Characterization and Hydrolysis Studies of a Prodrug Obtained as Ester Conjugate of Geraniol and Ferulic Acid by Enzymatic Way.

Ferulic acid (Fer) and geraniol (Ger) are natural compounds whose antioxidant and anti-inflammatory activity confer beneficial properties, such as antibacterial, anticancer, and neuroprotective effects. However, the short half-lives of these compounds impair their therapeutic activities after conventional administration. We propose, therefore, a new prodrug (Fer-Ger) obtained by a bio-catalyzed ester conjugation of Fer and Ger to enhance the loading of solid lipid microparticles (SLMs) designed as Fer-Ger delivery and targeting systems. SLMs were obtained by hot emulsion techniques without organic solvents. HPLC-UV analysis evidenced that Fer-Ger is hydrolyzed in human or rat whole blood and rat liver homogenates, with half-lives of 193.64 ± 20.93, 20.15 ± 0.75, and 3.94 ± 0.33 min, respectively, but not in rat brain homogenates. Studies on neuronal-differentiated mouse neuroblastoma N2a cells incubated with the reactive oxygen species (ROS) inductor H2O2 evidenced the Fer-Ger ability to prevent oxidative injury, despite the fact that it appears ROS-promoting. The amounts of Fer-Ger encapsulated in tristearin SLMs, obtained in the absence or presence of glucose, were 1.5 ± 0.1%, allowing the control of the prodrug release (glucose absence) or to sensibly enhance its water dissolution rate (glucose presence). These new "green" carriers can potentially prolong the beneficial effects of Fer and Ger or induce neuroprotection as nasal formulations.

Enzymatic Synthesis of New Acetoacetate-Ursodeoxycholic Acid Hybrids as Potential Therapeutic Agents and Useful Synthetic Scaffolds as Well.

Ursodeoxycholic acid (UDCA) and acetoacetate are natural compounds present in the human intestine and blood, respectively. A number of studies highlighted that besides their well-known primary biological roles, both compounds possess the ability to influence a variety of cellular processes involved in the etiology of various diseases. These reasons suggested the potential of acetoacetate-UDCA hybrids as possible therapeutic agents and prompted us to develop a synthetic strategy to selectively derivatize the hydroxyl groups of the bile acid with acetoacetyl moieties. 3α-acetoacetoxy UDCA was obtained (60% isolated yield) via the regioselective transesterification of methyl acetoacetate with UDCA promoted by the Candida antarctica lipase B (CAL-B). 3α,7ß-bis-acetoacetoxy UDCA was obtained instead by thermal condensation of methyl acetoacetate and UDCA (80% isolated yield). This bis-adduct was finally converted to the 7ß-acetoacetoxy UDCA (82% isolated yield) via CAL-B catalyzed regioselective alcoholysis of the ester group on the 3α position. In order to demonstrate the value of the above new hybrids as UDCA-based scaffolds, 3α-acetoacetoxy UDCA was subjected to multicomponent Biginelli reaction with benzaldehyde and urea to obtain the corresponding 4-phenyl-3,4-dihydropyrimidin-2-(1H)-one derivative in 65% isolated yield.

Solvent-Free Enzymatic Synthesis of Dietary Triacylglycerols from Cottonseed Oil in a Fluidized Bed Reactor.

The synthesis of structured lipids with nutraceutical applications, such as medium-long-medium (MLM) triacylglycerols, via modification of oils and fats represents a challenge for the food industry. This study aimed to synthesize MLM-type dietary triacylglycerols by enzymatic acidolysis of cottonseed oil and capric acid (C10) catalyzed by Lipozyme RM IM (lipase from Rhizomucor miehei) in a fluidized bed reactor (FBR). After chemical characterization of the feedstock and hydrodynamic characterization of the reactor, a 22 central composite rotatable design was used to optimize capric acid incorporation. The independent variables were cycle number (20-70) and cottonseed oil/capric acid molar ratio (1:2-1:4). The temperature was set at 45 °C. The best conditions, namely a 1:4 oil/acid molar ratio and 80 cycles (17.34 h), provided a degree of incorporation of about 40 mol%, as shown by compositional analysis of the modified oil. Lipozyme RM IM showed good operational stability (kd = 2.72 × 10-4 h-1, t1/2 = 2545.78 h), confirming the good reuse capacity of the enzyme in the acidolysis of cottonseed oil with capric acid. It is concluded that an FBR configuration is a promising alternative for the enzymatic synthesis of MLM triacylglycerols.

Microwave-assisted enzymatic synthesis of geraniol esters in solvent-free systems: optimization of the reaction parameters, purification and characterization of the products, and biocatalyst reuse.

Various geraniol esters act as insect pheromones and display pharmacological activities, especially as neuroprotective agents. Therefore, the search for synthetic strategies alternative to traditional chemical synthesis could help designing ecofriendly routes for the preparation of such bioactive compounds. Hence, this work aims at the microwave-assisted enzymatic synthesis of geranyl esters in solvent-free systems. The process variables were optimized for the synthesis of geranyl acetoacetate, achieving 85% conversion after 60 min using a 1:5 substrates molar ratio (ester to geraniol), 80 °C and 8.4% of Lipozyme 435 lipase without removal of the co-produced methanol. On the other hand, a 95% conversion was reached after 30 min using 1:6 substrates molar ratio, 70 °C and 7% lipase in the presence of 5Å molecular sieves for the methanol capture. In addition, the lipase showed good reusability, maintaining the same activity for five reaction cycles. Finally, under the above optimized conditions, other geraniol esters were successfully synthetized such as the geranyl butyrate (98%), geranyl hexanoate (99%), geranyl octanoate (98%), and geranyl (R)-3-hydroxybutyrate (56%). These results demonstrate the microwave-assisted lipase-catalyzed transesterification in a solvent-free system as an excellent and sustainable catalytic methodology to produce geraniol esters.

Biocatalysis and Bioactive Molecules: Future and Development.

Due to the increasing interest in molecules obtained by bioprocesses over the past decade, biocatalysis has gained momentum in a variety of industrial sectors [...].

The bioherbicidal potential of isolated fungi cultivated in microalgal biomass.

This study evaluated the bioherbicidal potential of wild fungi grown on microalgal biomass from the digestate treatment of biogas production. Four fungal isolates were used and the extracts were evaluated for the activity of different enzymes and characterized by gas chromatography coupled with mass spectrometry. The bioherbicidal activity was assessed by application on Cucumis sativus, and the leaf damage was visually estimated. The microorganisms showed potential as agents producing an enzyme pool. The obtained fungal extracts presented different organic compounds, most acids, and when applied to Cucumis sativus, showed high levels of leaf damage (80-100 ± 3.00%, deviation relative to the observed average damage). Therefore, the microbial strains are potential biological control agents of weeds, which, together with the microalgae biomass, offer the appropriate conditions to obtain an enzyme pool of biotechnological relevance and with favorable characteristics to be explored as bioherbicides, addressing aspects within the environmental sustainability.

Optimization, kinetic, and scaling-up of solvent-free lipase-catalyzed synthesis of ethylene glycol oleate emollient ester.

The use of enzymatic catalysts is an alternative to chemical catalysts as they can help to obtain products with less environmental impact, considered sustainable within the concept of green chemistry. The optimization, kinetic, lipase reuse, and scale-up of enzymatic production of ethylene glycol oleate in the batch mode were carried out using the NS 88011 lipase in a solvent-free system. For the optimization step, a 23 Central Composite Design was used and the optimized condition for the ethylene glycol oleate production, with conversions above 99%, was at 70 °C, 600 rpm, substrates molar ratio of 1:2, 1 wt% of NS 88011 in 32 H of reaction. Kinetic tests were also carried out with different amounts of enzyme, and it showed that by decreasing the amount of the enzyme, the conversion also decreases. The lipase reuse showed good conversions until the second cycle of use, after which it had a progressive reduction reaching 83% in the fourth cycle of use. The scale-up (ninefold increase) showed promising results, with conversion above 99%, achieving conversions similar to small-scale reactions. Therefore, this work proposed an environmentally safe route to produce an emollient ester using a low-cost biocatalyst in a solvent-free system.

Lipase-Catalyzed Esterification of Geraniol and Citronellol for the Synthesis of Terpenic Esters.

This article describes the synthesis of terpenic esters derived from geraniol and citronellol (geranyl and citronellyl alkanoates) through esterification reactions catalyzed by the immobilized lipases from Thermomyces lanuginosus (Lipozyme TL IM®) and Candida antarctica (Novozym 435®). Geraniol was esterified with oleic, lauric, and stearic acids; and citronellol was esterified with oleic and stearic acids. For all the synthesized flavor esters, the best conditions were 35 °C, and the molar ratio between acid and alcohol was 1:1. Geranyl and citronellyl alkanoates reached yields between 80-100% within 4 h of reaction. For the synthesis of the citronellyl and geranyl oleate, higher yields were obtained in the absence of organic solvents. For the esters from lauric and stearic acids, using solvent was indispensable to improve the miscibility between the substrates. The reuse of Novozym 435® and Lipozyme TL IM® was performed for two more cycles after the first use, with yields higher than 60%. The results demonstrated the efficiency of the reaction catalyzed by these two commercial enzymes and the feasibility of the methodology for the production of synthetic flavor esters through enzymatic catalysis. The flavor esters synthesized were not described in the literature up to the date, giving this research an innovative feature.

Benzyl propionate synthesis by fed-batch esterification using commercial immobilized and lyophilized Cal B lipase.

In this work, a fed-batch approach was adopted to overcome propionic acid lipase inactivation effects in the benzyl propionate direct esterification mediated by lipases. The ester synthesis was performed using commercial immobilized (Novozym 435) and lyophilized form Candida antarctica fraction B lipase (Cal B) as biocatalysts of the esterification between benzyl alcohol and propionic acid in a solvent-free system. The reaction involved the propionic acid-controlled addition during the first 5 h ensuring an excess of alcohol to dilute the media. The biocatalyst Novozym 435 showed a good performance in the first cycle of the fed-batch esterification, ensuring 90 and 99% of conversion at substrates molar ratio of 1:1 and 1:5 (acid:alcohol), respectively. However, the enzyme lost the activity and the conversions were sharply reduced at the second cycle. A novel qualitative protein content analysis by optical microscopy showed that the lipase was desorbed from the support after the esterification, and this behavior was strongly related to the presence of propionic acid in the reaction medium. The lyophilized Cal B was also tested as biocatalyst of the benzyl propionate esterification and showed a similar performance (related to the Novozym 435) in ester conversion and initial reaction rates for all substrates molar ratios tested. Since the substrates affected the performance of the Novozym 435, the lyophilized Cal B is the most suitable catalyst to the benzyl propionate esterification with conversions above 90%, considering a the fed-batch approach in a solvent-free system.

Effect of supercritical carbon dioxide processing on Vibrio parahaemolyticus in nutrient broth and in oysters (Crassostrea gigas).

This study aimed to evaluate the technical feasibility of supercritical carbon dioxide (sc-CO2) treatment for Vibrio parahaemolyticus inactivation in oysters (Crassostrea gigas) and in nutrient broth. For this purpose, a variable-volume reactor was used as experimental system and a 23 factorial design was adopted considering the mass ratio between carbon dioxide and the product, pressurization and depressurization rate and pressurization cycles. Through statistical analysis of the experimental data, the mass ratio of 1:0.8 (product:carbon dioxide), depressurization rate of 10.0 MPa/min and one cycle of pressurization was determined as the best process condition to eliminate V. parahaemolyticus, and this was the condition used for the inactivation kinetic analysis. Comparison between the inactivation kinetics of V. parahaemolyticus showed that the behavior of this microorganism inactivation depends on the environment in which it operates and its initial count. The results confirm that the supercritical carbon dioxide is effective in inactivating microorganisms in oysters, including pathogenic V. parahaemolyticus, demonstrating the potential of this technology in the food industry.

Biocatalysis of aromatic benzyl-propionate ester by different immobilized lipases.

Benzyl propionate is an aromatic ester that possesses a fruity odor and is usually found in nature in the composition of some fruits such as plums and melons. This work aimed for the benzyl propionate synthesis by esterification using a new immobilized enzyme preparation with low-cost material from Candida antarctica (NS 88011) and three commercial immobilized lipases (Novozym 435, Lipozyme TL-IM and Lipozyme RM-IM). Novozym 435 had the best performance even when the solvent tert-butanol was absent of the reaction medium. Results from a 22 factorial design showed that an increase in the enzyme amount led to a higher conversion, even when the temperature was kept at the low value. Currently, no research had synthesized successfully benzyl propionate via esterification mediated by lipases; and we reached an ester conversion of ~ 44% after 24 h indicating that it is a promising route for benzyl propionate biotechnological production.

Enzymatic Chemoselective Aldehyde-Ketone Cross-Couplings through the Polarity Reversal of Methylacetoin.

The thiamine diphosphate (ThDP) dependent enzyme acetoin:dichlorophenolindophenol oxidoreductase (Ao:DCPIP OR) from Bacillus licheniformis was cloned and overexpressed in Escherichia coli. The recombinant enzyme shared close similarities with the acetylacetoin synthase (AAS) partially purified from Bacillus licheniformis suggesting that they could be the same enzyme. The product scope of the recombinant Ao:DCPIP OR was expanded to chiral tertiary α-hydroxy ketones through the rare aldehyde-ketone cross-carboligation reaction. Unprecedented is the use of methylacetoin as the acetyl anion donor in combination with a range of strongly to weakly activated ketones. In some cases, Ao:DCPIP OR produced the desired tertiary alcohols with stereochemistry opposite to that obtained with other ThDP-dependent enzymes. The combination of methylacetoin as acyl anion synthon and novel ThDP-dependent enzymes considerably expands the available range of C-C bond formations in asymmetric synthesis.

Lipozyme TL IM as Catalyst for the Synthesis of Eugenyl Acetate in Solvent-Free Acetylation.

The ability of commercial immobilized lipase from Thermomyces lanuginosus (Lipozyme TL IM) to catalyze the acetylation of essential clove oil with acetic anhydride in a solvent-free system was studied, and the antimicrobial activity of the ester formed was evaluated as well. Experimental design based on two variables (eugenol to acetic anhydride molar ratio and temperature) was employed to evaluate the experimental conditions of eugenyl acetate ester production. The maximum conversion yield (92.86 %) was obtained using Lipozyme TL IM (5 wt%, based on the total amount of substrates), with eugenol to acetic anhydride molar ratio of 1:5 at 70 °C. The chemical structure of the eugenyl acetate ester obtained at the optimized condition, and purified, was confirmed by the proton nuclear magnetic resonance ((1)H-NMR) analysis. The antimicrobial activity of eugenyl acetate ester was proven effective on Gram-positive and Gram-negative bacteria, with means of 16.62 and 17.55 mm of inhibition halo.

Lipase-Catalyzed Glycerolysis of Soybean and Canola Oils in a Free Organic Solvent System Assisted by Ultrasound.

This work shows new and promising experimental data of soybean oil and canola oil glycerolysis using Novozym 435 enzyme as catalyst in a solvent-free system using ultrasound bath for the emulsifier, monoglyceride (MAG), and diacylglycerol (DAG) production. The experiments were conducted in batch mode to study the influence of process variables as temperature (40 to 70 °C), immobilized enzyme content (2.5 to 10 wt%, relative to substrates), molar ratio glycerol/oil (0.8:1 to 3:1), agitation (0 to 1200 rpm) and ultrasound intensity (0 to 132 W cm(-2)). Highest yields of DAG+MAG (75 wt%) were obtained with molar ratio glycerol/canola oil 0.8:1, 70 °C, 900 rpm, 120 min of reaction time, 10 wt% of enzyme concentration, and 52.8 W cm(-2) of ultrasound intensity. When soybean oil was used, the best results in terms of DAG+MAGs (65 wt%) were using molar ratio of glycerol/soybean oil 0.8:1, 70 °C, 900 rpm, 90 min of reaction time, 10 wt% of enzyme content, and 40 % of ultrasound intensity (52.8 W cm(-2)). The results showed that the lipase-catalyzed glycerolysis in a solvent-free system with ultrasound bath can be a potential route for high content production of DAGs and MAGs.

A review on lipase-catalyzed reactions in ultrasound-assisted systems.

The named "green chemistry" has been receiving increasing prominence due to its environmentally friendly characteristics. The use of enzymes as catalysts in processes of synthesis to replace the traditional use of chemical catalysts present as main advantage the fact of following the principles of the green chemistry. However, processes of enzymatic nature generally provide lower yields when compared to the conventional chemical processes. Therefore, in the last years, the ultrasound has been extensively used in enzymatic processes, such as the production of esters with desirable characteristics for the pharmaceutical, cosmetics, and food industry, for the hydrolysis and glycerolysis of vegetable oils, production of biodiesel, etc. Several works found in the open literature suggest that the energy released by the ultrasound during the cavitation phenomena can be used to enhance mass transfer (substrate/enzyme), hence increasing the rate of products formation, and also contributing to enhance the enzyme catalytic activity. Furthermore, the ultrasound is considered a "green" technology due to its high efficiency, low instrumental requirement and significant reduction of the processing time in comparison to other techniques. The main goal of this review was to summarize studies available to date regarding the application of ultrasound in enzyme-catalyzed esterification, hydrolysis, glycerolysis and transesterification reactions.

Comparative lethality kinetic curves and predictive models of F-value for Listeria monocytogenes using different sanitizers.

The objective of this work was to evaluate the kinetic of inactivation of Listeria monocytogenes using peracetic acid, chlorhexidine, and organic acids as active agent, determining the respective D-, Z-, and F-values. From our knowledge, these important results from an industrial view point are not available in the current literature, mainly for organic acids, pointing out the main contribution of the present work. Lower D-values were obtained for peracetic acid and chlorhexidine, compared with the organic acids. For the reduction of 6 log10 of L. monocytogenes using peracetic acid, at 0.2, 0.1, and 0.05% are necessary 7.08, 31.08, and 130.44 min of contact, respectively. The mathematical models of F-values showed that at concentrations lower than 0.15% one can verify an exponential increase in F-values, for both de chlorhexidine and peracetic acid. The organic acids presented a linear behavior, showing slight variation in F-values, is even more effective in under dosage. The results obtained are of fundamental importance in terms of industrial strategy for sanitization procedure, permitting to choose the best relation product concentration/exposure time, aiming at reducing costs without compromising the disinfectant efficiency.

Immobilization of inulinase from Kluyveromyces marxianus NRRL Y-7571 using modified sodium alginate beads.

An experimental design was carried out to evaluate the effect of the concentrations of sodium alginate, glutaraldehyde and activated coal on the immobilization of inulinase from Kluyveromyces marxianus NRRL Y-7571. The experimental condition of 20 g/L of sodium alginate, 50 mL/L of glutaraldehyde and 30 g/L of activated coal led to the highest specific activity (2,063.5 U/mg of protein), corresponding to an enhancement of about 26 times compared to the activity of the free enzyme (79.1 U/mg of protein). The effect of pH and temperature on the immobilized enzyme activity was also evaluated, showing optimal activities at pH of 5.5 and 55 °C. The study of storage of immobilized inulinase in different temperatures showed that the extract kept its initial activity after 43 days of storage at 40 and 50 °C and after 138 days of storage either at 4 or 25 °C.

Ultrasound-assisted lipase-catalyzed transesterification of soybean oil in organic solvent system.

This work reports the transesterification of soybean oil with ethanol using two commercial immobilized lipases under the influence of ultrasound irradiation. The experiments were performed in an ultrasonic water bath, following a sequence of experimental designs to assess the effects of temperature, enzyme and water concentrations, oil to ethanol molar ratio and output irradiation power on the reaction yield. Results show that ultrasound-assisted lipase-catalyzed transesterification of soybean oil with ethanol might be a potential alternative route to conventional alkali-catalyzed method, as high reaction yields (~90 wt.%) were obtained at mild irradiation power supply (~100 W), and temperature (60 °C) in a relatively short reaction time, 4h, using Lipozyme RM IM as catalyst. The repeated use of the catalyst under the optimum experimental condition resulted in a decay in both enzyme activity and product conversion after two cycles. The use of Novozym 435 led to lower conversions (about 57%) but the enzyme activity was stable after eight cycles of use, showing, however, a reduction in product conversion after the forth cycle.

Assessment of two immobilized lipases activity and stability to low temperatures in organic solvents under ultrasound-assisted irradiation.

Both stability and catalytic activity of two commercial immobilized lipases were investigated in the presence of different organic solvents in ultrasound-assisted system. In a general way, for Novozym 435, the use of ethanol as solvent led to a loss of activity of 35% after 10 h of contact. The use of iso-octane conducted to a gradual increase in lipase activity in relation to the contact time, reaching a maximum value of relative activity of 126%. For Lipozyme RM IM, after 5 h of exposure, the enzyme presented no residual activity when ethanol was used as solvent. The solvents tert-butanol and iso-octane showed an enhancement of about 20 and 17% in the enzyme activity in 6 h of exposure, respectively. Novozym 435 and Lipozyme IM presented high stability to storage after treatment under ultrasound-assisted system using n-hexane and tert-butanol as solvents.

Ultrasound-assisted enzymatic transesterification of methyl benzoate and glycerol to 1-glyceryl benzoate in organic solvent.

The aim of this work is to report the enzymatic transesterification production of 1-glyceryl benzoate under ultrasound irradiation, using a commercial immobilized lipase, Novozym 435. Firstly, a preliminary evaluation was carried out at 2, 4 and 6h, at constant temperature of 50 °C, methyl benzoate to glycerol molar ratio of 1:1 and 5.5 wt% of enzyme concentration. After analyzing the results obtained, the experimental design technique was used to evaluate the effects of temperature, substrates molar ratio, enzyme concentration, solvent volume and ultrasonic power on the 1-glyceryl benzoate production. The highest conversion, around 16%, was obtained at 65 °C, 1:1 of methyl benzoate to glycerol molar ratio, 15 wt% of enzyme concentration, 7 mL of solvent and 40% ultrasonic power in 4h of reaction. A preliminary kinetic experiment carried out varying the enzyme concentration (15 and 20 wt%) keeping fixed the temperature at 35 °C, 1:1 of substrates molar ratio, 3 mL of solvent and 40% of maximum ultrasonic power led to lower (around 15% after 12 h of reaction) conversions compared to that achieved in the experimental design.