Enzymatic post-consumer poly(ethylene terephthalate) (PET) depolymerization using commercial enzymes.

Poly(ethylene terephthalate) (PET) is a synthetic polymer widely used globally. The high PET resistance to biotic degradation and its improper destination result in the accumulation of this plastic in the environment, largely affecting terrestrial and aquatic animals. This work investigated post-consumer PET (PC-PET) degradation using five commercial hydrolase enzymes (Novozym 51032, CalB, Palatase, Eversa, Lipozyme TL). Humicola insolens cutinase (HiC, Novozym 51032) was the most active among the enzymes studied. Several important reaction parameters (enzyme type, dual enzyme system, enzyme concentration, temperature, ultrasound treatment) were evaluated in PC-PET hydrolysis using HiC. The concentration and the proportion (molar ratio) of hydrolysis products, terephthalic acid (TPA), mono(2-hydroxyethyl) terephthalate (MHET), and bis(2-hydroxyethyl) terephthalate (BHET), were significantly changed depending on the reaction temperature. The TPA released at 70 °C was 3.65-fold higher than at 50 °C. At higher temperatures, the conversion of MHET into TPA was favored. The enzymatic PET hydrolysis by HiC was very sensitive to the enzyme concentration, indicating that it strongly adsorbs on the polymer surface. The concentration of TPA, MHET, and BHET increased as the enzyme concentration increased, and a maximum was achieved using 40-50 vol % of HiC. The presented results add relevant data to optimizing enzyme-based PET recycling technologies.

Novel efficient enzymatic synthesis of the key-reaction intermediate of PET depolymerization, mono(2-hydroxyethyl terephthalate) - MHET.

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.

Experimental and mathematical modeling approaches for biocatalytic post-consumer poly(ethylene terephthalate) hydrolysis.

The environmental impact arising from poly(ethylene terephthalate) (PET) waste is notable worldwide. Enzymatic PET hydrolysis can provide chemicals that serve as intermediates for value-added product synthesis and savings in the resources. In the present work, some reaction parameters were evaluated on the hydrolysis of post-consumer PET (PC-PET) using a cutinase from Humicola insolens (HiC). The increase in PC-PET specific area leads to an 8.5-fold increase of the initial enzymatic hydrolysis rate (from 0.2 to 1.7 mmol L-1 h-1), showing that this parameter plays a crucial role in PET hydrolysis reaction. The effect of HiC concentration was investigated, and the enzymatic PC-PET hydrolysis kinetic parameters were estimated based on three different mathematical models describing heterogeneous biocatalysis. The model that best fits the experimental data (R2 = 0.981) indicated 1.68 mgprotein mL-1 as a maximum value of the enzyme concentration to optimize the reaction rate. The HiC thermal stability was evaluated, considering that it is a key parameter for its efficient use in PET degradation. The enzyme half-life was shown to be 110 h at 70 ºC and pH 7.0, which outperforms most of the known enzymes displaying PET hydrolysis activity. The results evidence that HiC is a very promising biocatalyst for efficient PET depolymerization.

Biocatalytic esterification of fatty acids using a low-cost fermented solid from solid-state fermentation with Yarrowia lipolytica.

This study aimed to evaluate the use of a lyophilized fermented solid (named solid enzymatic preparation, SEP), with lipase activity, as a low-cost biocatalyst for esterification reactions of fatty acids present in acid raw materials for biodiesel synthesis. The SEP was obtained by solid-state fermentation (SSF) of soybean bran using the strain of Yarrowia lipolytica IMUFRJ 50682 and contains the lipases secreted by this yeast. The esterification reaction of ethanol and the predominant fatty acids present in different acid oil sources for biodiesel production (oleic, linoleic, stearic and palmitic acids) was investigated. Oleic acid conversion of above 85% was obtained after 24 h, using 30 wt% of SEP and ethanol/oleic acid molar ratio of 1, at 30 °C, in a reaction medium with and without solvent (n-hexane). Similar results were achieved with stearic (79%), palmitic (82%) and linoleic (90%) acids. The reusability of SEP was investigated over ten successive batches by washing it with different solvents (ethanol, water or n-hexane) between the cycles of ethyl oleate synthesis. Washing with water allowed the SEP to be reused for six cycles maintaining over 80% of the conversion reached in the first cycle. These results show the potential of this biocatalyst to reduce the content of free fatty acids in acid oils for biodiesel synthesis with a potential to be applied in a broad plethora of raw materials.

Enzymatic biodiesel synthesis using a byproduct obtained from palm oil refining.

An alternative route to produce biodiesel is based on esterification of free fatty acids present in byproducts obtained from vegetable oil refining, such as palm oil fatty acid distillate (PFAD). PFAD is a byproduct of the production of edible palm oil, which contains 96 wt.% of free fatty acids. The purpose of this work was to study biodiesel synthesis via esterification of PFAD with methanol and ethanol, catalyzed by commercial immobilized lipases (Novozym 435, Lipozyme RM-IM, and Lipozyme TL-IM), in a solvent-free system. The effects of reaction parameters such as type of lipase, enzyme amount, type of alcohol, alcohol amount, and enzyme reuse were studied. Fatty acid conversion of 93% was obtained after 2.5 h of esterification reaction between PFAD and ethanol using 1.0 wt.% of Novozym 435 at 60°C.

Characterization of commercial amylases for the removal of filter cake on petroleum wells.

Drilling fluid has many functions, such as carry cuttings from the hole permitting their separation at the surface, cool and clean the bit, reduce friction between the drill pipe and wellbore, maintain the stability of the wellbore, and prevent the inflow of fluids from the wellbore and form a thin, low-permeable filter cake. Filter cake removal is an important step concerning both production and injection in wells, mainly concerning horizontal completion. The drilling fluids are typically comprised of starch, the most important component of the filter cake. A common approach to remove this filter cake is the use of acid solutions. However, these are non-specific reactants. A possible alternative is the use of enzymatic preparations, like amylases, that are able to hydrolyze starch. Wells usually operate in drastic conditions for enzymatic preparations, such as high temperature, high salt concentration, and high pressure. Thus, the main objective of this work was to characterize four enzymatic preparations for filter cake removal under open hole conditions. The results showed that high salt concentrations (204,000 ppm NaCl) in completion fluid decreased amylolytic activity. All enzymatic preparations were able to catalyze starch hydrolysis at all temperatures tested (30, 65, 80, and 95 degrees C). An increase of amylolytic activity was observed with the increase of pressure (100, 500 and 1,000 psi) for one commercial amylase.