SCREENING OF XYLANASE PRODUCING Bacillus spp. AND CHARACTERIZATION OF XYLANASE FROM SELECTED ISOLATE (Bacillus circulans)

• Main Author: SEPTININGRUM (NIM 20605007), KRISNA
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/10781
• Institution: Institut Teknologi Bandung

<p align="justify">Xylan is the major constituent of hemicelluloses and is the second most abundant renewable resource with a high potential for hydrolysis to useful end product. Xylanases (1,4-B-D-xylan xylanohydrolase, EC 3.2.1.8) are the preferred catalysts for xylan hydrolysis. One of the primary applications of these catalysts is in pre-bleaching stage in pulp and paper industry. Several Bacillus has the ability to produce thermostable and alkalistable xylanase. The objectives of this experiment are to obtain bacteria with the highest xylanase activity and lowest cellulase from the genus Bacillus and characterization of xylanase from the selected species. Bacterial screening using qualitative and quantitative methods was performed on eight different bacteria (B. cereus, B. circulans, B. firmus, Bac termofil, Bacillus termofilik RP1, B. circulans termofilik, B. stearothermophilus and Ko1 isolate). The chosen isolate was cultivated in xylan medium and its growth pattern and activity curve was made. The extracellular enzyme was partially purified by fractionation steps using ammonium sulphate (20-40% saturation) and ion exchange chromatography DEAE-ToyoPEARL. Optimum pH, temperature and enzyme kinetics of the partially purified enzyme were determined. The result of qualitative screening showed xylanase/cellulase ratio of B. cereus, B. circulans and Ko1 isolate have the highest ratio than other isolates with ratio values 3,280; 7,278 and 3,857. The results of the qualitative and quantitative screening showed Bacillus circulans was able to produce xylanase with the lowest cellulase activity. The age of the inoculum and production time of xylanase from B. circulans was 18 hours, with a specific activity 17,21 U/mg. Fractionation of the enzyme using ammonium sulphate (20-40% saturation) showed an increase in specific activity (585,12 U/mg) with 34 fold purification. Enzyme purification using ion exchange chromatography showed an increase in specific activity (805,48 U/mg) with 46,8 fold purification. Enzyme characterization of the partially purified enzyme showed an optimum pH 9,5 with optimum temperatures 50o and 80oC. These results indicated that optimum pH from partially purified xylanase showed highest optimum pH than xylanase from B. circulans AB 16 (Xyl A and Xyl B) (6,0-6,5), but showed the same temperature chacterictic (75o-80o C). The enzyme kinetics of the xylanase from B. circulans did not obey the Michaelis-Menten equation. Partially purified xylanase from B. circulans exhibited favorable potential for pre-bleaching stage in pulp and paper industry because of its activity in alkaline pH and high temperature.