Patent GB-804608-A (GB)
Title: Proteolytic enzyme
IPC: C12N9/58
Filed: 1955-10-05
Abstract: A proteolytic enzyme, which is an amorphous powder having a molecular weight of 30,000, an isoelectric point of 10.2, a sedimentation constant of 2.5 X 10-13, a diffusion constant of 8.2 X 10-7, an electrophoretic mobility of 0.08 X 10-5 in glycine buffer of pH 10.5 and of 0.43 X 10-5 in barbitol buffer of pH 8.5, optional proteolytic activity at pH 9.0 measured against casein, which is stable over pH 4.0 to 11.0 and gives a positive precipitin reaction against a 1 in 1000 dilution of specific rabbit antiserum, is produced by fermenting a fungus of the order Entomophthorales, family Entomophthoraceae in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen and preferably inorganic salts.  The fungi are of the genera Entomophthora, Bacidiobolus, Conidiobolus, Completoria, Massaspora and Ancyclistes.  Species specified are E. apiculata, E. coronata, E. sphaerosperma, B. ranarum, C. villosus and C. brefeldianus. Sources of carbon for the nutrient medium are pentoses such as arabinose, ribose and xylose, monosaccharides such as mannose, levulose and galactose, disaccharides such as trehalose, maltose, lactose, cellibiose and sucrose, polysaccharides such as starch, and alcohols such as glycerol, mannitol, sorbitol, inositol and ethyl alcohol. Nitrogen sources are animal or vegetable proteins, soybean meal, casein, peptones, polypeptides, amino acids, animal stick liquor, sodium nitrate, asparagine urea, histidine and guanine. Inorganic salts are phosphates, sulphates and chlorides of alkali and alkaline earth metals and trace metals, e.g. cobalt, iron, magnesium and manganese.  The medium is adjusted to pH 5.0 to 9.0 and preferably 6.0 to 8.0, the temperature of cultivation is 20 to 30 and especially 25 to 30 DEG  C. and the duration is 24 to 90 hours especially 48 to 90 hours.  Anti-foaming agents such as olein and brominated castor oil may be present.  The enzyme is recovered from the filtered or unfiltered broth by adsorption at pH 6.5 to 8.5. Specified adsorbents are diatomaceous earths, magnesium silicates, fuller's earth, activated charcoal, lamp black, titanium oxide, alumina silica catalyst and zeolites such as "Permutit" (Registered Trade Mark).  Elution is by dilute aqueous mineral acid such as hydrochloric or sulphuric at pH 9.0 to 11.5 especially 10.5, preferably in the presence of sodium chloride.  For stability the pH is then adjusted to 6.0 to 8.0 and may be used as such in dry cleaning, hide bating or desizing cloth.  For pharmaceutical use, bread-baking and baby foods it is further purified by adding acetone to an aqueous solution.  The final product may be (a) dissolved in 1 per cent aqueous sodium chloride, lyophilized, (c) dried by suspension in an anhydrous water-miscible solvent such as acetone, (d) mixed with protecting and/or bulking agents.  Specified protecting agents are sorbitol, corn syrup, lactose, dextrose, gelatin and gum acacia.  The bulking agents may be starch, wood pulp, magnesium silicate and diatomaceous earth.  For use in leather manufacture, the enzyme may be precipitated from the fermentation beer with a protein precipitant such as tannic acid, phosphotungstic acid, zinc chloride and ferric chloride.  A protective agent may be added. The tannic acid precipitate may be slurred in water and treated with a watermiscible solvent such as acetone or methyl, ethyl, propyl or butyl alcohols in order to precipitate the enzyme.