Lipolytic production from solid-state fermentation of the filamentous fungus Penicillium polonicum and its applicability as biocatalyst in the synthesis of ethyl oleate.

Lipases represent versatile biocatalysts extensively employed in transesterification reactions for ester production. Ethyl oleate holds significance in biodiesel production, serving as a sustainable alternative to petroleum-derived diesel. In this study, our goal was to prospect lipase and assess its efficacy as a biocatalyst for ethyl oleate synthesis. For quantitative analysis, a base medium supplemented with Rhodamine B, olive oil, and Tween 80 was used. Solid-state fermentation utilized crambe seeds of varying particle sizes and humidity levels as substrates. In the synthesis of ethyl oleate, molar ratios of 1:3, 1:6, and 1:9, along with a total enzymatic activity of 60 U in n-heptane, were utilized at temperatures of 30 °C, 37 °C, and 44 °C. Reactions were conducted in a shaker at 200 rpm for 60 min. As a result, we first identified Penicillium polonicum and employed the method of solid-state fermentation using crambe seeds as a substrate to produce lipase. Our findings revealed heightened lipolytic activity (22.5 Ug-1) after 96 h of fermentation using crambe cake as the substrate. Optimal results were achieved with crambe seeds at a granulometry of 0.6 mm and a fermentation medium humidity of 60%. Additionally, electron microscopy suggested the immobilization of lipase in the substrate, enabling enzyme reuse for up to 4 cycles with 100% enzymatic activity. Subsequently, we conducted applicability tests of biocatalysts for ethyl oleate synthesis, optimizing parameters such as the acid/alcohol molar ratio, temperature, and reaction time. We attained 100% conversion within 30 min at 37 °C, and our results indicated that the molar ratio proportion did not significantly influence the outcome. These findings provide a methodological alternative for the utilization of biocatalysts in ethyl oleate synthesis.

Development of a magnetic biocatalyst useful for the synthesis of ethyloleate.

Candida antarctica Lipase B was successfully immobilized on magnetite (Fe3O4) nanoparticles functionalized with chitosan and glutaraldehyde. The obtained magnetic catalyst was characterized and its performance was evaluated in solvent-free synthesis of ethyl oleate at room temperature. The performance of this biocatalyst was compared with the commercial Novozym 435, as a tool to estimate the efficiency of immobilization. It was found that using 33 mg of the biocatalyst it was possible to reach almost the same activity that was obtained using 12 mg of Novozym 435. Furthermore, this new biocatalyst presents the advantages of not being degraded by short alcohols, being easily recovered from the reaction media by magnetic decantation, and low fabrication cost. The possibility of reutilization was also studied, keeping a significant activity up to eight cycles. A special sampling protocol was also developed for the multiphasic reaction system, to assure accurate results. This novel biocatalyst is an interesting alternative for potential industrial applications, considering the above-mentioned advantages.

Culture conditions stimulating high gama-Linolenic acid accumulation by Spirulina platensis / Condições de cultura simulando o levado acúmulo de ácido gama-linolênico por Spirulina platensis

Gamma-linolenic acid (GLA) production by Spirulina platensis under different stress-inducing conditions was studied. Submerged culture studies showed that low temperature (25ºC), strong light intensity (6 klux) and primrose oil supplement (0.8 percentw/v) induced 13.2 mg/g, 14.6 mg/g and 13.5 mg linolenic acid per gram dry cell weight respectively. A careful observation of fatty acid profile of the cyanobacteria shows that, oleic acid and linoleic acid, in experiments with varying growth temperature and oil supplements respectively, helped in accumulating excess γ-linolenic acid. In addition, cultures grown at increasing light regimes maintained the γ-linolenic acid to the total fatty acid ratio(GLA/TFA) constant, despite any change in γ-linolenic acid content of the cyanobacteria.

Estudou-se a produção de ácido γ-linolênico por Spirulina platensis em diferentes condições de estresse. Culturas submersas indicaram que temperatura baixa (25ºC), forte intensidade de luz (6 klux) e suplementação com óleo de prímula (0,8 por cento p/v) induziram a produção de ácido linolênico de 13,2 mg/g, 14,6 mg/g e 13,5 mg/g peso seco, respectivamente. Uma observação cuidadosa do perfil de ácidos graxos da cianobacteria indica que os ácidos oléico e linoléico, em experimentos com diferentes temperaturas de crescimento e suplementos de óleo, auxiliaram no acúmulo de excesso de ácido γ-linolênico. Além disso, as culturas obtidas em intensidades crescentes de luz mantiveram a relação ácido γ-linolênico/ácidos graxos totais constante, independentemente de qualquer mudança no conteúdo de ácido γ-linolênico da cianobactéria.

Organic phase synthesis of ethyl oleate using lipases produced by solid-state fermentation.

This paper reports a study of the enzymatic esterification of oleic acid and ethanol. The reaction was catalyzed by lipases produced by solid-state fermentation with Rhizopus sp. Olive oil and perlite were used as an inducer and inert support, respectively. Synthesis of ethyl oleate was carried out in a 10-mL batch reactor with magnetic stirring. The effects of substrate ratios, biocatalyst concentration, and temperature on the reaction rate and conversion efficiency were evaluated. The highest reaction rate (1.64 mmol/L min) was reached with an oleic acid/ethanol mol ratio of 1:5 (oleic acid 50 mM:ethanol 250 mM) and 1 g of biocatalyst. Conversions approaching 100% were obtained after 60 min of reaction at 45 degrees C with n-hexane as a solvent. The initial reaction rate increased proportionally with respect to biocatalyst concentration, which suggests that the reaction rate was not controlled by mass transfer. The biocatalyst retained more than 80% of its catalytic activity after 7 months of storage at 4 degrees C. The results demonstrate that the biocatalyst produced by Rhizopus sp. in solid-state fermentation can be successfully used for ethyl oleate synthesis over short reaction periods under conditions when ethanol is in excess.

High performance enzymatic synthesis of oleyl oleate using immobilised lipase from Candida antartica

High performance enzymatic synthesis of oleyl oleate, a liquid wax ester was carried out by lipase-catalysed esterification of oleic acid and oleyl alcohol. Various reaction parameters were optimised to obtain high yield of oleyl oleate. The optimum condition to produce oleyl oleate was reaction time; 5 min, organic solvents of log P is greater than or equal to 3.5, temperature; 40-50 ºC, amount of enzyme; 0.2-0.4 g and molar ratio of oleyl alcohol to oleic acid; 2:1. The operational stability of enzyme was maintained at >90 percent yield up to 9 cycles. Analysis of the yield of the product showed that at optimum conditions, >95 percent liquid wax esters were produced.

Frequent analytical/experimental problems in lipase-mediated synthesis in solvent-free systems and how to avoid them.

Compared with chemical catalysis, enzymatic catalysis is a relatively new topic. Experimental work involving lipases deserves careful attention and accurate procedures still need to be implemented. A rapid but careful survey of published data immediately demonstrates that experiments performed under similar conditions with similar reagents have led to very different results. The aim of this work is to point out the importance of accurate and systematic procedures in order to ensure the reproducibility of experimental data. We strongly believe that different results found by different labs are due to problems detected in the procedures used. Quantification of the immobilisation efficiency of lipase on several supports through UV/visible methods and sampling methods used to obtain correct enzymatic activity values are specifically analysed. After a brief review which demonstrates the big discrepancies found in the literature, original data from Candida rugosa lipase adsorption on polypropylene powder and its use in the solvent-free synthesis of ethyl oleate are introduced in order to exemplify the difficulties found in these kinds of systems. Several procedures described in the literature are assayed and the accuracy of the results obtained is carefully analysed. The aim of the whole analysis performed is that it would be useful for any powdered solid to be used as a support for a lipase in a solvent-free system for any synthesis reaction, especially for those involving a volatile reagent. Throughout this contribution, special emphasis is placed on how catalytic reaction results using enzymes (free and immobilised) are reported so as to allow comparison between published data, something which is usually difficult since very different units are used and often complementary data are not included.

Role of delta 9 desaturase activity in the maintenance of high levels of monoenoic fatty acids in hepatoma cultured cells.

The incorporation and delta 9 desaturation of exogenous [14C]stearic acid were studied in HTC 7288c cells in suspension. We examined the uptake of the acid over a wide range of concentrations (0-160 microM) after incubating the cells for 6 h in a chemically-defined medium. Under this experimental condition, the uptake of the labeled acid was more extensive than that obtained from static cultures or from monolayer of isolated hepatocytes of rats. At an external concentration of 160 microM ca. 52 nmoles of acid per mg of cellular protein was taken up. The production of oleic acid from [14C]stearate (delta 9 desaturation) correlated well with the uptake curve between 0-80 microM concentration. For higher stearate concentrations, the biosynthesis of oleic acid declined substantially and a plateau of 22 nmoles/mg cellular protein was reached. The incorporation and desaturation of an initial exogeneous concentration of [14C]stearic acid (80 microM) was also studied from 0-6 h. The results obtained demonstrated that the uptake of the substrate into cellular lipids was fast and non saturable. Quantitative gas-liquid chromatography of total cellular lipids under the different experimental conditions demonstrated a negative correlation between the decrease in the palmitic and palmitoleic acids and the increase in the intracellular levels of stearic and oleic acids. These analytical modifications took place with no changes in the saturated/monoenoic fatty acid ratio. This work also demonstrated a significant contribution of the stearoyl-CoA desaturase system to the high levels of oleic acid present in this kind of hepatoma cells.

Los ácidos grasos esenciales y sus funciones / Essential fatty acids and their functions

Se realizó una recopilación sobre los ácidos grasos esenciales y su función en animales y en humanos. La composición de las series n-6, n-3, n-9 y n-7 fue descripta enfatizando la reacción de competición de las mismas, así como también las necesidades diarias de los ácidos n-6 y n-3 en humanos. También se señaló el mecanismo de inter-conversión de los distintos ácidos grasos esenciales. La contribución de las 5 y 6 desaturasas a la formación de los ácidos polinosaturados se ha comentado cuidadosamente, indicándose que nuevos trabajos descartarían la existencia de la 4 desaturasa. A su vez se detalla la estructura del sistema desaturante, su ubicación en el cuerpo del animal y su regulación por medio de factores dietarios y hormonales. Se comentaron la reacciones de elongación y retroconversión, así como también la formación de eicosanoides. La importancia de los ácidos grasos esenciales y sus productos de transformación en la bioquímica clínica se ha tratado detalladamente, focalizando el efecto sobre la epidermis, sistemas cardiovascular y reproductor, presión arterial, diabetes y cáncer

Los ácidos grasos esenciales y sus funciones / Essential fatty acids and their functions

Se realizó una recopilación sobre los ácidos grasos esenciales y su función en animales y en humanos. La composición de las series n-6, n-3, n-9 y n-7 fue descripta enfatizando la reacción de competición de las mismas, así como también las necesidades diarias de los ácidos n-6 y n-3 en humanos. También se señaló el mecanismo de inter-conversión de los distintos ácidos grasos esenciales. La contribución de las 5 y 6 desaturasas a la formación de los ácidos polinosaturados se ha comentado cuidadosamente, indicándose que nuevos trabajos descartarían la existencia de la 4 desaturasa. A su vez se detalla la estructura del sistema desaturante, su ubicación en el cuerpo del animal y su regulación por medio de factores dietarios y hormonales. Se comentaron la reacciones de elongación y retroconversión, así como también la formación de eicosanoides. La importancia de los ácidos grasos esenciales y sus productos de transformación en la bioquímica clínica se ha tratado detalladamente, focalizando el efecto sobre la epidermis, sistemas cardiovascular y reproductor, presión arterial, diabetes y cáncer

Stearic acid desaturation in rat liver microsomes: stimulation by fatty acid binding protein.

A soluble protein (Mr = 12,000) showing the characteristics of fatty acid binding protein is partially purified from rat liver cytosol (15-fold on the basis of its affinity for oleic acid) using ammonium sulphate precipitation. More oleate than stearate is removed from liver microsomes incubated with similar amounts of both fatty acids and the protein, indicating that it has a higher affinity for oleic than for stearic acid. When added to microsomes, a fraction enriched in this protein stimulates stearic acid desaturation. Such an effect is abolished if the protein is pre-saturated with oleic acid. It is suggested that the stimulation of stearic acid desaturation by fatty acid binding protein may involve a selective removal of the product, oleic acid, from the microsomal membranes.

Stearic acid desaturation in rat liver microsomes: stimulation by fatty acid biding protein

Una proteína soluble (Mr=12 000) similar a FABP es parcialmente purificada a partir de citosol de hígado de rata (15 veces, considerando su afinidad por ácido oleico) por precipitación con sulfato de amonio. Durante la incubación de microsomas de hígado de rata conteniendo cantidades similares de los ácidos esteátrico y oleico, con esta proteína, hubo una disociación selectiva del ácido oleico desde la membrana microsomal. Cuando se agrega a microsomas una fracción enriquecida en esta proteína se estimula la desaturación del ácido esteárico. Este efecto es eliminado si la proteína es presaturada con ácido oleico. Se sugiere que esta proteína estimula la desaturación del ácido esteárico por un mecanismo que involucra la remoción del producto de la reacción

Stearic acid desaturation in rat liver microsomes: stimulation by fatty acid biding protein

Una proteína soluble (Mr=12 000) similar a FABP es parcialmente purificada a partir de citosol de hígado de rata (15 veces, considerando su afinidad por ácido oleico) por precipitación con sulfato de amonio. Durante la incubación de microsomas de hígado de rata conteniendo cantidades similares de los ácidos esteátrico y oleico, con esta proteína, hubo una disociación selectiva del ácido oleico desde la membrana microsomal. Cuando se agrega a microsomas una fracción enriquecida en esta proteína se estimula la desaturación del ácido esteárico. Este efecto es eliminado si la proteína es presaturada con ácido oleico. Se sugiere que esta proteína estimula la desaturación del ácido esteárico por un mecanismo que involucra la remoción del producto de la reacción (AU)

[Essential fatty acids: its transformations and functions]. / Los ácidos grasos esenciales: sus transformaciones y funciones.

Essential fatty acids, in animals, pertain to two different fatty acid families: the linoleic and the linolenic. These, and the non-essential families of oleic and palmitoleic are produced by action of the enzymes proper. The lack of essential fatty acids produces typical symptoms that are accompanied by fatty acid compositions, also typical, utilized with diagnostic value. The biological effects of essential fatty acids can be specific and nonspecific. The latter manifest themselves particularly in the phospholipid composition and, therefore, in the structure and fluency of the membranes. In contrast, specific essential fatty acids act in the formation of prostaglandins, prostacyclins, tromboxans and leucotriens. Each essential fatty acid produces specific effects, depending on the prostanoids formed and the tissue in question.

[Inestability of fatty acid desaturation enzymes in liver slices (author's transl)]. / Inestabilidad de las enzimas desaturantes de acidos grasos en cortes de higado

A study was made on the microsomal oxidative desaturating activity of fatty acids in rat liver slices incubated in different media. [1-14C] linoleic acid desaturation activity decreased during the incubation in Krebs-Ringer-Bicarbonate. The addition of glucose or pyruvate in the incubation medium did not alter the decrease of the linoleic desaturation activity compared to the controls. Linoleic desaturation to gamma-linolenic acid descreased even when the slices were incubated in different media and at different temperatures. However, the inclusion of aminoacids in the media prevented the decrease of linoleic acid desaturation. Microsomal stearic acid desaturation to oleic acid in liver slices was less modified by incubation than linoleic acid desaturation. Glucose inclusion in the medium enhanced 9-desaturation. The stability of the desaturases in the liver slices is discussed. The results evidence once more that 9-desaturation and 6-desaturation are accomplished by different enzymes controlled by separate mechanisms.