Purification and characterisation of acetolactate decarboxylase from Leuconostoc lactis NCW1.
A two-step strategy involving DEAE-cellulose and POROS PI anion exchange chromatography has been developed for rapid purification of acetolactate decarboxylase (ALD) from Leuconostoc lactis NCW1. This results in 5333-fold purification with a yield of 30%. Purified ALD is a dimer of 49-kDa subunits, has a pH optimum of 6.0, a pI of 4.2 and its activity is independent of metals or branched chain amino acids. At the optimum pH, the K(m) for 2-acetolactate (ALA) was found to be 1.3 mM and the turnover number was 4000 min(-1). N-terminal sequence comparison with other ALDs showed little sequence conservation in this region. Purified ALD does not catalyse direct production of diacetyl from ALA, unlike the crude extract.
O'Sullivan
S M
SM
Department of Biochemistry, University College Cork, Ireland.
Condon
S
S
Cogan
T M
TM
Sheehan
D
D
D000837:Anion Exchange Resins; D002262:Carboxy-Lyases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D003931:Diacetyl / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007934:Leuconostoc / Q000201:enzymology* / Q000378:metabolism; D008970:Molecular Weight
D000837:Anion Exchange Resins; D002262:Carboxy-Lyases; C023746:acetolactate decarboxylase; D003931:Diacetyl