Molecular cloning of a thermostable raw starch digesting amylase gene form a Streptomyces sp.
2008
Kaneko, T.(Akita-ken. Research Inst. for Food and Brewing (Japan))
Raw starch digesting amylase (RSA) from Streptomyces sp. E-2248 is a thermostable enzyme with a large potential for starch liquefaction and saccharification. The gene encoding this unique enzyme was cloned by a simple two-step PCR method, and expressed in Escherichia coli. A single open reading frame consisting of 1374 base pairs (67.47 mol% of G+C content) that encoded a polypeptide composed of 458 amino acid residues (Mr 49,386) was found. The E. coli transformant carrying the gene overproduced the recombinant RSA and the enzyme was successfully expressed under the transcriptional control of the tac-promoter. The purified recombinant enzyme was indistinguishable from that of the authentic enzyme, in terms of molecular weight and products, temperature, and pH profiles. The relationships between the structure and properties of the enzymes are also discussed.
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