Biodiesel Synthesis Using Recombinant Thermostable Lipase LK3 as a Catalyst Titin Haryati1,2, Fida Madayanti Warganegara1, Made Puspasari Widhiastuty1, Akhmaloka1,3*
1Biochemistry Research Group, Faculty of Mathematics and Natural Science, Institut Teknologi Bandung, Indonesia. 2Indonesian Center for Agricultural Biotechnology & Genetic Resources, Bogor, Indonesia. 3Department of Chemistry, Faculty of Science and Computer, Universitas Pertamina, Indonesia.
INTRODUCTION The use of lipase as catalyst for biodiesel synthesis has several advantages compared to acid/base. Nowadays the study of biodiesel synthesis with lipase as catalyst usually uses commercial lipases, such as Novozyme®435 dan Lipozyme IM60. The problem with using lipase as a catalyst is a long catalysis time. Therefore high temperatures are needed for the reaction to proceed quickly. Industrial biodiesel requires thermostable lipase more. Therefore, application of local thermostable lipase in biodiesel synthesis is needed. The objective of this study is to find out the transesterification activity of recombinant thermostable lipase LK3 in biodiesel synthesis. The lipase used in this study is recombinant thermostable lipase which is over expressed on Escherichia coli BL321. A total of 900 uL (0.3 mg) LK3 lipase was added to 2 mL of oil. Biodiesel synthesis is conducted for 24 hours using palm oil and alcohol as substrates. The alcohol used was varied, i.e methanol, ethanol, isopropanol, and n-butanol. The molar ratio of oil:alcohol is 1:6 using 50% of n-hexane as solvent. The temperature of reaction was 550C. The content of FAMEs in the reaction mixture was analyzed using a GC-14B gas chromatograph equipped with an DB1 capillary column (0.25 mm×30 m) and an FID detector. The column temperature was set at 80°C for 0.5 min, raised to 200°C at 5°C/min and kept at this temperature for 20 min. The temperatures of the injector and detector were set at 200 and 210°C, respectively. The nonadecanoic acid (C19:0, Sigma) methyl ester at 2.0 mg/mL was used as the internal standard. The total yield from the biodiesel was finally calculated according to Elkady, et al. 2015. RESULTS AND DISCUSSION The benefits of using lipase as biocatalysts are to produce clean products (biodiesel) and by-products (glycerol). Glycerol as by-products is easily separated through centrifugation process. Figure 1 shows the total biodiesel yield on alcohol variations as an acyl acceptor. Maximum biodiesel yield of 62% was obtained with ethanol as acyl acceptor group. Figure 1. Effect of alcohol variation on % biodiesel yield The results showed that alcohol as acyl acceptor group could directly impact on the efficiencies of enzymatic transesterification. Short alcohol gives bad effect to lipase conformation, but this result showed that LK3 lipase is ethanol tolerance. Therefore thermostable lipase LK3 is a potential catalyst for biodiesel synthesis. The peak related to the ethyl ester showed in Figure 2. Figure 2. GC Spectra for Ethanol as Acyl acceptor Group. References Ã, N. D. (2008). Enzymatic production of biodiesel from canola oil using immobilized lipase. 32, 1274–1278. https://doi.org/10.1016/j.biombioe.2008.03.005 Cesarini, S., Pastor, F. I. J., Nielsen, P. M., & Diaz, P. (2015). Moving towards a Competitive Fully Enzymatic Biodiesel Process. 7884–7903. https://doi.org/10.3390/su7067884 Elkady, M. F., Zaatou