2001 Volume 18 Pages 91-95
The gene encoding an L-glutamate oxidase from Streptomyces sp. X -119 -6 was cloned in Escherichia coli. The nucleotide sequence of the L-glutamate oxidase gene revealed an open reading frame of 2, 103 bp. encoding a 701-amino acid protein. The deduced primary structure shares 15.8% sequence identity with the L-amino acid oxidase, including the consensus amino acid sequence, -GXGXXG-. which is contained in β α β-fold binding the ADP at N-terminal region.
We constructed an expression plasmid (pKK-LGOX) and E. coli harboring pKK-LGOX was expressed the L-glutamate oxidase precursor. The molecular mass of precursor was 76 kDa and it has homodimer structure, but an enzyme from Str. sp. X-119 -6 has α2β2γ2 subunit structure, and it has the different enzymological character with an L-glutamate oxidase from Str. sp. X-119-6. To improve of affinity to substrate of an L-glutamate oxidase precursor, enzyme was digested with trypsin and isolated by gel-filtration. Two fragment (58 kDa and 18 kDa) was detected by SDS-PAGE, and its Km value is 0.2 mM, but precursor’s Km is 5 mM. It is suggested that digesting the enzyme by trypsin is separated the β subunit from precursor, and originated affinity to substrate.
