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Effective immobilisation of lipase to enhance esterification potential and reusability

Ashok Kumar, Vikrant Sharma, Prachi Sharma, and Shamsher S. Kanwar

Department of Biotechnology, Himachal Pradesh University, Shimla, 171 005, India

 

E-mail: kanwarss2000@yahoo.com

Abstract: 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 Km and Vmax 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.

Keywords: Lipolase T100 – silica – glutaraldehyde treatment – immobilisation – esterification – DMSO – methyl salicylate – linalyl ferulate

Full paper is available at www.springerlink.com.

DOI: 10.2478/s11696-013-0377-x

 

Chemical Papers 67 (7) 696–702 (2013)

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