Abstract The search for new biocatalysts has aroused great interest due to the variety of micro-organisms and their role as enzyme producers. Native lipases from Aspergillus niger and Rhizopus javanicus were used to enrich the n-3 long-chain polyunsaturated fatty acids content in the triacylglycerols of soybean oil by acidolysis with free fatty acids from sardine oil in solvent-free media. For the immobilization process, the best lipase/support ratios were 1:3 (w/w) for Aspergillus niger lipase and 1:5 (w/w) for Rhizopus javanicus lipase using Amberlite MB-1. Both lipases maintained constant activity for 6 months at 4 °C. Reaction time, sardine-free fatty acids:soybean oil mole ratio and initial water content of the lipase were investigated to determine their effects on n-3 long-chain polyunsaturated fatty acids incorporation into soybean oil. Structured triacylglycerols with 11.7 and 7.2% of eicosapentaenoic acid + docosahexaenoic acid were obtained using Aspergillus niger lipase and Rhizopus javanicus lipase, decreasing the n-6/n-3 fatty acids ratio of soybean oil (11:1 to 3.5:1 and 4.7:1, respectively). The best reaction conditions were: initial water content of lipase of 0.86% (w/w), sardine-free faty acids:soybean oil mole ratio of 3:1 and reaction time of 36 h, at 40 °C. The significant factors for the acidolysis reaction were the sardine-free fatty acids:soybean oil mole ratio and reaction time. The characterization of structured triacylglycerols was obtained using easy ambient sonic-spray ionization mass spectrometry. The enzymatic reaction led to the formation of many structured triacylglycerols containing eicosapentaenoic acid, docosahexaenoic acid or both polyunsaturated fatty acids.

As lipases CAL-B, LPS, LPS-C, LPS-D e a de A. níger, livres ou imobilizadas em filmes de amido de cará (Discorea alata L.), inhame (Colocasia esculenta (L.) Schott) ou de gelatina, foram utilizadas na acilação da (RS)-sec-butilamina com diferentes doadores acila em vários solventes orgânicos sob aquecimento convencional (CH) ou irradiação por microondas (MW). Ao utilizar a lipase de A. niger livre, a conversão foi três vezes maior sob irradiação por MW em relação ao CH a 35 ºC. Usando a lipase de A. niger livre, a (R)-amida foi obtida com 21% de conversão, resultando em valores de ee p > 99% e de E (enantiosseletividade) > 200, em 1 min de reação em MW. Ao utilizar a lipase de A. niger imobilizada em filmes de amido de cará e inhame, em 3 ou 5 min de reação em MW, a amida foi obtida com conversões moderadas, sendo de 8 a 25%, porém com maior seletividade (ee p > 99% e E > 200) em comparação à forma livre (45% de conversão, ee p 81% e 18 para o valor de E).

The lipases CAL-B, PSL, PSL-C, PSL-D, and A. niger lipase, free or immobilized in starch (obtained from two types of yam, known in Brazil as “cará” (Discorea alata L.) and “inhame” (Colocasia esculenta (L.) Schott) or gelatin films, were used in the acylation of (RS)-sec-butylamine with different acyl donors in various organic solvents applying conventional heating (CH) or microwave (MW) irradiation. In the case of free A. niger lipase, the conversion degrees were three times higher using MW irradiation when compared to conventional heating at 35 ºC. Using free A. niger lipase, the (R)-amide was obtained with a conversion degree of 21%, resulting in ee p > 99% and E-value (enantioselectivity value) > 200, in 1 min of reaction under MW irradiation. When the A. niger lipase was immobilized in yam starch films, the (R)-amide was obtained in moderate conversions of 8-25% after 3 or 5 min of reaction under MW irradiation, but with higher selectivity (ee p > 99% and E > 200) in comparison with the free form (conversion degree of 45%, ee p 81% and E value of 18).

A enantioseletividade (E) das lipases nativas de Aspergillus niger, Aspergillus terreus, Fusarium oxysporum, Mucor javanicus, Penicillium solitum e Rhizopus javanicus na resolução dos enantiômeros do (R,S)-ibuprofeno e (R,S)-cetoprofeno na reação de esterificação com 1-propanol em isoctano foi comparada com as lipases comerciais de Candida rugosa (Sigma) e Candida antarctica (Novozym®435). A lipase de C. rugosa mostrou boa enantioseletividade (E = 12) comparada com as da Novozym®435 (E = 6.7), de A. niger (E=4.8) e com as outras lipases que foram muito menos seletivas (E por volta de 2.3 e 1.5) na resolução do (R,S)-ibuprofeno, dentro das condições testadas. Após uma otimização preliminar das condições da reação (conteúdo de água, concentração da enzima e presença de aditivos) a enantioseletividade da lipase de A. niger pôde ser substancialmente aumentada (E = 15). Todas as lipases testadas mostraram baixa seletividade na resolução de (R,S)-cetoprofeno, resultando baixos rendimentos de éster e de excesso enantiomérico do ácido não esterificado.

The enantioselectivity (E) of native lipases from Aspergillus niger, Aspergillus terreus, Fusarium oxysporum, Mucor javanicus, Penicillium solitum and Rhizopus javanicus in the resolution of (R,S)-ibuprofen and (R,S)-ketoprofenenantiomers by esterification reaction with 1-propanol in isooctane was compared with known commercial Candida rugosa (Sigma) and Candida antarctica (Novozym®435) lipases. In the resolution of (R,S)-ibuprofen, C. rugosa lipase showed good selectivity (E = 12) while Novozym®435 (E = 6.7) and A. niger (E = 4.8) lipases had intermediate selectivities. Other enzymes were much less selective (E around 2.3 and 1.5), under tested conditions. After preliminary optimization of reaction conditions (water content, enzyme concentration and presence of additives) the enantioselectivity of native A. niger lipase could be enhanced substantially (E = 15). All tested lipases showed low selectivity in the resolution of (R,S)-ketoprofen because poor ester yields and low enantiomeric excess of the acid remaining were achieved.

Several polyunsaturated fatty acids (PUFA) belonging to the ômega 6 series, such as cis-6,9,12 gamma-linolenic acid, as well as those of the ômega 3 series, such as cis-5,8,11,14,17-eicosapentaenoic acid and cis-4,7,10,13,16,19-docosahexaenoic acid are of considerable interest due to their nutritional and therapeutic properties. Methods used for the concentration of PUFA from natural sources include urea adduct formation, solvent winterization, supercritical fluid extraction and lipase-catalyzed reaction. Lipases are known to have little reactivity on PUFA and these acids can be enriched by selective hydrolysis, direct esterification of glycerol with PUFA and interesterification. Since lipase reactions are advantageous with respect to fatty acid, positional specificities and mild incubation condition, these enzymes are considered to be suitable for the production of PUFA concentrates for medical purposes.

Análises experimentais foram realizadas objetivando a composição quantitativa dos ácidos graxos Eicosapentaenóico (EPA) e Docosahexaenóico (DHA), em diferentes partes do corpo de espécies de peixes marinhos da costa brasileira (atum, bonito, olho de boi, cavalinha, sardinha e serra). Os teores de EPA e DHA foram analisados em duas partes distintas: olho (órbita ocular e material gorduroso da cavidade ocular) e filés, sendo significativas as diferenças entre as mesmas. Os teores de DHA para uma determinada espécie foram sempre superiores no olho em relação ao filé, sendo o mesmo observado para o EPA em quatro das espécies (olho de boi, cavalinha, sardinha e serra). Comparando-se a mesma espécie e partes do corpo dos peixes, observou-se que os teores de DHA foram superiores aos teores de EPA, exceto para a sardinha. A somatória dos níveis de EPA e DHA em filés foram maiores para as espécies sardinha e bonito, mostrando serem uma boa fonte alimentar destes ácidos, especialmente a sardinha por ser uma fonte com preço acessível no Brasil.

Quantitative compositions of two unsaturated fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were examined in fish oil from orbital cavity and fillets of fishes which captured from Brazilian coast. The experimental results demonstrated that quantities of DHA were extremely higher in oil from orbital cavity of Atum and Bonito as compared with fillet oil, whereas orbital cavity oil from Olho de Boi, Cavalinha, Sardinha, and Serra contained slightly higher DHA than fillet oil. On the other hand, fillet oil from Atum and Bonito contained more EPA than oil from orbital cavity, whereas orbital cavity oils from Olho de Boi, Cavalinha, Sardinha and Serra contained higher quantities of EPA than oils from fillets.

A linhagem de Mucor sp LB-54, considerada uma potencial produtora de ácido gama-linolênico (GLA), foi selecionada para o estudo de diferentes temperaturas de cultivo em agitador rotativo. A linhagem usada neste experimento era capaz de acumular uma quantidade alta de lipídeos intracelulares, 20,73 % do peso seco de biomassa e conteúdo de GLA de 15 % dentre os ácidos graxos totais de sua constituição após 5 dias de incubação à 28°C. Quando a temperatura de cultivo foi diminuída de 28oC para 12°C, o conteúdo de GLA aumentou de 15 para 24% dentre os ácidos graxos totais de sua constituição. Com o objetivo de otimizar as condições de cultivo para a produção rápida de biomassa e produção de lipídeos contendo conteúdo alto de GLA, o fungo foi cultivado em duas combinações de temperaturas associadas com a suplementação de fonte de carbono (glicose). A produção máxima de GLA (74mg/l) pela linhagem de Mucor sp LB-54 foi obtida após 5 dias de incubação à 28°C em meio base, seguida da adição de glicose (7% p/v) no meio de cultura e uma posterior incubação por mais 3 dias a 12°C. A identidade do GLA foi confirmada pelo sistema acoplado cromatógrafo à gás - espectrômetro de massa.

As a relatively prolific producer of GLA, the strain of Mucor sp LB-54 was selected for a study at different growth temperatures in shaker flask culture. The strain used in our experiment was capable to accumulate a relatively high amount of intracellular lipid, 20.73 % of dry cell weight, and GLA content of 15 % of total fatty acids after 5 days of incubation at 28°C. As the growth temperature was decreased from 28 to 12°C the percentage of GLA increased from 15 to 24 % of total fatty acids. In order to optimize the culture conditions for rapid biomass production and lipid production with a high proportion of GLA, the fungus was grown at two temperature combinations associated supplies of carbon source (glucose) in the culture medium. Maximal production of GLA (74 mg/l) was obtained from the Mucor sp LB-54 strain after 5 days of incubation at 28°C in basal medium following glucose addition (7 % w/v) and incubation for an additional 3 days at 12°C. The identity of GLA found in the strain of Mucor sp LB-54 was confirmed by the coupled gas chromatography-mass spectrometry