GLYCEROL DEHYDROGENASE from Cellulomonas sp.


Appearance White amorphous powder, lyophilized
Activity GradeⅢ 50U/mg-solid or more
(containing approx. 50% of stabilizers)
Contaminant NAD oxidase ≤1.0×10⁻³%
Stabilizer BSA
Stability Stable at -20°C for at least 6 months (Fig.1)
Molecular weight approx. 390,000
Isoelectric point 4.4±0.1
Michaelis constants 1.1×10⁻²M (Glycerol), 8.9×10⁻⁵M (NAD⁺)
Structure 10 subunits (42,000) per enzyme molecule
Inhibitors p-Chloromercuribenzoate, o-phenanthroline, monoiodoacetate, heavy metal ions (Co⁺⁺, Ni⁺⁺, Cu⁺⁺, Zn⁺⁺, Cd⁺⁺)
Optimum pH 10.0-10.5(Fig.4)
Optimum temperature 50°C(Fig.5)
pH Stability pH 7.5-10.5 (25°C, 20hr)(Fig.6)
Thermal stability below 55°C (pH 7.5, 15min)(Fig.7)
Substrate specificity This enzyme has the highest specificity for glycerol and 1,2-
propanediol, and also oxidizes glycerol-α-monochlorohydrin, ethylene glycol and 2,3-butanediol (Table 1). The oxidative reaction is stimulated by K⁺, NH⁺₄ and Rb⁺.
Effect of various chemicals  (Table 2)


This enzyme is useful for enzymatic determination of glycerol and of triglyceride when coupled with lipoprotein lipase (LPL-311, LPL-314) in clinical analysis.¹ ² ⁾



glycerol dehydrogenase

Glycerol+NAD⁺                                                   Dihydroxyacetone+NADH+H⁺

The appearance of NADH is measured at 340nm by spectrophotometry.

Unit definition:

One unit causes the formation of one micromole of NADH per minute under the conditions described below.


A. Carbonate-bicarbonate buffer, pH 11.0 0.2M (Prepare by mixing 0.2M K₂CO₃ and 0.2M NaHCO₃ to reach pH 11.0).
B. Glycerol solution 0.3M
C. Ammonium sulfate solution 1.0M
D. NAD⁺ solution 10mM [Weigh 143.5mg of NAD⁺(MW=717.45) and dissolve in 18.0ml
of HO and, after adjusting the pH to 7.0 with 0.5 N KOH, fill up to
20.0ml with HO] (Should be prepared fresh)
E. Enzyme diluent 20mM K-phosphate buffer pH 7.5.


Concentration in assay mixture
Carbonate buffer 0.10 M
Glycerol 0.10 M
NAD⁺ 1.0mM
Ammonium sulfate
33 mM

1. Prepare the following working solution, immediately before use.

30.0 ml Carbonate-bicarbonate buffer, pH 11.0 (A)
22.0 ml Substrate solution (B)
2.0 ml Ammonium sulfate solution (C)
6.0 ml NAD⁺ solution (D)

Be sure the pH in the range (pH 10.0-10.5). If not, adjust the pH to 10.5 with 1.0 N KOH or 1.0N HCl, and store on ice in a brownish bottle.

2. Pipette 2.9ml of the working solution into a cuvette (d=1.0cm) and equilibrate at 25°C for about 5 minutes.

3. Add 0.1ml of the enzyme solution* and mix by gentle inversion.

4. Record the increase in optical density at 340nm against water for 3 to 4 minutes in a spectrophotometer thermostated at 25°C and calculate theΔOD per minute from the initial linear portion of the curve (ΔOD test).
At the same time, measure the blank rate (ΔOD blank) by using the same method as the test except that the enzyme diluent is added instead of enzyme solution.

* Dissolve the enzyme preparation in ice-cold enzyme diluent (E), dilute to 0.10-0.25U/ml with the same buffer and store on ice.


Activity can be calculated by using the following formula :

ΔOD/min (ΔOD test−ΔOD blank ) ×Vt × df

Volume activity (U/ml) =                                                               =ΔOD/min×4.82×df


Weight activity (U/mg)=(U/ml)×1/C

: Total volume (3.0ml)
: Sample volume (0.1ml)
: Millimolar extinction coefficient of NADH (㎠/micromole)
: Light path length (cm)
: Dilution factor
: Enzyme concentration in dissolution (c mg/ml)


  1. T.Nishina; Rinsho Kensa(Japanese), 22, 1304 (1978).
  2. M.Sugiura, et al.; Clin, Chim.Acta, 81, 125 (1977).
  3. R.M.Burton; Methods in Enzymology, vol.1, p.397 (S.P.Colowick & N.O.Kaplan eds.), Academic Press, New York- London (1955).
Table 1. Substrate Specificity of Glycerol dehydrogenase
Substrate Relative activity(%) Substrate Relative activity(%)
Glycerol 100 2,3-Butanediol 52.6
Glycerol-α-monochlorohydrin 48.5
Ethylene glycol 7.8
1,2-Propanediol 132
1,3-Propanediol Methanol
1,3-Butanediol Ethanol

―, Not detected

Table 2. Effect of Various Chemicals on Glycerol dehydrogenase
Chemical Concn.(mM) Residual
Chemical Concn.(mM) Residual
None 100 PCMB 0.1 25
Metal salt 1.0   MIA 1.0 78
MgCl₂   100
NaN 1.0 110
  97 NAF 1.0 108
SrCl₂   98
EDTA 1.0 101
BaCl₂   95 KCN 1.0 94
MnCl₂ 94 o-Phenanthroline 1.0 13
CoCl₂   57      
NiCl₂   50
CuSO₄   2.4
ZnCl₂   1.5      
CdCl₂   11      

PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; EDTA, Ethylenediaminetetraacetate.

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