Produção, purificação e propriedades bioquímicas de lipase ácida de Candida viswanathii
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Data
2012-07-17
Autores
Almeida, Alex Fernando de [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Lipases são enzimas amplamente distribuídas nos seres vivos e apresentam a função biológica de hidrolisar ligações ésteres de triacilgliceróis, liberando diacilgliceróis, monoacilgliceróis, ácidos graxos e glicerol. Dependendo da atividade de água estas enzimas catalisam outras reações como esterificação, transesterificação, aminólise e lactonização, importantes em diversas aplicações biotecnológicas dessas enzimas. Neste trabalho, uma linhagem de Candida viswanathii foi selecionada, entre outras noventa linhagens de fungos filamentosos e leveduras, como produtora de lipase e suas condições de cultivo foram estudadas em cultivos submersos e em substratos sólido, visando o aumento da produção da enzima. Outro objetivo deste trabalho também foi purificar, imobilizar e caracterizar a lipase produzida nas condições estabelecidas em cultivos submersos. Os resultados obtidos nos cultivos submersos indicaram que, entre as substâncias puras testadas, trioleína e ácido oléico foram os melhores indutores da atividade lipase. Contudo, as melhores condições de cultivo e produção de lipase foram alcançadas utilizando azeite de oliva 1,5% (m/v), extrato de levedura 0,2% (m/v), agitação de 210 rpm, pH 6,0, temperatura de 27,5 ºC, após 72 h de cultivo (biomassa 19,0 g/L; atividade 100 U). A adição de 0,1% (m/v) de lecitina de soja aumentou em 1,5 vezes a produção de lipase (147,5 U). A caracterização bioquímica da lipase do caldo fermentado revelou que se tratava de uma lipase ácida com pH ótimo de 3,5, propriedade descrita pela primeira vez para uma lipase do gênero Candida. A temperatura ótima foi de 40 ºC. Esta enzima bruta mostrou-se altamente estável na faixa de pH 4,0 a 5,0 e na temperatura de 40 ºC após 24 h (100%). Nos cultivos sólidos...
Lipases are widely distributed enzymes in animals, plants, bacteria, yeasts and fungi, which hydrolyses ester bonds of triacylglycerols and fatty acids. In addition, these enzymes also catalyse other reactions as esterification, transesterification, aminolysis and lactonization, depending their water activity. The ability of lipases to perform very specific chemical transformation and their enantioselective properties has made them increasingly popular in many biotechnological applications. In this work, Candida viswanathii was selected among ninety filamentous fungi and yeast strains, due to as its lipase activity and culture conditions were studied aiming to increase the enzyme production. Furthermore, this study aimed to purify, immobilize and characterize the lipase produced in submerged cultures. The results showed that triolein and oleic acid are the best inducers of lipase activity. However, the highest lipase production was obtained from 1.5% (w/v) olive oil and 0.2% (w/v) yeast extract in rotary shaker experiments at 27.5 ºC, pH 6.0 and 210 rpm, after 72 h (19.0 g/L biomass; 100 U lipase activity). The addition of 0.1% (w/v) soy lecithin increased 1.5-fold the lipase production (147.5 U). Biochemical characterization of the crude enzyme revealed an acid lipase with optimum pH of 3.5, a property first described in Candida genus. Optimum temperature activity was 40 ºC. Crude lipase was highly stable at pH 4.0 to 5.0 and at temperature 40 ºC during 24 h (100%). Solid substrates cultures from industrial byproducts, xiv the highest lipase production was obtained in wheat bran and barley spent grain (1:1), 40% (w/w) chicken fat, 3.5% (w/w) yeast extract, humidity 40% after 5 days, at 30°C (153 U/gds). Under these culture conditions, the lipase showed optimum activity at pH 5.0 and 50 ºC, and thermal enzyme stability at pH... (Complete abstract click electronic access below)
Lipases are widely distributed enzymes in animals, plants, bacteria, yeasts and fungi, which hydrolyses ester bonds of triacylglycerols and fatty acids. In addition, these enzymes also catalyse other reactions as esterification, transesterification, aminolysis and lactonization, depending their water activity. The ability of lipases to perform very specific chemical transformation and their enantioselective properties has made them increasingly popular in many biotechnological applications. In this work, Candida viswanathii was selected among ninety filamentous fungi and yeast strains, due to as its lipase activity and culture conditions were studied aiming to increase the enzyme production. Furthermore, this study aimed to purify, immobilize and characterize the lipase produced in submerged cultures. The results showed that triolein and oleic acid are the best inducers of lipase activity. However, the highest lipase production was obtained from 1.5% (w/v) olive oil and 0.2% (w/v) yeast extract in rotary shaker experiments at 27.5 ºC, pH 6.0 and 210 rpm, after 72 h (19.0 g/L biomass; 100 U lipase activity). The addition of 0.1% (w/v) soy lecithin increased 1.5-fold the lipase production (147.5 U). Biochemical characterization of the crude enzyme revealed an acid lipase with optimum pH of 3.5, a property first described in Candida genus. Optimum temperature activity was 40 ºC. Crude lipase was highly stable at pH 4.0 to 5.0 and at temperature 40 ºC during 24 h (100%). Solid substrates cultures from industrial byproducts, xiv the highest lipase production was obtained in wheat bran and barley spent grain (1:1), 40% (w/w) chicken fat, 3.5% (w/w) yeast extract, humidity 40% after 5 days, at 30°C (153 U/gds). Under these culture conditions, the lipase showed optimum activity at pH 5.0 and 50 ºC, and thermal enzyme stability at pH... (Complete abstract click electronic access below)
Descrição
Palavras-chave
Enzimas imobilizadas, Lipase, Enzimas - Purificação
Como citar
ALMEIDA, Alex Fernando de. Produção, purificação e propriedades bioquímicas de lipase ácida de Candida viswanathii. 2012. 213 f. Tese - (doutorado) - Universidade Estadual Paulista, Instituto de Biociências de Rio Claro, 2012.