PURIFICATION AND CHARACTERIZATION OF DIACETYL REDUCTASE FROM CHICKEN LIVER AND STREPTOCOCCUS-LACTIS AND ENZYMATIC DETERMINATION OF DIACETYL AND DIKETONES

Two enzymes which correspond to the Enzyme Commission classification diacetyl reductase (acetoin: NAD oxidoreductase E.C 1.1.1.5) have been isolated and purified from chicken liver and Streptococcus lactis subspecies diacetylactis 18-16. Both enzymes reduced vicinal diketones and uncharged alpha-dicarbonyl compounds using NADH as cofactor; the liver enzyme also accepted NADPH as cofactor. The bacterial enzyme accepted acetoin as a substrate whereas the liver enzyme did not. The liver enzyme was used to quantify aqueous diacetyl or diketone solutions by enzymic reaction with reduced cofactor. The quantity of reduced cofactor remaining after reaction was inversely proportional to the original concentration of diacetyl or diketones. The relationship between absorbance and concentration was linear up to 50 mg dm-3 diacetyl, with a lower limit of sensitivity of 1 mg dm-3. Greater sensitivity (linearity up to 1.5 mg dm-3, lower limit of 0.05 mg dm-3) could be obtained by coupling the reaction to an enzymic cycling system.