Characteristics and application potential of an alchhol oxidase from the hydrocarbondegrading fungus aspergillus terrrus
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A hydrocarbon-degrading Aspergillus terreus was isolated from the oil contaminated soil. The organism could degrade a wide range of petroleum hydrocarbons including the immediate oxidation products of hydrocarbons, like alkanols and alkanals. Among all the linear chain carbon substrates, highest growth of 39 D 4 g lD1 (wet weight) was observed when n-hexadecane was used as the sole source of carbon and energy. Using SEM the morphological change of the hyphae during growth of the fungi on hydrocarbon substrates was demonstrated. In glucosegrown cells the hyphae were smooth with a thick cell wall, whereas, the cell wall of the n-hexadecane cells was uneven and thin. The adaptation of the cells for up taking the hydrophobic hydrocarbon substrates through sorption mechanism and accumulation of high lipid content in the cells, as evident from the nearly seven-fold more lipid production (4.4 g%) in the n-hexadecane-grown cells than the glucosegrown cells (0.62 g%), were attributed to the observed morphological change. Analyses of the fatty acid profile by ESI/MS in the isolated lipid showed that nine different fatty acids induced in the hexadecane-grown cells were void in the glucosegrown cells. The oleic acid and palmitic acid were the fatty acids with highest peak intensity in the spectral profile corresponding to the glucose-grown cells and hexadecane-grown cells, respectively. It was revealed by analyzing the percentage distribution of different fatty acids in each cell type that palmitic and stearic acids were the predominant fatty acids in the hexadecane-grown cells; whereas, oleic and linoleic acids were the predominant fatty acids observed in the glucose-grown cells. Unlike glucose-grown cells, considerably high amount of fatty acids with chain length C32 and C33 were present in n-hexadecane-grown cells and each accounted nearly 9% (based on peak intensity) of the total fatty acid content in the cells. A microsomal membrane bound alcohol oxidase enzyme was isolated from this fungus during its growth on n-hexadecane substrate. This oxidase could oxidize short chain alcohol-, iii long chain alcohol-, secondary alcohol-, and aryl alcohol- substrates and was localized in the microsomes of the cell. The optimal pH and temperature of the enzyme were found to be 8.1 D 0.5 and 27-33 oC, respectively. The stability of the alcohol oxidase was drastically decreased beyond 30 oC. The enzyme showed 33% enantiomeric excess for the R(-)2-octanol over S(+)2-octanol, which may be correlated with the lower Km values of the enzyme for the R(-)2-octanol than the S(+)2-octanol. The fluorescence emission spectrum of the protein (at 443 nm excitation) was similar to that obtained with authentic FAD. In this flavoenzyme, flavin was non-covalently but avidly associated. The native protein molecular mass was 269 D 5kDa and the subunit molecular masses were 85-, 63-, 43-, 27-, and 13- kDa. The enzyme showed highest affinity for n-heptanol (Km = 0.498 mM, Kcat = 2.7 x 102 s-1). The isoelectric point of the proteins was within 8.3-8.5. High aggregating property of the protein was demonstrated by AFM, DLS and TEM analyses. Chemical analysis showed the presence of oleic acid and palmitic acid at a ratio of 2:1 in the purified protein. The high aggregating property of the alcohol oxidase may be correlated with the observed lipoidic nat...
Supervisor: Pranab Goswami
BIOSCIENCES AND BIOENGINEERING