The Proteolytic Enzymes of Micro‐organisms

Abstract

Résumé.The formation of exo‐enzymes may be shown in the case of bacteria by centrifuging off the organisms, with or without filtration, through a suitable filter candle or Seitz asbestos filter, and incubating with the appropriate substrate at the required pH. Further concentration and purification may be carried out by evaporation in vacuo at low temperatures (Hladik, 1910), fractional precipitation with ammonium sulphate (London and Pakhotina, 1917), precipitation, dialysis and re‐precipitation with alcohol (Munter, 1910), adsorption on alumina or kaolin and fractional elution with suitable buffers of selected pH (Willstätter, 1928; Hopkins, 1930), dialysis (Harden and Young, 1906; Walter, 1917), and extracting with glycerine (Macfadyen, 1892).With moulds and actinomycetes the mycelium may be filtered off through paper and suitable methods of concentration applied to the filtrate. For the preparation of endo‐enzymes of bacteria, autolysis may be used, alternative freezing and thawing (Young, 1929), grinding in an agate mortar (Stevens and West, 1922, a, b), or lysis with bile (Avery and Cullen, 1923). For yeasts and fungi the “acetone‐dauerhefe” process employed by Dox (1913), combined grinding and extraction of juice by high pressure (Buchner, 1898), and low temperatures (Rowland, 1910), are available, after which further precipitation may be carried out. Halderer (1909, 1910, 1912) investigated the conditions influencing filtration of enzymes through porcelain filters. Two interesting recent methods of following protein hydrolysis are by Krebs (1930) and Gates (1930). The former dissolved the protein in bicarbonate solution in the cup of a Warburg manometer in the presence of 5 per cent. carbon dioxide. As hydrolysis proceeds the carbon dioxide tension changes and may be read off on the manometer. Gates measured the reduction in density of exposed photographic film in a photometer, progressive proteolysis of the gelatin leading to release of silver. Viscometric methods are well adapted to the study of breakdown of gelatin (von Gröer, 1912; Haines, 1933). A nephelometric method of following proteolysis has been developed by Rona and Kleinmann (1928). Foreman and Graham‐Smith (1928) worked out titration methods for following the changes in meat broth during growth of Staphylococcus aureus.