Effective immobilisation of lipase to enhance esterification potential and reusability
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A commercial lipase, “Lipolase T100”, was immobilised onto silica by means of physical adsorption. The silica-bound lipase was subsequently exposed to 1 vol. % glutaraldehyde (pentane-1,5-dial). The silica was loaded repeatedly with the Lipolase T100 in 0.05 M Tris buffer (pH 8.5) until saturation was achieved. During the 1st, 2nd, 3rd, 4th, and 5th cycles of loading of silica with the enzyme, the protein-binding on the silica achieved 51.73 %, 48.27 %, 26.92 %, 10.73 %, and 4.29 %, respectively. The synthesis of methyl salicylate (methyl 2-hydroxybenzoate) and linalyl ferulate (3,7-dimethylocta-1,6-dien-3-yl 4-hydroxy-3-methoxycinnamate) carried out at 45°C under shaking with mole ratios of 200 mM of acid and 500 mM alcohol in DMSO using 15 mg mL−1 of hyper-activated biocatalyst resulted in yield(s) of 77.2 % of methyl salicylate and 65.3 % of linalyl ferulate in the presence of molecular sieves. The hyper-activated biocatalyst was more efficient than the previously reported silica-bound lipase with minimum leaching of the enzyme from the reaction mixture. The K m and V max of the free (0.142 mM and 38.31 μmol min−1 mL−1, respectively) and silica-bound lipase (0.043 mM and 26.32 μmol min−1 mg−1, respectively) were determined for the hydrolysis of p-NPP. During repeated esterification studies using silica-bound lipase, yields of 50.1 % of methyl salicylate after the 5th cycle, and 53.9 % of linalyl ferulate after the 7th cycle of esterification were recorded. In the presence of molecular sieves (30 mg mL−1) in the reaction mixture, the maximum syntheses of methyl salicylate (77.2 %) and linalyl ferulate (65.3 %) were also observed. In a volumetric batch scale-up, when the reaction volume was increased to 50 mL, 44.9 % and 31.4 % yields of methyl salicylate and linalyl ferulate, respectively, were achieved.
KeywordsLipolase T100 silica glutaraldehyde treatment immobilisation esterification DMSO methyl salicylate linalyl ferulate
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- Bučko, M., Mislovičová, D., Nahálka, J., Vikartovská, A., Šefčovičová, J., Katrlík, J., Tkčá, J., Gemeiner, P., Lacík, B., Štefuca, V., Polakovič, M., Rosenberg, M., Rebroš, M., Šmogrovičová, D., & Švitel, J. (2012). Immobilization in biotechnology and biorecognition: from macro- to nanoscale systems. Chemical Papers, 66, 983–998. DOI: 10.2478/s11696-012-0226-3.CrossRefGoogle Scholar
- Kanwar, S. S., Kaushal, R. K., Verma, M. L., Kumar, Y., Chauhan, G. S., Gupta, R., & Chimni, S. S. (2005a). Synthesis of ethyl laurate by hydrogel immobilized lipase of Bacillus coagulans MTCC-6375. Indian Journal of Microbiology, 45, 187–193.Google Scholar
- Kanwar, S. S., Verma, H. K., Kaushal, R. K., Gupta, R., Chimni, S. S., Kumar, Y., & Chauhan, G. S. (2005b). Effect of solvents and kinetic parameters on synthesis of ethyl propionate catalysed by poly (AAc-co-HPMA-cl-MBAm)-matriximmobilized lipase ofPseudomonas aeruginosa BTS-2. World Journal of Microbiology & Biotechnology, 21, 1037–1044. DOI: 10.1007/s11274-004-7869-3.CrossRefGoogle Scholar
- Kumar, A., & Kanwar, S. S. (2012a). Lipase production in solid-state fermentation (SSF): Recent developments and biotechnological applications. Dynamic Biochemistry, Process Biotechnology and Molecular Biology, 6(1), 13–27.Google Scholar
- Kumar, A., Sharma, P., & Kanwar, S. S. (2012). Lipase catalyzed esters syntheses in organic media: a review. International Journal of Institutional Pharmacy and Life Sciences, 2(2), 91–119.Google Scholar
- Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. The Journal of Biological Chemistry, 193, 265–275.Google Scholar
- Winkler, U. K., & Stuckmann, M. (1979). Glucogen, hyaluronate, and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens. Journal of Bacteriology, 138, 663–670.Google Scholar