Molecular cloning of acid-stable glucose isomerase gene from Streptomyces olivaceoviridis E-86 by a simple two-step PCR method, and its expression in Escherichia coli
Kaneko, T. (Akita-ken. Inst. of Brewing (Japan)) | Saito, K. | Kawamura, Y. | Takahashi, S.
Glucose isomerase (GI) from Streptomyces olivaceoviridis E-86 is a unique enzyme, very acid-stable with a large potential for corn sweetener industries. 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 1164 base pairs (70.7 mol% of G+C content) that encoded a polypeptide composed of 388 amino acid residues (Mr 42,993) was found. The E. coli transformant carrying the gene overproduced the recombinant GI (rGI) and the enzyme was successfully expressed as a tetramer under the transcriptional control of the tac-promoter. The purified recombinant enzyme was indistinguishable from that of the authentic enzyme e.g. molecular weight, immunological properties, N-terminal amino acid sequences, subunit structures, and temperature and pH profiles. The relationships between structure and properties of the enzymes are also discussed.
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