Optimizing the enzymatic production of biolubricants by the Taguchi method: Esterification of the free fatty acids from castor oil with 2-ethyl-1-hexanol catalyzed by Eversa Transform 2.0.

The solvent-free esterification of the free fatty acids (FFAs) obtained by the hydrolysis of castor oil (a non-edible vegetable oil) with 2-ethyl-1-hexanol (a branched fatty alcohol) was catalyzed by different free lipases. Eversa Transform 2.0 (ETL) features surpassed most commercial lipases. Some process parameters were optimized by the Taguchi method (L16'). As a result, a conversion over 95% of the FFAs of castor oil into esters with lubricants properties was achieved under optimized reaction conditions (15 wt% of biocatalyst content, 1:4 molar ratio (FFAs/alcohol), 30 °C, 180 rpm, 96 h). The substrates molar ratio had the highest influence on the dependent variable (conversion at 24 h). FFAs/2-ethyl-1-hexanol esters were characterized regarding the physicochemical and tribological properties. Interestingly, the modification of the FFAs with 2-ethyl-1-hexanol by ETL increased the oxidative stability of the FFAs feedstock from 0.18 h to 16.83 h. The biolubricants presented a lower friction coefficient than the reference commercial mineral lubricant (0.052 ± 0.07 against 0.078 ± 0.04). Under these conditions, ETL catalyzed the oligomerization of ricinoleic acid (a hydroxyl fatty acid) into estolides, reaching a conversion of 25.15% of the initial FFAs (for the first time).

Biocatalytic production of biolubricants: Strategies, problems and future trends.

The increasing worries by the inadequate use of energy and the preservation of nature are promoting an increasing interest in the production of biolubricants. After discussing the necessity of producing biolubricants, this review focuses on the production of these interesting molecules through the use of lipases, discussing the different possibilities (esterification of free fatty acids, hydroesterification or transesterification of oils and fats, transesterification of biodiesel with more adequate alcohols, estolides production, modification of fatty acids). The utilization of discarded substrates has special interest due to the double positive ecological impact (e.g., oil distillated, overused oils). Pros and cons of all these possibilities, together with general considerations to optimize the different processes will be outlined. Some possibilities to overcome some of the problems detected in the production of these interesting compounds will be also discussed.

Biosynthesis of alkanes/alkenes from fatty acids or derivatives (triacylglycerols or fatty aldehydes).

This review summarizes the most relevant advances in the biological transformation of fatty acids (or derivatives) into hydrocarbons to be used as biofuels (biogasoline, green diesel and jet biofuel). Among the used enzymes, the fatty acid decarboxylase from Jeotgalicoccus sp. ATCC 8456 (OleTJE) stands out as a promising enzyme. OleTJE may be coupled in cascade reactions with metalloenzymes or reductases from the Old Yellow Enzymes (OYE) family to perform the hydrogenation of α-olefins into paraffins. The photodecarboxylase from Chlorella variabilis NC64A (CvFAP) is an example of coupling biocatalysis and photocatalysis to produce alkanes. Besides the (photo)decarboxylation of free fatty acids and/or triacyclglycerols to produce alkanes/alkenes, by enzymes has also been employed. The cyanobacterial aldehyde decarbonylase (cAD) from Nostoc punctiforme is an outstanding example of this kind of enzymes used to produce alkanes. Overall, these kinds of enzymes open up new possibilities to the production of biofuels from renewable sources, even if they have many limitations on the current situation. The possibilities of improving enzymes features via immobilization or coimmobilization, as well as the utilization of whole cells haves been also reviewed.

Modulation of lipase B from Candida antarctica properties via covalent immobilization on eco-friendly support for enzymatic kinetic resolution of rac-indanyl acetate.

In this study, the modulation of enzymatic biocatalysts were developed by the use of lipase B from Candida antarctica covalently immobilized on an eco-friendly support, cashew apple bagasse, activated with 10% glycidol-ethylenediamine-glutaraldehyde (GEG) under different immobilization strategies (5 mM or 100 mM ionic strength and in absence or presence of 0.5% (v/v) Triton X-100). The biocatalysts were characterized for thermal and organic solvents stabilities and compared with the soluble enzyme. The biocatalysts were then applied to the hydrolysis of the rac-indanyl acetate (2:1 ratio enzyme/substrate) at pH 7.0 and 30 °C for 24 h. For all the strategies evaluated, GEG promoted kinetic resolution of rac-indanyl acetate with maximum conversion (50%) and led to (R)-indanol with excellent enantiomeric excess (97%), maintaining the maximum conversion for five consecutive cycles of hydrolysis. Therefore, the use of cashew apple bagasse has proved to be a promising eco-friendly support for enzyme immobilization, since it resulted in stable biocatalysts for enzymatic kinetic resolution.

Lipase From Rhizomucor miehei Immobilized on Magnetic Nanoparticles: Performance in Fatty Acid Ethyl Ester (FAEE) Optimized Production by the Taguchi Method.

In this communication, it was evaluated the production of fatty acid ethyl ester (FAAE) from the free fatty acids of babassu oil catalyzed by lipase from Rhizomucor miehei (RML) immobilized on magnetic nanoparticles (MNP) coated with 3-aminopropyltriethoxysilane (APTES), Fe3O4@APTES-RML or RML-MNP for short. MNPs were prepared by co-precipitation coated with 3-aminopropyltriethoxysilane and used as a support to immobilize RML (immobilization yield: 94.7 ± 1.0%; biocatalyst activity: 341.3 ± 1.2 U p -NPB/g), which were also activated with glutaraldehyde and then used to immobilize RML (immobilization yield: 91.9 ± 0.2%; biocatalyst activity: 199.6 ± 3.5 U p -NPB/g). RML-MNP was characterized by X-Ray Powder Diffraction (XRPD), Fourier Transform-Infrared (FTIR) spectroscopy and Scanning Electron Microscope (SEM), proving the incorporation and immobilization of RML on the APTES matrix. In addition, the immobilized biocatalyst presented at 60°C a half-life 16-19 times greater than that of the soluble lipase in the pH range 5-10. RML and RML-MNP showed higher activity at pH 7; the immobilized enzyme was more active than the free enzyme in the pH range (5-10) analyzed. For the production of fatty acid ethyl ester, under optimal conditions [40°C, 6 h, 1:1 (FFAs/alcohol)] determined by the Taguchi method, it was possible to obtain conversion of 81.7 ± 0.7% using 5% of RML-MNP.

A new heterofunctional support for enzyme immobilization: PEI functionalized Fe3O4 MNPs activated with divinyl sulfone. Application in the immobilization of lipase from Thermomyces lanuginosus.

Lipase from Thermomyces lanuginosus (TLL) was immobilized onto a novel heterofunctional support, divinyl sulfone (DVS) superparamagnetic nanoparticles (SPMNs) functionalized with polyethyleneimine (PEI). Particle size and zeta potential measurements, elemental analysis, X-ray powder diffraction, magnetic measurements, and infrared spectroscopy analysis were used to characterize the TLL preparations. At pH 10, it was possible to achieve 100 % of immobilization yield in 1 h. The immobilization pH gives TLL preparations with different stabilities; indeed the TLL preparation immobilized at pH 5.0 was the most stable during the thermal inactivation at all pH values. For the hydrolysis of racemic methyl mandelate, the nanobiocatalysts immobilized at pH 5.0 and blocked with ethylenediamine (EDA) and ethanolamine (ETA) obtained good enantioselectivities (68 % and 72 %, respectively) with high catalytic activities in the reaction medium at pH 7.0. The operational stability of the systems was evaluated in the esterification reaction of benzyl alcohol, obtaining up to 61 % conversion after the seventh reaction cycle. These results show that SPMN@PEI-DVS support is a robust strategy for the easy and rapid recovery of the nanobiocatalyst by applying a magnetic field, showing great potential for industrial applications.

Sonohydrolysis using an enzymatic cocktail in the preparation of free fatty acid.

In this work, the concept of lipase cocktail has been proposed in the ultrasound-assisted hydrolysis of coconut oil. Lipase from Thermomyces lanuginosus (TLL), lipase from Rhizomucor miehei (RML), and lipase B from Candida antarctica (CALB) were evaluated as biocatalysts in different combinations. The best conversion (33.66%) was achieved using only RML; however, the best lipase cocktail (75% RML and 25% CALB) proposed by the triangular response surface was used to achieve higher conversions. At the best lipase cocktail, reaction parameters [temperature, biocatalyst content and molar ratio (water/oil)] were optimized by a Central Composite Design, allowing to obtain more than 98% of conversion in the hydrolysis of coconut oil in 3 h of incubation at 37 kHz, 300 W and 45 °C by using 20% of the lipase cocktail (w/w) and a molar ratio of 7.5:1 (water/oil). The lipase cocktail retained about 50% of its initial activity after three consecutive cycles of hydrolysis. To the authors' knowledge, up to date, this communication is the first report in the literature for the ultrasound-assisted hydrolysis of coconut oil catalyzed by a cocktail of lipases. Under ultrasound irradiation, the concept of lipase cocktail was successfully applied, and this strategy could be useful for the other types of reactions using heterogeneous substrates.

Ethyl Butyrate Synthesis Catalyzed by Lipases A and B from Candida antarctica Immobilized onto Magnetic Nanoparticles. Improvement of Biocatalysts' Performance under Ultrasonic Irradiation.

The synthesis of ethyl butyrate catalyzed by lipases A (CALA) or B (CALB) from Candida antarctica immobilized onto magnetic nanoparticles (MNP), CALA-MNP and CALB-MNP, respectively, is hereby reported. MNPs were prepared by co-precipitation, functionalized with 3-aminopropyltriethoxysilane, activated with glutaraldehyde, and then used as support to immobilize either CALA or CALB (immobilization yield: 100 ± 1.2% and 57.6 ± 3.8%; biocatalysts activities: 198.3 ± 2.7 Up-NPB/g and 52.9 ± 1.7 Up-NPB/g for CALA-MNP and CALB-MNP, respectively). X-ray diffraction and Raman spectroscopy analysis indicated the production of a magnetic nanomaterial with a diameter of 13.0 nm, whereas Fourier-transform infrared spectroscopy indicated functionalization, activation and enzyme immobilization. To determine the optimum conditions for the synthesis, a four-variable Central Composite Design (CCD) (biocatalyst content, molar ratio, temperature and time) was performed. Under optimized conditions (1:1, 45 °C and 6 h), it was possible to achieve 99.2 ± 0.3% of conversion for CALA-MNP (10 mg) and 97.5 ± 0.8% for CALB-MNP (12.5 mg), which retained approximately 80% of their activity after 10 consecutive cycles of esterification. Under ultrasonic irradiation, similar conversions were achieved but at 4 h of incubation, demonstrating the efficiency of ultrasound technology in the enzymatic synthesis of esters.

Immobilization of Lipase A from Candida antarctica onto Chitosan-Coated Magnetic Nanoparticles.

In this communication, lipase A from Candida antarctica (CALA) was immobilized by covalent bonding on magnetic nanoparticles coated with chitosan and activated with glutaraldehyde, labelled CALA-MNP, (immobilization parameters: 84.1% ± 1.0 for immobilization yield and 208.0 ± 3.0 U/g ± 1.1 for derivative activity). CALA-MNP biocatalyst was characterized by X-ray Powder Diffraction (XRPD), Fourier Transform Infrared (FTIR) spectroscopy, Thermogravimetry (TG) and Scanning Electron Microscope (SEM), proving the incorporation of magnetite and the immobilization of CALA in the chitosan matrix. Besides, the immobilized biocatalyst showed a half-life 8-11 times higher than that of the soluble enzyme at pH 5-9. CALA showed the highest activity at pH 7, while CALA-MNP presented the highest activity at pH 10. The immobilized enzyme was more active than the free enzyme at all studied pH values, except pH 7.