MODIFIED CHITOSAN AS A MATRIX FOR IMMOBILIZATION OF LIPASE ENZYME PPD2 FROM GEOBACILLUS THERMOLEOVORANS

• Main Author: FATNAH , NURWANTI
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/20378
• Institution: Institut Teknologi Bandung

Lipase is the enzyme which hydrolyzes fats into fatty acids and alcohols. In industrial application, lipase enzyme is used not only to change the oil and fat, but also to synthesize fatty esters. Lipase is used in various industry fields, because it has a wide availability, low cost and it does not require cofactors and has specificity against the substrate. There are many problems in industries that use enzymes, such as high operating costs, low stability and difficulties to recycle. Recently, a technique that can minimize the problem has been developed, namely immobilization of enzymes. The immobilized enzyme showed many advantages, namely those are good catalytic stability, can be used continuously, recycle ability, low operating costs, etc. Immobilization using covalent bond is one method that is widely used because of the bonds formed between the enzymes and a supporting matrix enzyme will prevent the release of enzyme into the environment. Various biopolymers especially polysaccharides that are insoluble in water such as cellulose, starch, agarose and chitosan have been widely used as a matrix in the immobilization of enzymes. Chitosan is the second largest polysaccharide in nature, non-toxic and can be used as an anti-microbial compound. In addition, there is reactive functional group for chemical modification, stable, renewable and easy preparation. Those make it suitable for biotransformation. In this study, thermophilic lipase enzyme was obtained from Papandayan crater. Lipase gene from local isolates PPD2 been cloned and expressed intracellularly in E. coli BL21(DE3) using pET30(a) as an expression vector are used in thermophilic recombinant lipase enzymes. The initial stage of cultivation is done by growing bacterial of E. coli BL21(DE3)-pET30(a)-lipITB1.2 from glycerol stocks in LB-kanamycin medium. Furthermore, the production of enzymes through induction use IPTG to obtain cell pellets, then lysised by using lysozyme and sonication (30 seconds on, 10 seconds off 11 times), which produces crude extract enzyme with a specific activity of 9,10 ?mol/mg.minutes. Crude extract enzyme is then partially purified by heating optimization at a temperature of 60, 70 and 80 oC, then it is fractionationed by using acetone in increments of 0-20%, 20-40% and 40-60%.The highest specific activity is obtained by heating at the temperature of 60 °C and the active fraction is obtained by 0-20%. The results are able to increase the specific activity of 2 and 3 times as compared with the crude extract enzyme. In addition, the results of SDS-PAGE on both of them also show protein bands which are thick enough to its target protein of about 40 kDa. Chitosan, which is successfully modified by using succinic anhydride through optimization of temperature, time and mole ratio between chitosan and succinic anhydride, is the supporting matrix that used. Modified chitosan produce N-succinyl chitosan which is indicated by the strong absorption band amide groups on the results of FTIR with wave numbers 1564.27 cm-1 and 1411.89 cm-1. Immobilization of lipase enzyme is done by adding the enzyme to the N-succinyl chitosan by stirring it for 8 hours. The efficiency of the immobilized enzyme is equal to 96% and increase the specific activity of 3 times compared to the active fraction of the enzyme. Immobilized enzymes are tested for their performance through pH, kinetic parameters and reusability. The stability of the enzyme after the immobilization did not change, it was shown by the optimum pH before and after immobilization which were at pH 9.0. The kinetic parameters that follow the curve of Lineweaver Burk generated the Km and Vmax values for the free enzyme, which are of 42.2 mM and 12.0 µmol/mg.minutes, respectively. Meanwhile, the Km and Vmax values for the immobilized enzyme are 10.5 mM and 8.7 µmol/mg.minutes, respectively. The reusability of the immobilized enzyme was capable to maintain its activity up to 81% after five times of usage.