Comparison of Cutinase Separation in Different Chromatographic Media

Abdul Wahab Mohammad(1*), Jamaliah Md.Jahim(2), Suhaila Johar(3), Osman Hassan(4)

(1) Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor.
(2) Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor.
(3) Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor.
(4) School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor.
(*) Corresponding Author


Cutinase is a hydrolytic enzyme that has both properties of lipase and esterase, thus finding its use in many areas. Previous studies have investigated both upstream and downstream processes for cutinase production from microbial source. However, no study has yet to address the use of membrane chromatography for cutinase purification, which is more favourable in terms of process resolution and product throughput as compared to the conventional gel chromatography. Hydrophobic interaction was chosen as the separation mechanism for cutinase purification in this study. The optimisation of cutinase purification in two different types of chromatographic media; conventional packed-gel and membrane matrix, were pre-determined by the best compromise between the recovery and purity of the purified cutinase. It was found that the optimised condition were of pH 4.0 and 1.0 M ammonium sulfate for the conventional column (50% recovery, 4.8-fold purity) and pH 6.0 with 1.5 M ammonium sulfate for the membrane–matrix column(87% recovery, 30-fold purity). Preferential interaction analysis was used to describe the protein chromatographic behaviour in each chromatographic media. Graph of natural algorithm of protein retention data to the function of salt concentration at pH 4.0 and 6.0 for each column were plotted. It was found that at the optimum pH condition for gel-packed column, a small amount of ammonium sulfate was sufficient to achieve maximum cutinase recovery and purity since the effect of salt at that particular pH were less significant. Consequently, the number of released water molecules were calculated and it was observed that for membrane column, larger number of water was released at pH 6.0 illustrating more protein were bounded to the stationary phase, thus explaining the optimum pH condition of the particular column.


Purification, Cutinase, Packed-bed, Membrane matrix, Hydrophobic interaction, Preferential interaction analysis

Full Text:



1. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72, 248-254.
2. Carvalho, C. M. L., Aires-Barros, M. R., Cabral, J. M. S. (1998). Cutinase structure, function and biocatalytic applications, Electron. J. Biotechnol., 1, 160-173.
3. Chen, J., Yang, T., Cramer, S. M. (2008). Prediction of protein retention times in gradient hydrophobic interaction chromatographic systems, J. Chromatogr., A, 1177, 207-214.
4. Dutta, K., Sen, S., Veeranki, V. D. (2009). Production, characterization and applications of microbial cutinases, Process Biochem., 44, 127-134.
5. Ghosh, R. (2001). Separation of proteins using hydrophobic interaction membrane chromatography, J. Chromatogr., A, 923, 59-64.
6. Ghosh, R., and Wang, L. (2006). Purification of humanized monoclonal antibody by hydrophobic interaction membrane chromatography, J. Chromatogr., A, 1107, 104-109.
7. Ghosh, R., and Wong, T. (2006). Effect of module design on the efficiency of membrane chromatographic separation processes, J. Membr. Sci., 281, 532-540.
8. Huang, R., Mah, K. Z., Malta, M., Kostanski, L. K., Filipe, C. D. M., Ghosh, 18 Comparison of Cutinase Separation in Different Chromatographic Media R. (2009). Chromatogrphic separation of proteins using hydrophobic membrane shielded with an environment-responsive hydrogel, J. Membr. Sci., 345, 177-182.

9. Kepka, C., Colleet, E., Roos, F., Tjerneld, F., Veide, A. (2005). Two-step recovery process for tryptophan tagged cutinase: Interfacing aqueous two- phase extracton and hydrophobic interaction chromatography, J. Chromatogr., A, 1075, 33-41.

10. Kolattukudy, P.E., Purdy, R.E., Maiti, I.B. (1981). Cutinases from fungi and pollen, Methods Enzymol., 71, 652-664.
11. Kumar, S., Kinon, K., Upadhyay, A., Kanwar, S. S., Gupta, R. (2005).
Production, purification, and characterization of lipase from thermophilic and alkaliphilic Bacillus coagulans BTS-3, Protein Expression Purif., 41, 38-44.
12. Lienqueo, M. E., Mahn, A., Salgado, J.C., Asenjo, J. A. (2007). Current insights on
protein behaviour in hydrophobic interaction chromatography, J. Chromatogr., B, 849, 53-68.
13. Lienqueo, M. E., Salazar, O., Calado, C. R. C., Fonseca, L. P., Cabral, J. M. S. (2008). Influence of tryptophan tags on the purification of cutinase, secreted by recombinant Saccharomyces cerevisiae, using cationic expended bed adsorption and hydrophobic
interaction chromatography, Biotechnol. Lett., 30, 1353-1358.
14. Nilsson, A., Neves-petersen, M. T., Johansson, H.-O., Jansson, J., Schillén, K., Tjerneld, F., Petersen, S. B. (2003). Tryptophan-tagged cutinase studied by steady state fluorescence for understanding of tag interactions in aqueous two-phase systems, Biochim. Biophys. Acta, 1646, 57-66.

15. Pereira, L. R., Prazeres, D. M. F., Mateus, M. (2010). Hydrophobic interacton membrane chromatography for plasmid DNA purification: Design and optimization, J. Sep. Sci., 33, 1175-1184.
16. Perkins, T. W., Mak, D. S., Root, T. W., Lightfoot, E. N. (1997). Protein retention in hydrophobic interaction chromatography: modelling variation with buffer ionic strength and column hydrophobicity, J. Chromatogr., A, 766, 1-14.
17. To, B. C. S., and Lenhoff, A. M. (2007). Hydrophobic interaction chromatography of proteins I. The effects of protein and adsorbents properties on retention and recovery, J.
Chromatogr., A, 1141, 191-205.
18. Wang, G.-Y., Michailides, T. J., Hammock, B. D., Lee, Y.-M., Bostock, R. M. (2000). Affinity purification and characterization of a cutinase from the fungal plant pathogen Monilinia
fructicola (Wint.) honey, Arch. Biochem. Biophys., 382, 31-38.
19. Xia, F., Nagrath, D., Garde, S., Cramer, S. M. (2004). Evaluation of selectivity changes in HIC systems using a preferential interaction based analysis. Biotechnol. Bioeng., 87, 354-363.
20. Yu, D., McLean, M. D., Hall, J. C., Ghosh, R. (2008). Purification of monoclonal antibody from tobacco extract using membrane-based bioseparation techniques, J. Membr. Sci., 323, 159-166.


Article Metrics

Abstract views : 1120 | views : 930


  • There are currently no refbacks.

ASEAN Journal of Chemical Engineering  (print ISSN 1655-4418; online ISSN 2655-5409) is published by Chemical Engineering Department, Faculty of Engineering, Universitas Gadjah Mada.