(Diagnostic Reagent Grade) ASAHI KASEI ENZYMES T-01REACH適合品

LIPASE [LP]

from Chromobacterium viscosum
(Triacylglycerol acylhydrolase, EC 3.1.1.3)
(Triacylglycerol lipase)

Triglyceride + 3 H₂O → Glycerol + 3 Fatty Acid

Preparation and Specification

Appearance: : White to off-white amorphous powder, lyophilized

Specific activity: : More than 2,500 U/mg solid

Contaminants: :

Cholesterol oxidase: Less than 0.01 % (U/U)

Catalase: Less than 0.01 % (U/U)

Properties

Substrate specificity: : See Table 1

Molecular weight: : 120 kDa (pH 3.7) (gel filtration)
30 kDa (pH 7.3) (gel filtration)

Isoelectric point: : pH 3.7 and pH 7.3

Optimum pH: : 3.0–10.0Figure 1

pH stability: : 4.0–10.0 (50℃, 60 min) Figure 2

Thermal stability: : Stable at 70℃ and below (pH 7.0, 10 hr) Figure3

Storage stability: : At least one year at –20℃Figure4

Activators: : Detergents

Applications for Diagnostic Test

This enzyme is useful for enzymatic determination of triglyceride when coupled with glycerophosphate oxidase (T–60) and glycerol kinase (T–64)

	LP
TG + 3 H₂O	→	Glycerol + 3 FFA
	GKZ
Glycerol + ATP	→	G-3-P + ADP
	GPOSP
G-3-P + O₂	→	DHAP + H₂O₂
	POD
2H₂O₂ + 4-AA + Phenol	→	Quinoneimine dye + 4 H₂O

TG: Triglyceride
FFA: Free fatty acid
DHAP: Dihydroxyacetone phosphate

Table 1. Substrate specificity

Substrate	Relative activity (%)
Triolein	100
Tripalmitin	22
Trimyristin	53
Trilaurin	103
Tricaprin	166
Tricaprylin	312
Tricaproin	156
Tributyrin	94
Tripropionin	22
Triacetin	38

Table 2. Effect of various chemicals on LP activity

Additives	Concentration	Relative activity (%)
None	-	100
NiCl	1mM	97
MnCl₂	1mM	98
(NH₄)₂ SO₄	1mM	100
MgCl₂	1mM	99
ZnCl	1mM	94
ZnSO₄	1mM	94
Ba(CH₃COO)₂	1mM	98
CaCl₂	1mM	100
MoSO₄	1mM	106
CuSO₄	0.5mM	26
CuCl₂	0.5mM	26
FeCl₃	1mM	103
CoCl₂	1mM	96
Li₂CO₃	1mM	93
EDTA	1mM	105
KCl	100mM	100
NaCl	100mM	98
NaN₃	0.05%	97
NaF	20mM	88

Fig.1 pH Optimum

▲: Mcllvaine buffer
□: Phosphate buffer
△: Borate buffer

Fig.2 pH Stability

●: 37℃, 24 hr
〇: 50℃, 1 hr
pH 3, 4, 5 Mcllvaine buffer
pH 6, 7, 8 Phosphate buffer
pH 9, 10, 11 Borate buffer

Fig.3 Thermal Stability

pH 7.0, 10 hr
〇: Lyophilized powder
●: Aqueous solution

Fig.4 Storage (Iyophilized powder)

〇: －20℃
□: 5℃
△: 30℃

Assay

Principle

The assay is based on the titration of fatty acids liberated in the following reactions:

	LP
Triglyceride+3 H₂O	→	Glycerol+3 Fatty acid
	( Titration)
Fatty acid+NaOH	→	Na-Fatty acid+H₂O

Unit definition

One unit is defined as the amount of enzyme which liberates 1 μ mole of fatty acid per minute at 37℃ under the conditions specified in the assay procedure.

Reagents

Substrate suspension (Olive oil and Adekatol SO–120) 50 g of Olive oil (Japanese Pharmacopoeia grade) and 50 g of Adekatol SO–120 are suspended with 150 ml of distilled water.
Reaction stopper
Mixture of ethanol and acetone (1:1)
Indicator
1% (W/V) Phenolphthalein-ethanol solution

Titration solution
50 mM NaOH solution
Enzyme dilution buffer
0.1 M KH₂PO₄–NaOH buffer, pH 8.0 containing 0.1% (W/V) BSA and 0.1% (W/V) NaN₃
Reagents
Olive oil: (Japanese Pharmacopoeia grade)
Ethanol: (Japanese Pharmacopoeia grade)
Adekatol SO–120 : ADEKA CORPORATION
BSA: Millipore Fraction V pH5.2 #81–053

Enzyme solution

Accurately weigh about 10 mg of the sample and add enzyme dilution buffer to make a total of 50 ml.
Dilute it with enzyme dilution buffer to adjust the concentration to within 2–4 U/ml.

Procedure

Pipette accurately 5 ml of substrate suspension and 2 ml of distilled water into a test tube (24 mm i.d. × 200 mmH) and mix to start the preincubation at 37℃.
After 10 min, add 0.5 ml of enzyme solution and mix to start the reaction.

※ In the case of a test blank, add 0.5 ml of enzyme dilution buffer in place of enzyme solution.

After 20 min, stop the reaction with 16 ml of reaction stopper.
Add 3 drops of indicator and titrate the whole mixture with under nitrogen gas bubbling.

※ End point of titration: Appearance of the same color as that of the blank

Titration volume sample : Vs

blank : Vc

△V = (Vs – Vc) ≦ 2.5 ml

Vc ≦ 0.6 ml

Calculation

Activity (U/mg of powder) = {(△V×F)/20)} × 50 × 1/0.5 × 1/x

20 :	reaction time (min)
F :	factor of titration solution (50 mM NaOH)
50 :	concentration (mM) of titration solution (50 mM NaOH)
0.5 :	the volume of enzyme solution (ml)
X :	concentration of the sample in enzyme solution (mg/ml)

Storage

Storage at –20℃ in the presence of a desiccant is recommended. Enzyme activity will be retained for at least one year under this condition (Figure 4) .

References

Yamaguchi, T., Muroya, N., Isobe, M. and Sugiura, M. (1973) Agric. Biol. Chem., 37, 999–1005.
Sugiura, M., Isobe, M., Muroya, N. and Yamaguchi, T. (1974) Agric. Biol. Chem., 38, 947–952.
Sugiura, M. and Isobe, M. (1974) Biochim. Biophys. Acta, 341, 195–200.
Sugiura, M. and Isobe, M. (1975) Chem. Pharm. Bull., 23, 1226–1230.
Horiuchi, Y., Koga, H. and Gocho, S. (1976) J. Biochem.
(Tokyo), 80, 367–370.
Saiki, T., Takagi, Y. Suzuki, T., Narasaki, T., Tamura, G. and Arima, K. (1969) Agric. Biol. Chem., 33, 414.

LP 活性測定法 (Japanese)

試薬液

基質懸濁液 (オリーブ油とアデカトールSO–120 の懸濁液)
「局方」オリーブ油50.0g とアデカトールSO–120
50.0g を精製水150ml に懸濁する。
反応停止液
エタノール－アセトン (1:1) 混液
指示液
1% (W/V) フェノールフタレン－エタノール溶液
滴定液
50mM NaOH 液
酵素溶解希釈用液
0.1% (W/V) BSA と0.1% (W/V) NaN 3 を含む0.1M
KH₂PO₄ –NaOH 緩衝液pH8.0
試薬
オリーブ油：「局方」
エタノール：「局方」
アデカトールSO–120：ADEKA 製
BSA: Millipore 製　Fraction V pH5.2 #81–053

酵素試料液

検品約10mg を精密に量り、酵素溶解希釈用液に溶解して全容50ml とする。
その液を酵素溶解希釈用液で2～4U/ml 濃度となるように適宜希釈する。

測定操作法

試験管 (24mm i.d. × 200mm H) に基質懸濁液5ml と精製水2ml を正確に分注して攪拌混和後、37℃で予備加温する。
10 分経過後、酵素試料液0.5ml を加えて混和し、37℃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0.5ml を加える。
20 分経過後、反応停止液16ml を加えて反応を停止する。
指示液3 滴を加えてN₂ ガスで攪拌しながら滴定液で滴定する。

※ 滴定の終点は盲検時と同色を呈した時点とする。
求められた滴定量を試料液はVs、盲検液はVc とする。

ΔV = (Vs – Vc) ≦ 2.5 ml

Vc ≦ 0.6 ml

計算

活性 (U/mg) = {(ΔV×F)/20) × 50 × 1/0.5 × 1/x

20 :	反応時間 (min)
F :	滴定液 (50mM NaOH) のFactor
50 :	滴定液 (50mM NaOH) の濃度 (mM)
0.5 :	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)

Titration volume sample :	Vs
blank :	Vc

△V =	(Vs – Vc) ≦ 2.5 ml
	Vc ≦ 0.6 ml

ΔV =	(Vs – Vc) ≦ 2.5 ml
	Vc ≦ 0.6 ml

LIPASE [LP] (T-01)