LIPASE [LP] (T-01)

(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
  1. 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
  1. 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
  1. 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.
  2. Reaction stopper
    Mixture of ethanol and acetone (1:1)
  3. Indicator
    1% (W/V) Phenolphthalein-ethanol solution
  1. Titration solution
    50 mM NaOH solution
  2. Enzyme dilution buffer
    0.1 M KH2PO4–NaOH buffer, pH 8.0 containing 0.1% (W/V) BSA and 0.1% (W/V) NaN3
  3. 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
  1. 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
  1. 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℃.
  2. 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.
  1. After 20 min, stop the reaction with 16 ml of reaction stopper.
  2. 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
  1. Yamaguchi, T., Muroya, N., Isobe, M. and Sugiura, M. (1973) Agric. Biol. Chem., 37, 999–1005.
  2. Sugiura, M., Isobe, M., Muroya, N. and Yamaguchi, T. (1974) Agric. Biol. Chem., 38, 947–952.
  3. Sugiura, M. and Isobe, M. (1974) Biochim. Biophys. Acta, 341, 195–200.
  4. Sugiura, M. and Isobe, M. (1975) Chem. Pharm. Bull., 23, 1226–1230.
  5. Horiuchi, Y., Koga, H. and Gocho, S. (1976) J. Biochem.
    (Tokyo), 80, 367–370.
  6. 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
  1. 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。
  2. 反応停止液
    エタノール-アセトン (1:1) 混液
  3. 指示液
    1% (W/V) フェノールフタレン-エタノール溶液
  4. 滴定液
    50mM NaOH 液
  5. 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
  6. 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
  1. 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
  1. 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。
  2. 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。
  3. 20 分経過後、反応停止液16ml を加えて反応を停止する。
  4. 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
  1. 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)