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.
Highlights
Microbial lipases have been extensively applied in several biotechnological processes including biofuel production and food biotechnology [1,2,3,4,5]
Aminated enzymes were mixed with a suspension of 6.0 g of Glyoxyl–agarose support (Gx) support in 60 mL of 50 mM sodium phosphate buffer at pH 7.0 with 20 mM of a given additive and the mixture thermostated at 25 ◦C; when needed, the addition of NaBH4 at 5 mg/mL for a 30 min duration defined the end-point of the enzyme immobilization process [31]; the resulting Gx derivatives were washed ten times with 50 mL of 25 mM sodium phosphate buffer at pH 7.0 and stored at 4 ◦C until further use
The aim of producing useful Gx derivatives at neutral pH was achieved by the strategy proposed in this study: solid-phase chemical modification of enzymes, the selection of adequate immobilization additives, and a later basification, contribute to obtaining active and robust biocatalysts in natural oil transformation
Summary
Microbial lipases have been extensively applied in several biotechnological processes including biofuel production and food biotechnology [1,2,3,4,5]. (ii) The addition of reducing agents such as cyanoborohydride or aminoboranes in some cases decrease the immobilization pH on Gx below 9.0 by reducing the initial reversible enzyme–Gx imine bonds to irreversible secondary amino bonds As these toxic agents are not selective, they reduce aldehyde glyoxyl groups which limits Gx reactivity during the immobilization process, producing poorly stabilized derivatives [30]. To extend the usefulness of Gx as an immobilization support for white biotechnology applications, it is necessary to find new strategies that surpass or complement those that currently exist Those eventual new strategies may take advantage of key organic synthesis tools such as nucleophilic catalysis: diverse aniline and anthranilic acid (AA) derivatives have been used as organocatalysts for imine bond formation during the synthesis of oxime or hydrazone protein conjugates under soft conditions (neutral pH and room temperature) [34,35]. Sci. 2017, 18, 2130 derivative, reaction parameters were compared with those of the conventional Gx–TLL derivative and the industrial reference biocatalyst Novozyme®435
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