Methodology Design Essay

849 words - 4 pages

The experimental design of this research can be divided into five main stages. The first stage is development of yeast cell colony on nutrient agar and inoculum preparation of Candida rugosa ATCC 14830. The microorganism used in this research is isolated from natural soil. Second stage is extraction of lipase enzyme from the growth medium that contain olive oil that act as a substrate. Third stage of this project is preparation of immobilized and free enzyme culture system. The immobilized enzyme culture system is prepared with Ramsay medium as fermentation medium and calcium alginate gel beads as immobilized support. The next stage is data analysis on lipase activity on both culture ...view middle of the document...

The flask containing inoculum was shaken at 200 rpm at 30°C for about 10 hours to get the 0.7-o.8 of OD, which read at 600 nm of wavelength.
Second stage is extraction of lipase enzyme from the growth medium that contain olive oil that act as a substrate. Lipase is extracted from the production medium after 48 hours of incubation time by centrifugation at 150 rpm for 30 min in a refrigerated centrifuge. The supernatant obtained contain extracellular lipase. To release the intracellular lipase, harvested cells were suspended in lysis buffer (0.05 M phosphate buffer, pH 7) and subjected to five rounds of cell disruption around 1 minute with the help of for recovery of maximum enzyme. The suspension was centrifuged rpm for 30 minutes and cell free lipase extract was collected.
Third stage of this project is preparation of immobilized and free enzyme culture system. The immobilized enzyme culture system is prepared with Ramsay medium as fermentation medium and calcium alginate gel beads as immobilized support. Lipase solution was mixed with sodium and then the mixture was stirred thoroughly to ensure complete mixing. As soon as the mixed solution was dripped into calcium chloride solution with a syringe, Calcium-alginate beads were formed. After 20 minutes of hardening the beads were collected by filtration, then washed with tris-HCl buffer (0.2 M pH 7.5) several time to remove the unbound enzyme (Won et al., 2005). The immobilized lipase and free lipase were added into 250 mL flask. The flasks were shaken...

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