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Lipases are an important group of biocatalysts for many industrial applications. Two new commercial low-cost lipases Eversa® Transform and Eversa® Transform 2.0 was immobilized on four different hydrophobic supports: Lewatit-DVB, Purolite-DVB, Sepabeads-C18, and Purolite-C18. The performance of immobilized lipases was investigated in the transesterification of sunflower oil solvent-free in an anhydrous medium. Interesting results were obtained for both lipases and the four supports, but with Sepabeads support the lipases Eversa showed high catalytic activity. However, the more stable and efficient derivative was Eversa® Transform immobilized on Sepabeads C-18. A 98 wt% of ethyl ester of fatty acid (FAEE) was obtained, in 3 h at 40ºC, ethanol/sunflower oil molar ratio of 3:1 and a 10 wt% of the immobilized biocatalyst. After 6 reaction cycles, the immobilized biocatalyst preserved 70 wt% of activity. Both lipases immobilized in Sepabeads C-18 were highly active and stable in the presence of ethanol. The immobilization of Eversa Transform and Eversa Transform 2.0 in hydrophobic supports described in this study appears to be a promising alternative to the immobilization and application of these news lipases still unexplored., (© 2022. The Author(s).)

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 2 3 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., (© 2020 International Union of Biochemistry and Molecular Biology, Inc.)

Nanobiocatalysts were produced via immobilization of CalB lipase on polyurethane (PU) based nanoparticles and their application on the synthesis of important industrial products was evaluated. Nanoparticles of polyurethane functionalized with poly(ethylene glycol) (PU-PEG) were synthetized through miniemulsion polymerization and the addition of crosslinking agents were evaluated. The nanoparticles were employed as support for CalB and the kinetic parameters were reported. The performance of new biocatalysts was evaluated on the hydrolysis reaction of p-NPB and on the enantioselective hydrolysis of (R,S)-mandelic acid. The esterification reaction was evaluated on the production of ethyl esters of Omega-3. The effect of poly(ethylene glycol) molar mass (400, 4000 or 6000 Da)on the biocatalyst activity was also analyzed. The PU-PEG6000-CalB showed the highest value of the kinetic parameters, highlighting the high reaction rate. The addition of trehalose as crosslinking agent improved the thermal stability of the biocatalysts. PU-PEG400-CalB was the most active nanobiocatalyst, exhibiting a ethyl esters production of 43.72 and 16.83 mM.U -1 using EPA and DHA, respectively. The nanobiocatalyst was also applied in enantiomeric resolution of mandelic acid, showing promising enantiomeric ratios. The results obtained in this work present alternative and sustainable routes for the synthesis of important compounds used on food and pharmaceutical industries., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In this work, the free lipase Eversa ® Transform 2.0 was used as a catalyst for enzymatic glycerolysis reaction in a solvent-free system. The product was evaluated by nuclear magnetic resonance ( 1 H NMR) and showed high conversion related to hydroxyl groups. In sequence, the product of the glycerolysis was used as stabilizer and biopolyol for the synthesis of poly(urea-urethane) nanoparticles (PUU NPs) aqueous dispersion by the miniemulsion polymerization technique, without the use of a further surfactant in the system. Reactions resulted in stable dispersions of PUU NPs with an average diameter of 190 nm. After, the formation of the PUU NPs in the presence of concentrated lipase Eversa ® Transform 2.0 was studied, aiming the lipase immobilization on the NP surface, and a stable enzymatic derivative with diameters around 231 nm was obtained. The hydrolytic enzymatic activity was determined using ρ-nitrophenyl palmitate (ρ-NPP) and the immobilization was confirmed by morphological analysis using transmission electron microscopy and fluorescence microscopy.

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.

Lipase B from Candida antarctica (CalB) is the most widely used lipase, including in many industrial sectors, such as in biodiesel and pharmaceuticals production. CalB has been produced by heterologous expression using Pichia pastoris under PGK constitutive promoter (named LipB). Here, we have studied the structural features of commercial CalB and LipB enzymes using circular dichroism and fluorescence under different conditions. In the presence of denaturing agents CalB was more stable than LipB, in contrast, at increasing temperatures, LipB was more thermostable than CalB. Mass spectrometry data indicates that both enzymes have an insertion of amino acids related to α-factor yeast signal, however LipB enzyme showed the addition of nine residues at the N-terminal while CalB showed only four residues. Molecular modeling of LipB showed the formation of an amphipathic α-helix in N-terminal region that was not observed in CalB. This data suggests that this new α-helix possess could be involved in LipB thermostability. These results associated with new structural studies may provide information to the design of novel biocatalysts., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The total or partial substitution of fossil raw materials by biobased materials from renewable resources is one of the great challenges of our society. In this context, the reaction under mild condition as enzyme-catalyzed esterification was applied to investigate the esterification of the biobased 10-undecenoic acid with 2-hydroxyethyl methacrylate (HEMA) to obtain a new diene ester monomer. The environmentally friendly enzymatic reaction presented up to 100% of conversion; moreover, the production of possible by-products was minimized controlling reaction time and amount of enzyme. Furthermore, the presence of chloroform was evaluated during the enzymatic reactions and despite high conversions with higher enzyme concentration, the solvent-free system showed fast kinetics even with 1.13 U/g substrates. In addition, the commercial immobilized lipases Novozym 435 and NS 88011 could be applied for up to 10 cycles keeping conversions about 90%. The scale-up of the reaction was possible and a purification procedure was applied in order to isolate the diene ester monomer 2-(10-undecenoyloxy)ethyl methacrylate, preserving its double bonds, which could allow a potential use of this product in the synthesis of new renewable polymers through techniques as metathesis, thiol-ene, or free-radical polymerization.

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.

The use of green sources for materials synthesis has gained popularity in recent years. This work investigated the immobilization of lipase NS-40116 (Thermomyces lanuginosus lipase) in polyurethane foam (PUF) using a biopolyol obtained through the enzymatic glycerolysis between castor oil and glycerol, catalyzed by the commercial lipase Novozym 435 for the PUF formation. The reaction was performed to obtain biopolyol resulting in the conversion of 64% in mono- and diacylglycerol, promoting the efficient use of the reaction product as biopolyol to obtain polyurethane foam. The enzymatic derivative with immobilized lipase NS-40116 presented apparent density of 0.19 ± 0.03 g/cm 3 and an immobilization yield was 94 ± 4%. Free and immobilized lipase NS-40116 were characterized in different solvents (methanol, ethanol, and propanol), temperatures (20, 40, 60 and 80 °C), pH (3, 5, 7, 9 and 11) and presence of ions Na + , Mg ++ , and Ca ++ . The support provided higher stability to the enzyme, mainly when subjected to acid pH (free lipase lost 80% of relative activity after 360 h of contact, when the enzymatic derivative lost around 22%) and high-temperature free lipase lost 50% of relative activity, while the immobilized remained 95%. The enzymatic derivative was also used for esterification reactions and conversions around 66% in fatty acid methyl esters, using abdominal chicken fat as feedstock, were obtained in the first use, maintaining this high conversion until the fourth reuse, proving that the support obtained using environmentally friendly techniques is applicable.

Alternatives to improve the stability and activity of enzymes have been rising in the last years due to the potential industrial application of these catalysts. However, the enzymes characteristics in terms of stability and catalytic efficiency can reduce, in some cases, due to the reaction conditions. Due to a lack in the literature concerning structural information related to the new commercial Eversa® Transform 2.0 enzyme (NS-40116) we investigated the conformational structure by spectroscopic and mass spectrometry techniques after exposure in permanent magnetic flux density (0.7 and 1.34 T) in recirculation mode (1, 2, and 4 h) at 0.06 L·min -1 . The influence of pH on the enzymatic solution associated with the magnetic flux (pH 5, 7, and 9) was also evaluated. Under the best reaction condition (pH 7 after 4 h in a recirculation mode at 1.34 T), enzyme activity 77% higher than the control sample was obtained. Mass spectrometry techniques showed changes in the NS-40116 tertiary structure. Thus, the application of magnetic fields as an enzymatic pre-treatment showed to be a promising technique and a viable alternative to increase the enzymatic activity since it is a low cost, environmentally friendly, and ease operation process., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The performance of two new commercial low-cost lipases Eversa® Transform and Eversa® Transform 2.0 immobilized in different supports was investigated. The two lipases were adsorbed on four different hydrophobic supports. Interesting results were obtained for both lipases and for the four supports. However, the most active derivative was prepared by immobilization of Eversa® Transform 2.0 on Sepabeads C-18. Ninety-nine percent of fatty acid ethyl ester was obtained, in 3 h at 40 °C, by using hexane as solvent, a molar ratio of 4:1 (ethanol/oil), and 10 wt% of immobilized biocatalyst. The final reaction mixture contained traces of monoacylglycerols but was completely free of diacylglycerols. After four reaction cycles, the immobilized biocatalyst preserved 75% of activity. Both lipases immobilized in Sepabeads C-18 were very active with ethanol and methanol as acceptors, but they were much more stable in the presence of ethanol.

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 2 2 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.

The demand for environmentally friendly products allied with the depletion of natural resources has increased the search for sustainable materials in chemical and pharmaceutical industries. Polyesters are among the most widely used biodegradable polymers in biomedical applications. In this work, aliphatic polyesters (from globalide and ω-pentadecalactone) were synthesized using a new commercial biocatalyst, the low-cost immobilized NS 88011 lipase (lipase B from Candida antarctica immobilized on a hydrophobic support). Results were compared with those obtained under the same conditions using a traditional, but more expensive, commercial biocatalyst, Novozym 435 (lipase B from C. antarctica immobilized on Lewatit VP OC). When NS 88011 was used in the polymerization of globalide, longer reaction times (240 min)-when compared to Novozym 435-were required to obtain high yields (80-90 wt%). However, higher molecular weights were achieved. When poly(ω-pentadecalactone) was synthesized, high yields and molecular weights (130,000 g mol -1 ) were obtained and the enzyme concentration showed strong influence on the polyester properties. This is the first report describing NS 88011 in polymer synthesis. The use of this cheaper enzymatic preparation can provide an alternative for polyester synthesis via enzymatic ring-opening polymerization.

The oleaginous yeast Moniliella spathulata R25L270 was the first yeast able to grow and produce extracellular lipase using Macaúba ( Acrocomia aculeate ) cake as substrate. The novel lipase was recently identified, and presented promising features for biotechnological applications. The M. spathulata R25L270 lipase efficiently hydrolyzed vegetable and animal oils, and showed selectivity for generating cis -5,8,11,15,17-eicosapentaenoic acid from sardine oil. The enzyme can act in a wide range of temperatures (25-48 °C) and pH (6.5-8.4). The present study deals with the immobilization of M. spathulata R25L270 lipase on hydrophobic, covalent and ionic supports to select the most active biocatalyst capable to obtain omega-3 fatty acids (PUFA) from sardine oil. Nine immobilized agarose derivatives were prepared and biochemically characterized for thermostability, pH stability and catalytic properties (K M and V max ). Ionic supports improved the enzyme-substrate affinity; however, it was not an effective strategy to increase the M. spathulata R25L270 lipase stability against pH and temperature. Covalent support resulted in a biocatalyst with decreased activity, but high thermostability. The enzyme was most stabilized when immobilized on hydrophobic supports, especially Octyl-Sepharose. Compared with the free enzyme, the half-life of the Octyl-Sepharose derivative at 60 °C increased 10-fold, and lipase stability under acidic conditions was achieved. The Octyl-Sepharose derivative was selected to obtain omega-3 fatty acids from sardine oil, and the maximal enzyme selectivity was achieved at pH 5.0., Competing Interests: The authors declare that they have no competing interests.

The objective of this study is to evaluate the batch enzymatic production of biodiesel in solvent-free system under ultrasound using as substrates ethanol, soybean oil and macauba fruit oil. For this purpose, a Plackett & Burman experimental design was carried out for soybean oil while a 2 4-1 design was conducted for macauba oil in order to maximize the biodiesel conversion for each system. Good conversions to fatty acid ethyl esters (FAEE), 88% for soybean oil and 75.2% for macauba oil, was obtained thus demonstrating the potential use of ultrasound for this reaction system., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Antioxidants are substances that defend cells against damage, kidnapping and destroying free radicals. They have been largely used in the food industry due the possibility to control the oxidation process, aimed to increase shelf life. Thus, esterification reaction to obtain ascorbyl linoleate catalyzed by Novozym 435 lipase assisted by ultrasound bath was investigated. In this work, molecular sieve (4 Å) was added to the reaction medium to remove the water formed during the esterification reaction to improve the process performance. According to the results, ascorbyl linoleate production up to 90 % was reached after 1 h of reaction time carried out using ultrasound bath, 1:9 molar ratio of substrates L-ascorbic acid to linoleic acid, 20 mL of tert-butanol as organic solvent, 5 wt% of Novozym 435 lipase, 10 wt% of molecular sieve at 70 °C.

Poly(urea-urethane) (PUU) nanoparticles with encapsulated superparamagnetic magnetite (Fe 3 O 4 ) were obtained by interfacial miniemulsion polymerization and used as support for immobilization of lipase B from Candida antarctica (CALB). CALB enzyme was immobilized on magnetic PUU nanoparticles in two steps. The enzyme was immobilized in the lyophilized nanoparticles (magnetic PUU) after the support synthesis in phosphate buffer (pH 7.6) containing CALB, by the contact between nanoparticles and enzymatic solution. The mixture was incubated at 30 °C in an orbital shaker during 0.5 until 6 h to determine the time for maximum immobilization efficiency. The enzyme activity was determined by esterification reactions between lauric acid and propanol. Residual activities above 95 % in relation to free enzyme were obtained in 1 h of immobilization with enzyme concentration of 0.55 mg/mL. FTIR spectrum and SEM-FEG images were used to confirm the presence of CALB on magnetic support after immobilization and stability of support even after immobilization process, respectively. Thermal (40, 60, and 80 °C) and pH (pH 4, 7, and 10) stabilities, storage stability, and reuse were evaluated. CALB immobilized derivatives showed high stabilities with residual activities of 95, 100, and 100 % at 40, 60, and 80 °C, respectively, in 6 h of incubation. After incubation in different pH values, CALB immobilized derivative presented activities of 81, 76, and 69 % in relation to activities in the beginning of the stabilization process in pH 4, 7, and 10, respectively. Furthermore, CALB immobilized derivative reduces only 15 % of its activity after 30 days of storage at 4 °C. Reuse results showed that immobilized CALB on magnetic PUU nanoparticles led to 95 % of geranyl oleate conversion after 8 cycles of application demonstrating high stability of the CALB immobilized derivative under different conditions.

The diacylglycerols (DAG) are emulsifiers with high added value used as functional additives in food, medicine, and cosmetic industries. In glycerolysis of oils for the production of DAG, the immiscibility between the substrates (glycerol and oil phases) has to be overcome, for example, by the addition of a food grade surfactant like Tween 65. The main objective of this work was to optimize the process conditions for obtaining diacylglycerols rich in the omega-3 eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, through the enzymatic glycerolysis of fish oil, in systems with Tween 65 and without this surfactant, using Lipozyme TL(®) IM as biocatalyst. The experiments were performed according to predetermined conditions varying the temperature, enzyme load, the oil to glycerol molar ratio and, when added, the surfactant load. After 6 h of reaction, it was possible to produce up to 20.76 and 13.76% of diacylglycerols from fish oil in the reactions with and without Tween 65, respectively.

With the aim of studying the best method for the interaction of polyurethane (PU) foam and Candida antarctica lipase B, different methods of CalB immobilization were studied: adsorption (PU-ADS), bond (using polyethyleneimine) (PU-PEI), ionic adsorption by PEI with cross-linking with glutaraldehyde (PU-PEI-GA) and entrapment (PU). The characterization of immobilized enzyme derivatives was performed by apparent density and Fourier transform infrared spectroscopy. The free enzyme and enzyme preparations were evaluated at different pH values and temperatures. The highest enzyme activity was obtained using the PU method (5.52 U/g). The methods that stood out to compare the stabilities and kinetic parameters were the PU and PU-ADS. Conversions of 83.5 and 95.9 % for PU and PU-ADS derivatives were obtained, in 24 h reaction, using citronella oil and propionic acid as substrates.

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.

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.

This work reports the transesterification of soybean oil with ethanol using a commercial immobilized lipase, Novozym 435, under the influence of ultrasound irradiation, in a solvent-free s. The experiments were performed in an ultrasonic water bath, following a sequence of experimental designs to evaluate the effects of temperature, enzyme and water concentrations, oil to ethanol molar ratio and output irradiation power on the reaction yield. Besides, a kinetic study varying the substrates molar ratio and enzyme concentration was also carried out. Results show that ultrasound-assisted lipase-catalyzed transesterification of soybean oil with ethanol in solvent-free system might be a potential alternative route to conventional alkali-catalyzed and/or traditional enzymatic methods, as high reaction yields (~78 wt%) were obtained at mild irradiation power supply (~132 W), and temperature (63 °C) in a relatively short reaction time, 1 h. Additionally, a study regarding the enzyme reuse was carried out at the experimental condition that afforded the best reaction yield.

With the objective to obtain immobilized Candida antarctica lipase B (CalB) with good activity and improved utilization rate, this study evaluated the influence of enzyme and crodamol concentrations and initiator type on the CalB enzyme immobilization in nanoparticles consisting of poly(methyl methacrylate) (PMMA) obtained by miniemulsion polymerization. The kinetic study of immobilized CalB enzyme in PMMA nanoparticles was evaluated in terms of monomer conversion, particle size, zeta potential, and relative activity. The optimum immobilization condition for CalB was compared with free enzyme in the p-NPL hydrolysis activity measurement. Results showed a higher CalB enzyme stability after 20 hydrolysis cycles compared with free CalB enzyme; in particular, the relative immobilized enzyme activity was maintained up to 40%. In conclusion, PMMA nanoparticles proved to be a good support for the CalB enzyme immobilization and may be used as a feasible alternative catalyst in industrial processes.

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.

This work reports the maximization of eugenyl acetate production by esterification of essential oil of clove in a solvent-free system using Novozym 435 as catalyst. The antimicrobial and antioxidant activities of clove essential oil and eugenyl acetate produced were determined. The conditions that maximized eugenyl acetate production were 60 °C, essential oil of clove to acetic anhydride ratio of 1:5, 150 rpm, and 10 wt% of enzyme, with a conversion of 99.87 %. A kinetic study was performed to assess the influence of substrates' molar ratio, enzyme concentration, and temperature on product yield. Results show that an excess of anhydride, enzyme concentration of 5.5 wt%, 50 °C, and essential oil of clove to acetic anhydride ratio of 1:5 afforded nearly a complete conversion after 2 h of reaction. Comparing the antibacterial activity of the essential oil of clove before and after esterification, we observed a decrease in the antimicrobial activity of eugenyl acetate, particularly with regard to minimum inhibitory concentration (MIC). Both eugenyl acetate and clove essential oil were most effective to the gram-negative than gram-positive bacteria group. The results showed a high antioxidant potential for essential oil before and particularly after the esterification reaction thus becoming an option for the formulation of new antioxidant products.

Lipases produced by a newly isolated Sporidiobolus pararoseus strain have potential catalytic ability for esterification reactions. After production, the enzymatic extracts (conventional crude and precipitated, 'CC' and 'CP', and industrial crude and precipitated, 'IC' e 'IP') were partially characterized. The enzymes presented, in general, higher specificity for short chain alcohols and fatty acids. The precipitated extract showed a good thermal stability, higher than that for crude enzymatic extracts. The 'CC' and 'CP' enzymes presented high activities after exposure to pH 6.5 and 40 °C. On the other hand, the 'IC' and 'IP' extracts kept their activities in a wide range of pH memory but presented preference for higher reaction temperatures. Preliminary studies of application of the crude lipase extract in the enzymatic production of geranyl propionate using geraniol and propionic acid as substrates in solvent-free system led to a reaction conversion of 42 ± 1.5%.

This work investigates the continuous production of alkyl esters from soybean fatty acid (FA) charges using immobilized Novozym 435 as catalyst. The experiments were performed in a packed-bed bioreactor evaluating the effects of FA charge to alcohol (methanol and ethanol) molar ratio, from 1:1 to 1:6, substrate flow rate in the range of 0.5-2.5 mL/min and output irradiation power up to 154 W, at fixed temperature of 65 °C, on the reaction conversion. Results showed that almost complete conversions to fatty acids ethyl esters were achieved at mild ultrasonic power (61.6 W), FA to ethanol molar ratio of 1:6, operating temperature (65 °C) and remained nearly constant for long-term reactions without negligible enzyme activity losses.

Enzymatic esterification of eugenol is a matter of great scientific and technological interest due to the well-known drawbacks of the chemical-catalyzed route as well as the potential use of produced compounds as natural antimicrobials. This work reports the maximization of eugenil acetate production by esterification of eugenol and acetic anhydride in a solvent-free system using Novozym 435 as catalyst. The antimicrobial activity of eugenol and eugenil acetate was also determined. The operating conditions that maximized eugenil acetate production were 50 °C, eugenol to acetic anhydride of 1:3, 150 rpm, and 5.5 wt% of enzyme, with a conversion of 99 %. A kinetic study was performed to assess the influence of substrates molar ratio, enzyme concentration, and temperature on eugenil acetate yield. Results show that an excess of anhydride, low enzyme concentration (1 wt%), and 60 °C afforded nearly complete conversion after 6 h of reaction. The highest antimicrobial activity of eugenil acetate was observed against Acinetobacter sp. (48.66 mm) at concentration of 20 μL. Results indicate that the esterification of eugenol improved its antimicrobial properties. New experimental data on enzymatic esterification of eugenol and acetic anhydride are reported in this work, showing a promising perspective to overcome the inconvenient of the chemical-catalyzed route for obtaining antimicrobial natural compounds.

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

This work reports experimental kinetic data of solvent-free glycerolysis of olive oil using a commercial immobilized lipase (Novozym 435) under the influence of ultrasound irradiation. The experiments were performed in a mechanically stirred reactor under ultrasound irradiation, evaluating the effects of temperature (50-70 °C), enzyme concentration (2.5-10 wt%) and glycerol to oil molar ratio (0.8:1-3:1). Results show that ultrasound-assisted lipase-catalyzed glycerolysis might be a potential alternative route to conventional methods, as high contents of reaction products, especially monoglycerides, were achieved at mild irradiation power supply (~130 W) and temperature, in a relatively short reaction time (2h) and low enzyme content (7.5 wt%). To completeness, two simplified kinetic modeling approaches, based on the ordered-sequential bi bi mechanism and reaction stoichiometry, were employed to represent the experimental data, thus allowing a better understanding of the reaction kinetics., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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.

This work is focused on the optimization of reaction parameters for the synthesis of ascorbyl palmitate catalyzed by Candida antarctica lipase in different organic solvents under ultrasound irradiation. The sequential strategy of experimental design proved to be useful in determining the optimal conditions for reaction conversion in tert-butanol system using Novozym 435 as catalyst. The optimum production was achieved at 70°C, ascorbic acid to palmitic acid molar ratio of 1:9, enzyme concentration of 5 wt% at 3h of reaction, resulting in an ascorbyl palmitate conversion of about 27%. Reaction kinetics for ascorbyl palmitate production in ultrasound device showed that satisfactory reaction conversions (∼26%) could be achieved in short reaction times (2h). The empirical kinetic model proposed is able to satisfactorily represent and predict the experimental data., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The objective of this study was to provide some features on immobilization and partial characterization of lipases from wheat seeds. The optimum pH and temperature were found to be 5.5 and 32-37 °C, respectively. The stability of the concentrated enzymatic extract to high temperatures (25, 35, 45, and 55 °C) showed that the incubation of the extract at 55 °C led to its complete inactivation. The concentrated enzymatic extract kept 90% of its hydrolytic and esterification activities until 70 and 40 days of storage at 4 °C, respectively. The extract presented higher hydrolytic specificity to substrates of medium and long chains and higher esterification affinity to fatty acids of short and medium chains and alcohols with two and three carbon atoms. After the immobilization process using activated coal and sodium alginate as supports, an enhancement of about threefold in lipase activity was observed. The development of the present work permitted us to point out some characteristics of lipases from wheat seeds necessary for the proposition of new future industrial applications for this important biocatalyst.

One relevant limitation hindering the industrial application of microbial lipases has been attributed to their production cost, which is determined by the production yield, enzyme stability among other. The objective of this work was to evaluate the concentration and immobilization of lipase extracts from Penicillium brevicompactum obtained by solid-state fermentation of babassu cake and castor bean cake. The precipitation with ammonium sulfate 60% of saturation of crude extract obtained with babassu cake as raw material showed an enhancement in hydrolytic and esterification activities from 31.82 to 227.57 U/g and from 170.92 to 207.40 U/g, respectively. Concentrated lipase extracts showed preference to medium-chain triglycerides and fatty acids. It is shown that the enzyme activity is maintained during storage at low temperatures (4 and -10°C) for up to 30 days. Higher esterification activities were achieved when the lipase extract was immobilized in sodium alginate and activated coal.

This study reports the maximization of geranyl oleate production by esterification of geraniol and oleic acid in a solvent-free system using a commercial lipase as catalyst. The operating conditions that maximized geranyl oleate production were determined to be 40 °C, geraniol to oleic acid molar ratio of 5:1, 150 rpm and 10 wt% of enzyme, with a resulting reaction conversion of about 93%. After determining the best reaction parameters, a kinetic study was performed and the results obtained in this step allow to conclude that an excess of alcohol (alcohol to acid molar ratio of 5:1), relatively low enzyme concentration (5 wt%) and temperature of 50 °C afforded nearly complete reaction conversion after 1 h of reaction. New experimental data on enzymatic esterification of geraniol and oleic acid for geranyl oleate production are reported in this work, showing a promising perspective of the technique to overcome the inconvenience of the chemical-catalyzed route.

This work reports the optimization of geranyl propionate production by esterification of geraniol and propionic acid in a solvent-free system using a commercial lipase as catalyst. For this purpose, a sequential strategy was performed applying two experimental designs. The operating conditions that optimized geranyl propionate production were determined to be 40 °C, geraniol to propionic acid molar ratio of 3:1, 150 rpm and 10 wt% of enzyme, with a resulting reaction conversion of about 93%. After determining the optimum reaction parameters, a kinetic study was carried out evaluating the influence of substrates molar ratio, enzyme concentration and temperature on reaction conversion. Results obtained in this step allow to conclude that an excess of alcohol (acid to alcohol molar ratio of 1:6), relatively low enzyme concentration (5 wt%), temperature of 40 °C and substrates molar ratio of 1:1 afforded nearly complete reaction conversion after 30 min of reaction. New experimental data on enzymatic esterification of geraniol and propionic acid for geranyl propionate production are reported in this work, showing a promising perspective of the technique to overcome the well-known drawbacks of the chemical-catalyzed route.

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., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The main objective of this work was to optimize lipase production, in terms of hydrolytic and esterification activities, by Penicillium brevicompactum and Penicillium verrucosum in solid state fermentation using agroindustrial residues as raw material. Maxima hydrolytic activities of 48.6 and 87.7 U/g were achieved when P. brevicompactum was cultured in babassu cake and castor meal, respectively. Higher esterification activities (around 244 U/g) were achieved when P. brevicompactum was used as microorganism and babassu cake as raw material. Different experimental conditions led to these promising values, clearly showing that no correlation can be attributed between hydrolytic and esterification activities. In spite of the several applications of lipases which are capable of catalyze synthesis reactions, only few works in this subject are presented in the literature, especially when low cost raw materials are used.

The main objective of this work was the isolation and screening of microorganisms with potential for producing lipases for the synthesis of fatty esters as well as evaluating the specificity of the enzymes produced, using different alcohols (methanol, ethanol, n-propanol, and butanol) and fatty acids (oleic and lauric acids) as substrates. Promising biocatalysts for organic synthesis were obtained in this work. The isolated strains 69F and 161Y showed ability to efficiently catalyze the reaction for production of n-propyl oleate. Other strains can also be considered of potential interest, as 74F, 111Y, and 186Y. The future development of production using different substrates could result in cheap crude lipase of high importance to industrial applicability.

This work reports solvent-free enzymatic glycerolysis of olive oil with an immobilized lipase (Novozym 435) using Tween 40, Tween 65, Tween 80, Tween 85, Triton X-100, and soy lecithin as surfactants. The first step was the screening of two potential surfactants for Monoacylglycerol (MAG) and Diacylglycerol (DAG) production with a pre-established operating condition and 2 h of reaction time. Afterwards, a sequential experimental design strategy was carried out in order to optimize MAG and DAG production using Tween 65 and Triton X-100 as surfactants. The operating conditions that optimized MAG and DAG yields were 70 degrees C, stirring rate of 600 rpm, glycerol:olive oil molar ratio of 6:1, 16 wt% of surfactant Tween 65 and 9.0 wt% of Novozym 435, leading to a content of 26 and 17 wt% of MAG and DAG, respectively.

This work investigated the influence of temperature, enzyme concentration, substrates molar ratio, in the absence and presence of organic solvent, at two molar ratios of the substrates on the enzymatic production of linalil esters using the immobilized lipase Novozym 435 as catalyst, different acids and linalool and Ho-Sho essential oil as substrates. The best reaction conversion was obtained at the highest temperature (70 degrees C), for both solvent free (3.81%) and with solvent addition (2.25%), for a solvent to substrates molar ratio of 2:1, enzyme concentration of 5 wt% and acid to alcohol molar ratio of 1:1. The reaction kinetics revealed that Ho-Sho essential oil afforded the greatest conversions when compared with pure linalool. Higher linalil esters production were achieved after 10 h reaction (5.58%) in 2:1 solvent to substrates molar ratio, with enzyme concentration of 5 wt%, at 70 degrees C and anhydride to alcohol molar ratio of 1:1 using Ho-Sho essential oil as substrate.

This work reports new experimental data and mathematical modeling of lipase-catalyzed biodiesel production using soybean oil and ethanol as substrates and pressurized n-propane as solvent. The experiments were carried out in a batch reactor, recording the reaction kinetics and evaluating the effects of temperature in the range of 45-70 degrees C, enzyme content from 1 to 20 wt% and oil to ethanol molar ratios of 1:3, 1:6, 1:9 and 1:15. The solvent to substrates mass ratio and pressure were set at 2:1 and 50 bar, respectively. Results showed that lipase-catalyzed alcoholysis in propane medium might be a potential alternative to conventional techniques for biodiesel production, since good conversions were obtained at mild temperature and pressure conditions. The semi-empirical mathematical model based on balance equations, adopted to describe the transesterification kinetics in pressurized n-propane, yielded relative deviations between experimental and calculated values lower than 10%, thus allowing a satisfactory representation of experimental results and a better understanding of the transesterification reaction., (2010 Elsevier B.V. All rights reserved.)

This work reports the application of a lipase in the 2-ethylhexyl palmitate esterification in a solvent-free system with an immobilized lipase (Lipozyme RM IM). A sequential strategy was used applying two experimental designs to optimize the 2-ethylhexyl palmitate production. An empirical model was then built so as to assess the effects of process variables on the reaction conversion. Afterwards, the operating conditions that optimized 2-ethylhexyl palmitate production were established as being acid/alcohol molar ratio 1:3, temperature of 70 degrees C, stirring rate of 150 rpm, 10 wt.% of enzyme, leading to a reaction conversion as high as 95%. From this point, a kinetic study was carried out evaluating the effect of acid:alcohol molar ratio, the enzyme concentration and the temperature on product conversion. The results obtained in this step permit to verify that an excess of alcohol (acid to alcohol molar ratio of 1:6), relatively low enzyme concentration (10 wt.%) and temperature of 70 degrees C, led to conversions next to 100%.

The aim of this work is to report the production of mono- and diglycerides from olive oil at ambient condition and in pressurized n-butane as solvent medium. For this purpose, a commercial immobilized lipase (Novozym 435) was employed as catalyst and sodium (bis-2-ethyl-hexyl) sulfosuccinate (Aerosol-OT or AOT) as surfactant. The experiments were conducted in batch mode varying the temperature, pressure, and AOT concentration. Results showed that lipase-catalyzed glycerolysis either with compressed n-butane or in solvent-free system with AOT as surfactant might be a potential alternative route to conventional methods, as high contents of reaction products, especially monoglycerides ( approximately 60 wt.%), were achieved at mild temperature and pressure with a relatively low solvent to substrates mass ratio (4:1) in short reaction times (2 h).

This work reports the optimization of 2-ethylhexyl palmitate production by esterification reaction in a solvent-free system using a commercial lipase as catalyst. For this, a sequential strategy was performed applying three experimental designs. An empirical model was built so as to assess the effects of process variables on the reaction conversion. Afterward, the operating conditions that optimized 2-ethylhexyl palmitate production were determined to be acid to alcohol molar ratio of 1:5.5, 70 degrees C, 150 rpm and 10.5 wt% of enzyme, leading to a reaction conversion as high as 93%. From this point, a kinetic study was carried out evaluating the influence of acid to alcohol molar ratio, enzyme concentration and temperature on product yield. Results obtained in this step allow to conclude that an excess of alcohol (acid to alcohol molar ratio of 1:6), relatively low enzyme concentration (10 wt%) and temperature of 70 degrees C led to nearly complete reaction conversion.

This work investigates the continuous production of fatty acid ethyl esters from soybean oil in compressed fluids, namely carbon dioxide, propane and n-butane, using immobilized Novozym 435 as catalyst. The experiments were performed in a packed-bed bioreactor evaluating the effects of temperature in the range of 30-70 degrees C, from 50 to 150 bar, oil to ethanol molar ratio of 1:6-1:18 and solvent to substrates mass ratio of 4:1-10:1. In contrast to the use of carbon dioxide and n-butane, results showed that lipase-catalyzed alcoholysis in a continuous tubular reactor in compressed propane might be a potential route to biodiesel production as high reaction conversions were achieved at mild temperature (70 degrees C) and pressure (60 bar) conditions in short reaction times.

This work aimed to evaluate the effects of particle size, solid/solvent ratio (w/v), and reaction time on hydrolytic and esterification activities of a lipase extract from wheat seeds. The higher hydrolytic activity was 5.9 U/g with a particle size of 425 microm, solid/solvent ratio of 30:60 (w/v), and reaction time of 15 h, with maximum hydrolytic activity of 14.47 U/g after 24 h of precipitation. For esterification activity the best result was 57.88 U/g with a particle size of 425 microm and solid/solvent ratio of 30:120 (w/v) for 5 h, with a maximum value after 10 h of precipitation reaching 208.20 U/g. The partial characterization showed that the optimal pH and temperature were found to be 5.5 and 32-37 degrees C, respectively. The extract stability at low temperatures was kept after 48 h of storage in terms of esterification activity. The hydrolytic activity was kept constant at -10 degrees C during 72 h and diminished considerably after 24 h at 4 degrees C.

This work reports experimental data and kinetic modeling of solvent-free glycerolysis of olive oil using a commercial immobilized lipase (Novozym 435) in the presence of Triton X-100 surfactant for the production of monoacylglycerols (MAG) and diacylglycerols (DAG). The experiments were performed in batch mode evaluating the effects of temperature (30-70 degrees C), enzyme concentration (2.5-18 wt %), Triton X-100 concentration (10-20 wt %), and glycerol to oil molar ratio (3:1, 6:1, and 9:1). Experimental results showed that lipase-catalyzed solvent-free glycerolysis with the addition of Triton X-100 might be a potential alternative route to conventional organic solvent methods, as good conversions were obtained with relatively low enzyme concentrations (9 wt %) in short reaction times (240 min). The glycerolysis and hydrolysis parallel reactions were considered with rate constants estimated by minimizing a maximum likelihood function. A very satisfactory agreement between experimental data and model results was obtained, thus allowing a better understanding of the reaction kinetics.

Aims: The objective of this work was to investigate the lipase production by a newly isolated Penicillium sp., using experimental design technique, in submerged fermentation using a medium based on peptone, yeast extract, NaCl and olive oil, as well as to characterize the crude enzymatic extracts obtained., Methods and Results: Lipase activity values of 9.5 U ml(-1) in 96 h of fermentation was obtained at the maximized operational conditions of peptone, yeast extract, NaCl and olive oil concentrations (g l(-1)) of 20.0, 5.0, 5.0 and of 10.0 respectively. The partial characterization of crude enzymatic extract obtained by submerged fermentation showed optimum activity at pH range from 4.9 to 5.5 and temperature from 37 degrees C to 42 degrees C. The crude extract maintained its initial activity at freezing temperatures up to 100 days., Conclusions: A newly isolated strain of Penicillium sp. used in this work yielded good lipase activities compared to the literature., Significance and Impact of the Study: The growing interest in lipase production is related to the potential biotechnological applications that these enzymes present. New lipase producers are relevant to finding enzymes with different catalytic properties of commercial interest could be obtained, without using genetically modified organisms (GMO).

Current studies about lipase production by solid-state fermentation involve the use of agro-industrial residues towards developing cost-effective systems directed to large-scale commercialization of enzyme-catalyzed processes. In this work, lipase production and partial characterization of the crude enzymatic extracts obtained by Penicillium verrucosum using soybean bran as substrate was investigated. Different inductors were evaluated and the results showed that there is no influence of this variable on the lipase production, while temperature and initial moisture were the main factors that affected enzyme production. The optimized cultivation temperature (27.5 degrees C) and initial moisture of substrate (55%) were determined using the response surface methodology. Kinetics of lipase production was followed at the optimized growth conditions. Optimum lipase yield was 40 U/g of dry bran. The crude enzymatic extract showed optimal activity in the range from 30 to 45 degrees C and in pH 7.0.