Dissertation
Dissertation > Industrial Technology > Chemical Industry > Other chemical industries > Fermentation industry > Enzyme preparation ( enzyme ) > Lipase

Studies on Fermentation Optimization, Purification and Enzyme Characteristics of Lipase from Aspergillus Oryzae FS-1

Author DengLinFen
Tutor ZhengYi
School Fujian Normal University
Course Biochemistry and Molecular Biology
Keywords Aspergillus oryzae lipase fermentation optimization purification enzyme characteristics
CLC TQ925.6
Type Master's thesis
Year 2011
Downloads 22
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The fermentation medium of Aspergillus oryzae FS-1 producing lipase was optimized.The single-factor test was carried out and the result indicated that the optimum carbon source was corn flour, nitrogen source was peptone. Then the Plackett-Burman design was taken to investigate which factors contributed to lipase fermentation most and 3 most significant influence factors were found, those were peptone, olive oil and Na2HPO4. In the following experiments, Response surface methodology of Box-Behnken design was carried out to optimize the 3 significant factors and the optimum levels of those were found. The formulation of the optimum fermentation medium(g/L) was corn flour 0.5, peptone 50, olive oil 15, NaNO3 10, Na2HPO42.5, KCl 0.5, MgSO4 0.5, FeSO4 0.01. The optimum fermentation condition was obtained: the optimal amount of inoculation was 2%, the optimum initial pH was 6.8 and the optimal liquid volume were 50mL culture in 250mL shake flasks, the fermentation temperature was 28℃, the optimum initial fermentation pH was 8.0, the fermentation time was 48 hours. After the optimization, the fermentation level of lipase was increased from 3.25U/mL to 18.75U/mL.The purification of FS-1 lipase was carried out. Collecting fermentation supern atant fluid, The proteinase was purified by using ammonium sulfate, dialysis and DEAE Sepharose Fast Flow. After these purification processes, the purification mul-tiple reached 9.21 and the rate of recovery was 26.39%. Its fineness was checked to be about a single band by SDS-PAGE.Enzyme characteristics research indicated that the best enzymatic reaction temperature was 36℃, the enzymatic reaction was relative stable under 40℃; the optimum pH of enzymatic reaction was 7.0, enzymatic reaction was stable between pH 3.0-9.0. Olive oil and glycerol tributyrate were used as the substrate and the enzymatic reaction fit the Michealis equation, the maximum reaction initial velocity Vm were 23.70μmol/mL/min and 24.33μmol/mL/min. Michaelis constant (Km) were 48.44μmol/mL and 20.43 u mol/mL each. The activation energy(Ea) were 41.78 kJ/mol and 31.68kJ/mol.Research on the effects of several effectors on the FS-1 liapse activity, (1)the metal ion’s influence on the enzyme activity indicated that Na+, K+, Li+, Ba2+ion had no any effects on the enzyme activity, Ca2+ion had a few stimulative effects on the enzyme activity, while Mg2+, Cu2+, Fe2+, Hg2+, Fe3+, Al3+inhibited the enzyme.(2) Organic solvent influence on the enzyme activity indicated that methonal, ethanol, formaldehyde and isopropyl alcohol inhibited the enzyme. Formaldehyde is most strong inhibition. N-pentane had no effect on the enzyme.(3)Chemically modified agents, include cis-butenedioic, monobromo-acetic, acet-imidazole,β-mercaptoethanol, acetylacetone, chloramines-T, N-bromo-succinimide and benzyl sulfonicacid fluoride, influence on the enzyme activity showed thatβ-mercaptoethanol and benzyl sulfonicacid fluoride inhibited the enzyme. Preliminary judgment was that methionine residues and serine residues closely related with catalytic activity.

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