DEVELOPMENT of HydroMIP as A FUNCTIONAL MATERIAL for LIPASE ITB1.2 PURIFICATION

• Main Author: Arsiwan, Ruswandi
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/38273
• Institution: Institut Teknologi Bandung

Lipases (acylglycerol hydrolases, 3.1.1.3) are highly chemo-, regio-, and enantio- selective enzyme and have capabality to catalize the hydrolysis, alcoholysis, esterification, and transesterification reactions. Due to their potential and capability, lipases have been used in foods, cosmetics, soaps and leather industry. Lipases also used in pharmaceuticals, chemicals and agrochemicals. Due to wide uses, lipases demand in market are very high. Lipases can be produced from microorganisms, one of that is bacteria. ITB has developed bacteria that was able to produce a thermostable lipase called Lipase ITB1.2. The purification method to purify the enzyme has been established. The steps of purification were conducted based on temperature, charge, size and affinity of the enzyme to certain metal. But the method was not efficient due to time consumption, power and materials. Because of that, the alternative purification method needs to develop by using MIP. The advantages of using MIP are chemical and thermal stability, reusable, cheap, easy to synthesis, robust and high selectivity to target molecules. In addition, the separation method is friendlier to environment and health due to reusable and little solvents used. In the preliminary research, two kinds of MIP were synthesized which are HydroMIP and DryMIP. HydroMIP was synthesized using acrylamide as monomer functional, N,N'-methylenebisacrylamide as crosslinker and ammonium persulfate as initiator polymerization in present of lipase ITB1.2. The lipase ITB1.2 which is used in polymerization was isolated from E. coli BL21(DE3)-pET30(a)-lipITB1.2 cells. The isolation method covered four steps which were heating in 70 oC, anion exchange DEAE-Cellulose, filtration using vivaspin6 30 kDa and IMAC Ni-NTA. The specific activity of isolated lipase ITB1.2 was 17 times higher than crude enzyme after heating treatment. The value of specific activity was 34,96 unit/mg. The synthesized MIPs were characterization by SEM and FTIR. Base on the FTIR analysis results, the new bond was created in MIP which is signed by the loss of double bond =C-H in 3035,96 cm-1. Whereas, from the SEM analysis in 500 times zooming in 10 kV, the morphology of MIP is rougher than NIP. In order to get best adsorption condition, the adsorption capacity was optimized. The parameters optimization were pH, time, mass of adsorbent and the concentration of lipase ITB1.2. Base on the optimization results, the optimum conditions were in 50 mM Na-phosphate buffer pH 9,5, contact time 60 minutes, mass adsorbent 200 mg and the enzyme concentration 0,046 mg/mL. Performance of HydroMIP in purifying the enzyme was tested. The Adsorption of pure enzyme was in optimum condition. Whereas, the desorption of lipase ITB1.2 used buffer of 50 mM Na-phosphate pH 8,6 as elution solution and two types of washing solutions which were buffer of 50 mM Na-phosphate pH 9,5 and demineralized water. The persentage adsorption of lipase ITB1.2 using optimum condition was 8,09%. The elution by buffer 50 mM Na-phosphate pH 9,5 as washing solution produced lipase ITB1.2 with highest consentration in the second elution. The concentration was 2,13 mg/mL. While the elution by demineralized water as washing solution, there was no enzyme found in the elution solution, all the enzyme were released in the washing steps.