(Diagnostic Reagent Grade) ASAHI KASEI ENZYMES T-63REACH適合品

LIPASE [LPBP]

from Microorganism
(Triacylglycerol acylhydrolase, EC 3.1.1.3)
(Triacylglycerol lipase)

Triglyceride + 3H₂O → Glycerol + 3 Fatty Acid

★	Advantages
	①	Low adsorption onto cuvette
	②	Stability in solution

Preparation and Specification

Appearance: : White to light brownish amorphous powder, lyophilized

Specific activity: : More than 800 U/mg solid

Properties

Substrate specificity: : See Table 1

Molecular weight: : 55 kDa (SDS–PAGE)

Isoelectric point: : pH 4.9±0.2

Optimum pH: : 4.2Figure 1

pH stability: : pH 3.5–7.0 (45℃, 60 min) Figure 2

Optimum temperature: : 37℃ (Phosphate buffer) Figure3

Thermal stability: : Stable at 37℃ and below (pH 7.5, 30 min) Figure4

Effect of metal ions: : See Table 2

Low adsorption: : See Figure 5

Liquid stability: : See Figure 6

High reactivity after long storage: : See Figure 7

Effect of various chemicals: : See Table 3

Effect of detergents: : See Table 4

Applications for Diagnostic Test

This enzyme is useful for enzymatic determination of triglyceride.

	LPBP
TG + 3H₂O	→	Glycerol + 3 Fatty acid
	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
DHAP : Dihydroxyacetone phosphate

Table 1. Substrate specificity

Substrate	Relative activity (%)
Triolein	100
Trimyristin	14.8
Trilaurin	34.9
Tricaprylin	92.2
Tricaprin	87.1
Tricaproin	13.5
Tributyrin	38.0
Triacetin	0

Table 3. Effect of detergents on LPBP activity

Substrate	Relative activity (%)
None	100
Adekanol NP695	91.9
Adekanol NP720	96.2
Adekanol SO120	86.7
Adekanol B795	88.1
Triton X305	91.9
Emulgen B66	87.9

Table 2. Efect of various chemicals on LPBP activity

Additives	Concentration	Relative activity (%)
None	-	100
NiCl	1mM	94
MnCl₂	1mM	90
(NH₄)₂ SO₄	1mM	92
MgCl₂	1mM	98
ZnCl	1mM	100
ZnSO₄	1mM	100
Ba(CH₃COO)₂	1mM	101
CaCl₂	1mM	97
MoSO₄	1mM	100
CuSO₄	0.5mM	103
CuCl₂	0.5mM	99
FeCl₃	1mM	97
CoCl₂	1mM	106
Li₂CO₃	1mM	105
EDTA	1mM	102
KCl	100mM	103
NaCl	100mM	100
NaN₃	0.05%	98
NaF	20mM	4

Fig.1 pH Optimum

〇: Macllvaine buffer

Fig.2 pH Stability

45℃, 60 min.
〇: Citrate buffer
●: 3,3-Dimethylglutarate-NaOH buffer
□: Phosphate buffer

Fig.3 Optimum temperature

pH 6.8
40 mM Phosphate buffer

Fig.4 Thermal Stability

pH 7.5, 30 min.
40 mM Phosphate buffer

Fig.5 Influence on the measurement of NEFA after TG assay demonstrating low LPBP adsorption

Autoanalyzer : HITACHI 7150

Fig.6 Stability of reconstituted LPBP

■: detergent not added
●: Adekanol B-795
▲: Adekatol SO-120

Storage conditions :	20mM Good’s buffer, pH 5.5
	0.06% 4-amino antipyrine
	0.05% NaN3, 0.05% detergent

Fig.7 Reactivity of LPBP after long-term storage in liquid form

■: initial
●: at 25℃,4 weeks later

Storage conditions: 250U/ml LPBP, 0.25 U/ml MGLP
0.05% Tween 20 or Adekatol SO-120

Assay

Principle

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

	LPBP
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 μmole of fatty acid per minute at 37℃ under the conditions specified in the assay procedure.

Reagents

McIlvaine buffer pH 4.2
Mix 0.2 M Na₂HPO₄ and 0.1 M citrate solution and adjust pH to 4.2 at 25℃.
Substrate suspension (Olive oil and Adekatol SO–120)
50g of Olive oil (Japanese Pharmacopoeia grade and 50g 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 6.0 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 10 ml. Dilute it with enzyme dilution buffer to adjust the concentration as required.

Procedure

Pipette accurately 5 ml of substrate suspension and 2 ml of McIlvaine buffer into test tube (24 mm i.d. × 200 mmH) and mix to preincubate at 37℃.
After 10 min, add 0.50 ml of enzyme solution and mix to start the reaction.

※ In the case of a test blank, add 0.50 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 under nitrogen gas bubbling.

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

Titration volume :Vs

blank : Vc

△V = (Vs－Vc) ≦ 1.5 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.

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) Biochem. 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., 80, 367–370.
Saiki, T., Takagi, Y. Suzuki, T., Narasaki, T., Tamura, G. and Arima, K. (1969) Agric. Biol. Chem., 33, 414.

LPBP 活性測定法 (Japanese)

試薬液

McIlvaine 緩衝液 pH4.2
0.2M Na₂HPO₄ 溶液と0.1M クエン酸溶液を混合してpH4.2 (25℃) に調整する。
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) NaN3 を含む
0.1M KH₂PO₄–NaOH 緩衝液 pH6.0
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、酵素溶解希釈用液で溶解して全容10ml とする。
その液を酵素溶解希釈用液で適宜希釈する。

測定操作法

試験管 (24mm i.d × 200mm H) に基質懸濁液5.0mlとMcIlvaine 緩衝液2.0ml 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0.50ml 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0.50ml を加える。
20 分経過後、反応停止液16.0ml 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 (淡赤色) 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) ≦ 1.5 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 :Vs
	blank : Vc

LIPASE [LPBP] (T-63)