Bacitracin production

A subsequent patent, U.S. Patent 2,828,246 described a commercial process for bacitracin production. A 1,230 gallon portion of a medium containing 10% soybean oil meal, 2.50% starch and 0.50% calcium carbonate having a pH of 7.0 was inoculated with a culture of bacitracin-producing bacteria of the Bacillus subtilis group and the inoculated medium incubated for a period of 24 hours with aeration such that the superficial air velocity was 12.1. An assay of the nutrient medium following the fermentation revealed a yield of bacitracin amounting to 323 units/ml. This was more than twice the yields previously obtained. 

Use Animal feeds, adhesives, medium for bacitracin production. 

The activity of bacitracin production was the highest when whole cells were immobilized in the gel prepared with 5 % total acrylamide (95 % of acrylamide monomer and 5 % BIS). On the other hand, no effect of BIS content on production of bacitracin was observed. However, the best productivity of bacitracin by immobilized whole cells was only 20-25 % of that by washed cells. These results suggest that the lower rate of bacitracin production is mainly caused by the inactivation of enzymes in whole cells with polymerization reagents (especially AA and APS) and may be partly due to hindered diffusion of the substrates and/or products through the gel. 

Effect of Air, Media, Bacteria Content on Bacitracin Production. The effect of air (oxygen) on bacitracin production by immobilized whole cells was examined. The incubation of immobilized whole cells was carried out under aerobic conditions and under nitrogen. Bacitracin produced under anaerobic condition was only 30 % of that under aerobic condition. 

Preliminary experiments indicated that the activity of bacitracin production by immobilized whole cells was remarkably reduced at the second reaction when a fermentation medium was used. Furthermore, the growth of leaked bacteria was observed when a fermentation medium (starch-bouillon) was employed for bacitracin production. Therefore, the effect of medium composition on bacitracin productivity was examined. As shown in Table III, bacitracin productivity in a medium... 

Since bacitracin production in the 1 % peptone medium was higher than that in the 0.5 % meat extract medium, the 1% peptone medium (containing 0.25 % saline and 1 x lO M MnSO-) was employed for further work. The growth of the leaked and contaminating bacteria was not observed during the incubation time. 

The effect of the cell content in the gel on the bacitracin production was examined. The rate of bacitracin production increased with increasing the amount of whole cells in the gel. 

A lag in bacitracin production was observed in the case of the gel containing the smallest amount of bacteria. However, the activity of each gel came to the same level with successive uses. 

Bacitracin Production from Amino Acids and ATP. Bacitracin... 

The results are shown in Figure 3. No activation of bacitracin production was observed under these conditions. The amount of bacitracin produced from amino acids and ATP was lower than that from 1 % peptone. 

The activity of bacitracin production increased with increasing use cycles of immobilized whole cells and reached a steady state maximum. Furthermore, apparent half-life of bacitracin production was estimated to be at least one week. 

On the other hand, the activation of bacitracin production was not observed when amino acids and ATP were used as substrates. 

In addition, the rate of activation increased with increasing amount of bacteria in the gel. These results suggested that the activation of bacitracin production was caused by the growth of bacteria in the gel. Therefore, the gel was examined by electron microscopy. 

The apparent growth of cells in the gel (especially at the surface layer) was observed after successive uses (total 56 h of reaction time) while the amount of cells evidently had decreased in the center of the gel. The diffusion of oxygen through the gel matrix may be a limiting factor of bacitracin production. The growth of the bacteria during incubation provides an obvious explanation for the increase in the activity of bacitracin production with successive uses. A steady state of bacitracin production could mean that the interstitial space of the gel was filled with active bacteria, or may be controlled by the diffusion rate of substrates and/or products through the gel. 

The rate of bacitracin production by immobilized whole cells was slightly higher than that of common fermentation. 

Figure 3. Bacitracin production from amino acids and ATP by immobilized cells (O) amino acids (2.5mM) and ATP (2mM),(%) % peptone.

Figure 4. Electron micrographs of immobilized cells in the gel (bacitracin production) (a) gel immediately after immobilization (b) gel after 56-hr incubation.

As a model system we would like to describe the biosynthesis of bacitracin, a commercially important peptide antibiotic. Its mechanism of formation has been studied in our laboratory as well as by others. A cell free system for bacitracin production by B. Hcheniformis ATCC 10716 was reported by Ishihara et al.(29). The methods used were modified (Ref. 31) and a partially purified enzyme complex which performed de novo synthesis of bacitracin from the L-isomers of the constituent amino acids, ATP and Mg2 was isolated (Ref. 31 - 34). D-glutamic acid and D-phenylala-nine which occur in bacitracin support its synthesis (Ref. 31). This was also the case for D-aspartic acid (unpublished results). 

Amin, A., Khan, MA., and Ahmad, T. (2013) Optimized antimicrobial peptide (Bacitracin) production by immobilized and free cells and of Bacillus spp. GU215 using Wood chips and silicon polymer beads. Pak. J. Pharm Sci., 26, 1077-1082. 

Under various conditions, whole cells of strain A-5 appear to produce only 5 to 10% of the quantity of bacitracin formed by cells of strain 10716 (Bernlohr and Novelli, i960, 1963 Snoke, 196O and Snoke and Cornell, 1965). However, the biosynthetic capacity of the two strains has not been directly compared to determine to what extent differences in conditions of culture and assay in different laboratories might be responsible for the apparent disparity in yield. Published information concerning possible variation in bacitracin yield among large numbers of strains of B. licheniformis is not available (Hickey, 1964). In view of possible associations between bacitracin production and sporu-lation (vide infra), it would be of interest to determine if asporogenous mutants form more, or less, antibiotic than do wild strains. 

Bernlohr, R. W., and G. D. Novelli Some characteristics of bacitracin production by Bacillus licheniformis. Arch. Biochem. Biophys. 87, 232 (i960). 

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