RESUMO
Immobilization of enzymes has many advantages for their application in biotechnological processes. In particular, the cross-linked enzyme aggregates (CLEAs) allow the production of solid biocatalysts with a high enzymatic loading and the advantage of obtaining derivatives with high stability at low cost. The purpose of this study was to produce cross-linked enzymatic aggregates (CLEAs) of LipMatCCR11, a 43 kDa recombinant solvent-tolerant thermoalkaliphilic lipase from Geobacillus thermoleovorans CCR11. LipMatCCR11-CLEAs were prepared using (NH4)2SO4 (40% w/v) as precipitant agent and glutaraldehyde (40 mM) as cross-linker, at pH 9, 20 °C. A U10(56) uniform design was used to optimize CLEA production, varying protein concentration, ammonium sulfate %, pH, glutaraldehyde concentration, temperature, and incubation time. The synthesized CLEAs were also analyzed using scanning electron microscopy (SEM) that showed individual particles of <1 µm grouped to form a superstructure. The cross-linked aggregates showed a maximum mass activity of 7750 U/g at 40 °C and pH 8 and retained more than 20% activity at 100 °C. Greater thermostability, resistance to alkaline conditions and the presence of organic solvents, and better durability during storage were observed for LipMatCCR11-CLEAs in comparison with the soluble enzyme. LipMatCCR11-CLEAs presented good reusability by conserving 40% of their initial activity after 9 cycles of reuse.
Assuntos
Proteínas de Bactérias/química , Geobacillus/enzimologia , Lipase/química , Agregados Proteicos , Proteínas de Bactérias/genética , Reagentes de Ligações Cruzadas/química , Estabilidade Enzimática , Geobacillus/genética , Lipase/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
The fatty acid profile of hepatocytes and adipocytes is determined by the composition of the dietary lipids. It remains unclear which fatty acid components contribute to the development or reduction of insulin resistance. The present work examined the fatty acid composition of both tissues in sucrose-induced obese rats receiving fish oil to determine whether the effect of dietary (n-3) polyunsaturated fatty acids (PUFAs) on the reversion of metabolic syndrome in these rats is associated to changes in the fatty acid composition of hepatocyte and adipocyte membrane lipids. Animals with metabolic syndrome were divided into a corn-canola oil diet group and a fish oil diet group, and tissues fatty acids composition were analyzed after 6 weeks of dietary treatment. Fatty acid profiles of the total membrane lipids were modified by the fatty acid composition of the diets fed to rats. N-3 PUFAs levels in animals receiving the fish oil diet plus sucrose in drinking water were significantly higher than in animals under corn-canola oil diets. It is concluded that in sucrose-induced obese rats, consumption of dietary fish oil had beneficial effects on the metabolic syndrome and that such effects would be conditioned by the changes in the n-3 PUFAs composition in hepatic and adipose tissues because they alter membrane properties and modify the type of substrates available for the production of active lipid metabolites acting on insulin resistance and obesity.
Assuntos
Gordura Abdominal/metabolismo , Ácidos Graxos Ômega-3/administração & dosagem , Ácidos Graxos/análise , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/metabolismo , Obesidade/dietoterapia , Obesidade/metabolismo , Gordura Abdominal/química , Gordura Abdominal/efeitos dos fármacos , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Sacarose Alimentar , Ácidos Graxos Monoinsaturados/administração & dosagem , Ácidos Graxos Monoinsaturados/metabolismo , Óleos de Peixe/administração & dosagem , Óleos de Peixe/metabolismo , Fígado/efeitos dos fármacos , Masculino , Síndrome Metabólica/induzido quimicamente , Síndrome Metabólica/dietoterapia , Síndrome Metabólica/metabolismo , Obesidade/induzido quimicamente , Óleo de Brassica napus , Ratos , Ratos WistarRESUMO
The gene for a Geobacillus thermoleovorans CCR11 thermostable lipase was recovered by PCR and cloned. Four genetic constructions were designed and successfully expressed in E. coli: (i) the lipase structural gene (lipCCR11) in the PinPoint Xa vector; (ii) the lipase structural gene (lipACCR11) in the pET-28a(+) vector; (iii) the lipase structural gene minus the signal peptide (lipMatCCR11) in the pET-3b vector; and (iv) the lipase structural gene plus its own promoter (lipProCCR11) in the pGEM-T cloning vector. The lipase gene sequence analysis showed an open reading frame of 1,212 nucleotides coding for a mature lipase of 382 residues (40 kDa) plus a 22 residues signal peptide. Expression under T7 and T7lac promoter resulted in a 40- and 36-fold increase in lipolytic activity with respect to the original strain lipase. All recombinant lipases showed an optimal activity at pH 9.0, but variations were found in the temperature for maximum activity and the substrate specificity among them and when compared with the parental strain lipase, especially in the recombinant lipases that contained fusion tags. Therefore, it is important to find the appropriate expression system able to attain a high concentration of the recombinant lipase without compromising the proper folding of the protein.