(Diagnostic Reagent Grade) ASAHI KASEI ENZYMES T-250REACH compliant product

CHOLESTEROL ESTERASE [CENII]

from Pseudomonas sp.
(Sterol esterase)

Cholesterol ester + H₂O → Cholesterol + Fatty acid

Preparation and Specification

Appearance: : White to pale brownish amorphous powder, lyophilized

Specific activity: : More than 100 U/mg solid

Properties

Substrate specificity: : See Table 1

Molecular weight: : 30kDa (SDS–PAGE)

Isoelectric point: : pH 5.28 (estimated from amino acid sequence)

Optimum pH: : 6.5Figure 1

pH stability: : 6.0–11.0 (37°C, 60 min, in 0.1% BSA)Figure 2

Optimum temperature: : 35-40°C (Phosphate Buffer)Figure 3

Thermal stability: : Stable at 45°C and below (pH 8.0, 30min)Figure 4

Storage stability: : At least one year at –20°CFigure 6

Effect of coexisting ions: : See Table 2

Effect of detergents: : See Table 3

Activator: : Adekatol TN-100, Adekatol PC-8, Adelatol SO-120, Adekatol SO-135, Triton X-100

Applications for Diagnostic Test

This enzyme is useful for enzymatic determination of total cholesterol, HDL-C, and LDL-C coupled with cholesterol oxidase (T–101).

CENII
Cholesterol ester + H₂O	→	Cholesterol + FFA
CONII-FD
Cholesterol + O₂	→	Cholestenone + H₂O₂
POD
2 H₂O₂ + 4-AA + Phenol	→	Quinoneimine dye + 4 H₂O

FFA : Free fatty acid

Table 1. Substrate specificity

Substrate	Relative activity (%)
	CENII	CEN (T-18)
Cholesterol Acetate C 2:0	22.6	21.1
Cholesterol Propionate C 3:0	27.7	28.1
Cholesterol Butyrate C 4:0	78.3	78.9
Cholesterol Palmitate C 16:0	37.9	42.2
Cholesterol Stearate C 18:0	13.2	13.0
Cholesterol Oleate C 18:1	100.0	100.0
Cholesterol Linolate C 18:2	80.9	78.1

Table 2. Effect of detergents on CENII activity

Additives	Concentration	Relative activity (%)
None	-	100
NiCl	1mM	67
MnCl₂	1mM	101
(NH₄)₂SO₄	1mM	107
MgCl₂	1mM	106
ZnCl	1mM	87
ZnSO₄	1mM	81
Ba(CH₃COO)₂	1mM	106
CaCl₂	1mM	114
MoSO₄	1mM	69
CuSO₄	0.5mM	3
CuCl₂	0.5mM	0
FeCl₃	1mM	102
CoCl₂	1mM	86
Li₂CO₃	1mM	100
CH₃COOT	1mM	104
EDTA	1mM	101
KCl	0.1M	97
NaCl	0.1M	98
NaN₃	0.05%	102
NaF	20mM	98

Table 3. Effect of detergents on CENII activity

Detergent (0.3%)	Relative activity (%)
None	100
TritonX-100	147
Adekatol TN-100	146
Adekatol SO-120	142
Newcol 707	146
Newcol 710	139
Emulgen 120	144
Emulgen 705	159
Sodium cholate	103
Deoxycholic acid	128
Sodium dodecyl sulfate	1.1
Sucrose fatty acid ester	144

Table 4. Effect of detergents on CENⅡ activity when switching from Triton X-100 to other detergents

Detergent (0.3%)	Relative activity (%)
TritonX-100	100
Adekatol SO-120	61
Adekatol SO-135	69
Adekatol TN-100	82
Adekatol PC-8	81
Tween 20	1
Tween 40	1
Tween 80	1
Sodium Cholate	0
Brij 35	2
CHAPSO	0
Benzyltriethylammonium Chloride	1
Sodium dodecyl sulfate	0
Emulgen LS-110	30
Newcol 710	13
None	0

Fig. 1 pH Optimum pH

〇: Citrate buffer
●: Phosphate buffer
□: Tris-HCl buffer
■: Glycine-NaOH buffer

Fig. 2 pH Stability

37°C, 60min. in 0.1% BSA
〇: Citrate buffer
●: Phosphate buffer
□: Tris-HCl buffer
■: Glycine-NaOH buffer

Fig. 3 Optimum temperature

pH6.8
40mM Phosphate buffer

Fig. 4 Thermal stability

Tris-HCl buffer pH8.0 30min
CEN II
〇: None
●: + 0.1% Newcol 710
□: + 1mM CaCl₂
■: + 0.1% Newcol 710 + 1mM CaCl₂
CEN(T-18)
* ： None

Fig. 5 Thermal stability

Tris-HCl buffer pH8.0
+0.1% Newcol 710
+1mM CaCl₂ 30min
■: CEN II
△: CE from Pseudomonas sp.

Fig. 6 Storage (lyophilized powder)

－20°C

Assay

Principle

The assay is based on the increase in absorbance at 493 nm as the formation of quinoneimine dye proceeds in the following reactions:

CENII
Cholesterol ester+H₂O	→	Cholesterol+Fatty acid
CO
Cholesterol+O₂	→	△⁴–Cholesten–3–one+H₂O₂
POD
2H₂O₂ +4 –AA +Phenol	→	Quinoneimine dye + 4 H₂O

CO: Cholesterol oxidase

Unit definition

One unit is defined as the amount of enzyme which liberates 1 μmole of cholesterol per minute at 37°C under the conditions specified in the assay procedure.

Reagents

Reaction mixture

0.2M KH₂PO₄–NaOH buffer pH 6.8	0.60 ml
0.35%(W/V)4–AA solution	0.30 ml
0.2%(W/V)Phenol solution	0.30 ml
100 U/ml POD solution₁₎	0.30 ml
3%(W/V)Triton X–100 solution	0.30 ml
0.2 U/ml CONⅡ solution²⁾	0.60 ml
Substrate solution³⁾	0.30 ml
Distilled water	0.30 ml

1):

100 U/ml POD solution
Dissolve 1000 U(PPU)of POD with 10 ml of distilled water.

2):

0.2 U/ml CONII solution
Dissolve 2 U of CONII with CONII dilution buffer ^※)

※):	CONII dilution buffer 0.1 M KH₂PO₄–Na₂HPO₄ buffer pH 7.0 containing 0.05%(W/V)Triton X–100.

3):

Substrate solution
Calf serum

Enzyme dilution buffer
10 mM KH₂PO₄–NaOH buffer pH 7.5 containing 0.1%(W/V)bovine serum albumin(BSA).
Reagents
Triton X–100 : The Dow Chemical Company
CONII : Asahi Kasei Pharma Corporation #T– 84
Calf serum: GIBCO Co.(USA)
BSA: Millipore Fraction V pH 5.2 #81–053
4– AA: NACALAI TESQUE, INC. Special grade #01907–52
POD: Sigma Chemical Co. Type II #P–8250

Enzyme solution

Accurately weigh about 20 mg of the sample and add enzyme dilution buffer to make a total of 20 ml. Dilute it with enzyme dilution buffer to adjust the concentration to within 0.3–0.5 U/ml.

Procedure

Pipette accurately 3.0 ml of reaction mixture into a small test tube and preincubate it at 37°C.
After 10 min, add 50 μl of enzyme solution and mix to start the reaction at 37°C.

※ In the case of a test blank, add 50 μl of enzyme dilution buffer in place of enzyme solution.
After starting the reaction, measure the rate of increase per minute in absorbance at 493 nm. The rate must be measured within the linear portion of the absorbance curve.
Absorbance sample : As/min

blank : Ab/min

△A/min =(As/min−Ab/min)≦ 0.050 Abs/min

Calculation

Activity(U/mg of powder)= {(△A/min)/(12.0×1/2)} × 3.05/0.05 × 1/x

12.0 :	millimolar extinction coefficient of quinoneimine dye at 493 nm(cm²/μmole)
1/2 :	a multiplier derived from the fact that 2 mole of H₂O₂ produce 1 mole of quinoneimine dye
3.05 :	final volume(ml)
0.05 :	volume of enzyme solution(ml)
X :	concentration of the sample in enzyme solution(mg/ml)

Storage

Storage at –20°C in the presence of a desiccant is recommended. Enzyme activity will be retained for at least one year under this condition(Figure 5)

References

Bradford, M. B.,(1976)Anal. Biochem., 72, 248–254.
Allain, C. C., Poon, L. S., Chan, C. S. G., Richmond, W.and Fu, P.C.(1974)Clin. Chem., 20, 470–475.
Kameno, Y., Nakano, N. and Baba, S.(1976)Japanese
Journal of Clinical Pathology, 24, 650.

CENII 活性測定法（Japanese）

試薬液

反応試薬混合液

0.2M KH2PO4–NaOH 緩衝液	0.60 ml
0.35%(W/V)4–AA 溶液	0.30 ml
0.2%(W/V)フェノール溶液	0.30 ml
100U/ml POD 溶液¹⁾	0.30 ml
3%(W/V)トリトン X–100 溶液	0.30 ml
0.2U/ml CONII溶液²⁾	0.60 ml
基質溶液³⁾	0.30 ml
精製水	0.30 ml

1):

100U/ml POD 溶液
POD 1,000 単位(PPU)を精製水 10ml で溶解する。

2):

0.2U/ml CONII溶液
CONII 2 単位(U)を CONII溶解用液^※)10ml で溶解する。

※):	CONII溶解用液 0.05%(W/V)トリトン X–100 を含む 0.1M KH₂PO₄–Na₂HPO₄ 緩衝液 pH7.0

3):

基質溶液
仔牛血清液

酵素溶解希釈用液
0.1%(W/V)BSA を含む 10mM KH₂PO₄–NaOH
緩衝液 pH7.5
試薬
トリトン X–100：Dow Chemical 製
CONII(コレステロール酸化酵素)：旭化成ファーマ製 #T–84
仔牛血清液(Calf serum)：GIBCO(USA)製
BSA: Millipore 製 Fraction V pH5.2 #81–053
4–AA：ナカライテスク製特級 #01907-52
POD：シグマ製 Type II #P–8250

酵素試料液

検品約 20mg を精密に量り、酵素溶解希釈用液に溶解して全容 20ml とする。
その液を酵素溶解希釈用液で 0.3～0.5U/ml 濃度となるように適宜希釈する。

測定操作法

小試験管に反応試薬混合液を 3.0ml 正確に分注して37°Cで予備加温する。
10 分経過後、酵素試料液 50 μl を正確に加えて混和し、37°Cで反応を開始する。

※ 盲検は酵素試料液の代わりに酵素溶解希釈用液50 μl を加える。
反応開始後、493nm における吸光度を測定して直線的に反応している 1 分間当たりの吸光度変化を求める。
求められた吸光度変化を試料液は As/min、盲検液は Ab/min とする。
ΔA/min =(As/min−Ab/min)≦ 0.050 Abs/min

計算

活性(U/mg)= {(△A/min)/(12.0×1/2)} × 3.05/0.05 × 1/x

12.0 :	キノンイミン色素の 493nm におけるミリモル分子吸光係数(cm² /μmole)
1/2 :	H₂O₂2 モルからキノンイミン色素 1 モルが生成することによる係数
3.05 :	反応総液量(ml)
0.05 :	反応に供した酵素試料液量(ml)
X :	酵素試料液中の検品濃度(mg/ml)

Absorbance sample :	As/min
blank :	Ab/min

CHOLESTEROL ESTERASE [CENⅡ] (T-250)