Influence of Dlutaraldehyde Cross-Linking Modes on the Recyclability of Immobilized Lipase B from Candida antarctica for Transesterification of Soy Bean Oil

Abstract

Lipase B from Candida antarctica (CAL-B) is largely employed as a biocatalyst for hydrolysis, esterification, and transesterification reactions. CAL-B is a good model enzyme to study factors affecting the enzymatic structure, activity and/or stability after an immobilization process. In this study, we analyzed the immobilization of CAL-B enzyme on different magnetic nanoparticles, synthesized by the coprecipitation method inside inverse micelles made of zwitterionic surfactants, with distinct carbon chain length: 4 (ImS4), 10 (ImS10) and 18 (ImS18) carbons. Magnetic nanoparticles ImS4 and ImS10 were shown to cross-link to CAL-B enzyme via a Michael-type addition, whereas particles with ImS18 were bond via pyridine formation after glutaraldehyde cross-coupling. Interestingly, the Michael-type cross-linking generated less stable immobilized CAL-B, revealing the influence of a cross-linking mode on the resulting biocatalyst behavior. Curiously, a direct correlation between nanoparticle agglomerate sizes and CAL-B enzyme reuse stability was observed. Moreover, free CAL-B enzyme was not able to catalyze transesterification due to the high methanol concentration; however, the immobilized CAL-B enzyme reached yields from 79.7 to 90% at the same conditions. In addition, the transesterification of lipids isolated from oleaginous yeasts achieved 89% yield, which confirmed the potential of immobilized CAL-B enzyme in microbial production of biodiesel.

Keywords: biodiesel synthesis; cross-linking types; enzyme immobilization; lipase; magnetic nanoparticles.

Scheme 1

Immobilized CAL-B catalyzed transesterification reaction of triacylglycerol from soy bean oil in methanol to obtain fatty acid methyl ester (FAME also called “biodiesel”).

Figure 1

(A) Scheme of NP-ImSn synthesis using reverse micelles made of zwitterionic surfactants ImSn (n = 4, 10 and 18); (B) Typical sizes obtained for the particles using different analyses.

Figure 2

Analysis of the parameters evaluated in the immobilization protocol. (A) Effect of the amount of nanoparticles used; (B) Load of immobilized CAL-B on the amount of magnetic nanoparticles used; (C) Effect of the glutaraldehyde concentration used in the reaction yield (left axis) and in CAL-B loading (right axis- dotted lines) and (D) effect of the immobilization time. NP-ImS4/CAL-B (black line, □), NP-ImS10/CAL-B (red line, ○) and NP-ImS18/CAL-B (blue line, △).

Figure 3

Infrared spectra of free CAL-B (black line), NP-ImS₄/CAL-B (red line), NP-ImS₁₀/CAL-B (blue line), NP-ImS₁₈/CAL-B (green line). (A) Region from 4000 cm⁻¹ to 400 cm⁻¹ and (B) region from 1850 cm⁻¹ to 400 cm⁻¹.

Figure 4

Surface representation of the crystal structure of CAL-B enzyme from Candida antartica (PDB ID: 4K6G) showing the location of the exposed lysines (in blue)/arginines (in orange) residues and the catalytic site (in red).

Figure 5

Influence of the transesterification reaction time on the yield of FAMEs production at different temperatures: 27 °C (black line), 32 °C (red line) and 37 °C (blue line) for each of the immobilized systems: (A) NP-ImS4-CALB, (B) NP-ImS10-CAL-B and (C) NP-ImS18-CALB.

Figure 6

(A) Recycling of immobilized CAL-B for transterification reaction. The reaction percentage yield for each system is represented in colored bars: NP-ImS4/CAL-B (black bars), NP-ImS10/CAL-B (red bars) and NP-ImS18/CAL-B (blue bars). (B) Infrared spectra of NP-ImS4/CAL-B before (black line) and after (red line) the three reaction cycles.