Optimized Conditions for In Situ Immobilization of Lipase in Aldehyde-silica Packed Columns

  • Seo Woo Yong (Department of Environmental Engineering and Biotechnology, Myongji University) ;
  • Lee Kisay (Department of Environmental Engineering and Biotechnology, Myongji University)
  • Published : 2004.11.01

Abstract

Optimal conditions for the in situ immobilization of lipase in aldehyde-silica packed columns, via reductive amination, were investigated. A reactant mixture, containing lipase and sodium borohydride (NaCBH), was recirculated through an aldehyde-silica packed column, such that the covalent bonding of the lipase, via amination between the amine group of the enzyme and the aldehyde terminal of the silica, and the reduction of the resulting imine group by NaCBH, could occur inside the bed, in situ. Mobile phase conditions in the ranges of pH $7.0{\~}7.8$, temperatures between $22{\~}28^{circ}C$ and flow rates from $0.8{\~}1.5\;BV/min$ were found to be optimal for the in situ immobilization, which routinely resulted in an immobilization of more than 70 mg­lipase/g-silica. Also, the optimal ratio and concentration for feed reactants in the in situ immobilization: mass ratio [NaCBH]/[lipase] of 0.3, at NaCBH and lipase concentrations of 0.75 and 2.5 g/L, respectively, were found to display the best immobilization characteristics for concentrations of up to 80 mg-lipase/g-silica, which was more than a 2-fold increase in immobilization compared to that obtained by batch immobilization. For tributyrin hydrolysis, the in situ immobilized lipase displayed lower activity per unit mass of enzyme than the batch-immobilized or free lipase, while allowing more than a $45\%$ increase in lipase activity per unit mass of silica compared to batch immobilization, because the quantity of the immobilization on silica was aug­mented by the in situ immobilization methodology used in this study.

References

  1. Wang, T. H. and W. C. Lee (2003) Immobilization of proteins on magnetic nanoparticles. Biotechnol. Bioprocess Eng. 8: 263-267. https://doi.org/10.1007/BF02942276
  2. Regan, D. L., P. Dunnill, and M. D. Lilly (1974) Immobilized enzyme reaction stability: Attrition of the support material. Biotechnol. Bioeng. 16: 333-343 https://doi.org/10.1002/bit.260160304
  3. Domenici, E., C. Bertucci, P. Salvadori, G. Felix, I. Cahagne, S. Motellier, and I. W. Wainer (1990) Synthesis and chromatographic properties of an HPLC chiral stationary phase based upon human serum albumin. Chromatographia 29: 170-176 https://doi.org/10.1007/BF02268706
  4. Larsson, P. O. (1984) High-performance liquid affinity chromatography. Meth. Enzymol. 104: 212-223 https://doi.org/10.1016/S0076-6879(84)04091-X
  5. Fessenden, R. J., J. S, Fessenden, and M. W. Logue (1998) Organic Chemistry. 6th ed., pp. 771-772. Brooks/ Cole, CA, USA.
  6. Sigma-Aldrich, Inc. (2004) http://www.sigmaaldrich.com, L3126 Specification sheet
  7. Cheetham, P. S. J. (1995) Principles of industrial biocatalysis and bioprocessing. pp. 206-218. In: A. Wiseman (ed.). Handbook of Enzyme Biotechnology, 3rd eds., Ellis Horwood, UK
  8. Massolini, G., E. Galleri, E. de Lorenzi, M. Pregnolato, M. Terreni, G. Felix, and C. Gandini (2001) Immobilized penicillin G acylase as reactor and chiral selector in liquid chromatography. J. Chromatogr. A 921: 147-160 https://doi.org/10.1016/S0021-9673(01)00850-0
  9. Mutty, V. R., J. Bhat, and P. K. A. Muniswaran (2002) Hydrolysis of rice bran oil using immobilized lipase in a stirred batch reactor. Biotechnol. Bioprocess Eng. 7: 367-370 https://doi.org/10.1007/BF02933523
  10. Brodelius, P. (1978) Industrial applications of immobilized biocatalysts. Adv. Biochem. Eng. 10: 76-129
  11. R. A. Messing (1978) Carriers for immobilized biologically active systems. Adv. Biochem. Eng. 10: 51-73 https://doi.org/10.1017/S0266078400007549
  12. Murty, V. R., J. Bhat, and P. K. A. Muniswaran (2002) Hydrolysis of oils by using immobilized lipase enzyme: A review. Biotechnol. Bioprocess Eng. 7: 57-66 https://doi.org/10.1007/BF02935881
  13. Chibata, I., T. Tosa, T. Sato, and T. Mori (1976) Production of L-amino acids by aminoacylase adsorbed on DEAE-Sephadex. Meth. Enzymol. 44: 746-759 https://doi.org/10.1016/S0076-6879(76)44053-3
  14. Rapp, P. (1995) Production, regulation, and some properties of lipase activity from Fusarium oxysporum f. sp. vasinfectum. Enzyme Microb. Technol. 17: 832-838 https://doi.org/10.1016/0141-0229(94)00114-7
  15. Erlandsson, P., L. Hansson, and R. Isaksson (1986) Direct analytical preparative resolution of enantiomers using albumin adsorbed to silica as a stationary phase. J. Chromatogr. 370: 470-483
  16. Pitcher, W. H. Jr. (1978) Design and operation of immo-bilized enzyme reactors. Adv. Biochem. Eng. 10: 1-26 https://doi.org/10.1007/BFb0004469
  17. Uhlig, H. (1998) Industrial Enzyme and Their Applications. pp. 179-190. John Wiley & Sons, NY, USA