ISOLATION AND CHARACTERIZATION OF ORGANIC SOLVENT STABILISED LIPASE FROM HERMETIA ILLUCENS PUPAE

• Main Author: MARCHANTIA KARIMA (NIM:10512039), ELFINA
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/26899
• Institution: Institut Teknologi Bandung

Hermetia illucens is one type of fly that can degrade organic waste. The life cycle of H. illucens <br /> <br /> which is mostly dried without consuming air is expected to be an indication of an enzyme in the body <br /> <br /> of H. illucens that is stable in organic solvents, one of which is lipase. Lipase is an enzyme that can <br /> <br /> generally catalyze the long hydrolysis/synthesis of triglycerides. Lipase which has resistance in <br /> <br /> organic solvents is very much needed in the industrialized world which is starting to use lipase as an <br /> <br /> organic synthesis catalyst. In this study, lipase isolation from H. illucens was carried out in the pupa <br /> <br /> phase which is expected to have resistance in organic solvents. The pupa phase or dormant phase is <br /> <br /> chosen as lipase because it has a higher lipase process than the other phases. Preparation of lipase <br /> <br /> isolation is done by first eroding the H. illucens pupa phase which is then dissolved in a solution of <br /> <br /> NaCl, KCl, MgCl2, and Sucrose. This solution was homogenized in 300 &#956;L tris-HCl buffer (pH 8) <br /> <br /> and added 1% Triton X-100. The solution was incubated for 10 minutes then centrifuged at 8000 rpm <br /> <br /> for 25 minutes at 40oC. The supernatant found from the results of this process is a solution containing <br /> <br /> lipase. Furthermore, optimization of pH, temperature, incubation time, substrate type, and type of <br /> <br /> buffer. Lipase fractionation was carried out using three levels of ammonium sulfate salt, namely <br /> <br /> fractions 0-20%, 20-40%, and 40-60%. Lipase activity was carried out by measuring the absorbance <br /> <br /> of the p-nitrofenol (pNP) compound at a wavelength of 405 nm using a UV-Vis spectrophotometer. <br /> <br /> Lipase characterization was carried out on pH, temperature, incubation time, organic solvents, metal <br /> <br /> ions, and inhibitors. Also determined the kinetic parameters of the enzyme reaction. The results <br /> <br /> showed that lipase had the highest activity at 37 oC and pH 8 with an incubation time of 20 minutes. <br /> <br /> Lipases can also be used with the use of pNPP substrates and tris-HCl buffers. Isolation and <br /> <br /> fractionation of lipase gave the best results in fractions of 20-40% with values for each fraction in a <br /> <br /> row 0.95 U / mg, 1.90 U / mg, and 0.52 U / mg. The addition of K+ and Mg2+ ions was observed to <br /> <br /> significantly increase lipase activity. Lipase activity was also observed using the incubation of organic <br /> <br /> solvents methanol, 2-propanol, ethanol, and acetone. Observed lipase has a Michaelis-Menten kinetics <br /> <br /> model with Vmax value = 0,0027 mM / minute and KM = 3,4460 mM. This research has successfully <br /> <br /> isolated lipase from H. illucens pupa phase that is stable in organic solvents and characterization of <br /> <br /> this enzyme was also successfully carried out.