RESUMO
The hydrolysis reaction of p-nitrophenyl butyrate catalyzed by lipases was followed with in situ UV/vis diode array spectrophotometry. Five enzymes - Candida antarctica lipase B and Fusarium solani pisi cutinase wild-type and three single-mutation variants - were tested as catalysts in homogeneous conditions and immobilized on zeolite NaY, on a polyacrylate support and as cross-linked aggregates. Using deconvolution techniques and kinetic modeling, the thermal stability of the different biocatalysts was compared in operational conditions and the results were supported by steady-state enzyme fluorescence measurements. We concluded that both the mutagenesis and the immobilization on zeolite NaY had a positive effect on the thermal stability of F. solani pisi cutinase.
Assuntos
Lipase/análise , Mutagênese , Espectrometria de Fluorescência/métodos , Zeolitas/metabolismo , Resinas Acrílicas/química , Candida/enzimologia , Catálise , Proteínas Fúngicas , Fusarium/metabolismo , Hidrólise , Cinética , Lipase/química , Modelos Estatísticos , Espectrofotometria Ultravioleta/métodos , Temperatura , Fatores de TempoRESUMO
The kinetics of the hydrolysis reaction of p-nitrophenyl butyrate catalyzed by a single-site mutant of Fusarium solani pisi cutinase, supported on zeolite NaY by adsorption, was followed using ultraviolet-visible (UV/Vis) in situ diode array spectrophotometry. Spectral band-fitting techniques allowed the use of in situ diode array spectrophotometry to obtain quantitative data on the concentrations of the various species present in an aqueous reaction medium even in the presence of the solid catalyst particles, which interfere with the absorption measurements. This technique overcame the problems arising from the variable light scattering produced by these highly dispersible catalyst particles. Kinetic modeling of the reaction system was used to compute estimates of the reaction rate constants involved.
Assuntos
Butiratos/química , Hidrolases de Éster Carboxílico/química , Fusarium/enzimologia , Catálise , Hidrólise , Espectrofotometria Ultravioleta/métodos , Zeolitas/químicaRESUMO
The reactivity, stability and unfolding of wild-type (WT) Fusarium solani pisi cutinase and L153Q, S54D and T179C variants were studied in the absence and presence of the dioctyl sulfosuccinate sodium salt (AOT) surfactant. In the absence of surfactant the S54D variant catalytic activity is similar to that of the WT cutinase, whereas L153Q and T179C variants show a lower activity. AOT addition induces an activity reduction for WT cutinase and its variants, although for low AOT concentrations a small increase of activity was observed for S54D and T179C. The enzyme deactivation in the presence of 0.5 mM AOT is relatively slow for the S54D and T179C variants when compared to wild-type cutinase and L153Q variant. These results were correlated with secondary and tertiary structure changes assessed by the CD spectrum and fluorescence of the single tryptophan and the six tyrosine residues. The WT cutinase and S54D variant have similar secondary and tertiary structures that differ from those of T179C and L153Q variants. L153Q, S54D and T179C mutations prevent the formation of hydrophobic crevices responsible for the unfolding by anionic surfactants, with the consequent decrease of the AOT-cutinase interactions.
Assuntos
Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Substituição de Aminoácidos , Naftalenossulfonato de Anilina/metabolismo , Butiratos/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Dicroísmo Circular , Estabilidade Enzimática/efeitos dos fármacos , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fusarium/enzimologia , Fusarium/genética , Hidrólise , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Fluorescência , Succinatos/farmacologia , Tensoativos/farmacologia , TermodinâmicaRESUMO
Cutinase is an enzyme suitable for detergent applications as well as for organic synthesis in non-aqueous solvents. However, its inactivation in the presence of anionic surfactants is a problem which we have addressed by creating a complete saturation library. For this, the cutinase gene from Fusarium solani pisi was mutated to incorporate all 19 possible amino acid exchanges at each of the 214 amino acid positions. The resulting library was screened for active variants with improved stability in the presence of the anionic surfactant dioctyl sulfosuccinate sodium salt (AOT). Twenty-four sites in cutinase were discovered where amino acid replacements resulted in a 2-11-fold stability increase as compared to the wild-type enzyme.
Assuntos
Hidrolases de Éster Carboxílico/química , Mutagênese , Sequência de Aminoácidos , Hidrolases de Éster Carboxílico/genética , Estabilidade Enzimática , Fusarium/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Conformação ProteicaRESUMO
The activity of various lipases was compared, in both free and immobilized forms, using the kinetics of the hydrolysis reaction of p-nitrophenyl butyrate, which was followed with in situ UV/Vis diode array spectrophotometry. Several enzymes were used to catalyze the reaction, namely Candida antarctica lipase B and Fusarium solani pisi cutinase wildtype and three single-mutation variants. The enzymes were tested in three different forms: free, immobilized as cross-linked aggregates and supported on zeolite NaY. A simple kinetic model was used to allow a quantitative comparison of the behavior of the different catalysts. It was concluded that although immobilization reduces the activity of the enzyme, the zeolite offers a much higher specific activity when compared to the cross-linked aggregates, thus supplying a heterogeneous catalyst with promising catalytic properties.
