RESUMEN
Xyloglucan-specific endo-ß-1,4-glucanases (Xegs, EC 3.2.1.151) exhibit high catalytic specificity for ß-1,4 linkages of xyloglucan, a branched hemicellulosic polysaccharide abundant in dicot primary cell walls and present in many monocot species. In nature, GH12 Xegs are not associated with carbohydrate-binding modules (CBMs), and here, we have investigated the effect of the fusion of the xyloglucan-specific CBM44 on the structure and function of a GH12 Xeg from Aspergillus niveus (XegA). This fusion presented enhanced catalytic properties and conferred superior thermal stability on the XegA. An increased k cat (chimera, 177.03 s(-1); XegA, 144.31 s(-1)) and reduced KM (chimera, 1.30 mg mL(-1); XegA, 1.50 mg mL(-1)) resulted in a 1.3-fold increase in catalytic efficiency of the chimera over the parental XegA. Although both parental and chimeric enzymes presented catalytic optima at pH 5.5 and 60 °C, the thermostabilitiy of the chimera at 60 °C was greater than the parental XegA. Moreover, the crystallographic structure of XegA together with small-angle X-ray scattering (SAXS) and molecular dynamics simulations revealed that the spatial arrangement of the domains in the chimeric enzyme resulted in the formation of an extended binding cleft that may explain the improved kinetic properties of the CBM44-XegA chimera.
Asunto(s)
Aspergillus/enzimología , Endo-1,3(4)-beta-Glucanasa/química , Endo-1,3(4)-beta-Glucanasa/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Glucanos/metabolismo , Xilanos/metabolismo , Secuencia de Aminoácidos , Aspergillus/química , Aspergillus/genética , Endo-1,3(4)-beta-Glucanasa/genética , Proteínas Fúngicas/genética , Glucanos/química , Cinética , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Ingeniería de Proteínas , Estructura Terciaria de Proteína , Dispersión del Ángulo Pequeño , Especificidad por Sustrato , Difracción de Rayos X , Xilanos/químicaRESUMEN
A bifunctional enzyme has been created by fusing two Bacillus subtilis enzymes: the ß-1,3-1,4-glucanase (BglS, EC 3.2.1.73) that hydrolyzes plant cell wall ß-glucans and the copper-dependent oxidase laccase (CotA, EC 1.10.3.2) that catalyzes the oxidation of aromatic compounds with simultaneous reduction of oxygen to water. The chimeric laccase/ß-1,3-1,4-glucanase was created by insertion fusion of the bglS and cotA genes, and expressed in Escherichia coli. The affinity-purified recombinant chimeric enzyme showed both laccase and glucanase activities, with a maximum laccase activity at pH 4.5 and 75°C that showed a V(max) 30% higher than observed for the parental laccase. The maximum glucanase activity in the chimeric enzyme was at pH 6.0 and 50°C, with a slight reduction in V(max) by â¼10% compared with the parental glucanase. A decreased K(M) resulted in an overall increase in the K(cat)/K(M) value for the glucanase activity of the chimeric enzyme. The hydrolytic activity of the chimera was 20% higher against natural milled sugarcane bagasse as compared with equimolar mixtures of the separate parental enzymes. Molecular dynamics simulations indicated the approximation of the two catalytic domains in the chimeric enzyme, and the formation of an inter-domain interface may underlie the improved catalytic function.
Asunto(s)
Bacillus subtilis/enzimología , Celulosa/metabolismo , Endo-1,3(4)-beta-Glucanasa/metabolismo , Lacasa/metabolismo , Ingeniería de Proteínas/métodos , Saccharum/metabolismo , Celulosa/química , Endo-1,3(4)-beta-Glucanasa/química , Endo-1,3(4)-beta-Glucanasa/genética , Cinética , Lacasa/química , Lacasa/genética , Simulación de Dinámica Molecular , Oxidación-Reducción , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharum/químicaRESUMEN
Trichoderma species are readily isolated from Brazilian cerrado soil by conventional methods and some of them were characterized as Trichoderma koningii. The effect of carbon source on the production of beta-1,3-glucanases in the culture filtrates of a specific Trichoderma koningii strain (ALL 13) was investigated. Enzyme activity was detected in all carbon sources tested and only one band of beta-1,3-glucanase was detected in non-denaturing PAGE. This enzyme was purified by Sephacryl S-200 gel filtration and Phenyl Sepharose CL 4B chromatography. A typical procedure provided 105-fold purification with 13.4% yield. The molecular weight of the purified enzyme was 75 kDa as estimated by SDS-PAGE. The enzyme hydrolyzed laminarin in an endo-like fashion to form small oligosaccharides and glucose. The Km and Vmax values for beta-1,3-glucanase, using laminarin as substrate, were 0.148 mg.mL-1 and 0.159 U.min-1, respectively. The pH optimum for the enzyme was pH 4.6 and maximum activity was obtained at 50 degrees C. Hg2+ inhibited the purified enzyme.