Recombinant expression, biochemical characterization and stabilization through proteolysis of an L-glutamate oxidase from Streptomyces sp. X-119-6.
L-glutamate oxidase (LGOX) from Streptomyces sp. X-119-6 is a protein of 150 kDa that has hexamer structure alpha2beta2gamma2. The gene encoding LGOX was cloned and heterologously expressed in Escherichia coli. LGOX isolated from the E. coli transformant had the structure of a one chain polypeptide. Although the recombinant LGOX exhibited catalytic activity, it was inferior to the LGOX isolated from Streptomyces sp. X-119-6 in catalytic efficiency. The recombinant LGOX exhibited low thermostability compared to the LGOX isolated from Streptomyces sp. X-119-6 and was an aggregated form. Proteolysis of the recombinant LGOX with the metalloendopeptidase from Streptomyces griseus (Sgmp) improved its catalytic efficiency at various pH. Furthermore, the Sgmp-treated recombinant LGOX had a subunit structure of alpha2beta2gamma2 and nearly the same enzymological character as the LGOX isolated from Streptomyces sp. X-119-6. A higher molecular species observed for the recombinant LGOX was not detected for the Sgmp-treated recombinant LGOX. These results prove that proteolysis by Sgmp is involved in the stabilization of the recombinant LGOX.
Arima
Jiro
J
Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University, Okayama 700-8530.
Tamura
Takashi
T
Kusakabe
Hitoshi
H
Ashiuchi
Makoto
M
Yagi
Toshiharu
T
Tanaka
Hidehiko
H
Inagaki
Kenji
K
D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D004792:Enzyme Precursors / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D004795:Enzyme Stability / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics*
D001426:Bacterial Proteins; D004792:Enzyme Precursors; D011994:Recombinant Proteins; D000594:Amino Acid Oxidoreductases; C023061:L-glutamate oxidase; D010447:Peptide Hydrolases