| Lipase, which catalyses the hydrolysis of long chain triglycerides played an important role in industrial catalyzation. It is widely used in the hydrolyzation, alcohol sis and synthesis of ester interface of oil-water and Glycerol and fatty acidsbe produced. Lipase is widely used in food, medicine, scour, leather and paper industry. Many researches have been carried out on resources of domestic and foreign barley lipase. Recently, because of the increased demand for lipase in dometic and the defect of low production, few species, relying on imports of lipase, screening and selection strains of high yield advantage for the future for the industrial production of lipase has some significance.Therefore, In this paper, screening of lipase-producing strains, optimization of fermentation conditions, mutation breeding, and other aspects of enzymatic properties of lipase were carried out. Then we collect matericals and provide evidences for the breeding work and molecular studying. The main results of this paper were as follows.1. A fungi which produces alkaline lipase was isolated from soil on a selective medium that contained olive oil as the only source of carbon from Ya'an. The lipase activity reached maximal level of 6.40U/mL. According to the morphological, physiological, biochemical identification and ITS sequence analysis, the strain belongs to Penicillium chrysogenum and named Penicillium chrysogenum-J23.2. Single factor experiments, orthogonal design, Plackeet-Burman design and the corresponding surface analysis are used in optimization experiments.The optimization experiment about fermentation medium composition and culture have showed that the optimum medium composition and cultural condition were:sucrose 0.1%, peptone 2.0%, olive oil 1.27%, soybean meal 2.97%, K2HPO40.75%, NaH2PO4 0.1%, MgSO4·7H2O 2.06%, NaH2PO4 0.1%, Tween80 0.5%, pH 9.0. The initial inoculation volume was 1.0%, speed of agitation was 200 r/min, incubation time was 48 h, the optimal temperature of the lipase were 30℃. Under such conditions, the lipase activity in culture supernatant was 2.21 times high as the original one.3. In this research work, two ways of mutagenesis treatment, such as UV-irradiation, MW-irradiation, were carried to study the effectiveness of increasing the productivity of lipase-producing strain P. Chrysogenum-J23 which was conserved by our laboratory. Finally, seven mildew whose diameter ratio between digest zone and colony were found, and the high yield mutant(obtained from UV-irradiation)was selected and produced alkaline lipase activity 53.8% higher than that of parent strain. Stability test demonstrated that the lipase activity remained the same level after five generations.4. Lipase characterization showed that:its maximal activity was observed at 33℃and pH 7.5. The lipase was highly stable in the pH range from 6.0 to 10.0, and the enzyme activity kept at 30% when the lipase was preserved at 50℃. High concentration (50mmol/L) of common detergent ions as Ca2+,Mg2+,K+could activate the lipase produced by J23. However, Fe2+,Mn2+,Cu2+,Pb2+,Li2+could inhibit it. It indicated that lipases from P. chrysogenum-J23 would be applied widely in as it showed high thermo stabilities, alkaline and stabilities of wide pH range (from 6.0 to 10.0)... |
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