Effect Of Environmental Growth Conditions On Antimicrobial Protein Produced By Bacillus Subtilis Atcc21332 In The Presence Of Cymbopogon Flexuosus Essential Oil

Abstract

Exposure of bacteria towards environmental stress factors such as a mild concentration of antimicrobial substances could affect the bacterial growth. The bacteria cells may activate the target genes that are related to stress responses pathway in which will further enhance the secretion of proteins. However, the bacterial growth and the bioactive proteins synthesis rate are also depended on other factors, including pH and nutrients availability. Therefore, this present study focused on the effect of environmental stress factors (Cymbopogon Flexuosus essential oil) and different growth conditions (pH and nutritional factors) onBacillopeptidase F or bioactive proteins secreted by B. subtilis ATCC21332. The bacterial cells were cultivated in media with 0.01 MIC of C. flexuosus esselitial oil as stress inducer and grown under different pH (pH 6,7 and 8) and 1 % (w/w) of nutritional factors, including carbon (glucose, sucrose and starch), nitrogen (urea, casein, gelatin) and inorganic salts (calcium chloride, sodium nitrate and sodium dihydrogen phosphate) sources by one-variable- at-a-time approach or combination of two and four parameters. SDS-PAGE analysis showed that new extracellular proteins with approximate size of 30.43 kDa, 30.66 kDa, 3 1.92 kDa, 3 1.35 kDa and 30.80 kDa were synthesized by B. subtilis ATCC21332 after being exposed to C. .flex-trostls essential oil and grown in each different media consist of either starch, gelatin, casein, sodium dihydrogen phosphate or calcium chloride. Meanwhile, stressed induced B. subtilis ATCC21332 cultured in two growth parameters (pH 6 and differ nutritional factors) produced Bacillopeptidase F only. However, stressed induced B. subtilis ATCC21332 cultured in four growth parameters (pH 6, starch, calcium chloride and gelatin) secreted 32 kDa new protein. MALDI-TOFITOF analysis on the 32 kDa protein produced was recognized as Vpr and Bacillopeptidase F. The extracellular proteins produced by B. subtilis ATCC21332 revealed antimicrobial activity against Gram positive bacteria (B. cereus and S. epidermidis) compared to Gram-negative bacteria. Hence, B. subtilis ATCC21332 in the presence of C.flexuosus essential oil as stress inducer and cultured in various growth condition by one-variable-at-a-time approach as well as combination of two and four parameters could enhance the secretion of Bacillopeptidase F and new extracellular proteins with antimicrobial activity.