SOME CONDITIONS AFFECTING THE PRODUCTION OF GELATINASE BY PROTEUS BACTERIA

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Abstract
The method used for determination of gelatinase was that of Fermi, and Palitzsch and Walbum, as modified by Clark (1920). Details are described and the equation K = (log ts[long dash]C)/T is used, in which C= logarithm of constant time in minutes required for gelatin control without enzyme to set to the selected state; ts, time required for mixture of gelatin and enzyme to set in ice water; T, time of incubation of gelatin-enzyme mixture; and K, a constant characterizing enzyme strength. The optimum reaction of the digesting mixture is [plus or minus] p H 8.4, and portions of the gelatin mixture after incubation at 30[degree] for varying periods of time are brought to constant pH before chilling in ice water for the test. Production of gelatinase by Proteus, strain acx, grown in peptone medium, was increased by aeration, and this increase appears to be due to other factors than differences in numbers of bacteria or p H of cultures. Since a variation in brand of peptone used caused wide variation in gelatinase production, even under apparently the same conditions, a synthetic culture medium was developed in which the strain of Proteus studied not only grew well but produced considerable gelatinase. This medium gave uniform results and can be easily duplicated. The basic medium, reaction of which is adjustable to p H 7.0, was made as follows: 10.0 gm. Na2HPO4 2H2O, 2.0 gm. KH2PO4, 10 gm. NH4Cl, 10 gm. glucose, distilled water to 1000 cc. Proteus grew in this basic medium under aerobic conditions but produced no noticeable amounts of gelatinase. When 0.05% CaCO3 or CaSO4 was added, they grew more luxuriantly and produced considerable gelatinase; but when MgSO4 was added, and no Ca salt, the bacteria grew luxuriantly, but gelatinase production was "small." When both Ca and Mg were added, the best production of gelatinase was obtained. The effect of Ca and Mg salts on gelatinase production was not due to difference in numbers of bacteria or to pH of culture. When lactic acid neutralized by NaOH was substituted for glucose, and the NH4Cl decreased, the same effects of Ca and Mg salts on gelatinase production were obtained. The effect of Ca and Mg was not due to an effect on liquefaction of gelatin by gelatinase, but on production of gelatinase in the cultures containing the salts. Autolysis of cultures killed by phenol did not result in further production of enzyme on addition of salts left out during growth. When Proteus was grown in synthetic media under anaerobic conditions, it was found that gelatinase was produced when glucose was the only source of C. When lactate was the only source of C, a "fair" production of gelatinase was obtained if small amounts of NaNO3 were present.