Stabilization of Candida antarctica Lipase B (CALB) Immobilized on Octyl Agarose by Treatment with Polyethyleneimine (PEI)

Abstract

Lipase B from Candida antarctica (CALB) was immobilized on octyl agarose (OC) and physically modified with polyethyleneimine (PEI) in order to confer a strong ion exchange character to the enzyme and thus enable the immobilization of other enzymes on its surface. The enzyme activity was fully maintained during the coating and the thermal stability was marginally improved. The enzyme release from the support by incubation in the non-ionic detergent Triton X-100 was more difficult after the PEI-coating, suggesting that some intermolecular physical crosslinking had occurred, making this desorption more difficult. Thermal stability was marginally improved, but the stability of the OCCALB-PEI was significantly better than that of OCCALB during inactivation in mixtures of aqueous buffer and organic cosolvents. SDS-PAGE analysis of the inactivated biocatalyst showed the OCCALB released some enzyme to the medium during inactivation, and this was partially prevented by coating with PEI. This effect was obtained without preventing the possibility of reuse of the support by incubation in 2% ionic detergents. That way, this modified CALB not only has a strong anion exchange nature, while maintaining the activity, but it also shows improved stability under diverse reaction conditions without affecting the reversibility of the immobilization.

Keywords: PEI modification; enzyme physical intermolecular crosslinking; enzyme stabilization; interfacial adsorption; reversible immobilization.

Figure 1

Immobilization courses of CALB at pH 7 on octyl agarose at low ionic strength. Rhombi, solid line: suspension; triangles, dashed line: supernatant; circles, solid line: reference.

Figure 2

Thermal inactivation courses of the OCCALB and OCCALB-PEI biocatalysts. Panel (a): 80 °C and pH 5; panel (b): 70 °C and pH 7; panel (c): 60 °C and pH 9. Open circles, solid line: OCCALB; closed circles, solid line: OCCALB PEI; dashed line: free CALB.

Figure 2

Thermal inactivation courses of the OCCALB and OCCALB-PEI biocatalysts. Panel (a): 80 °C and pH 5; panel (b): 70 °C and pH 7; panel (c): 60 °C and pH 9. Open circles, solid line: OCCALB; closed circles, solid line: OCCALB PEI; dashed line: free CALB.

Figure 3

Effect on enzyme activity of the incubation of the immobilized CALB in the presence of different organic solvents. Enzyme preparations were incubated in mixtures of organic solvents/100 mM Tris-HCl pH 7 at 40 °C. Solid line: OCCALB-PEI biocatalyst; dashed line: OCCALB biocatalyst; circles: 80% 1,4-dioxane; squares: 45% acetonitrile; triangles: 70% ethanol.

Figure 4

Desorption of the CALB from the OCCALB (circles) and OCCALB-PEI (triangles) derivatives with Triton X-100.

Figure 5

SDS-PAGE analysis of OCCALB and OCCALB-PEI derivatives under different treatments. Lane 1: low molecular weight protein standard from GE Healthcare. Lane 2: OCCALB; lane 3: OCCALB-PEI; lane 4: OCCALB and lane 5: OCCALB-PEI incubated at pH 5 and 80 °C. Lane 6: OCCALB and lane 7: OCCALB-PEI incubated at pH 7 and 70 °C. Lane 8: OCCALB and lane 9: OCCALB-PEI incubated at pH 9 and 60 °C.

Figure 6

SDS-PAGE analysis of OCCALB and OCCALB-PEI derivatives with organic solvents. Lane 1: low molecular weight protein standard from GE Healthcare. Lane 2: OCCALB; lane 3: OCCALB-PEI; lane 4: OCCALB and lane 5: OCCALB-PEI incubated in 90% dioxane; lane 6: OCCALB and lane 7: OCCALB-PEI incubated in 70% ethanol; lane 8: OCCALB and lane 9: OCCALB-PEI incubated in 45% acetonitrile.