RESUMO
Candida rugosa lipase has been covalently immobilized on ferromagnetic azide polyethyleneterepthalate (Dacron) with specific activity retention of 16% for 4-nitrophenyl palmitate and 24% for hydrolysis of triolein in hexane. The immobilized enzyme was more thermal stable than the soluble one, retaining 78.8% of the activity after 1 h at 60 degrees C. Also, this immobilized derivative was stable at the storage at 4 degrees C. It has been used 5 cycles for pNPP hydrolysis without loss of activity. Soluble and immobilized Candida rugosa lipase showed a Michaelian behavior for fatty acid 4-nitrophenyl esters and different apparent K(M) values: 0.110 mM and 0.124 mM (4-nitrophenyl palmitate - C16); 0.193 mM and 0.235 mM (4-nitrophenyl laurate - C12) and 0.206 mM and 0.119 mM (4-nitrophenyl butyrate - C4), respectively. The immobilized lipase was more efficient for catalyzing the hydrolysis of 4-nitrophenyl esters with short chain length fatty acid (4-NPB - C4) than soluble enzyme. The ferromagnetic Dacron-lipase derivative was able to catalyze the synthesis of triolein from glycerol and oleic acid with 50% of conversion after 72 h at 40 degrees C.
Assuntos
Biotecnologia/métodos , Candida/enzimologia , Enzimas Imobilizadas/metabolismo , Lipase/metabolismo , Polietilenotereftalatos , 2-Propanol , Ésteres/metabolismo , Temperatura Alta , Cinética , Magnetismo , Solventes , Especificidade por SubstratoRESUMO
Chitin deacetylase (CDA) is an enzyme that catalyzes the hydrolysis of acetamine groups of N-acetyl-D: -glucosamine in chitin, converting it to chitosan in fungal cell walls. In the present study, the activity in batch culture of CDA from six Mucoralean strains, two of them wild type, isolated from dung of herbivores of Northeast Brazil, was screened. Among the strains tested, Cunninghamella bertholletiae IFM 46114 showed a high intracellular enzyme activity of 0.075 U/mg protein after 5 days of culture, and a wild-type strain of Mucor circinelloides showed a high intracellular enzyme activity of 0.060 U/mg protein, with only 2 days of culture, using N-acetylchitopentaose as substrate. This enzyme showed optimal activity at pH 4.5 in 25 mM glutamate-sodium buffer at 50 degrees C, and was stable over 1 h preincubation at the same temperature. The kinetic parameters of CDA did not follow Michaelis-Menten kinetics, but rather Hill affinity distribution, showing probable allosteric behavior. The apparent K(HILL) and Vmax of CDA were 288+/-34 nmol/l and 0.08+/-0.01 U mg protein(-1) min(-1), respectively, using N-acetylchitopentaose as substrate at pH 4.5 at 50 degrees C.
Assuntos
Amidoidrolases/metabolismo , Cunninghamella/enzimologia , Cunninghamella/crescimento & desenvolvimento , Mucor/enzimologia , Mucor/crescimento & desenvolvimento , Ativação Enzimática , Microbiologia Industrial , Cinética , Técnicas MicrobiológicasRESUMO
Chitosan from a native Mucoralean strain, Syncephalastrum racemosum, isolated from herbivorous dung (Northeast-Brazil), was used as a film support for lipase immobilization. S. racemosum showed highest chitosan yield (152 mg g dry mycelia weight(-1); 15.2% of dry mycelia weight) among the nine strains screened, which presented 89% D-glucosamine. A chitosan film was used for lipase (EC 3.1.1.3) immobilization using glutaraldehyde as a bifunctional agent. The immobilized lipase retained 47% (12.6 micromol s(-1) m(-2)) of its initial catalytic activity after four cycles of reaction. This result is comparable (same order of magnitude) to that of the enzyme immobilized on film made from commercially available crustacean chitosan.
Assuntos
Quitina/análogos & derivados , Quitina/metabolismo , Enzimas Imobilizadas/metabolismo , Fungos/química , Lipase/metabolismo , Membranas Artificiais , Animais , Quitina/biossíntese , Quitina/química , Quitosana , Crustáceos/química , Estabilidade Enzimática , Fungos/metabolismo , GlutaralRESUMO
Lipase (Glycerol ester hydrolase EC 3.1.1.3.) from a Brazilian strain of Fusarium solani FSI has been investigated. The effect of different carbon sources and trace elements added to basal medium was observed with the aim of improving enzyme production. Lipase specific activity was highest (0.45 U mg(-1)) for sesame oil. When this medium was supplemented with trace elements using olive oil, corn oil and sesame oil the lipase specific activity increased to 0.86, 1.89 and 1.64 U mg(-1), respectively, after 96 h cultivation without any considerable biomass increase. The Km of this lipase using pNPP (p-nitrophenylpalmitate) as substrate, was 1.8 mM with a Vmax of 1.7 micromol min(-1) mg protein(-1). Lipase activity increased in the presence of increasing concentrations of hexane and toluene. In contrast, incubation of this enzyme with water-soluble solvents decreased its activity after 10% concentration (v/v) of the solvent. The lipase activity was stable below 35 degrees C but above this temperature activity losses were observed.
Assuntos
Fermentação , Fusarium/enzimologia , Indústrias , Lipase/química , Reatores Biológicos , Óleo de Milho/metabolismo , Hexanos/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Lipase/metabolismo , Azeite de Oliva , Palmitatos/metabolismo , Óleos de Plantas/metabolismo , Óleo de Gergelim/metabolismo , Temperatura , Fatores de Tempo , Tolueno/metabolismoRESUMO
A lipases (glycerol ester hydrolases E. C. 3.1.1.3) from a brazilian strain of Penicillium citrinum has been investigated. When the microorganism was cultured in the simple medium (1.0% olive oil and 0.5% yeast extract), using olive oil in as carbon source in the inocula, the enzyme extracted showed maximum activity (409 IU/mL). In addition, decrease of yeast extract concentration also reduces the lipase activity. Nevertheless, when yeast extract was replaced by ammonium sulfate, no activity was detected. Purification by precipitation with ammonium sulfate showed best activity in the 40-60% fraction. The optimum temperature for enzyme activity was found in the range of 34-37 degrees C. However, after 30 min at 60 degrees C, the enzyme was completely inactivated. The enzyme showed optimum at pH 8.0. The dried concentrated fraction (after dialysis and lyophilization) maintained its lipase activity at room temperature (28 degrees C) for 8 mo. This result in lipase stability suggests an application of lipases from P. citrinum in detergents and other products that require a high stability at room temperature.
Assuntos
Lipase/isolamento & purificação , Penicillium/enzimologia , Brasil , Meios de Cultura , Indução Enzimática/efeitos dos fármacos , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Lipase/biossíntese , Lipase/química , Azeite de Oliva , Penicillium/genética , Óleos de Plantas/farmacologia , TemperaturaRESUMO
Batch culture experiments involving ammonia uptake in Saccharomyces cerevisiae BC55 pCYG4 have been carried out. This strain carries the plasmid pCYG4 that directs substantial overproduction of NADP-GDH, conferring an 11-fold increase in activity. The wild type cells had a specific growth rate greater than BC55 pCYG4. The ammonia uptake was practically the same until 15 h of growth. However, the amount of ammonia hydroxide added during growth (60 h) was two and half times greater in the BC55 pCYG4 than wild type cells. The results suggest that the presence of the plasmid pCYG4 can increase the amount of ammonia taken by the cells, but not the amount of biomass.
Assuntos
Amônia/metabolismo , Plasmídeos , Saccharomyces cerevisiae/metabolismo , Transporte Biológico , Meios de Cultura , Cinética , Saccharomyces cerevisiae/genéticaRESUMO
The gene (GDH1) coding for the NADP-linked glutamate dehydrogenase system (NADP-GDH) has been cloned from Saccharomyces cerevisiae strain. Cells being transformed by the NADP-GDH gene on a 2 micron bared vector (pCYG4) plasmid confering 11-fold higher level on expressed GDH activity over the wild-type cells. The behavior of these cells was investigated under chemostatic growth with a carbon rate-limiting nutrient. Specific growth rates of cells carrying plasmid pCYG4 were found to be slightly slower than wild type cells. Furthermore, the NADP-GDH activity increases proportionally with the dilution rate. In addition, oscillations in the NADP-GDH activity, especially at a dilution rate up to 0.15/h, are probably consequential on the appearance of a changing mixed population (cells with and without plasmids).
Assuntos
Genes Fúngicos/fisiologia , Glutamato Desidrogenase/genética , Plasmídeos/genética , Saccharomyces cerevisiae/genética , Carbono , Ecologia , Glutamato Desidrogenase/metabolismo , Desidrogenase de Glutamato (NADP+) , Engenharia de Proteínas , Saccharomyces cerevisiae/crescimento & desenvolvimento , Transformação GenéticaRESUMO
Batch culture experiments of three different strains of Saccharomyces cerevisiae have been carried out. The first strain was transformed by a plasmid pCYG4, which carries the glutamate dehydrogenase (NADP-GDH, E.C. 1.4.14) gene conferring an 11-fold increase in activity. The second was transformed by the same plasmid, but without NADP-GDH, and the third was the wild type. The specific growth rates of the two recombinant DNA strains were below that of the wild type, which can be related to extra plasmid protein production.