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.

Antimicrobials agents at low concentration could stress out bacteria by inducing proteins production via regulating the transcription and translation process. The interruption of bacterial quorum sensing (QS), or cell-to-cell communication is known to have the potential to weaken the bacterial pathogenicity by inhibiting their communication. This study aims to explore the potential of synthetic antimicrobial compound, ethyl pentanoate in stimulating proteins production by Bacillus subtilis ATCC11774 as well as to determine the anti-QS activity of microbial proteins produced. The bacterial cells were exposed to 0.01 MIC of ethyl pentanoate in fermentation process at 37°C for 48 h and 72 h respectively. The proteins produced were further isolated and analyzed by using Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). Results showed that a new extracellular protein with approximate size of 15 kDa was produced by B. subtilis ATCC11774 after being treated with ethyl pentanoate at 37°C for 48 h and 72h. Despites of new protein band production, there was a deletion of protein band with approximate size of 18 kDa on protein synthesized at 72 h of fermentation. Whilst, the anti-QS activity of microbial proteins produced by B. subtilis at 37°C for 48 h and 72 h was determined by agar- wells diffusion assay, resulting “halo” inhibition zone ranged from 10.00 ± 1.00 to 10.33 ± 0.56 in diameter. Therefore, B. subtilis ATCC11774 in the presence of ethyl pentanoate at 0.01 MIC could regulate the extracellular protein production and expression. The isolated protein also exhibited anti-QS activity.

Blood clotting is an emerging problem that seriously threatens the health of human beings such as strokes and heart attacks. For the treatment, anti-coagulants are used in which it could prevent and interrupt the process involved in the formation of blood clots. Microbial proteases have now been attracted since it is cost effective and no side effects than typical anti-coagulant agents. The protease enzymes were discovered from many microorganisms including Bacillus sp. Previous study reported that B. subtilis could produce a serine protease, known as Bacillopeptidase F with anti-coagulant activity. However, study on anti-coagulant activity of microbial protein secreted by B. subtilis after being induced with Cymbopogonflexuosus essential oil and cultured at difference pH media is still limited. In this present study, B. subtilis ATCC21332 cells were treated with a low concentration (0.01 MIC) of C. flexuosus essential oil and cultured in difference pH media (pH 6, pH 7 and pH 8) at 30°C for 72 h of fermentation. The extracellular proteins were then extracted and precipitated by using 80% of ammonium sulfate before being further identified using Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis(SDS-PAGE) and tested for anti-coagulant activity. SDS-PAGE analysis exhibited a proteins profile with a band in approximate size of 30 kDa was appeared for the treated bacteria with C. flexuosus essential oil and cultivated at three difference pH of media. The extracellular proteins secreted by B. subtilis ATCC21332 after being treated with 0.01 MIC of C. flexuosus essential oil and cultivated either at pH 6 or pH 7 or pH 8 could also prevent blood clotting. However, the extracellular proteins produced by B. subtilis ATCC21332 without inducing with 0.01 MIC of C. flexuosus essential oil only exhibited anti-coagulant activity when the bacterial cells were cultured at pH 7. As a conclusion, B. subtilis ATCC21332 can be enhanced to produce anti-coagulant enzymes after being treated with low concentration of C. flexuosus essential oil and cultivated in natural pH media or even in slightly basic or slightly acidic environment. Further study should be done to purify, identify and analyze the anti-coagulant enzymes from B. subtilis ATCC21332. Keywords: Cymbopogonflexuosus Essential Oil, pH effect, Anti-Coagulants Activity, Extracellular Proteins, Bacillus subtilis ATCC21332.