Modulation of the Biocatalytic Properties of a Novel Lipase from Psychrophilic Serratia sp. (USBA-GBX-513) by Different Immobilization Strategies

Abstract

In this work, the effect of different immobilization procedures on the properties of a lipase obtained from the extremophilic microorganism Serratia sp. USBA-GBX-513, which was isolated from Paramo soils of Los Nevados National Natural Park (Colombia), is reported. Different Shepharose beads were used: octyl-(OC), octyl-glyoxyl-(OC-GLX), cyanogen bromide (BrCN)-, and Q-Sepharose. The performance of the different immobilized extremophile lipase from Serratia (ESL) was compared with that of the lipase B from Candida antarctica (CALB). In all immobilization tests, hyperactivation of ESL was observed. The highest hyperactivation (10.3) was obtained by immobilization on the OC support. Subsequently, the thermal stability at pH 5, 7, and 9 and the stability in the presence of 50% (v/v) acetonitrile, 50% dioxane, and 50% tetrahydrofuran solvents at pH 7 and 40 °C were evaluated. ESL immobilized on octyl-Sepharose was the most stable biocatalyst at 90 °C and pH 9, while the most stable preparation at pH 5 was ESL immobilized on OC-GLX-Sepharose supports. Finally, in the presence of 50% (v/v) tetrahydrofuran (THF) or dioxane at 40 °C, ESL immobilized on OC-Sepharose was the most stable biocatalyst, while the immobilized preparation of ESL on Q-Sepharose was the most stable one in 40% (v/v) acetonitrile.

Keywords: USBA-GBX-513; hyperactivation; immobilization; interfacial activation; lipase from psychrophilic microorganism.

Figure 1

Different immobilization protocols used in this research. (A): Immobilization on octil-Sepharose; (B): Immobilization on octyl-glyoxyl Sepharose; (C): Immobilization in BrCN-Sepharose; (D): Immobilization in Q-Sepharose.

Figure 1

Different immobilization protocols used in this research. (A): Immobilization on octil-Sepharose; (B): Immobilization on octyl-glyoxyl Sepharose; (C): Immobilization in BrCN-Sepharose; (D): Immobilization in Q-Sepharose.

Figure 2

Immobilization courses of USBA-GBX-513 lipase on (A) OC-Sepharose; (B) OCGLX-Sepharose (C) CNBr-Sepharose; (D) Q-Sepharose; and (E) CALB on OC-Sepharose. The experiments were performed using 1 mg of enzyme/g of support. Other specifications are described in the methods. Orange triangles: supernatant and Blue circles: suspension.

Figure 3

SDS-PAGE analysis of USBA-GBX-513 biocatalyst. PM: Protein Molecular mass marker, Lane 1. Free USBA-GBX-513 extract, Lane 2. OC- USBA-GBX-513, Lane 3. OCGLX- USBA-GBX-513, Lane 4. CNBr-USBA-GBX-513.

Figure 4

Effect of pH on the activity of different preparations of USBA-GBX-513 lipase on: Empty rhombi: free USBA-GBX-513; blue circles: OC-USBA-GBX-513; orange triangles: OC-GLX-USBA-GBX-513; green squares: Q-Sepharose-USBA-GBX-513; pink rhombi: CNBr- USBA-GBX-513 versus p-NPB. Experiments were performed as described in the methods.

Figure 5

Thermal inactivation at pH (A) 5, (B) 7, and (C) 9 of different USBA-GBX-513 lipase preparations at 90 °C. Empty rhombi: free USBA-GBX-513; dotted line OC-CALB; blue circles: OC-USBA-GBX-513; orange triangles: OCGLX-USBA-GBX-513; green squares: Q-Sepharose-USBA-GBX-513; pink rhombi: CNBr- USBA-GBX-513.

Figure 6

Stability in organic cosolvents of different lipase preparations: (A) ACN, (B) THF, and (C) 1,4-dioxane. Experiments were performed at 50% (v/v) in 50 mM Tris-HCl pH 7 and 40 °C. Dotted line OC-CALB; blue circles: OC-USBA-GBX-513; orange triangles: OC-GLX-USBA-GBX-513; green squares: Q-Sepharose- USBA-GBX-513; pink rhombi: CNBr- USBA-GBX-513.