RESUMO
Site-directed mutagenesis was performed to define the active site of the Schwanniomyces occidentalis glucoamylase. The mutated GAM1 genes were expressed in Saccharomyces cerevisiae, and enzymatic and growth properties of the transformants were determined. Mutants were transcribed and translated similar to the wild-type glucoamylase. Therefore, all effects on enzymatic activity could be referred to single amino acid substitutions. Asp470 was shown to be essential for the enzyme activity. Replacement of Asp470 by glycine led to a complete loss of activity. We suppose that Asp470 serves as a general acid-base and stabilizes the formation of the intermediate carbenium ion. Substitution of Trp468 by alanine affected predominantly the alpha-1,6 activity and not the alpha-1,4 activity of the enzyme. The exchange impaired substrate binding as well as enzymatic catalysis. An influence of amino acid 474 on the substrate specificity could not be demonstrated. Exchanges at position 474 exhibited K(m) and Vmax values similar to wild-type glucoamylase.
Assuntos
Glucana 1,4-alfa-Glucosidase/química , Saccharomycetales/enzimologia , Sequência de Aminoácidos , Ácido Aspártico/metabolismo , Sítios de Ligação , Glucana 1,4-alfa-Glucosidase/genética , Glucana 1,4-alfa-Glucosidase/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Saccharomycetales/genética , Triptofano/metabolismoRESUMO
The exploitation of recombinant DNA technology to engineer expression systems for heterologous proteins represented a major task within the field of biotechnology during the last decade. Yeasts attracted the attention of molecular biologists because of properties most favourable for their use as hosts in heterologous protein production. Yeasts follow the general eukaryotic posttranslational modification pattern of expressed polypeptides, exhibit the ability to secrete heterologous proteins and benefit from an established fermentation technology. Aside from the baker's yeast Saccharomyces cerevisiae, an increasing number of alternative non-Saccharomyces yeast species are used as expression systems in basic research and for an industrial application. In the following review a selection from the different yeast systems is described and compared.
Assuntos
Engenharia de Proteínas , Proteínas Recombinantes/biossíntese , Leveduras/genética , Leveduras/metabolismo , Expressão Gênica , Vetores Genéticos , Kluyveromyces/genética , Kluyveromyces/metabolismo , Pichia/genética , Pichia/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
The Schwanniomyces occidentalis glucoamylase (GAM)-encoding gene (GAM1) was isolated from a lambda Charon4A genomic library using synthetic oligodeoxynucleotides as probes. GAM1 contains an ORF of 2874 nucleotides (nt) coding for 958 amino acids. S1 mapping revealed that the transcript has only a very short 5'-untranslated leader of 8-12 nt. Disruption and displacement of the GAM1 gene in Sc. occidentalis resulted in loss of the ability to grow on starch efficiently. The gam1 strains still exhibit low GAM activity suggesting that at least a second weakly expressed GAM-encoding gene (GAM2) is present in Sc. occidentalis. Expression of the Sc. occidentalis GAM1 gene in Saccharomyces cerevisiae was achieved after promoter exchange. S. cerevisiae cells transformed with centromere plasmids carrying the GAM1 gene fused to promoters of different S. cerevisiae genes, namely GAL1, PDC1 and ADH1, efficiently secrete GAM and are able to grow with soluble starch as a sole carbon source. The essential enzymatic properties of the GAMs secreted from S. cerevisiae and Sc. occidentalis are identical, although the modifications of the proteins are different.
