Role of pyruvate in enhancing pyruvate decarboxylase stability towards benzaldehyde.
Biotransformation of benzaldehyde and pyruvate into (R)-phenylacetylcarbinol (PAC) catalysed by Candida utilis pyruvate decarboxylase (PDC) at low buffer concentration (20 mM MOPS) was enhanced by maintenance of neutral pH through acetic acid addition. PDC was very stable in this buffer (half-life 138 h at 6 degrees C), however a benzaldehyde emulsion (400 mM) caused rapid deactivation. The inclusion of 2M glycerol did not protect PDC from inactivation by benzaldehyde but initial rates were increased by 50% and the final PAC level was enhanced from 40 to 51 g l(-1). Low levels of by-products acetaldehyde (0.1-0.15 g l(-1)) and acetoin (1.1-1.3 g l(-1)) were formed in both the presence and absence of 2 M glycerol. Interestingly PDC was more stable towards benzaldehyde when pyruvate was present: no activity was lost during the first hour of biotransformation (2 M glycerol, benzaldehyde concentration decreased from 400 to 345 mM, pyruvate from 480 to 420 mM) but PDC was completely inactivated in less than 30 min when exposed to the same concentrations of benzaldehyde in the absence of pyruvate. Thus the enzyme in catalytic action was more stable than the resting enzyme.
Rosche
Bettina
B
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia. b.rosche@unsw.edu.au
Breuer
Michael
M
Hauer
Bernhard
B
Rogers
Peter L
PL
D000096:Acetone / Q000031:analogs & derivatives* / Q000138:chemical synthesis*; D001547:Benzaldehydes / Q000737:chemistry*; D002175:Candida / Q000201:enzymology*; D002384:Catalysis; D004789:Enzyme Activation; D004795:Enzyme Stability; D005990:Glycerol / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011767:Pyruvate Decarboxylase / Q000737:chemistry*; D019289:Pyruvic Acid / Q000737:chemistry*
D001547:Benzaldehydes; D000096:Acetone; D019289:Pyruvic Acid; C035293:1-hydroxy-1-phenyl-2-propanone; D011767:Pyruvate Decarboxylase; D005990:Glycerol; C032175:benzaldehyde