Ernandes B. Pereira

Kinetic studies of lipase from Candida rugosa: a comparative study between free and immobilized enzyme onto porous chitosan beads.

The search for an in expensive support has motivated our group to undertake this work dealing with the use of chitosan as matrix for immobilizing lipase. In addition to its low cost, chitosan has several advantages for use as a support, including its lack of toxicity and chemical reactivity, allowing easy fixation of enzymes. In this article, we describe the immobilization of Canada rugosa lipase onto porous chitosan beads for the enzymatic hydrolysis of oliveoil. The binding of the lipase onto the support was performed by physicalad sorption using hexane as the dispersion medium. A comparativestudy between free and immobilized lipase was conducted in terms of pH, temperature, and thermal stability. A slightly lower value for optimum pH (6.0) was found for the immobilized form in comparison with that attained for the soluble lipase (7.0). The optimum reaction temperature shifted from 37°C for the free lipase to 50°C for the chitosan lipase. The patterns of heat stability indicated that the immobilization process tends to stabilize the enzyme. The half-life of the soluble free lipase at 55°C was equal to 0.71 h (Kd=0.98 h−1), whereas for the immobilized lipase it was 1.10 h (Kd=0.63 h−1). Kinetics was tested at 37°C following the hydrolysis of olive oil and obeys the Michaelis-Menten type of rate equation. The Km was 0.15 mM and the Vmax was 51 μmol/(min·mg), which were lower than for free lipase, suggesting that the apparent affinity toward the substrate changes and that the activity of the immobilized lipase decreases during the course of immobilization.

Evaluation of different approaches for lipase catalysed synthesis of citronellyl acetate

Three different approaches for lipase catalysed synthesis of citronellyl acetate by a commercial available immobilized lipase have been studied: a) direct esterification reaction of citronellol with acetic acid; b) alcoholysis of butyl acetate with citronellol and c) transesterification of citronellyl butyrate with butylacetate. Heptane was used as solvent for all the experiments. The extent of reaction occurred in the order alcoholysis>transesterification>esterification. Substrate partitioning between the immobilization support and the organic medium seems to greatly influence the catalytic performance of this enzyme preparation. Production of citronellyl acetate, was found to be dependent on the partition coefficient of the acyl donor which account the greatest value for the acetic acid.

Esterification Activity and Stability of Candida rugosa Lipase Immobilized into Chitosan

Microbial lipase from Candida rugosa immobilized into porous chitosan beads was tested for esterification selectivity with butanol and different organic acids (C2–C12), and butyric acid and different aliphatic alcohols (C2–C10). After 24 h, the acids tested achieved conversions of about 40–45%. Acetic acid was the only exception, and in this case butanol was not consumed. Different alcohols led to butyric acid conversions >40%, except for ethanol, in which case butyric acid was converted only 26%. The system’s butanol and butyric acid were selected for a detailed study by employing an experimental design. The influence of temperature, initial catalyst concentration, and acid:alcohol molar ratio on the formation of butyl butyrate was simultaneously investigated, employing a 23 full factorial design. The range studied was 37–50°C for temperature (X1), 1.25–2.5% (w/v) for the catalyst concentration (X2), and 1 and 2 for the acid:alcohol molar ratio (X3). Catalyst concentration (X2) was found to be the most significant factor and its influence was positive. Maximum ester yield (83%) could be obtained when working at the lowest level for temperature (37°C), highest level for lipase concentration (2.5% [w/v]), and center level of acid:alcohol molar ratio (1.5). The immobilized lipase was also used repeatedly in batch esterification reactions of butanol with butyric acid, revealing a half-life of 86 h.

Kinetic Studies of Lipase from Candida rugosa

The search for an inexpensive support has motivated our group to undertake this work dealing with the use of chitosan as matrix for immobilizing lipase. In addition to its low cost, chitosan has several advantages for use as a support, including its lack of toxicity and chemical reactivity, allowing easy fixation of enzymes. In this article, we describe the immobilization of Candida rugosa lipase onto porous chitosan beads for the enzymatic hydrolysis of olive oil. The binding of the lipase onto the support was performed by physical adsorption using hexane as the dispersion medium. A comparative study between free and immobilized lipase was conducted in terms of pH, temperature, and thermal stability. A slightly lower value for optimum pH (6.0) was found for the immobilized form in comparison with that attained for the soluble lipase (7.0). The optimum reaction temperature shifted from 37°C for the free lipase to 50°C for the chitosan lipase. The patterns of heat stability indicated that the immobilization process tends to stabilize the enzyme. The half-life of the soluble free lipase at 55°C was equal to 0.71 h (K d = 0.98h-1), whereas for the immobilized lipase it was 1.10 h (K d = 0.63h-1). Kinetics was tested at 37°C following the hydrolysis of olive oil and obeys the Michaelis- Menten type of rate equation. The K m was 0.15 mM and the V max was 51 µmol/ (min-mg), which were lower than for free lipase, suggesting that the apparent affinity toward the substrate changes and that the activity of the immobilized lipase decreases during the course of immobilization.

Anaerobic biodegradability of dairy wastewater pretreated with porcine pancreas lipase

Lipids-rich wastewater was partial hydrolyzed with porcine pancreas lipase and the efficiency of the enzymatic pretreatment was verified by the comparative biodegradability tests (crude and treated wastewater). Alternatively, simultaneous run was carried out in which hydrolysis and digestion was performed in the same reactor. Wastewater from dairy industries and low cost lipase preparation at two concentrations (0.05 and 0.5% w.v-1) were used. All the samples pretreated with enzyme showed a positive effect on organic matter removal (Chemical Oxygen Demand-COD) and formation of methane. The best results were obtained when hydrolysis and biodegradation were performed simultaneously, attaining high COD and color removal independent of the lipase concentration. The enzymatic treatment considerably improved the anaerobic operational conditions and the effluent quality (lower content of suspended solids and less turbidity). Thus, the use of enzymes such as lipase seemed to be a very promising alternative for treating the wastewaters having high fat and grease contents, such as those from the dairy industry.

Improvement of the enzymatic synthesis of ethyl valerate by esterification reaction in a solvent system

ABSTRACT The present study reports the improved enzymatic synthesis of ethyl valerate (green apple flavor) by esterification reaction of ethanol and valeric acid in heptane medium. Lipase from Thermomyces lanuginosus (TLL) was immobilized by physical adsorption on polyhydroxybutyrate (PHB) particles and used as a potential biocatalyst. The effect of certain parameters that influence the ester synthesis was evaluated by factorial design. The experimental conditions that maximized the synthesis of ethyl valerate were 30.5°C, 18% m/v of biocatalyst (TLL–PHB), absence of molecular sieves, agitation of 234 rpm, and 1,000 mM of each reactant (ethanol and valeric acid). Under these conditions, conversion percentage ≈92% after 105 min of reaction was observed. Soluble TLL was also used as biocatalyst and the highest conversion was of 82% after 120 min of reaction. Esterification reaction performed in a solvent-free system exhibited conversion of 13% after 45 min of reaction catalyzed by immobilized lipase, while the soluble lipase did not exhibit catalytic activity. The synthesis of the ester was confirmed by Fourier transform infrared spectroscopy and gas chromatography–mass spectrometry analyses. After six consecutive cycles of ethyl valerate synthesis, the prepared biocatalyst retained ≈86% of its original activity.

SÍNTESE DE BIODIESEL A PARTIR DO ÓLEO DE AÇAÍ EMPREGANDO LIPASE COMERCIAL IMOBILIZADA EM SUPORTE DE BAIXO CUSTO

Para suprir a crescente demanda mundial energetica, em um contexto de grandes preocupacoes sobre as questoes ambientais e a escassez das reservas dos combustiveis fosseis e, alem do interesse pela independencia energetica, investe-se na pesquisa e em formas alternativas de energia. Dentre essas alternativas, o biodiesel destaca-se por ser um combustivel renovavel produzido utilizando-se fontes naturais. As analises da materia-prima, como a densidade, a viscosidade e a massa molar media, foram realizadas previamente ao processo de sintese. Este trabalho consiste na producao de biodiesel a partir de oleo de acai empregando lipase comercial imobilizada em suporte de baixo custo. A sintese de biodiesel alcancou um rendimento de 51,49% em 12h de reacao.

OTIMIZAÇÃO E CARACTERIZAÇÃO DE MICROPARTÍCULAS DE QUITOSANA IMOBILIZADA EM LIPASE PANCREÁTICA COMERCIAL DOI: http://dx.doi.org/10.5892/ruvrd.v15i1.3714

A aplicacao de enzimas em atividades industriais vem crescendo dia a dia, tornando-se indispensavel para processos como tratamentos continuos, controle automatizado, melhoria dos rendimentos por unidade de enzima, obtencao de derivados mais puros e diminuicao de necessidades energeticas, entre outras. Como a utilizacao de enzimas nativas apresenta limitacoes, por exemplo, o alto custo, pode-se optar pela imobilizacao de biocatalisadores a fim de minimizar tais limitacoes, proporcionando altos rendimentos industriais. Dentre os suportes utilizados na imobilizacao, destacam-se polimeros naturais de baixo custo e de facil obtencao, como a quitosana, usada no preparo de particulas para diferentes propositos, o que permite, dentre outras vantagens, incrementarem as cineticas de adsorcao e promover maior facilidade de manuseio e operacao. Neste trabalho foram testadas diferentes concentracoes de quitosana, sendo as micro e/ou nanoparticulas analisadas morfologicamente por microscopia eletronica de varredura (MEV). Alem disso, foram realizadas a caracterizacao fisico-quimica e a determinacao da atividade da enzima livre e imobilizada. A tecnologia de imobilizacao de lipases em particulas de quitosana tem sido alvo de muitas investigacoes visando aperfeicoar a obtencao de produtos de interesse biotecnologico, abrindo novas perspectivas de mercado.