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Butyl oleate synthesis by the lipase B from Candida antarctica (CalB) under extreme halophilic conditions was investigated in the present research through the AOT/Water/Isooctane reverse micellar system. The impact of aqueous content (Wo=H2OSurfactant) and NaCl variation on the enzymatic activity of CalB in the butyl oleate reaction in reverse micelles was explored. The results indicated that, based on the increase of NaCl, it is remarkable to achieve higher enzymatic activity up to 444.85 μmolmin at 5 M NaCl and Wo = 10, as the best esterification conditions at pH 7.2 and 30 °C. However, it was clear that butyl oleate synthesis by lipase CalB increased based on the reduction in the average reverse micelle size, where reverse micelle sizes were determined by dynamic light scattering (DLS). This increase in butyl oleate synthesis demonstrated the potential of reverse micelles as systems that enhance mass transport phenomena in heterogeneous biocatalysis. Furthermore, reverse micelles are promising systems for extreme halophilic lipases research.

Background: Hydroxycinnamic acids and some of their derivatives are molecules with interesting biological activities; for instance, hydroxylated hydroxycinnamic esters have proved to have antifungal properties, and thus the generation of these molecules is of industrial importance. In this study, the direct esterification capacity of the pure recombinant type B feruloyl esterase from Aspergillus terreus (AtFAE B) was evaluated by its ability to catalyze the synthesis of isobutyl o- coumarate, an interesting antifungal molecule. A ternary solvent system (isooctane/isobutanol/water) was employed to improve the synthesis of isobutyl o- coumarate, assessing different substrate concentrations, enzyme load, water percentages and pH and temperature values. Results: AtFAE B showed the highest initial rate at 18% (v/v) isobutanol and 50 mM o- coumaric acid, 0.04 mg/ml of enzyme, 4% (v/v) water without buffer and 40°C. AtFAE B half-lives at 30°C, 40°C and 50°C were 16.5 h, 1.75 h and 3.5 min, respectively. Thus, we decided to evaluate the bioconversion yield at 30°C, where the enzyme showed the highest operational stability. At this temperature, we obtained a yield of ~80% after only 8 h of reaction, using a 78:18:4 isooctane:isobutanol:water ternary solvent system, with 50 mM of o- coumaric acid. Conclusions: Under these improved conditions, the productivity was 1.06 g isobutyl o -coumarate/L*h with a biocatalyst yield of 209.6 kg isobutyl o -coumarate/kg free AtFAE B, demonstrating the promising potential of AtFAE B to accept the non-canonical o- coumaric acid as the substrate and to achieve the synthesis of isobutyl o- coumarate. Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Tabla normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0cm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}

In-Cell Crosslinked Enzymes (InCLEs) is a procedure to improve the thermal and operational stability of Bacillus megaterium intracellular decarboxylase activity, allowing to obtain enhanced whole-cell biocatalyst to produce 4-vinyl guaiacol (4VG). InCLEs were achieved using a fixed amount of biomass in a two-step procedure: 1) permeation of the membrane cell for reactants diffusion, and 2) intracellular proteins crosslinking. To optimize InCLEs preparation, 7.68 mg of biomass in the presence of different ethanol concentrations (10–50% v/v) were permeated, subsequently, the intracellular proteins were crosslinked with a glutaraldehyde/biomass ratios of 0, 1.63 and 3.26 μmol/mg. At the best conditions (10% v/v ethanol and 1.63 μmol/mg), thermostability increases 4.7-fold against the permeated uncrosslinked biocatalyst (PUCB). To further enhance the stability, the internalization of different molecular weight polyethyleneimine (PEI, 1.09 mg/mL) was evaluated. PEI-InCLEs using 25 kDa PEI, showed a half-life 68-fold higher than PUCB (1 min) at 50 °C. After 7 cycles (26.3 mM ferulic acid, pH 7), PEI-InCLEs retained 83 and 69% of the decarboxylase activity at 40 and 50 °C, producing 32.7 and 29.8 μmol of 4VG, respectively. Here we demonstrated that the InCLEs procedure is an effective strategy to obtain stable whole-cell biocatalysts for the production of high-valued biotechnological molecules.

Feruloyl esterases (FAEs) are versatile enzymes able to release hydroxycinnamic acids or synthesize their ester derivatives, both molecules with interesting biological activities such as: antioxidants, antifungals, antivirals, antifibrotic, anti-inflammatory, among others. The importance of these molecules in medicine, food or cosmetic industries provides FAEs with several biotechnological applications as key industrial biocatalysts. However, FAEs have some operational limitations that must be overcome, which can be addressed through different protein engineering approaches to enhance their thermal stability, catalytic efficiencies, and selectivity. This review aims to present a brief historical tour through the mutagenesis strategies employed to improve enzymes performance and analyze the current protein engineering strategies applied to FAEs as interesting biocatalysts. Finally, an outlook of the future of FAEs protein engineering approaches to achieve successful industrial biocatalysts is given.

Feruloyl esterases synthesize butyl hydroxycinnamates, molecules possessing interesting biological properties, nonetheless, they exhibit a low stability under synthesis conditions in organic solvents, restricting its use. To enhance its operational stability in synthesis, we immobilized type A feruloyl esterase from Aspergillus niger (AnFAEA) using several carrier-bound and carrier-free strategies. The most active biocatalysts were: 1) AnFAEA immobilized on epoxy-activated carriers (protein load of 0.6 mgenzyme x mg−1carrier) that recovered 91 % of the initial hydrolytic activity, and 2) AnFAEA aggregated and cross-linked in the presence of 5 mg of BSA and 15 mM of glutaraldehyde (AnFAEA-amino-CLEAs), which exhibited 385 % of its initial hydrolytic activity; both using 4-nitrophenyl butyrate as substrate. The AnFAEA-amino-CLEAs were 12.7 times more thermostable at 60 °C than the AnFAEA immobilized on epoxy-activated carrier, thus AnFAEA-amino-CLEAs were selected for further characterization. Interestingly, during methyl sinapate hydrolysis (pH 7.2 and 30 °C), AnFAEA-amino-CLEAs KM was 15 % higher, while during butyl sinapate synthesis the KM was reduced in 63 %, both compared with the soluble enzyme. The direct esterification of butyl sinapate at solvent free conditions using sinapic acid 50 mM, reached 95 % conversion after 24 h employing AnFAEA-amino-CLEAs, which could be used for 10 cycles without significant activity losses, demonstrating their outstanding operational stability., We acknowledge the funding of Fundación Hergar (F), Foundation ARAID, Aragon Government, Spain (E37-17R) for funding the work by Fernando López Gallego. This work was supported by the programa Atención a Problemas Nacionales from CONACyT-México [project number 2017-01-6267]. Daniel Grajales and Susana Velasco acknowledge the scholarships received from CONACyT-México.

Amycolatopsis sp. ATCC 39116 catabolizes ferulic acid by the non-oxidative deacetylation and β-oxidation pathways to produce vanillin and vanillic acid, respectively. In submerged culture, vanillin productivity decreased more than 8-fold, when ferulic, p-coumaric, and caffeic acids were employed in pre-cultures of the microorganism in order to activate the ferulic acid catabolic pathways, resulting in a carbon redistribution since vanillic acid and guaiacol productivities increased more than 5-fold compared with control. In contrast, in surface culture, the effects of ferulic and sinapic acids in pre-cultures were totally opposite to those of the submerged culture, directing the carbon distribution into vanillin formation. In surface culture, more than 30% of ferulic acid can be used as carbon source for other metabolic processes, such as ATP regeneration. In this way, the intracellular ATP concentration remained constant during the biotransformation process by surface culture (100 μg ATP/mg protein), demonstrating a high energetic state, which can maintain active the non-oxidative deacetylation pathway. In contrast, in submerged culture, it decreased 3.15-fold at the end of the biotransformation compared with the initial content, showing a low energetic state, while the NAD+/NADH ratio (23.15) increased 1.81-fold. It seems that in submerged culture, low energetic and high oxidative states are the physiological conditions that can redirect the ferulic catabolism into β-oxidative pathway and/or vanillin oxidation to produce vanillic acid.

Halophilic microorganisms are potentially capable as platforms to produce low-cost biosurfactants. However, the robustness of bioprocesses is still a challenge and, therefore, it is essential to understand the effects of microbiological culture conditions through bioreactor engineering. Based on a design of experiments (DOE) and a response surface methodology (RSM) tailored and taken from the literature, the present work focuses on the evaluation of a composite central design (CCD) under batch cultures in stirred-tank bioreactors with the halophilic bacteria Salibacterium sp. 4CTb in order to determine the operative conditions that favor mass transfer and optimize the production of a lipopeptide. The results obtained showed profiles highlighting the most favorable culture conditions, which lead to an emulsification index (E24%) higher than 70%. Moreover, through the behavior of dissolved oxygen (DO), it was possible to experimentally evaluate the higher volumetric coefficient of mass transfer in the presence of lipopeptide (kLa = 31 1/h) as a key criterion for the synthesis of the biosurfactant on further cell expansion.

En los últimos años la preocupación por la conservación y optimización de los recursos naturales y del medio ambiente, así como los demás problemas territoriales existentes, hacen necesario la recopilación, tratamiento y análisis de la información para el desarrollo de una investigación, que puede resultar laboriosa, compleja y costosa, es por esto que los sistemas de información geográfica (SIG) han tomado gran relevancia al momento de analizar problemas relacionados con información espacial, ya que permiten la automatización, visualización y manipulación de datos geográficos, convirtiéndose en una herramienta fundamental para la toma de decisiones Los SIG además de efectuar operaciones de manera confiable, proveen al usuario la información relacionada con fenómenos espaciales, la cual es de gran importancia en el ámbito investigativo, teniendo en cuenta que gran parte de la información sobre recursos naturales y ambientales que se maneja está vinculada con la información geográfica o espacial. El presente libro es una compilación de resultados de investigaciones desarrolladas durante los años 2018 – 2019, enfocadas a las aplicaciones de los SIG en diversas áreas de la ingeniería civil., In recent years the concern for the conservation and optimization of natural resources and the environment, as well as existing territorial problems, make it necessary to collect, process and analyze information for the development of research, which can be laborious, complex and expensive, that is why Geographic Information Systems (GIS) have acquired significant relevance when analyzing problems related to spatial information, since they allow the automation, visualization and manipulation of geographic data, becoming a fundamental tool for the decision making. GIS, besides to performing operations reliably, provide the user with information related to spatial phenomena, which has great importance in the research field, considering that a large part of the information on natural and environmental resources that is handled is linked with geographic or spatial information. This book is a compilation of research results developed during the years 2018 - 2019, focused on the applications of GIS in various areas of civil engineering.

In order to search viable alternatives for detoxifying the castor bean toxic waste, and harnessing for diverse applications, the objective of this study was evaluate the thermal-detoxified method and determinate the castor bean cake nutraceutical potential properties. Autoclaving (15 psi, 15 min, 121 ° C) process in presence of humidity (30 %) was performed as detoxified method. Presence of ricin was evaluated using ELISA and the castor bean cake lethal dose (LD50) determinate by acute toxicity assay in male Wistar rats. Additionally, the castor bean cake chemical and phytochemical characterization was evaluated and its nutraceutical potential determinate throughout antioxidant and hypoglycemic effects. The results showed that after the thermal-detoxified treatment, ricin concentration decreased over 99 %, and the castor bean cake LD50 was greater than 5000 mg/kg of body weight. After detoxified process, protein content increases 17.5 % and lipids decreases 10 %. In addition, a greater content of insoluble fraction (13 %) and 2.6-fold times more of resistant starch, as compared to un-detoxified cake, was observed. Furthermore, in comparison with untreated cake, thermal process led to an upsurge of gallic acid (3.5-fold times) and 4-O- caffeoylquinic acid (8.8-fold times). About nutraceutical potential properties, a lower antioxidant capacity and a hypoglycemic effect, in comparison with the control, were observed. The thermal-detoxified method eliminates ricin of castor bean cake, providing to be a potential source of proteins, lipids, fiber, and bioactive compounds as well as exhibiting antioxidant and hypoglycemic effects.

Feruloyl esterases (FAEs) are a diverse group of hydrolases widely distributed in plants and microorganisms which catalyzes the cleavage and formation of ester bonds between plant cell wall polysaccharides and phenolic acids. FAEs have gained importance in biofuel, medicine and food industries due to their capability of acting on a large range of substrates for cleaving ester bonds and synthesizing highadded value molecules through esterification and transesterification reactions. During the past two decades extensive studies have been carried out on the production, characterization and classification of FAEs, however only a few reports of suitable High Throughput Screening assays for this kind of enzymes have been reported. This review is focused on a concise but complete revision of classical to High Throughput Screening methods for FAEs, highlighting its advantages and disadvantages, and finally suggesting future perspectives for this important research field.

To date, several sensitive methods, based on radiolabeled elements or sterically hindered fluorochrome groups, are usually employed to screen lipase and phospholipase A (PLA) activities. Here, a new ultraviolet spectrophotometric assay for lipase or PLA was developed using natural triglycerides or synthetic glycerophosphatidylcholines containing α-eleostearic acid (9Z, 11E, 13E-octadecatrienoic acid) purified from Aleurites fordii seed oil. The conjugated triene present in α-eleostearic acid constitutes an intrinsic chromophore and consequently confers strong UV absorption properties of this free fatty acid as well as of lipid substrates harboring it. The substrate was coated into the wells of a microplate, and the lipolytic activities were measured by the absorbance increase at 272 nm due to the transition of α-eleostearic acid moiety from the adsorbed to the soluble state. This continuous assay is compatible with a high-throughput screening method and can be applied specifically to the screening of new potential lipase, PLA

Solid-state fermentation (SSF) has been largely employed during the last three decades to produce different biomolecules of industrial interest, particularly enzymes. Through the use of agroindustrial wastes as SSF substrates, an economic process of lipases production can be achieved. In this chapter we describe a comprehensive SSF method for producing an economical preparation of Rhizomucor miehei lipase, employing sugarcane bagasse and used vegetal oil as substrates. To demonstrate the usefulness of the lipase produced by this method, we utilized directly the dried fermented solid, as a heterogeneous biocatalyst for the ethanolysis of different fats and oils. Final ethyl ester conversions (90%, 24 h) were similar with those obtained using a commercial immobilized Rhizomucor miehei lipase at our best conditions. In this work we demonstrated that SSF is an easy and economical method for the production of lipases that can be used directly as heterogeneous biocatalysts for biodiesel production, employing low-cost feedstocks.

Carbohydrate esterases are a group of enzymes which release acyl or alkyl groups attached by ester linkage to carbohydrates. The CAZy database, which classifies enzymes that assemble, modify, and break down carbohydrates and glycoconjugates, classifies all carbohydrate esterases into 16 families. This chapter is an overview of the research for nearly 50 years around the main groups of carbohydrate esterases dealing with the degradation of polysaccharides, their main biochemical and molecular traits, as well as its application for the synthesis of high added value esters.

To date, several sensitive methods, based on radiolabeled elements or sterically hindered fluorochrome groups, are usually employed to screen lipase and phospholipase A (PLA) activities. Here, a new ultraviolet spectrophotometric assay for lipase or PLA was developed using natural triglycerides or synthetic glycerophosphatidylcholines containing α-eleostearic acid (9Z, 11E, 13E-octadecatrienoic acid) purified from Aleurites fordii seed oil. The conjugated triene present in α-eleostearic acid constitutes an intrinsic chromophore and consequently confers strong UV absorption properties of this free fatty acid as well as of lipid substrates harboring it. The substrate was coated into the wells of a microplate, and the lipolytic activities were measured by the absorbance increase at 272 nm due to the transition of α-eleostearic acid moiety from the adsorbed to the soluble state. This continuous assay is compatible with a high-throughput screening method and can be applied specifically to the screening of new potential lipase, PLA1 and PLA2 inhibitors.

Solid-state fermentation (SSF) has been largely employed during the last three decades to produce different biomolecules of industrial interest, particularly enzymes. Through the use of agroindustrial wastes as SSF substrates, an economic process of lipases production can be achieved. In this chapter we describe a comprehensive SSF method for producing an economical preparation of Rhizomucor miehei lipase, employing sugarcane bagasse and used vegetal oil as substrates. To demonstrate the usefulness of the lipase produced by this method, we utilized directly the dried fermented solid, as a heterogeneous biocatalyst for the ethanolysis of different fats and oils. Final ethyl ester conversions (>90%, 24 h) were similar with those obtained using a commercial immobilized Rhizomucor miehei lipase at our best conditions. In this work we demonstrated that SSF is an easy and economical method for the production of lipases that can be used directly as heterogeneous biocatalysts for biodiesel production, employing low-cost feedstocks.

Carbohydrate esterases are a group of enzymes which release acyl or alkyl groups attached by ester linkage to carbohydrates. The CAZy database, which classifies enzymes that assemble, modify, and break down carbohydrates and glycoconjugates, classifies all carbohydrate esterases into 16 families. This chapter is an overview of the research for nearly 50 years around the main groups of carbohydrate esterases dealing with the degradation of polysaccharides, their main biochemical and molecular traits, as well as its application for the synthesis of high added value esters.

Carica papaya lipase is a versatile biocatalyst that is employed for many biotechnological purposes. Its lipase activity was first observed to be tightly linked to the insoluble fraction of latex. Nevertheless, recent studies have shown that this activity is also present in the fruit peel and seeds, suggesting that the lipase activity occurs in other parts of the plant. In the present work, the hydrolytic activity on trioctanoin was determined in various plant by-products, including latex, leafs, petioles, meristems, fruits, and the stem. The most hydrolytic activity was found in the latex (11 U/mL), followed by the petioles (1.7 U/mL). The hydrolytic selectivity was determined using triacetin, tripropionin, tributyrin, and trioctanoin. The enzymes present in the latex showed a higher rate of hydrolysis of tributyrin, while those present in the petioles had a preference for tripropionin, possibly indicating the occurrence of at least two different triacylglycerol hydrolases. Five self-immobilized ...

Capsaicin, the major component of chili peppers, has shown potential therapeutic effect against metabolic disease inducing weight loss. Its effect is exerted by a particular molecular mechanism and pharmacodynamics, distinct to current obesity treatments. However, its high irritating taste or pungency has limited its use to some clinical trials. Capsaicin-like molecules, known as capsaicin synthetic analogs, contain the majority of capsaicin domains but without those of pungency. Recently, they have received much attention by their potent antiobesity effect. The study of their structure-activity relationship would unravel the mechanisms responsible of this effect. This review summarizes much of the current experimental evidence of the potential effect in metabolism regulation of natural and synthetic capsaicin analogs discovered to date.

The aim of this work is to propose and evaluate a catalytic profile of biocatalysts with lipase activity based on four characteristic reactions of lipases. For that, the catalytic profiles of three lipolytic biocatalysts from strains of the genus Rhizopus produced by solid-state fermentation (SSF) were determined based on acyl-, regio-, enantio- and chemoselectivity. Four commercial lipases were also evaluated according to their catalytic selectivity. p-nitrophenol esters from C-4 to C-16 were used to evaluate the acyl selectivities of the biocatalysts. The three biocatalysts produced by SSF presented the highest hydrolytic activity on triglycerides of medium chain length (C-8). The use of high performance thin layer chromatography (HPTLC) allowed us to demonstrate the biocatalysts produced by SSF presented sn-1,3-regioselectivity. Based on the Quick E method, the biocatalysts preferentially acted on the S-enantioisomer of glycidyl butyrate esters. Although their E values were insufficiently high ...

The growing incidence of obesity is a worldwide public health problem leading to a risk factor for non-alcoholic fatty liver disease, which extends from steatosis to steatohepatitis and cirrhosis. We investigated whether the aqueous extract of Hibiscus sabdariffa L. (Hs) reduces body weight gain and protects the liver by improving lipid metabolism in high fat diet-induced obese C57BL/6NHsd mice. We found that oral administration of the Hs extract reduced fat tissue accumulation, diminished body weight gain and normalized the glycemic index as well as reduced dyslipidemia compared to the obese mice group that did not receive Hs treatment. In addition, Hs treatment attenuated liver steatosis, down-regulated SREBP-1c and PPAR-γ, blocked the increase of IL-1, TNF-α mRNA and lipoperoxidation and increased catalase mRNA. Our results suggest that the anti-obesity, anti-lipidemic and hepatoprotective effects of the Hs extract are related to the regulation of PPAR-γ and SREBP-1c in the liver.