Characterization and partial purification of L-asparaginase from Corynebacterium glutamicum.
A high L-asparaginase (L-asparagine amidohydrolase: EC 3.5.1.1) activity was found under conditions of lysine overproduction in cultures of Corynebacterium glutamicum. L-Asparaginase was purified 98-fold by protamine sulphate precipitation. DEAE-Sephacel anion exchange, ammonium sulphate precipitation and Sephacryl S-200 gel filtration. The asparaginase protein was subjected to PAGE under non-denaturing conditions, identified by an in situ reaction and eluted from the gel in an active form. The estimated Mr from gel filtration and SDS-PAGE was 80,000. The L-asparaginase activity was inhibited by the L-asparagine analogue 5-diazo-4-oxo-L-norvaline. Neither D-asparagine nor L-glutamine was a substrate for the enzyme. L-Asparaginase was produced constitutively: its role may be that of an overflow enzyme, converting excess asparagine into aspartic acid, the direct precursor of lysine and threonine.
Mesas
J M
JM
Facultad de Biología, Universidad de León, Spain.
Gil
J A
JA
Martín
J F
JF
D001215:Asparaginase / Q000302:isolation & purification*; D001224:Aspartic Acid / Q000096:biosynthesis* / Q000494:pharmacology; D003352:Corynebacterium / Q000187:drug effects / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D013379:Substrate Specificity
D001224:Aspartic Acid; D001215:Asparaginase; D008274:Magnesium; D011188:Potassium