FANKI ANNET 17/U/6987/CHE/PE MUTAWATA STELLA 17/U/15117/CHD/GV CHELANGAT MAUREEN 17/U/6982/CHE/PE NKUGWA MARK WILLIAM 17/U/7029/CHE/PE SEGAWA JOHN 17/U/15118/CHD/GV IROTA EMMANUEL JOSHUA 17/U/6988/CHE/PE EGESSA ANTHONY 17/U/15116/CHD/GV ATUHWERE ANDREW 17/U/6975/CHE/PE AHEREZA JANITH 17/U/6967/CHE/PE NAKATO HARRIET 17/U/7016/CHE/PE PRODUCTION AND CHARACTERISATION OF LIPASE ENZYME FROM LACTOBACILLUS ACIDOPHILUS . PROBLEM STATEMENT The demand for lipase enzyme increases daily world wide, this has been majorly due to the high cost of production of the enzyme, due to this reason, this project is aiming at putting lactobacillus acidophilus as a cheaper raw material for the production of lipase enzyme since lactobacillus acidophilus is largely found in milk. MAIN OBJECTIVE
Production and characterisation of lipase
enzyme from lactobacillus acidophillus SPECIFIC OBJECTIVES • To purify and characterise the enzyme for its maximum activity. • To isolate, identify and screen lactobacillus acidophilus from curd sample • To obtain the optimum activity by using temperature, pH, V max, Km, activators, and inhibitors using standard methods METHODOLOGY
Collection of raw material
collection of raw materials (Cont’d)
• The raw material to be used for the
production of lipase is curd that will be obtained from fermented milk samples from cows in the local area of Banda (Kampala). • The milk will be collected in a sterile stainless steel container placed in an ice box, brought to the laboratory and carried the isolation of Lactobacillus acidophilus. Isolation of lactobacillus acidophilus from curd sample
• The Lactobacillus acidophilus will be isolated
by serial dilution plate agar method from curd sample. • 1g of the curd sample will be weighed and inoculated in 9ml sterilized distilled water and the dilution will be made up to 10ml. The sample will be mixed in vortex shaker , transferred to 1ml to a second dilution and 1ml to sterilized Petri plates . Isolation of lactobacillus acidophilus from curd sample (Cont’d) • The sterilized nutrient agar media will be poured in a plate agar, swirled in clockwise and anti-clockwise direction. The plates will be solidified and incubated at 37oC for 24-48hrs. After incubation ,the sample will be observed for growth of small, circular, creamish colonies of Lactobacillus. Screening of Lactobacillus acidophilus • The isolated strains will be screened for the production of lipase enzyme. 250 ml of Lactobacillus will be prepared in a selective agar base media in a conical flask, sterilized at 15 psi for 15minutes. • The media will be cooled and poured into sterilized Petri plates. The plates will be kept for solidification and then the isolated strains will be streaked. The plates will be incubated at 37°C for 24 hours. After incubation the plates will be observed for growth of large and whitish colonies of Lactobacillus. Maintenance of culture
• The isolated bacterial culture of Lactobacillus
will be maintained on a nutrient agar media (NAM) slants and stored at 4° C in refrigerator. • Development of the inoculums and production of lipase from Lactobacillus in selective media (Lactobacillus selective broth). For the development of inoculum 1ml culture of Lactobacillus will be transferred from stock to 100 ml sterile nutrient broth and Lactobacillus selective broth Maintenance of culture (Cont’d) • For the production of lipase 100ml of nutrient broth will be taken and Lactobacillus selective broth in a 250 ml conical flask. The media will be sterilized at 15 psi for 15 minutes, cooled and 1% inoculum (A410= 0.5) of Lactobacillus will be added. The flask will be incubated at 37 °C and at 150rpm for 72hrs . The lipase production will be checked for every 24 hours. Development of the inoculum in a Lactobacillus selective broth
• The development of inoculum 1 ml culture of
Lactobacillus will be transferred from stock to 100 ml sterilized nutrient broth and Lactobacillus selective broth. For the production of lipase 100ml will be taken of nutrient broth and Lactobacillus selective broth in a 250 ml conical flask. Development of the inoculum in a Lactobacillus selective broth (Cont’d) • The media will be sterilized at 15 psi for 15 minutes, cooled and 1% inoculum (A410= 0.5) of Lactobacillus will be added. The flask will be incubated at 37 °C and at 150 rpm for 72hrs. The lipase production will be checked for every 24 hours. Lipase enzyme assay
• Lipase activity will be assayed in the
Lactobacillus broth by using p-nitro phenol as a standard curve. The Lactobacillus broth will be centrifuged at 10,000rpm for 10 min and collects the supernatant. Lipase enzyme assay (Continued) • To the supernatant, lipase activity will be carried out by using 0.05 M Tris-Hydrochloric Acid buffer, pH 8.5. To 2.9 ml of Tris -Hydrochloric Acid buffer (0.05 M, pH 8.5), added 60 μl of the substrate (p-NPP, 9 mM). • The reaction mixture will be incubated at 55OC in a water bath for 10 min in order to remove the turbidity and 40 μl of enzyme will be added thereafter. Lipase enzyme assay (Cont’d) • The reaction mixture will again be incubated at 55 degrees Celsius in water bath for 10 min. The reaction will be stopped by chilling at -40 degrees Celsius. A standard curve of p-Nitro-phenol will be plotted at the selected concentrations (100-1000 μg/mL) of Absorbance 410nm of test sample. Lipase enzyme assay (Cont’d) • One unit (U) of lipase activity will be defined as amount of enzyme required to release one micromole of p-NPP from the substrate (p-NPP) per minute by one mL of the enzyme preparation.