CHOLESTEROL ESTERASE from Schizophyllum commune

CEO-301, 302

Steryl-ester acylhydrolase (EC 3.1.1.13)

PREPARATION and SPECIFICATION
Appearance Light brown amorphous powder, lyophilized
Activity GradeIII 2.0 U/mg-solid or more
(containing approx. 20% of stabilizers)
Stabilizer Na-Cholate
PROPERTIES
Stability Stable at -20°C for at least One year (Fig.1,2)
Molecular weight approx. 130,000
Isoelectric point 4.1±0.1
Michaelis constants 3.9×10⁻⁵M (Linoleate), 9.2×10⁻⁵M (Palmitate),
6.3×10⁻⁵M (Decylate), 8.8×10⁻⁵M (Propionate)
Inhibitors Heavy metal ions (Hg⁺⁺, Ag⁺, Fe⁺⁺⁺)
Optimum pH 4.8-8.0 (Cholesterol linoleate), 5.0 (serum)(Fig.5)
Optimum temperature: 55-60℃(Fig.6)
pH Stability pH 2.5-7.5 (25℃, 20hr)(Fig.7)
Thermal stability  below 55℃ (pH 5.5, 10min)(Fig.8)
Substrate specificity (Table 1)

APPLICATIONS

This enzyme is useful for enzymatic determination of total cholesterol when coupled with cholesterol oxidase (COO-311, COO-321,COO-331) in clinical analysis.

ASSAY

Principle:

cholesterol esterase

Cholesterol ester                                 Cholesterol+Fatty acid

cholesterol oxidase (COD)

Cholesterol                                  ►Cholest-4-en-3-one+HO

 

The appearance of indamine is measured at 590nm by spectrophotometry.

Unit definition:

One unit causes the hydrolysis of one micromole of cholesterol ester per minute under the conditions described below.

Method:

Reagents
A.COD-POD solution 14.4g Na₂HPO₄・12H₂O, 31g boric acid, 15,000 U COD (GradeIII), 25,000 PU POD, 290mg sodium cholate/1,000ml of H₂O (adjusting the pH to 6.5) (stable for a month if stored at 0-5℃)
B. DMA-MBTH solution 400mg EDTA-Na₂, 0.7ml DMA, 150mg MBTH/1,000ml of 0.5M acetate buffer, pH 4.7 (stable for a week if stored at 0-5℃ in a brownish bottle)
C. Cholesterol linoleate solution 0.6mM [Dissolve 40mg of cholesterol linoleate in 8.0ml of absolute ethanol on a hot plate, mix 90ml of 1.0% (V/V) of Triton X-100 solution, keep at 70℃ in a hot water bath for 15 minutes and, after cooling down to room temperature in running water, fill up to 100ml with 1.0% (V/V) Triton X-100 (Should be prepared fresh)]
D. HCl solution 1.0 N
E. Enzyme diluent 10mM Phosphate buffer, pH 7.0.

Procedure

1. Prepare the following reaction mixture in a test tube and equilibrate at 37℃ for about 5 minutes.

1.0ml COD-POD solution(A)
1.0ml DMA-MBTH solution (B)
0.1ml enzyme solution*

2. Add 1.0ml of the substrate solution (C) and mix.

3. After exactly 10 minutes at 37°C, add 1.0ml of HCl solution (D) to stop the reaction and measure the optical
density at 590 nm against water (OD test).
At the same time, prepare the blank by first mixing the reaction mixture (1.0ml of substrate solution is used instead of the enzyme solution) with 1.0ml of HCl solution (D) after 10 min-incubation at 37°C, followed by the
addition of the enzyme of the enzyme solution (OD blank).

* Dissolve the enzyme preparation in ice-cold enzyme diluent and dilute to 0.02~0.05U/ml with the same buffer.

Calculation

Activity can be calculated by using the following formula :

ΔOD (ΔOD test−ΔOD blank ) × Vt × df

Volume activity (U/ml) =                                                               =ΔOD × 0.105×df

39.0× t ×Vs

 

Weight activity (U/mg) = (U/ml) × 1/C

Vt
: Total volume (4.1ml)
Vs
: Sample volume (0.10ml)
39.0
: Millimolar extinction coefficient of indamine dye under the assay conditions(㎠/micromole)
t
: Reaction time (10 minutes)
df
: Dilution factor
C
: Enzyme concentration in dissolution (c mg/ml)

REFERENCES

  1. W.Richmond; Clin.Chem., 19, 1350 (1973).
  2. H.M.Flegg; Ann.Clin.Biochem., 10, 79 (1973).
  3. C.C.Allain et al.; Clin.Chem., 20, 470 (1974).
  4. P.N.Tarbutton and C.R.Gunter; Clin.Chem., 20, 724 (1974).
  5. S.Nomoto; Rinsho Kensa, 20, 688 (1976).
  6. Y.Kameno et al.; Jap.J.Clin.Path., 24, 650 (1976).

Table 1. Substrate Specificity of Cholesterol esterase
[The reaction was carried out at 37℃ for 10min in 0.1M acetate buffer, pH 5.0, contg. 0.2mM each cholesterol ester and 0.33% Triton X-100.]
Cholesterol ester Relative activity (%) Cholesterol ester Relative activity (%)
Linoleate (18 :2) 100 Laurate (12 :0) 108
Acetate (2 :0) 9 Tridecanoate (13 :0) 59
Propionate (3 :0) 40 Myristate (14 :0) 57
Butyrate (4 :0) 38 Pentadecanote (15 :0) 76
Crotonate (4 :1) 0 Palmitate (16 :0) 111
Valerate (5 :0) 18 Heptadecanoate (17 :0) 99
Caproate (6 :0) 63 Stearate (18 :0) 39
Heptanoate (7 :0) 32 Oleate (18 :1) 59
Caprylate (8 :0) 94 Lelaidate (18 :3) 43
Nonanoate (9 :0) 120 Linolenate (18 :3) 91
Decylate (10 :0) 143 Arachidonate
(20 :4) 3
10-Undecenoate (11 :1) 141      

Number of carbon atoms and number of double bonds are given in parentheses.

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