59-05-032 Proceeding

52 Proceedings of the Princess Maha Chakri Sirindhorn Congress C. Cellulase activity assay Dinitrosalicylic acid (DNS) assaywas performedwith the standardmethod to determine amounts of reducing sugars in the samples [7]. The reducing sugar amounts of hydrolysates were observed by using the spectrophotometer at 540 nm. Endoglucanase (CMCase) activity was determined by using CMC as substrate. 0.2% w/v of substrates were mixed in 500 µl of 50 mM phosphate buffer (pH 7.0) and 500 ml of each enzyme fraction. Each samples were incubated in shaking incubator at 45°C for 2 h, 200 rpm according to standard cellulase assay [8]. Then the hydrolysis reaction was stopped by heating the sample to 95°C for 10 min. The amounts of reducing sugars in the samples were calculated based on the relationship of absorbance value and glucose amount in the glucose standard curve based on DNS assay. III. Results and Discussion The extracellular fraction of Bacillus sp. strain MSL2-S2 culture grown in CMC broth media was collected as crude enzyme. Then the enzyme was concentrated by using ammonium precipitation method. The dialyzed enzymes were loaded into sephacryl S-100 HR column. Each enzyme fractions were collected separately and aliquoted to test their activities with CMC substrates. The absorbance from DNS assay of each fractions were observed as shown in Fig. 1. From total 30 cellulase fractions, fraction NO. 13 has the highest absorbance at 540 nm compared to other fractions suggesting that it has the highest CMCase activity. Therefore, fraction NO. 13 was selected to be used in the next experiment. Figure 1 The CMCase activity of Bacillus sp. strain MSL2-S2 in each cellulase fraction. Then fraction NO. 13 was concentrated again by using concentrator spin column with MWCO 10 kDa. The size of enzyme molecule of concentrated NO. 13 sample was determined in SDS-PAGE analysis. In the same time, the concentrated NO. 13 sample was loaded in CMC- zymogram gel to confirm CMCase activity in situ (Figure 2). In SDS-gel, a single protein band was observed at 50 kDa molecular weight, and at the same position, a clear band in congo-red background was also detected. This result suggested that a 50 kDa protein in the fraction NO. 13 has CMCase activity.