SUMMARY: The subsp. 712 gene encoding phospho-β-galactosidase was isolated from the lactose mini-plasmid pMG820 and cloned and expressed in and The low phospho-β-galactosidase activity in transformed with high-copy-number plasmids containing the gene contrasted with the high activity found in containing the original, low-copy-number lactose plasmid pMG820, and indicated that the original lactose promoter was absent from the cloned DNA. In the phospho-β-galactosidase could be overproduced using the strong inducible δ P promoter, which allowed a rapid purification of the active enzyme. The complete nucleotide sequence of the gene and its surrounding regions was determined. The deduced amino acid sequence was confirmed by comparison with the amino acid composition of the purified phospho-β-galactosidase and its amino-terminal sequence. This also allowed the exact positioning of the gene and identification of its characteristic Gram-positive translation initiation signals. The homologous expression data and the sequence organization of the gene indicate that the gene is organized into a large lactose operon which contains an intergenic promoter located in an inverted repeat immediately preceding the gene. The organization and sequence of the gene were compared with those of the highly homologous gene from A remarkable bias for leucine codons was observed in the genes of these two species. Heterogramic homology was observed between the deduced amino acid sequence of the phospho-β-galactosidase, that of the functionally analogous phospho-β-glucosidase, and that of an β-glucosidase (cellobiase).


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