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.
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.
Kaneko
T
T
Department of Bioengineering, Akita Research Institute of Food and Brewing, Japan. kaneko@sake.arif.pref.akita.jp
Saito
K
K
Kawamura
Y
Y
Takahashi
S
S
D000143:Acids / Q000737:chemistry; D019747:Aldose-Ketose Isomerases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology*
D000143:Acids; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase