RESUMO
Lipase B from Candida antarctica (CalB) is one of the biocatalysts most used in organic synthesis due to its ability to act in several medium, wide substrate specificity and enantioselectivity, tolerance to non-aqueous environment, and resistance to thermal deactivation. Thus, the objective of this work was to treat CalB in supercritical carbon dioxide (SC-CO2) and liquefied petroleum gas (LPG), and measure its activity before and after high-pressure treatment. Residual specific hydrolytic activities of 132% and 142% were observed when CalB was exposed to SC-CO2 at 35 â, 75 bar and 1 h and to LPG at 65 â, 30 bar and 1 h, respectively. Residual activity of the enzyme treated at high pressure was still above 100% until the 20th day of storage at low temperatures. There was no difference on the residual activity loss of CalB treated with LPG and stored at different temperatures over time. Greater difference was observed between CalB treated with CO2 and flash-frozen in liquid nitrogen (- 196 â) followed by storage in freezer (- 10 â) and CalB stored in freezer at - 10 â. Such findings encourage deeper studies on CalB as well as other enzymes behavior under different types of pressurized fluids aiming at industrial application.
Assuntos
Enzimas Imobilizadas , Lipase , Dióxido de Carbono , Proteínas FúngicasRESUMO
Poly(ethylene terephthalate) (PET) is one of the main synthetic plastics produced worldwide. The extensive use of this polymer causes several problems due to its low degradability. In this scenario, biocatalysts dawn as an alternative to enhance PET recycling. The enzymatic hydrolysis of PET results in a mixture of terephthalic acid (TPA), ethylene glycol (EG), mono-(2-hydroxyethyl) terephthalate (MHET) and bis-(2-hydroxyethyl) terephthalate (BHET) as main products. This work developed a new methodology to quantify the hydrolytic activity of biocatalysts, using BHET as a model substrate. The protocol can be used in screening enzymes for PET depolymerization reactions, amongst other applications. The very good fitting (R2 = 0.993) between experimental data and the mathematical model confirmed the feasibility of the Michaelis-Menten equation to analyze the effect of BHET concentration (8-200 mmol L-1) on initial hydrolysis rate catalyzed by Humicola insolens cutinase (HiC). In addition to evaluating the effects of enzyme and substrate concentration on the enzymatic hydrolysis of BHET, a novel and straightforward method for MHET synthesis was developed using an enzyme load of 0.025 gprotein gBHET-1 and BHET concentration of 60 mmol L-1 at 40 °C. MHET was synthesized with high selectivity (97 %) and yield (82 %). The synthesized MHET properties were studied using differential scanning calorimetry (DSC), thermogravimetry (TGA), and proton nuclear magnetic resonance (1H NMR), observing the high purity of the final product (86.7 %). As MHET is not available commercially, this synthesis using substrate and enzyme from open suppliers adds new perspectives to monitoring PET hydrolysis reactions.
Assuntos
Polietilenotereftalatos , Prótons , Etilenoglicol/química , Etilenos , Hidrólise , Ácidos Ftálicos , Plásticos/química , Polietilenotereftalatos/química , PolímerosRESUMO
This work studied the effect of the cation alkyl chain length of 1-alkyl-n-methylimidazolium chloride ([Cnmim]Cl)-based ILs on the activity of Aspergillus niger lipase. First, the lipase activity in the presence of different ILs concentration over time was determined. ILs with shorter cation alkyl side chain length, namely [C4mim]Cl and [C6mim]Cl, promoted an increase of lipase activity; while, [C8mim]Cl, depending on its concentration, maintained or decreased the enzyme activity. In the presence of ILs with longer cation alkyl chain length, i.e., [C10mim]Cl and [C12mim]Cl, the lipase relative activity was reduced with 0.1 (%v/v) and until suppressed ([C12mim]Cl at 0.3 (%v/v)) as a result of irreversible changes in its secondary structure. Fluorescence and circular dichroism spectroscopy analysis confirmed the results achieved. These findings show that [Cnmim]Cl-based ILs can exert different behavior on the lipase' activity (enhance, maintain or even inhibit) and structural conformation, depending on the cation alkyl chain length and their relative concentration.
Assuntos
Aspergillus niger/enzimologia , Proteínas Fúngicas/química , Imidazóis/química , Líquidos Iônicos/química , Lipase/químicaRESUMO
This work evaluated a wild-type Streptomyces clavuligerus strain as a whole-cell lipase (Sc-WCL) producer by submerged fermentation. In an orbital shaker, lipase hydrolytic activity of 3000 U L-1, measured at pH 9.0 and 37 °C by using p-nitrophenyl palmitate as substrate, was achieved after 36 h fermentation using glycerol-free production medium in a baffled Erlenmeyer flask at 28 °C and pH 6.8. Maximum productivity of 52.5 U L-1 h-1 was achieved after 24 h in bioreactor using glycerol-free production medium at pH 6.8 and 28 °C, with agitation at 400 rpm and aeration at 1 vvm. Sc-WCL was shown to be more active at 60 °C and pH 10.7, while higher activity retention was observed at 30-40 °C after 1 h incubation at pH 10. Sc-WCL showed to have potential to be used as biocatalyst in hydrolysis and esterification reactions. In the hydrolysis of p-nitrophenyl palmitate, lyophilized Sc-WCL expressed a hydrolytic activity (330 units g-1 solid, measured at 37 °C and pH 9.0) around 100-fold higher than the ones declared by a supplier of lyophilized powders of mixtures of intracellular lipases from Thermus thermophiles and Thermus flavus (≥3.0 units g-1 solid, measured at 65 °C and pH 8.0). In the synthesis of butyl butyrate in anhydrous medium, 85% ester conversion was achieved at 37 °C after 8 h reaction. Thus, Sc-WCL showed to be a promising biocatalyst because it is cheaper than the isolated and purified lipases.