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 |
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/x20 : | 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)