Optimal Conditions for Extraction and Purification of Penicillinase Enzyme

 The present study aimed to evaluate the optimum conditions for extracting and purifying penicillinase enzyme. The enzyme source was Bacillus licheniformis 6346 obtained from the Iranian Research Organization for Science and Technology.

 B. licheniformis was cultured in a medium containing mineral salts, nitrogen compounds, and cultured carbon sources. Following the harvesting and preparation of bacterial suspension, the destruction of the bacterial cell wall and crude cell extraction was completed using a combination of lysozyme and weak ultrasonication. Afterward, enzyme amount was determined utilizing chromatography and electrophoresis. Furthermore, the optimum growth temperature and incubation time were assessed based on the absorbance of samples at 240 nm, enzyme activity, aerification intensity optimization, enzyme production time, and optimum pH.

 Approximately 8 g of cells were retrieved in one liter of culture. The optimum pH was determined as 6.8. The heat stability evaluation of the enzyme at different times revealed that the enzyme was stable for more than 1 h at 20°C-45°C, while it is inactivated in 10 min at 60°C. Moreover, the best incubation time for this bacterium was obtained as 9 h at 30°C. In terms of aerification intensity, the highest enzyme production rate of 2394 U/mL was achieved in a 250 mL Erlenmeyer flask with a 50 mL culture medium at 200 RPM. More aerification led to the inhibitory effect of oxygen and reduced enzyme activity.

 Our findings showed that the bacterium B. licheniformis 6346 can be used for producing penicillinase enzyme in optimum conditions.