Exploring Metagenomic Enzymes: A Novel Esterase Useful for Short-Chain Ester Synthesis

Enzyme-mediated esterification reactions can be a promising alternative to produce esters of commercial interest, replacing conventional chemical processes. The aim of this work was to verify the potential of an esterase for ester synthesis. For that, recombinant lipolytic enzyme EST5 was purified and presented higher activity at pH 7.5, 45 °C, with a Tm of 47 °C. Also, the enzyme remained at least 50% active at low temperatures and exhibited broad substrate specificity toward p-nitrophenol esters with highest activity for p-nitrophenyl valerate with a Kcat/Km of 1533 s−1 mM−1. This esterase exerted great properties that make it useful for industrial applications, since EST5 remained stable in the presence of up to 10% methanol and 20% dimethyl sulfoxide. Also, preliminary studies in esterification reactions for the synthesis of methyl butyrate led to a specific activity of 127.04 U·mg−1. The enzyme showed higher esterification activity compared to other literature results, including commercial enzymes such as LIP4 and CL of Candida rugosa assayed with butyric acid and propanol which showed esterification activity of 86.5 and 15.83 U·mg−1, respectively. In conclusion, EST5 has potential for synthesis of flavor esters, providing a concept for its application in biotechnological processes.

Immobilization of Lipase CALB in Organically Modified Silica

In order to improve the activity of enzymes immobilized in silica, additives such as polyethylene glycol (PEG) can be introduced in the sol-gel process. This addition aims to protect the enzymes from denaturing effects by forming protection between the protein and the reaction medium. Thus, the aim of the work was to evaluate the effect of the use of the PEG additive in the process of immobilization of the commercial lipase of Candida antarctica B (CALB) in xerogel and aerogel obtained by the sol-gel technique. The mathematical model for the process was validated, and the optimum points determined were 0.09 g/ml of enzyme and 0.15 g/ml of polyethylene glycol additive for the xerogel and 0.12 g/ml of enzyme and 0.20 g/mL of polyethylene glycol additive for the aerogel. The maximum esterification activity and yield values were 544 U/g, 585%, and 266 U/g, 140% for xerogel and aerogel, respectively. Polyethylene glycol showed better performance in the esterification activity and stability as an additive when used in the xerogel, that is, when the process of obtaining the support uses the removal of the solvent only by evaporation. Regarding aerogel, a reduction in enzyme activity was observed, which may be due to the interaction of PEG with CO2 in the drying process.

Enzymatic Biocatalyst using enzymes from Pineapple (Ananas comosus) Peel Immobilized in Hydrogel Beads

The enzymatic extract from pineapple peels, considering the time factor, low cost and ease of preparation of the reaction system, makes the use of this plant material scrap an economical alternative for some reactions of synthetic interest. Therefore, this work aimed to prepare pineapple peel enzymatic extract containing bromelain, with and without mechanical grinding for a period of up to 9-days of extraction, and then immobilizing those extracts on hydrogel beads for their application as a biocatalyst to produce energy interest esters. The best protein content obtained was 1.95 mg mL-1 for 6-days with mechanical grinding, as for the hydrolysis of p-NPP (p-nitrophenyl palmitate), 0.0125 U.mL-1 for 1-day extraction with trituration. The best index esterification activity achieved for the lauric acid as a substrate was 1.8 U mL-1 at 1-day extraction without grinding. For the hydrogel beads immobilization, a maximum protein yield of 28.8% was obtained with the 1-day extract and mechanical grinding, and in terms of p-NPP activity, a yield of 40.6% for the immobilized with 9-day extract without mechanical grinding. The immobilized and dehydrated beads with 1-day extract without grinding took the best esterification activity, 7.2 U g-1 of biocatalyst. The best conversion performance in the biocatalysis of fatty esters was by esterification of the dodecanoate n-propyl, with 95.1% for a period of 48 hours of reaction. For the transesterification reaction, the methyl oleate yield reached 47.3% after 120 hours of reaction.

Probing the Catalytic Efficiency of Supported Heteropoly Acids for Esterification: Effect of Weak Catalyst Support Interactions

Supported heteropoly acids are an interesting class of solid acid catalysts which possess flexible structure and super acidic properties essentially required for the oil-based biodiesel production. In this study, a series of catalysts containing 25 wt.% of heteropolytungstate (HPW) supported on various clays or SiO2 were prepared, and their catalytic efficiency was evaluated for esterification of acetic acid with heptanol. The as-prepared catalysts were characterized by various techniques including FT-IR spectroscopy, thermogravimetric analysis, X-ray diffraction, scanning electron microscopy, and BET. The catalytic efficiency of both bulk and supported HPW catalysts for the esterification activity strongly depends on the type of support and amount of catalyst; the bulk HPW catalyst and the catalyst supported by kaolinite with 25 wt.% of HPW exhibited highest activity. In order to study the effect of temperature on conversion, all the catalysts were subjected to different reaction temperatures. It was revealed that esterification activity of both bulk and supported HPW catalysts strongly depends upon the temperature variations of the reaction. Besides, the effect of leaching of active sites on the catalysts performance for biodiesel production was also evaluated by inductively coupled plasma studies (ICP). The kaolinite-supported catalyst (25% HPW/kaolinite) demonstrated higher amount of leaching which is also confirmed by the significant decrease in its catalytic activity when it is used for the second time. However, the higher activity demonstrated by HPW/kaolinite maybe because of some homogeneous reaction indicating a weak catalyst support interaction (WCSI) resulting in the leaching of the catalyst during the test. Furthermore, the effects of other reaction variables such as catalyst loading and reaction time on the conversion of acetic acid were also studied.