Production and characterization of lipase from local fungal isolate by solid-state fermentation

number: 
755
English
Degree: 
Imprint: 
Biotechnology
Author: 
Thikra A. Al-Wahab Mustafa
Supervisor: 
Dr. Khulood W. Al-Samarraei
Dr. Shatha S. . Al-Azawi
year: 
2002
Abstract:

Fourty five local fungal isolates belong to different species were tested for their ability to produce lipase, one of these isolates was selected as the best lipase producer.lt was identified as a strain of Aspergillus oryzae (T4). The optimum conditions for the production of the lipase by solid-state fermentation included culturing of the fungus on cotton seed meal hydrated with 1% ammonium sulfate and 1% olive oil as production medium 'with an initial pH 7.0 with 1:2 (w/v) hydration ratio and incubated for 7 days at 30 °c. The best extraction solution for the enzyme was 0.2M phosphate buffer pH 7.0, the productivity reached (2U/g) of dry weight under these conditions. The enzyme purification was carried out through two steps including concentration the enzyme with 80% ammonium sulfate saturation followed by gel filtration using Sephadex G-75.The overall purification fold was 88.7 with 23% yield. Some characters of partially purified enzyme were studied, the optimum pH for enzyme activity was 8.0, the enzyme is more stable at pH (5-8). the maximum enzyme activity was observed at 35 °c and the optimum thermal stability of the enzyme was at (30-35°c), the enzyme returned more than 60% of its activity at 50°c for 30 min. Treatment of the enzyme with heavy metal ions copper, ferric, zinc and mercuric ions at 5mM concentration, different levels of inhibition were observed ranging from (39%-85%). The chelating agent such as EDTA in a concentration of 2 and 5 mM and the reducing agents, cysteine and 2- mercaptoethnol have no effect on the enzyme activity, while the activity of the enzyme decreased after treatment with PMSF, this results indicated that the lipase ofA.oryzae T4 is a serine lipase. The enzyme was able to hydrolyze different kinds of oils, a higher enzyme activity appeared toward olive oil and sesame oil(6U/ml). The enzyme gave high activity when the substrate was emulsified with gum acacia (5.1U/ml) and PVA (4.8U/ml) comparing with sodium taurocholate (2.6U/ml).