59-05-032 Proceeding

51 Proceedings of the Princess Maha Chakri Sirindhorn Congress rate and can produce high efficiency cellulase [4]. Moreover, bacteria is amenable to be genetically modified to have specific characters that can be applied in various industrial applications [5]. Previously, in our lab, the Bacillus sp. strain MSL2-S2 was newly isolated from screening of cellulase-producing bacteria at 60 oC [6]. Here, this research focused on the purification and characterization of a cellulase enzyme produced by Bacillus sp. strain MSL2-S2. II. Materials and Methods A. Production of extracellular cellulase enzyme Bacillus sp . strain MSL2-S2 was previously screened and isolated from soil samples in the rice paddle field in Ayutthaya province, Thailand. This bacterium is able to grow in carboxymethyl cellulose (CMC) agar plates (0.5% CMC, 0.1% NaNO 3 , 0.1% K 2 HPO 4 , 0.1% KCl, 0.05% MgSO 4 , 0.05% yeast extract, 1.5% agar) at 60 o C. Therefore, it can be categorized to be a thermo-tolerance bacterium. The Bacillus sp. strainMSL2-S2 was inoculated in 500 ml of CMC broth and incubated in shaking incubator at 45˚C for 48 h, 200 rpm. Crude extracellular cellulase was collected by centrifugation at 5,000 rpm for 15 min to obtain the cell-free supernatant. B. Purification of cellulase enzyme The crude cellulase was precipitated by adding ammonium sulfate to 80% saturation and kept in 4˚C for 24 h. The cellulase precipitates were collected by centrifugation at 6,000 rpm for 15 min at 4˚C then dissolved in 5 ml sodium phosphate buffer (pH 7.0) followed by dialysis for 24 h. The concentrated protein was collected by using spin column with 10 kDa MWCO. The concentrated cellulase was separated by using sephacryl S-100 HR column. The column was equilibrated by 50 mM sodium phosphate buffer (pH 7.0). The fraction was collected at 1 ml/min flow rate. Concentration cellulase fraction was analyzed by 12% gel SDS-PAGE to determine the molecular weight of purified enzyme sample by comparing with standard proteinmarker (BLUeye Protein Ladder, RBC Bioscience, China). Protein samples were stained with Coomassie Brilliant Blue R-250. To generate the zymogram pattern, concentrated cellulase fraction was applied to 12% gel SDS-PAGE (containing 1% CMC). Then, SDS was removed by soaking the gel in wash buffer (30mMsodiumphosphate buffer containing 40% isopropanol (pH7.2)) for 1 h.The gel was soaked in equilibrated buffer (30 mM sodium phosphate buffer (pH 7.2)) for 1 h, and transferred into renaturing buffer (30 mM sodium phosphate buffer, 5 mM β -mercaptoethanol, 1 mMEDTA (pH 7.2)) at 4 oC overnight. The renatured gel was stained with 1% congo red solution for 15 min, and washed with 1MNaCl solution.The band of cellulase was seen as a clear zone against background.