The influence of the medium on the production of bacterial gelatinase

Abstract

IN a previous communication [Haines, 1931] it was shown that bacterial proteases are produced by organisms growing in simple synthetic media made up from carefully purified substances, and the influence of salts of calcium and magnesium on growth and protease formation was discussed. The methods used to estimate protease formation, namely, liquefaction of gelatin as evidenced by inability to set in ice-water and lack of coagulation of caseinogen on addition of acid, were however too crude to yield quantitative data as to the relative efficiency of the media in stimulating or inhibiting enzyme formation. In particular, it is to be expected that small amounts of gelatinase will be formed on certain media, insufficient in quantity to cause entire liquefaction, which will escape detection by such a method. More sensitive ways of measurement were therefore sought, and changes in viscosity of a mixture of gelatin and enzyme seemed to offer a ready means of following gelatinase action.Manning [1924] showed that the viscosity of 5 % gelatin could be measured in the ordinary way in an Ostwald viscometer provided that the determinations were carried out at 350 or above. Below 350 the gelatin is in gel form and possesses appreciable rigidity, but at 35°and above it is a sol. The shape of the viscosity-time curve also depends on the previous treatment of the gelatin, but is reproducible if the gelatin be made up under standard conditions. EXPERIMENTAL.The organisms used were freshly isolated from dung. In the previous work [1931] the strains of Proteus obtained would not grow in synthetic media. One strain has subsequently been isolated, however, which grew well, and the experiments described in this paper have been conducted with it and a strain of Pseudomonas. The characteristics of these organisms are listed in Appendix I. In general the organisms were grown on nutrient agar, PH 7 4, overnight at 200, the surface growth was scraped off, and washed and centrifuged in sterile saline three times. 1 cc. of the final suspension was used for inoculation purposes. The routine technique was the same as described in the previous paper, all substances being purified in a similar manner and carefully cleaned

The production and inactivation of pyocines

“…aeruginosa, a strain of Proteus vulgaris, and the proteolytic enzyme trypsin, provide some evidence that the inactivating substances were proteinases. It was also shown that calcium, which enhances the proteinases of pseudomonas and proteus cultures (Haines, 1932), has a similar effect on the pyocine inhibitors. Hamon & Peron (1962) found that some of the bacteriocines of Ps.…”

Inhibition of proteinases of certain clostridia by serum

“…As was pointed out by Haines (1933), the viscosity properties of gelatin change somewhat with repeated liquefaction and gelation. To avoid this, the stock solution was divided into smaller lots and stored in the refrigerator.…”

“…As Haines (1932Haines ( , 1933 has shown that equation (A) holds for the liquefaction of gelatin by filtrates of a variety of microorganisms. It was found to hold also for the bacterial enzymes No non-pathogenic organism in the above list possesses proteolytic enzymes which are able to resist inhibition by normal serum.…”

“…The method used for the determination of proteolytic activity was a modification of the method of Haines (1932Haines ( , 1933. The activity of the enzyme solution was calculated from the rate of gelatin liquefaction, measured in Ostwald viscosity tubes.…”

The proteolytic enzymes of Proteus vulgaris

“…Haines (14) showed that NH 4 could be used as the only source of nitrogen in the production of the·enzyme and was as effective as certain amino acids used singly.…”