RESUMO
A cell surface lectin was isolated and purified to homogeneity from the cell walls of a highly flocculent strain of Saccharomyces cerevisiae (NCIM 3528) by chromatography on DEAE-cellulose, phenyl Sepharose and Sephacryl S-300. It showed a molecular mass of 40 kDa on SDS-PAGE. It is an acidic protein with a pI of 4.0 and contains 44% hydrophobic amino acids. The N-terminal sequence up to 10 amino acid residues showed at least 70% homology with the predicted N-terminal sequence of the putative FLO1 as well as FLO5 gene products. The mannose-binding nature of the lectin was indicated by its high affinity and specificity towards the branched trisaccharide of mannose, a ligand which also inhibits the flocculation of yeast cells. Immunofluorescence studies confirmed the presence of lectin on the yeast cell surface and lectin-specific IgGs prevented flocculation of the cells. This cell surface mannose-specific lectin probably plays an important role in flocculation, with the branched trimannoside on the cell wall being the apparent carbohydrate receptor. The N-terminal sequence data gives a primary indication that the lectin could be a product of one of the FLO genes.
Assuntos
Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Parede Celular/química , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Aminoácidos/análise , Sequência de Carboidratos , Carboidratos/química , Carboidratos/farmacologia , Proteínas de Transporte/química , Colectinas , Floculação , Imunofluorescência , Testes de Inibição da Hemaglutinação , Concentração de Íons de Hidrogênio , Lectinas/química , Lectinas/isolamento & purificação , Lectinas/metabolismo , Lectinas de Ligação a Manose , Proteínas de Membrana , Metais/farmacologia , Dados de Sequência Molecular , Saccharomyces cerevisiae/genética , TemperaturaRESUMO
Conditions were optimized for rapid release and improved regeneration of protoplasts of Saccharomyces cerevisiae NCIM 3458. Rapid protoplast release was also obtained with representatives of several other yeast genera under the modified conditions of treatment. The application of the procedure in construction of a highly flocculent Saccharomyces cerevisiae with a killer character is described. Fusion was effected between UV-killed protoplasts of S. cerevisiae NCIM 3578 with a killer character and live protoplasts of the highly flocculent S. cerevisiae NCIM 3528 in the presence of polyethylene glycol (PEG) 6000. Fusants were selected using benomyl resistance as marker, the killer toxin producer rather than the highly flocculent yeast being resistant to the fungicide at a concentration of 100 micrograms ml-1. Fusants were also characterized by their DNA contents, capacity for ethanolic fermentation of molasses sugar and levels of invertase, alcohol dehydrogenase and pyruvate decarboxylase activities.
