Bacterial Cellulase from a Local Isolate, Bacillus Pumilus EB3
Ariffin, Hidayah (2006) Bacterial Cellulase from a Local Isolate, Bacillus Pumilus EB3. Masters thesis, Universiti Putra Malaysia.
Cellulase production from bacteria can be an advantage as the enzyme production rate is normally higher due to bacterial high growth rate. Screening of bacteria, optimisation of fermentation conditions and selection of substrates are important for the successful production of cellulase. This study is conducted to produce cellulase from our local isolate Bacillus pumilus EB3, using oil palm empty fruit bunch (EFB) and carboxymethyl cellulose (CMC) as substrate. The effect of physical, chemical and thermal pretreatment on the EFB chemical composition and physical structure was studied, aimed at reducing lignin and hemicellulose, and making EFB structure more amorphous. The effect of pretreatment on reducing sugars production using commercial cellulase (Celluclast 1.5L) was also examined. Production of cellulase was conducted in shake flask and 2L stirred tank reactor (STR). The effect of initial pH, temperature, nitrogen source and carbon source on cellulase production in the shake flask was investigated. Following that, cellulase produced from B. pumilus EB3 was purified using ion exchange chromatography with anion exchanger (HiTrap QXL) for characterisation of the cellulase. The results of EFB pretreatment revealed that combination of pretreatments involving physical, chemical and thermal treatment was most suitable to affect the chemical composition and physical structure of the EFB. Initial cellulose, hemicellulose and lignin content in untreated EFB were 51%, 28% and 15% respectively. After combination of pretreatments, the cellulose composition increased to 67% while hemicellulose and lignin content decreased to 17% and 10% respectively. The physical structure of the EFB was altered after pretreatments as based on the SEM micrograph. Alteration of EFB was due to removal of lignin and hemicellulose. Combination of pretreatments increased the hydrolysis of the EFB with the yield of 0.53 g reducing sugars / g EFB as compared to the untreated EFB where only 0.07 g reducing sugars being produced from 1 g of EFB. Study on cellulase production confirmed that fermentation parameters such as initial pH, temperature, carbon source and nitrogen source affected cellulase production. Cellulase from B. pumilus EB3 was found to be secreted the most at temperature 37°C, initial pH 7.0, 1% CMC as carbon source and 2 g/L of yeast extract as organic nitrogen source. The activity recorded during the fermentation was 0.006 U/mL, 0.076 U/mL and 0.032 U/mL respectively for FPase, CMCase and β-glucosidase. As production in the shake flask showed that EFB gave a competitive cellulase production as CMC, EFB was tested as carbon source in 2L STR. Due to hydrophobic characteristic of the treated EFB, the experiment was not so successful. Comparison of cellulase production using CMC as substrate in shake flask and 2L STR revealed that cellulase productivity was higher in the 2L STR than in the shake flask although overall, the maximum cellulase activity recorded was almost similar. Purification of cellulase from B. pumilus EB3 using ion exchange chromatography showed that 98.7% of total CMCase was recovered. Protein separation was however based on subtractive separation where the contaminants were bound to the column instead of CMCase. Characterisation of the enzyme found that CMCase from B. pumilus EB3 has a molecular weight range from 30-65 kDa and was optimally active at pH 6.0 and temperature 60°C. The CMCase also retained its activity over a wide pH range (pH 5.0–9.0) and temperature range (30-70°C).
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