Molecular cloning of acid-stable glucose isomerase gene from Streptomyces olivaceoviridis E-86 by a simple two-step PCR method, and its expression inEscherichia coli

Glucose isomerase (GI) from Streptomyces olivaceoviridis E-86 is a unique e nzyme, very acid-stable with a large potential for corn sweetener industrie s. 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 con sisting of 1164 base pairs (70.7 mol% of G + C content) that encoded a poly peptide composed of 388 amino acid residues (M-r 42,993) was found. The E, coli transformant carrying the gene overproduced the recombinant GI (rGI) a nd the enzyme was successfully expressed as a tetramer under the transcript ional control of the tac-promoter. The purified recombinant enzyme was indi stinguishable from that of the authentic enzyme e.g, molecular weight, immu nological properties, N-terminal amino acid sequences, subunit structures, and temperature and pH profiles. The relationships between structure and pr operties of the enzymes are also discussed.