

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Cited By (6)

• 1961
  • 1961-11-30 US US156143A patent/US3140984A/en not_active Expired - Lifetime
• 1962
  • 1962-11-26 GB GB44626/62A patent/GB1022026A/en not_active Expired
  • 1962-11-29 DK DK515262AA patent/DK104292C/da active

Non-Patent Citations (1)

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