Characterization, expression in Streptomyces lividans, and processing of the amylase of Streptomyces griseus IMRU 3570: two different amylases are derived from the same gene by an intracellular processing mechanism.
Extracellular amylase in Streptomyces lividans was undetectable in starch-supplemented medium. However, S. lividans produced fivefold-higher levels of amylase than Streptomyces griseus IMRU 3570 when transformed with the S. griseus amy gene. Two major proteins of 57 and 50 kDa with amylase activity accumulated in the culture broths of the donor S. griseus and S. lividans transformed with the amy gene. Both proteins were also present in protoplast lysates in the same relative proportion; they gave a positive reaction with antibodies against the 57-kDa amylase. They did not differ in substrate specificity or enzyme kinetics. The two amylases were purified to homogeneity by a two-step procedure. Both proteins showed the same amino-terminal sequence of amino acids, suggesting that both proteins are derived from the same gene. The deduced signal peptide has 28 amino acids with two positively charged arginines near the amino-terminal end. When an internal NcoI fragment was removed from the amy gene, the resulting S. lividans transformants did not synthesize any of the two amylase proteins and showed no reaction in immunoblotting. Formation of the 50-kDa protein was observed when pure 57-kDa amylase was treated with supernatants of protoplast lysates but not when it was treated with membrane preparations, indicating that the native 57-kDa amylase could be processed intracellularly.
Garcia-Gonzalez
M D
MD
Department of Ecology, Genetics and Microbiology, Faculty of Biology, University of León, Spain.
Martin
J F
JF
Vigal
T
T
Liras
P
P
D000595:Amino Acid Sequence; D000681:Amylases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D012689:Sequence Homology, Nucleic Acid; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism; D014169:Transformation, Bacterial
D000681:Amylases