New Strategy for the Immobilization of Lipases on Glyoxyl–Agarose Supports: Production of Robust Biocatalysts for Natural Oil Transformation

Abstract

Immobilization on Glyoxyl–agarose support (Gx) is one of the best strategies to stabilize enzymes. However, the strategy is difficult to apply at neutral pH when most enzymes are stable and, even when possible, produces labile derivatives. This work contributes to overcoming this hurdle through a strategy that combines solid-phase amination, presence of key additives, and derivative basification. To this end, aminated industrial lipases from Candida artarctica (CAL), Thermomyces lunuginosus (TLL), and the recombinant Geobacillus thermocatenulatus (BTL2) were immobilized on Gx for the first time at neutral pH using anthranilic acid (AA) or DTT as additives (immobilization yields >70%; recovered activities 37.5–76.7%). The spectroscopic evidence suggests nucleophilic catalysis and/or adsorption as the initial lipase immobilization events. Subsequent basification drastically increases the stability of BTL2–glyoxyl derivatives under harsh conditions (t1/2, from 2.1–54.5 h at 70 °C; from 10.2 h–140 h in 80% dioxane). The novel BTL2-derivatives were active and selective in fish oil hydrolysis (1.0–1.8 μmol of polyunsaturated fatty acids (PUFAs) min−1·g−1) whereas the selected TLL-derivative was as active and stable in biodiesel production (fatty ethyl esters, EE) as the commercial Novozyme®-435 after ten reaction cycles (~70% EE). Therefore, the potential of the proposed strategy in producing suitable biocatalysts for industrial processes was demonstrated.

Bioconjugation as a smart immobilization approach for α-amylase enzyme using stimuli-responsive Eudragit-L100 polymer: a robust biocatalyst for applications in pharmaceutical industry

“…At 5 and 25 C, the E-AC retained the initial specific activity for 15 d. As represented in Figure 6, free a-amylase retained 50 and 37% of its initial activity after 60 d of storage at 5 and 25 C, respectively, whereas, the immobilized enzyme retained 90 and 69% residual activity after 2 months storage at 5 and 25 C, respectively. Improvement of storage stability of different enzymes upon immobilization has been observed before by several authors [33][34][35]. In conclusion, stability results demonstrated the advantage of covalent conjugation of a-amylase onto Eudragit L-100 polymer whereas conjugation resulted in improved shelf life stability.…”

Novel Combi-lipase Systems for Fatty Acid Ethyl Esters Production

“…Negative effects (CF s < 1), when inhibitors and/or inactivators (e.g., glycerol or ethanol [34]) for each biocatalyst are accumulated along the reaction time in a higher degree for the presence of the other component with regard to the situation when each biocatalyst was alone. In the case of the semiCL studied here, that type of differences must emerge from the different orientation on the surface and microenvironment that the lipase acquires after immobilization on the support [7,16]: It is expected that in LW the lipase adsorbs through the open lid surface [20] while in PU through the surface with the higher content of anionic residues. As the CFs for RML and TLL in their respective semiCL shown an opposite behavior with regard to additivity (Figure 3), it is clear that even when the orientation on each support type may be similar for these lipases, the exact effect in catalytic complementarity is lipase-specific [25,35], and since the composition media changes during the reaction course, also dynamic.…”

“…, where X SI and X CS indicate the total protein or activity content after 24 h in the supernatants that were in contact before the measures with and without support, respectively [16].…”

“…In further experiments, this implied to choose derivatives obtained with higher amounts of offered protein (~50 mg for RML and~100 mg for CALB-LW) to obtain a %EE closer to those of the highly loaded biocatalysts ( Table 2). The fact that TLL allowed a higher protein saving may be related to its higher activity in transesterification compared to CALB or RML [16,25,27]. This makes the reaction to be more dominant on the external TLL-derivative surface, which implies that a higher proportion of the enzyme immobilized within the particle cannot process substrate (higher k cat , higher Thiele modulus and lower enzyme utilization [28]).…”

“…To this end, CL based on the combination of derivatives of the same enzyme (semiCL), conventional CL (where different enzymes are present) and co-immobilized lipases in different supports (coCL), were produced and compared with regard to its constituents. Thus, commercial enzymes from Novozyme ® (CALB, RML and TLL) and supports of different nature from Lewatit ® VP OC 1600 (LW) and Purolite ® ECR 1604 (PU) were used to obtain mono-enzymatic derivatives for the production of fatty acid ethyl esters (EE) from palm olein under very mild reaction conditions [16]. The effect of the enzyme loading in EE production for the most active derivatives was determined.…”