Optimization of the enzymatic hydrolysis of Moringa oleifera Lam oil using molecular docking analysis for fatty acid specificity.

Alternative strategies are required to develop the optimized production of fatty acids using biocatalysis; molecular docking and response surface methodology are efficient tools to achieve this goal. In the present study, we demonstrate a novel and robust methodology for the sustainable production of fatty acids from Moringa oleifera Lam oil using lipase-catalyzed hydrolysis (without the presence of emulsifiers or buffer solutions). Seven commercial lipases from Candida rugosa (CRL), Burkholderia cepacia (BCL), Thermomyces lanuginosus (TLL), Rhizopus niveus (RNL), Pseudomonas fluorescens (PFL), Mucor javanicus (MJL), and porcine pancreas (PPL) were used as biocatalysts. Initial screening showed that CRL had the highest hydrolytic activity (hydrolysis degree of 81%). Molecular docking analysis contributed to the experimental results, showing that CRL displays more stable binding free energy with oleic acid (C18:1), which is the fatty acid of highest concentration in Moringa oleifera Lam oil. To evaluate and optimize the hydrolysis process, response surface methodology (RSM) was used. The effect of temperature, mass ratio oil:water, and hydrolytic activity on enzymatic hydrolysis was evaluated by central composite design using RSM. Under the optimized conditions (temperature of 37 °C, mass ratio oil:water of 25%, and hydrolytic activity of 550 U goil -1 ), the maximum hydrolysis degree (100%) was achieved. The present study provides a robust method for the enzymatic hydrolysis of different oils for efficient and sustainable fatty acid production.

Improvement of the enzymatic synthesis of ethyl valerate by esterification reaction in a solvent system.

The present study reports the improved enzymatic synthesis of ethyl valerate (green apple flavor) by esterification reaction of ethanol and valeric acid in heptane medium. Lipase from Thermomyces lanuginosus (TLL) was immobilized by physical adsorption on polyhydroxybutyrate (PHB) particles and used as a potential biocatalyst. The effect of certain parameters that influence the ester synthesis was evaluated by factorial design. The experimental conditions that maximized the synthesis of ethyl valerate were 30.5°C, 18% m/v of biocatalyst (TLL-PHB), absence of molecular sieves, agitation of 234 rpm, and 1,000 mM of each reactant (ethanol and valeric acid). Under these conditions, conversion percentage ≈92% after 105 min of reaction was observed. Soluble TLL was also used as biocatalyst and the highest conversion was of 82% after 120 min of reaction. Esterification reaction performed in a solvent-free system exhibited conversion of 13% after 45 min of reaction catalyzed by immobilized lipase, while the soluble lipase did not exhibit catalytic activity. The synthesis of the ester was confirmed by Fourier transform infrared spectroscopy and gas chromatography-mass spectrometry analyses. After six consecutive cycles of ethyl valerate synthesis, the prepared biocatalyst retained ≈86% of its original activity.

Immobilization of lipase on chitin and its use in nonconventional biocatalysis.

Chitin was functionalized with hexamethylenediamine followed by glutaraldehyde activation, and its capacity to bind Candida rugosa lipase was investigated. The loading of 250 units g(-1) support showed to be effective, resulting in a uniform enzyme fixation with high catalytic activity. Both free and immobilized lipases were characterized by determining the activity profile as a function of pH, temperature, and thermal stability. For the immobilized lipase, the influence of the reaction temperature and substrate polarity in nonconventional biocatalysis was also analyzed. Production of butyl esters was found to be dependent on the substrate partition coefficient, which accounts the greatest value for the system butanol and butyric acid. The highest enzyme activity was found for the system butanol and caprylic acid at a reaction temperature of 40 degrees C. Under such conditions, the operational stability tests indicated that a small enzyme deactivation occurs after 12 batches, revealing a biocatalyst half-life of 426.7 h.

Esterification activity and stability of Candida rugosa lipase immobilized into chitosan.

Microbial lipase from Candida rugosa immobilized into porous chitosan beads was tested for esterification selectivity with butanol and different organic acids (C2-C12), and butyric acid and different aliphatic alcohols (C2-C10). After 24 h, the acids tested achieved conversions of about 40-45%. Acetic acid was the only exception, and in this case butanol was not consumed. Different alcohols led to butyric acid conversions >40%, except for ethanol, in which case butyric acid was converted only 26%. The system's butanol and butyric acid were selected for a detailed study by employing an experimental design. The influence of temperature, initial catalyst concentration, and acid:alcohol molar ratio on the formation of butyl butyrate was simultaneously investigated, employing a 2(3) full factorial design. The range studied was 37-50 degrees C for temperature (X1), 1.25-2.5% (w/v) for the catalyst concentration (X2), and 1 and 2 for the acid:alcohol molar ratio (X3). Catalyst concentration (X2) was found to be the most significant factor and its influence was positive. Maximum ester yield (83%) could be obtained when working at the lowest level for temperature (37 degrees C), highest level for lipase concentration (2.5% [w/v]), and center level of acid:alcohol molar ratio (1.5). The immobilized lipase was also used repeatedly in batch esterification reactions of butanol with butyric acid, revealing a half-life of 86 h.