Production, characterization and immobilization of lipases and their application in the synthesis of alkyl esters of fatty acids

The use of industrial palm oil processing residues as a source of carbon and inducer for microbial lipase production can be an attractive way to add value to these residues and help reduce the costs of their production. In addition, the immobilization of these enzymes on solid supports is a way of improving their operational stability and facilitating their recovery and reuse in the process. The characteristics of the carrier and the immobilization technique employed can modulate the catalytic properties of lipases, in some cases altering their regio- and/or stereospecificities. In this context, this work had two main objectives: (i) to investigate the viability of the use of industrial palm oil residues as raw material for the production of lipase in different culture systems; (ii) to evaluate the effect of immobilization support and post-immobilization modification techniques on the activity, stability and specificity of immobilized lipase. Regarding lipase production, after the preliminary screening of 18 fungi from the Embrapa collection, the Aspergillus niger C strain was selected to evaluate lipase production by solid state (SSF) and submerged (SF) fermentation. The lipolytic activities achieved by SSF and SF of A. niger C were 15.41 and 10.46 IU/mL, respectively. The lipase produced by A. niger C by SSF showed maximum lipolytic activity at pH between 4.0 and 6.5 and at temperature between 37 and 55°C. This enzyme presented good stability at 60°C (half-life around 12 h) and greater specificity towards long carbon chain substrates, especially palm and sunflower oils. As for the immobilization step, the immobilization of a commercial lipase of Thermomyces lanuginosus (LTL) on different hydrophobic commercial supports and post-immobilization modification (glycosylation, hydroxylation and PEGylation) was first evaluated in order to verify the effect of the support and the post-immobilization modification technique on lipase activity, stability and specificity. Immobilization of LTL in Purolite C18 (support containing octadecyl groups) under different experimental conditions allowed the preparation of immobilized derivatives with different regiospecificities and reaction rates. Immobilization at pH 8.5 and 30°C in the presence of CTAB (cetyltrimethylammonium bromide) produced a derivative with perfect sn-1.3 regiospecificity. On the other hand, immobilization at pH 5.0 and 4°C in the absence of CTAB produced a nonspecific derivative which catalyzed the ethanolysis of acyl groups from the sunflower oil at sn-1 and sn-3 positions very quickly as well as at sn-2 position continuously. After the chemical modification of LTL adsorbed on Purolite C18 (Purolite-LTL) with PEG, the modified derivative showed high stability in an organic solvent free system (half-life of 6 days at 40ºC) and in hexane (100% activity after 20 days at 40°C). Subsequently, the immobilization of partially purified A. niger C lipase was evaluated on the supports Purolite C18 and octyl-silica (octyltriethoxysilane-modified silica). Immobilization yields were low (28-69%, in terms of activity), compared to those obtained with LTL immobilized on the same supports (approximately 100%). However, the derivative produced by the immobilization of A. niger C lipase on Purolite C18 (Purolite-LAN) showed similar performance to the Purolite-LTL derivative in the esterification of oleic acid with n-octanol, achieving oleic acid conversions of approximately 80% after 24 h of reaction. Therefore, this work stands out in an economic-environmental context, by the use of an agroindustrial residue of the processing of palm oil as a substrate for the production of an enzyme of great economic interest in different industrial sectors and by obtaining a competitive biocatalyst with a commercial lipase in important biotechnological applications.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)O uso de resíduos do processamento industrial de óleo de palma como fonte de carbono e indutor para produção de lipase microbiana pode ser uma forma atrativa de agregar valor a esses resíduos e contribuir para reduzir os custos de sua produção. Além disso, a imobilização dessas enzimas em suportes sólidos é uma forma de melhorar sua estabilidade operacional e facilitar a sua recuperação e reutilização no processo. Adicionalmente, as características do suporte e a técnica de imobilização empregada podem modular as propriedades catalíticas das lipases, alterando em alguns casos suas régio- e/ou estereoespecificidades. Neste contexto, este trabalho teve dois objetivos centrais, a saber: (i) investigar a viabilidade do uso de resíduos industriais de óleo de palma como matéria-prima para a produção de lipase em diferentes sistemas de cultivo; (ii) avaliar o efeito do suporte de imobilização e de técnicas de modificação pós-imobilização sobre a atividade, estabilidade e especificidade da lipase imobilizada. Quanto à produção de lipases, após etapa preliminar de triagem de 18 fungos da coleção da Embrapa, selecionou-se a linhagem de Aspergillus niger C para se avaliar a produção de lipase por fermentação em estado sólido (FES) e submersa (FSm). As atividades lipolíticas alcançadas por FES e FSm de A. nicer C foram de 15,41 e 10,46 UI/mL, respectivamente. A lipase bruta produzida por A. niger C por FES apresentou atividade lipolítica máxima em pH entre 4,0 e 6,5 e em temperatura entre 37 e 55ºC. Essa enzima apresentou boa estabilidade a 60°C (meia-vida em torno de 12 h) e maior especificidade na hidrólise substratos de cadeia carbônica longa, destacando-se óleo de palma e girassol. Quanto à etapa de imobilização, primeiramente avaliou-se a imobilização de uma lipase comercial de Thermomyces lanuginosus (LTL) em diferentes suportes comerciais hidrofóbicos e modificação pós-imobilização (glicosilação, hidroxilação e PEGuilação) a fim de se verificar o efeito do suporte e da técnica de modificação pós-imobilização sobre a atividade, estabilidade e especificada da lipase. A imobilização de LTL em Purolite C18 (suporte contendo grupos octadecil) em diferentes condições experimentais permitiu a preparação de derivados imobilizados com diferentes regioespecificidades e taxas de reação. A imobilização a pH 8,5 e 30°C na presença de CTAB (brometo de cetiltrimetilamónio) produziu um derivado com regioespecificidade sn-1,3 perfeita. Por outro lado, a imobilização em pH 5,0 e 4°C na ausência de CTAB produziu um derivado não específico, catalisando a etanólise de grupos acil do óleo de girassol rico em ácido oleico das posições sn-1 e sn-3 de forma muito rápida e também continuamente da posição sn-2. Após a modificação química da LTL adsorvida em Purolite C18 (Purolite-LTL) com PEG, o derivado modificado apresentou alta estabilidade em sistema livre de solvente orgânico (meia-vida de 6 dias a 40ºC) e em hexano (100% de atividade após 20 dias a 40ºC). Posteriormente, avaliou-se a imobilização da lipase de A. niger C parcialmente purificada nos suportes Purolite C18 e octil-silica (silica modificada com octiltrietoxisilano). Os rendimentos de imobilização foram baixos (28 a 69%, em termos de atividade), comparados aos obtidos com a imobilização de LTL nos mesmos suportes (aproximadamente 100%). Entretanto, o derivado produzido pela imobilização da lipase de A. niger C em Purolite C18 (Purolite-LAN) apresentou desempenho similar ao derivado Purolite-LTL na esterificação de ácido oleico com n-octanol, atingindo conversões de ácido oleico de aproximadamente 80% após 24 h de reação. Destaca-se, portanto, a contribuição deste trabalho em um contexto econômico-ambiental, pelo aproveitamento de um resíduo agroindustrial do processamento do óleo de dendê como substrato para a produção de uma enzima de grande interesse econômico em diferentes setores industriais e pela obtenção de um biocatalisador imobilizado competitivo com uma lipase comercial em aplicações importantes de base biotecnológica.FAPESP: 2013/20826-0FAPESP: 2015/10530-2FAPESP: 2016/10636-

Caracterização bioquímica de leveduras industriais produtoras de etanol cultivadas em diferentes açúcares

Para o aprimoramento do processo industrial de produção de bioetanol, é fundamental o conhecimento da bioquímica e fisiologia dos microrganismos envolvidos. Assim, este estudo tem o objetivo de obter informações bioquímicas de leveduras industriais, principalmente ao que se refere à hidrólise da sacarose e maltose pela enzima invertase (β-frutofuranosidase, EC 3.2.1.26) e maltase (α- glicosidase, EC 3.2.1.20) respectivamente. As linhagens industriais Ethanol RedTM (Fermentis/Lasaffre - linhagem francesa) PE-2, SA-1, CAT-1 e BG (linhagens brasileiras) foram cultivadas em meios contendo diferentes fontes de carbono (sacarose, glicose, frutose, maltose e galactose), suplementados com peptona e extrato de levedo, e avaliadas com relação a produção de biomassa, consumo da fonte de carbono, viabilidade celular e níveis de atividade invertásica e maltásica. Resultados mostraram que todas as linhagens apresentam crescimento rápido e intenso em sacarose, glicose e frutose, porém apresentam comportamento diferente em meios contendo maltose e galactose. Todas as linhagens crescem lentamente em galactose. Ethanol RedTM é mais adaptada para o crescimento em maltose do que as linhagens brasileiras PE-2 e SA-1. As outras linhagens brasileiras (CAT-1 e BG) não crescem em maltose devido à ausência de atividade maltásica. Com relação aos níveis de atividade invertásica, foi identificada a presença de três categorias de linhagens: uma com altos níveis de atividade (Ethanol RedTM), outra com níveis mais baixos (PE-2, CAT-1), e uma terceira categoria com atividade intermediária entre estas duas primeiras categorias (SA-1 e BG). Além do valor acadêmico, os resultados obtidos têm importância aplicada na medida em que indicam que as linhagens industriais produtoras de etanol combustível apresentam diferentes características fisiológicas, que podem ser exploradas para aperfeiçoar o processo de fermentação alcoólicaFor the improvement of the industrial ethanol fuel-producing process, it is crucial to understand the biochemistry and physiology of the microorganisms involved. This study aims to obtain biochemical information of industrial yeasts, especially when it refers to the hydrolysis of sucrose and maltose by the enzyme invertase (β-frutofuranosidase, EC 3.2.1.26) and maltase (α-glicosidase, EC 3.2.1.20) respectively. The industrial strains Ethanol RedTM (Fermentis/Lasaffre - French strain) PE-2, SA-1, CAT-1 and BG (Brazilian strains) were cultured in media containing different carbon sources (sucrose, glucose, fructose, maltose and galactose) supplemented with peptone and yeast extract, and evaluated relative to biomass production, carbon source consumption, cell viability and levels of invertase and maltase activities. Results showed that all strains exhibit rapid and intense growth in presence of sucrose, glucose and fructose, but they have differing behavior in media containing maltose and galactose. All strains grow slowly on galactose. Ethanol RedTM is more adapted for growing in maltose than Brazilian PE-2 and CAT- 1. The remaining Brazilian strains (BG and CAT-1) do not grow in maltose, due to the absence of maltase activity. As far as the levels of invertase activity is concerned, three categories of strains have been identified: with high activity levels (Ethanol RedTM), with lower levels (PE-2, CAT-1), and a third category with intermediate activity levels between these first two categories (SA-1 and BG). Besides academic value, the results obtained have applied significance in indicating that industrial ethanol fuel-producing strains exhibit different physiological characteristics that can be exploited to improve the fermentation proces

Modulation of the regioselectivity of Thermomyces lanuginosus lipase via biocatalyst engineering for the Ethanolysis of oil in fully anhydrous medium

[Background] Enzymatic ethanolysis of oils (for example, high oleic sunflower oil containing 90% of oleic acid) may yield two different reaction products depending on the regioselectivity of the immobilized lipase biocatalyst. Some lipase biocatalysts exhibit a 1,3-regioselectivity and they produced 2 mols of fatty acid ethyl ester plus 1 mol of sn2-monoacylglycerol (2-MAG) per mol of triglyceride without the release of glycerol. Other lipase biocatalysts are completely non-regioselective releasing 3 mols of fatty acid ethyl ester and 1 mol of glycerol per mol of triglyceride. Lipase from Thermomyces lanuginosus (TLL) adsorbed on hydrophobic supports is a very interesting biocatalyst for the ethanolysis of oil. Modulation of TLL regioselectivity in anhydrous medium was intended via two strategies of TLL immobilization: a. - interfacial adsorption on different hydrophobic supports and b.- interfacial adsorption on a given hydrophobic support under different experimental conditions.[Results] Immobilization of TLL on supports containing divinylbenezene moieties yielded excellent 1,3-regioselective biocatalysts but immobilization of TLL on supports containing octadecyl groups yielded non-regioselective biocatalysts. On the other hand, TLL immobilized on Purolite C18 at pH 8.5 and 30 °C in the presence of traces of CTAB yielded a biocatalyst with a perfect 1,3-regioselectivity and a very interesting activity: 2.5 μmols of oil ethanolyzed per min per gram of immobilized derivative. This activity is 10-fold higher than the one of commercial Lipozyme TL IM. Immobilization of the same enzyme on the same support, but at pH 7.0 and 25 °C, led to a biocatalyst which can hydrolyze all ester bonds in TG backbone.[Conclusions] Activity and regioselectivity of TLL in anhydrous media can be easily modulated via Biocatalysis Engineering producing very active immobilized derivatives able to catalyze the ethanolysis of triolein. When the biocatalyst was 1,3-regioselective a 33% of 2-monoolein was obtained and it may be a very interesting surfactant. When biocatalyst catalyzed the ethanolysis of the 3 positions during the reaction process, a 99% of ethyl oleate was obtained and it may be a very interesting drug-solvent and surfactant. The absence of acyl migrations under identical reaction conditions is clearly observed and hence the different activities and regioselectivities seem to be due to the different catalytic properties of different derivatives of TLL.This work was sponsored by the Spanish Ministry of Economy, Industry and Competitiveness (projects BIO2012–36861 and CTQ2015–70348). Javier Rocha-Martin is grateful for the Juan de la Cierva fellowship (IJCI-2014-19,260) funded by the Spanish Ministry of Economy, Industry and Competitiveness. We also thank Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for granting the scholarship to Erick Abreu Silveira.Peer reviewe

Synthesis of omega-3 ethyl esters from chia oil catalyzed by polyethylene glycol-modified lipases with improved stability

Enzymatic synthesis of fatty acid ethyl esters (FAEE) from chia (Salvia hispanica L.) oil has been performed with different modified derivatives and compared with commercial immobilized lipases to produce omega-3 rich FAEE. Therefore, the main objective was to synthesize omega-3 esters from chia oil catalysed by polyethylene glycol-modified lipases using a biocatalyst with higher stability than commercial derivatives. Lipase from Thermomyces lanuginosus (TLL) was immobilized by hydrophobic adsorption on Sepabeads C-18 followed by a physicochemical coating of lipase surface with a dense layer of PEG. Ethanolysis reactions were carried out using pressurized liquid extracted chia seed oil and with different lipase derivatives to compare the omega-3 FAEE yield and ratio of reaction products, which were analysed by HPLC-ELSD. Furthermore, reutilization of lipase derivatives was studied to evaluate the stability after several reaction cycles to minimize decreasing of catalytic activity and develop a feasible enzymatic process for food industrial applications.Sin financiación6.306 JCR (2019) Q2, 5/71 Chemistry, Applied1.775 SJR (2019) Q1, 8/127 Analytical ChemistryNo data IDR 2019UE

Biocatalyst engineering of Thermomyces Lanuginosus lipase adsorbed on hydrophobic supports: Modulation of enzyme properties for ethanolysis of oil in solvent-free systems

Different immobilized biocatalysts of Thermomyces lanuginosus lipase (TLL) exhibited different properties for the ethanolysis of high oleic sunflower oil in solvent-free systems. TLL immobilized by interfacial adsorption on octadecyl (C-18) supports lost its 1,3-regioselectivity and produced more than 99% of ethyl esters. This reaction was influenced by mass-transfer limitations. TLL adsorbed on macroporous C-18 supports (616 Å of pore diameter) was 10-fold more active than TLL adsorbed on mesoporous supports (100–200 Å of pore diameter) in solvent-free systems. Both derivatives exhibited similar activity when working in hexane in the absence of diffusional limitations. In addition, TLL adsorbed on macroporous Purolite C-18 was 5-fold more stable than TLL adsorbed on mesoporous Sepabeads C-18. The stability of the best biocatalyst was 20-fold lower in anhydrous oil than in anhydrous hexane. Mild PEGylation of immobilized TLL greatly increased its stability in anhydrous hexane at 40 °C, fully preserving the activity after 20 days. In anhydrous oil at 40 °C, PEGylated TLL-Purolite C-18 retained 65% of its initial activity after six days compared to 10% of the activity retained by the unmodified biocatalyst. Macroporous and highly hydrophobic supports (e.g., Purolite C-18) seem to be very useful to prepare optimal immobilized biocatalysts for ethanolysis of oils by TLL in solvent-free systems.Spanish Ministry of Economy, Industry and Competitiveness (BIO2012-36861; CTQ2015-70348; IJCI-2014-19260)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) (2016/10636-8; 2015/10530-2; 2013/20826-0)3.503 JCR (2019) Q2, 47/156 Biotechnology & Aplied Microbiology0.992 SJR (2019) Q1, 60/359 BiotechnologyNo data IDR 2019UE

NEOTROPICAL XENARTHRANS: a data set of occurrence of xenarthran species in the Neotropics

Xenarthrans—anteaters, sloths, and armadillos—have essential functions for ecosystem maintenance, such as insect control and nutrient cycling, playing key roles as ecosystem engineers. Because of habitat loss and fragmentation, hunting pressure, and conflicts with domestic dogs, these species have been threatened locally, regionally, or even across their full distribution ranges. The Neotropics harbor 21 species of armadillos, 10 anteaters, and 6 sloths. Our data set includes the families Chlamyphoridae (13), Dasypodidae (7), Myrmecophagidae (3), Bradypodidae (4), and Megalonychidae (2). We have no occurrence data on Dasypus pilosus (Dasypodidae). Regarding Cyclopedidae, until recently, only one species was recognized, but new genetic studies have revealed that the group is represented by seven species. In this data paper, we compiled a total of 42,528 records of 31 species, represented by occurrence and quantitative data, totaling 24,847 unique georeferenced records. The geographic range is from the southern United States, Mexico, and Caribbean countries at the northern portion of the Neotropics, to the austral distribution in Argentina, Paraguay, Chile, and Uruguay. Regarding anteaters, Myrmecophaga tridactyla has the most records (n = 5,941), and Cyclopes sp. have the fewest (n = 240). The armadillo species with the most data is Dasypus novemcinctus (n = 11,588), and the fewest data are recorded for Calyptophractus retusus (n = 33). With regard to sloth species, Bradypus variegatus has the most records (n = 962), and Bradypus pygmaeus has the fewest (n = 12). Our main objective with Neotropical Xenarthrans is to make occurrence and quantitative data available to facilitate more ecological research, particularly if we integrate the xenarthran data with other data sets of Neotropical Series that will become available very soon (i.e., Neotropical Carnivores, Neotropical Invasive Mammals, and Neotropical Hunters and Dogs). Therefore, studies on trophic cascades, hunting pressure, habitat loss, fragmentation effects, species invasion, and climate change effects will be possible with the Neotropical Xenarthrans data set. Please cite this data paper when using its data in publications. We also request that researchers and teachers inform us of how they are using these data