Production and Characterization of Lipase form Aspergillus oryzae ST11, Immobilization with Magnetic Nanoparticles and Nanofibers, and Application for Biodiesel Production

Abstract

Seven isolates of fungi were screened for the ability to produce an extracellular lipase. The tested isolates were cultivated in the submerged fermentation with the basal medium containing 1% (w/v) palm oil, 2% peptone, 0.2% NaNO3, KH2PO4, 0.05% MgSO4 and pH 6 at 37°C and 150 rpm for 3 days. Only isolate ST11 grew and produced an extracellular lipase (12.2 U/ml). The ST11 was selected for optimization of the lipase production. The optimum conditions for lipase production was 1% olive oil, 1% lactose, 2% peptone, 1% NaNO3 (w/v) at pH 6. The inoculum (10' spores/ml) was applied into 100 ml of the medium. The fermentation was carried out at 37°C and 150 rpm for 4 days. The lipase unit after optimization was 31 U/ml The molecular and morphological techniques were used to identify the selected fungus (ST11). In this study, the ITS 1/ITS4 primers were used for amplifying the ITS region. It showed that the ST11 belonged to Aspergillus group. However, this technique could not differentiate the A. flavus from A. oryzae. So, the fungal morphology was studied. The morphological growth of Aspergillus could be differentiated on the malt extract agar and Czapek yeast agar. From the observation of conidia and conidiophore of the fungus ST11, it confirmed that this isolate was Aspergillus oryzae. Therefore, it was designated as Aspergillus oryzae ST11. After cultivating 4. oryze ST11 in the optimized medium, the crude lipase was purified by chilled acetone precipitation, anion exchange column chromatography (Q-HiTrap) and hydrophobic column chromatography (Butyl Toyopearl 650M). The yield of the purified lipase was 7.9% with the 13 purification folds. The molecular mass of the purified lipase was 25 kDa. The purified lipase was stable between pH 7-8 with the optimum activity at pH 7.5, it showed a good stability in the temperature between 30- 37°C and the maximum activity was at 37°C. The activity drastically dropped at 55°C.For the effect of metal ions on the lipase activity, it showed that in the presence of Ca2+, K+ and Mg2+ promoted the lipase activity whereas in the presence of Hg2+, Zn2+ and Cu2+ decreased the lipase activity. For the effect of surfactants and inhibitors on the lipase activity, it showed that all surfactants tested including Triton X-100, Tween-20, Tween-80, SDS and Arabic gum decreased the lipase activity. In contrast, the inhibitors (EDTA, PMSF and B-mercaptoethanol) did not decrease the lipase activity. Next study was the effect of solvents on the lipase stability. The hydrophilic solvents such as methanol and ethanol showed the lower in the activity compared to the hydrophobic solvents such as isooctane and hexane. For the substrate specificity, many natural oils were tested, it showed that the olive oil gave the highest activity. The partially purified lipase was prepared by precipitation with chilled acetone. It was immobilized on different materials for biodiesel production. The first material was the magnetic nanoparticle. The lipase was immobilized via the cross- linked enzyme aggregate technique with bovine serum albumin (BSA) (6 mg/ml) and 20 mM glutaraldehyde. The immobilized lipase on magnetic nanoparticle produced the highest biodiesel conversion (95%) after 24 h of reaction with the stepwise addition of methanol. The immobilized lipase was reused for 5 times with 60% remained activity. The next material was the electrospun nanofibrous membrane prepared by co-solvent between polystyrene and trimethylolpropane tris [poly (propylene glycol) amine terminated] ether. The 0.15 U/mg-support was obtained at the optimum immobilization conditions. It was stable to the wide range of pH and temperature compared to the free lipase. The highest biodiesel conversion was 95% with stepwise addition of methanol. It retained the ability to produce biodiesel at 81% at the 10th cycle. The last material used for immobilization was the electropun polyacrylonitrile (PAN) nanofibrous membrane. The lipase was precipitated on the surface of modified PAN nanofibrous membrane by ammonium sulfate with the addition of BSA. After glutaraldehyde cross linking, the highest immobilized activity was 87% compared to 42% of non-BSA addition. The immobilized lipase on PAN nanofibrous membrane was used for biodiesel production and compared with the commercially immobilized lipase (Novozym 435). The biodiesel conversion from immobilized lipase was 95% which was higher than that of Novozym 435 (52%) at 24 h. Moreover, the immobilized lipase on PAN nanofibrous membrane catalyzed the transesterification reaction with the one step addition of methanol (3.5 mole of methanol per 1 mole of palm oil). The immobilized lipase retained 83% activity after 10 cycles of use. Aspergillus oryzae ST11 lipase shows the potential for biodiesel production. It could be used to replace the chemical catalysts. The lipase could be immobilized on many different materials for reducing the cost of the biodiesel production process. The use of lipase also reduced the environment problem from the waste of biodiesel production process.