การผลิตและสมบัติของสารลดแรงตึงผิวชีวภาพจาก Bacillus subtilis BBK-1 / นิรันดร์ รุ่งสว่าง = Production and characterization of biosurfactant from Bacillus subtilis BBK-1 / Niran Roongsawang
Halotolerant biosurfactant producing bacteria were isolated from various sources including soil, sand, marine water and fermented food. Among these, a strain designated BBK-1 which was later identified as Bacillus subtilis gave the best biosurfactant activity. The strain gave high biosurfactant yield upon cultivated by using 0.5% w/v sucrose as carbon source, 0.2% w/v NH4NO3 as nitrogen source, 0.5% w/v yeast extract as notrogen source and trace mineral, 3% w/v Nacl, pH 7.5 at room temperature (30+-2 ํC), 200 rpm agitation for 24 hours. Under such condition Bacillus subtilis BBK-1 could produce biosurfactant with a relative biosurfactants concentration at 40. The methanol extracted biosurfactant is found stable to temperature ranging from 50 to 100 ํC upto 5 hours while at 121 ํC for 30 min. The biosurfactant showed activity in the present of 15% w/v NaCl as well as to pH from 5.0-10.0. The CMC value of product was at 12 mg/l which was lower than that of chemical surfactants such as sodium dodecyl sulfate, cetylpyridinium chloride, tween 80 and triton X-100. The results from HPLC, LC-MS and amino acid composition analyses indicated that Bacillus subtilis BBK-1 produced 3 types of lipopeptide biosurfactants identified as bacillomycin L, plipastatin and surfactin. Regulatory gene for biosynthesis of these biosurfactants in Bacillus subtilis BBK-1 was cloned by Southern and colony hybridization using sfp ํ gene from surfactin nonproducing Bacillus subtilis MI113 as probe. The recombinant plasmid obtained containing 4 kb-DNA insert which was the EcoRI fragment of the BBK-1 chromosomal DNA could transform Bacillus subtilis MI113 to a surfacin producing ability of Bacillus subtilis MI113. Nucleotide sequence between SacI site was determined upon which a one large open reading frame (672 bp., 224 amino acid residues) was identified. The deduced amino acid sequence of this open reading frame shares high identity with regulatory protein of lipopeptide biosurfactants production in Bacillus subtilis strains.