M. Nohýnek

Estimation of lipase activity by the diffusion plate method.

A diffusion plate method for the assay of the activity of lipases in large series of samples was worked out. When using this method the maximum deviation was found to be ±3.8%.

Esterases in mycobacteria. I. Carboxylic ester hydrolases in a submerged culture of Mycobacterium phlei.

It was found that a submerged culture ofMycobacterium phlei degrades simple esters (ethylacetate and ethylbutyrate) as well as synthetic lipids (triacetine and tributyrine). The effect of pH on the rate of degradation of tributyrine was investigated and the maximum activity of esterases found within a wide range of pH. The activity of esterases was followed during growth of a submerged culture ofMycobacterium phlei. Esterases were not released into the cultivation medium during growth or even during the early stationary phase. Only a low steady activity of esterases could be demonstrated in a filtrate of the cultivation liquid. The total activity of esterases reached its maximum after a 6–11 day incubation. The specific activity of esterases reached a maximum on the 6th day of incubation; its value decreased to about one half and did not change substantially on prolonged incubation. Changes in the specific activity of esterases were found to be time-related with changes of pH and a decrease of the specific activity was associated with a release of macromolecular compounds into the incubation medium. Esterases as well as other macromolecular compounds were isolated from the filtrate of the cultivation medium ofMycobacterium phlei. The isolated preparation contained 60–72% total activity of esterases present in the filtrate of the cultivation liquid.

Propionate and the production of monensins in Streptomyces cinnamonensis

Variants resistant to propionate wore prepared from a mutant strain ofStreptomyces cinnamonensis producing predominantly monensin A. Using selected resistants the production of monensins (in media with higher concentrations of propionate) was examined. Stimulation of monensin synthesis by propionate. was observed with 70% of the resistants studied. Propionate did not influence the ratio between monensin A and B production.

Esterases in mycobacteria. II. The isolation of carboxylic ester hydrolases from a submerged culture of Mycobacterium phlei.

A procedure for the isolation of a crude enzyme preparation of esterases fromMycobacterium phlei was worked out. The procedure consists in breaking cells in 1% KCl by ultrasonication, ultracentrifugation at 40,000 r.p.m. and isolation of acetone and ether dried enzyme preparation. Specific activity increased 2.8-fold after completion of the procedure. Esterases fromMycobacterium phlei were separated on Sephadex of G series to two enzymes with different substrate specificity. The first enzyme, acetic ester acetyl-hydrolase (E.C. 3.1.1.6) was found to be relatively specific for ethylacetate, the second, carboxylic ester hydrolase (E.C. 3.1.1.1) for ethylbutyrate and tributyrine. Preparations of both enzymes were made from the crude extracts of cells and from a mixture of macromolecular compounds isolated from the culture filtrate ofMycobacterium phlei.

Mutant strains of Streptomyces cinnamonensis protoplasts. Cultural and physiological conditions.

Mycelium ofStreptomyces cinnamonensis mutant strains cultivated in a synthetic medium with glycine produced protoplasts after lysis of cell walls with lysozyme. The protoplast yield was up to 95 %. The protoplasts could revert and mycelial forms were thus regenerated. In a sucrose-containing medium the protoplasts stored at 4 °C were stable for 2 d.Mycelium of Streptomyces cinnamonensis mutant strains cultivated in a synthetic medium with glycine produced protoplasts after lysis of cell walls with lysozyme. The protoplast yield was up to 95%. The protoplasts could revert and mycelial forms were thus regenerated. In a sucrose-containing medium the protoplasts stored at 4 degrees C were stable for 2 d.

Utilization of a lipid substrate for submerged fermentation ofStreptomyces albus

When investigating the effect of aeration on the utilization of a lipid substrate from the cultivation broth and production of salinomycin inStreptomyces albus it was demonstrated that a higher aeration results in a better utilization of soya oil and a higher production of the antibiotic.

Alizarin glucosyl transferase activity in Streptomyces aureofaciens mutants.

Alizarin glucosyl transferase activity was found in five mutant strains ofStreptomyces aureofaciens. The activity bears no direct relationship to the final products of tetracycline biosynthesis.

Esterases in mycobacteria. IV. The effect of macromolecular compounds isolated from a submerged culture of Mycobacterium phlei on the temporary activation of esterases.

Esterases ofMycobacterium phlei isolated by means of Sephadex G-100 chromatography could be temporarily activated by adding calcium ions. This activation could not be brought about in crude enzyme preparations from cells or in crude extracts from the culture filtrate. It was demonstrated that compounds (or a compound) without the esterase activity isolated after the separation of crude enzyme preparations on Sephadex G-100 (peak C) are responsible for the above difference. It was found that the suppression by compounds from the “C peak” of the temporary activation of esterases with calcium ions depends most probably on the ratio of these compounds to the quantity of the enzyme rather than on their concentration. In addition, the compounds of the C peak themselves were found to activate esterases. The activation was lower than the temporary activation with calcium ions. The possible mechanisms of the temporary activation of esterases and the importance of these findings, from the point of view of regulation of the activity of esterases during submerged cultivation ofMycobacterium phlei, are discussed.

Vitamins as effectors of monensin production by Streptomyces cinnamonensis

Vitamins added to submergedStreptomyces cinnamonensis cultures stimulated the production of monensins. Vitamins B2, B3, B5 and B12 enhanced the production by about 50%, vitamins B1 and B6 by 100%. The addition of biotin in optimal concentration resulted in more than 3-fold increase in total production.

Deacylation of acetylated amino acids by the esterase fromMycobacterium phlei

In addition to typical acetyl esters, acetic ester aoetyl-hydrolase fromMycobacterium phlei can degrade aliphatic acetylated amino acids under suitable conditions.