RESUMO
Following immunization of rabbits, the antiserum was initially analyzed for antiendoglucanase activity using dot-blot ELISA methods. When compared with the preimmune serum, the antiserum showed strong response even at 25 ng concentration. The specificity of the polyclonal antibodies, raised against the partially purified endoglucanase component of Penicillium funiculosum, was determined by Oüchterlony double diffusion. Rocket electrophoresis confirmed the presence of only two types of antigens in the serum. The two rockets obtained were attributable to endo I and the merging of immunologically identical (but mobility wise different) endo II and endo III. This antibody preparation was used as a probe. The deduced M1 of the cloned E. coli endo I was found to be 58 Kd by Western blotting.
Assuntos
Celulase/imunologia , Penicillium/enzimologia , Animais , Formação de Anticorpos/imunologia , Western Blotting , Celulase/genética , Ensaio de Imunoadsorção Enzimática , Imunodifusão , Imunoeletroforese , Penicillium/genética , Penicillium/imunologia , CoelhosRESUMO
A gene of Penicillium funiculosum encoding an endoglucanase was cloned and expressed in Escherichia coli using the lacZ promoter of vector pUC 18. The gene product hydrolyzed carboxymethyl cellulose and showed strong cross reactivity with P. funiculosum anticellulases.
Assuntos
Celulase/genética , Genes Fúngicos , Penicillium/genética , Celulase/imunologia , Clonagem Molecular , Escherichia coli/genética , Regulação Enzimológica da Expressão Gênica , Vetores Genéticos , Penicillium/enzimologiaRESUMO
Pennisetin, the alcohol soluble storage protein of pearl millet (Pennisetum americanum), was isolated in a homogeneous state. The intrinsic viscosity [n] of this protein was found to be in the range of 16.5-17.7 ml/g in 70% (v/v) aqueous ethanol. The [eta] changed marginally when temperature was increased from 20 to 70 degrees C and also in the presence of 10 mM NaCl. The data indicated that pennisetin was a rigid, rod shaped asymmetric hydrodynamic particle with molecular dimensions in the range of 301 x 14.4 A - 317.7 x 14.2 A. During denaturation with guanidine hydrochloride (Gdn.HCl), the intrinsic viscosity of pennisetin increased from 16 to 25ml/g with a mid point at 3.6 M of the denaturant. The native protein structure was unfolded in 6 M Gdn.HCl as shown by the exposure of aromatic amino acid residues buried in the native state and this transition was found to be reversible. The intrinsic viscosity of pennisetin in 5.9 M Gdn.HCl corresponded to Mr 25,000 which was comparable to that determined by SDS-PAGE.
Assuntos
Grão Comestível/química , Panicum/química , Proteínas de Plantas , Eletroforese em Gel de Poliacrilamida , Peso Molecular , Proteínas de Plantas/isolamento & purificação , Prolaminas , Conformação Proteica , Espectrometria de Fluorescência , ViscosidadeRESUMO
The endoglucanase component (CMCase I) ofPenicillium funiculosum cellulase was purified to apparent homogeneity by ultrafiltration and gel chromatography. It consists of a single polypeptide chain with a molecular weight of 56000 and is a glycoprotein. Viscometric and end-product analysis revealed the randomness of enzyme action. Multifunctional characteristic of CMCase I was studied with various carbohydrate substrates.
