Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Patent
1993-12-28
1996-02-06
Marx, Irene
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
435198, 424 946, C12N 120, C12N 5102
Patent
active
054895308
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to the use of specific lipases for producing drugs.
Lipases play an important part in the digestion of fats because they catalyze the elimination of fatty acids from them. Lipases are therefore employed in therapy for treating digestive disorders based on enzyme deficiency. The latter is found, for example, in cases of pancreatic insufficiency, chronic pancreatitis and following gastric resection. The lipases used for this purpose are mostly products based on pig pancreas which has been defatted, dried and ground. However, such preparations of pig pancreas have several serious disadvantages: an amount of up to 5-10 g per day. no lipolytic activity on passage through the stomach. therefore be administered either in acid-resistant form or in very high doses. contain proteases and amylases and are thus contraindicated for certain forms of pathological maldigestion.
It has already been proposed to prepare lipases for the therapy of maldigestion by cultivating fungi of the genus Aspergillus, Penicillium, Mucor, Candida or Rhizopus. DE-A 16 42 654 and EP-A 387 945 describe the preparation and purification of a lipase for therapeutic purposes by fermentation of the fungus Rhizopus arrhizus.
The present invention relates to the use of bacterial lipases which show an immunological cross-reaction with the antibodies to the lipase produced from the microorganism Pseudomonas spec. DSM 6483 and/or Pseudomonas cepacia IAM 1057 for producing drugs for the therapy of maldigestion. Pseudomonas spec. was deposited on May 27, 1991, at the International Depository Authority, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-3300, Braunschweig, Germany, as Deposit No. DSM 6535. In order to differentiate the bacterial lipases, antibodies against lipase from said microorganisms are required. The lipases can be obtained from bacteria by cultivating them in a nutrient medium, and isolating the enzyme from the culture broth. Suitable nutrient media contain sources of carbon and of nitrogen, inorganic salts and, where appropriate, small amounts of trace elements and vitamins. Nitrogen sources which can be used are inorganic or organic nitrogen compounds or materials which contain these compounds. Examples are: ammonium salts, nitrates, corn steep liquor, yeast autolysate, yeast extract and hydrolyzed casein. Carbon sources which can be used are sugars such as glucose, polyols such as glycerol or organic acids such as citric acid or fatty acids. Particularly suitable carbon sources are vegetable oils such as soybean, linseed or olive oil. Examples of inorganic salts are the salts of calcium, magnesium, manganese, potassium, zinc, copper, iron and other metals. Particularly suitable anions in the salts are phosphate and nitrate ions. The cultivation is preferably carried out at from 25.degree. to 33.degree. C. The pH of the medium is maintained at 6-7.5, preferably 6.5-7 using 2N sulfuric acid or ammonia to keep it constant during the fermentation. Submerged cultivation is carried out with vigorous aeration and stirring. Fermentation is continued, measuring the enzyme activity at intervals of three hours; until two consecutive measurements show constant activity. An incubation time of 40-60 hours is generally sufficient.
It is possible in this way to obtain, using bacterial strains which have been isolated directly from natural habitats, enzyme yields of 50-500 mg per 1 of culture broth. The enzyme yield can be increased by mutation with chemical agents or UV light followed by selection for improved lipase productivity.
The enzyme is removed from the culture broth in a conventional way. The broth is centrifuged or filtered to remove microorganisms and insoluble material. The liquid phase is then collected in order to obtain the lipase. This takes place by precipitation with a water-miscible organic solvent (eg. alcohol or acetone) or by adding a salt such as ammonium sulfate. The specific activity can be increased, and the content of impurities can be further reduced, by r
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"Bergey's Manual of Systematic Bacteriology", 1986, Krieg et al, ed. pp. 362-365, Williams & Wilkins.
Braatz Reinhard
Friedrich Thomas
Kurth Roland
Menkel-Conen Elke
Rettenmaier Hansjoerg
BASF - Aktiengesellschaft
Marx Irene
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