RESUMO
The cDNA of cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium has been cloned and sequenced. The 5' end was obtained by PCR amplification. The cDNA contains 2310 translated bases excluding the poly(A) tail. The deduced mature protein contains 770 amino acid residues and is preceded by a 18 residue long signal peptide. The regions of the amino acid sequence corresponding to the heme and FAD domains of CDH were identified as well as the nucleotide-binding motif, the disulfide pairing and a methionine residue chelating the heme iron. No homologous sequences were found for the heme domain, however, the FAD domain appears to be distantly related to the GMC oxidoreductase family.
Assuntos
Basidiomycota/genética , Desidrogenases de Carboidrato/genética , Genes Fúngicos/genética , Sequência de Aminoácidos , Aminoácidos/análise , Sequência de Bases , Basidiomycota/enzimologia , Desidrogenases de Carboidrato/química , Desidrogenases de Carboidrato/isolamento & purificação , Clonagem Molecular , DNA Complementar/genética , DNA Fúngico/genética , Flavina-Adenina Dinucleotídeo , Heme , Dados de Sequência Molecular , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
We have cloned and expressed a bacterial thermostable alpha amylase gene in Pichia pastoris using the methanol-controlled alcohol oxidase (AOX1) promoter. Two integrative vectors were constructed with two different secretion signal sequences in order to obtain efficient secretion of the protein. One vector contains the structural gene encoding the mature alpha amylase fused to the SUC2 gene signal sequence from Saccharomyces cerevisiae. In the other vector, the alpha amylase is expressed with its own signal sequence. In both cases, the alpha amylase were secreted into the culture medium with high efficiency, around 2.5 and 0.9 g/l respectively.
