LIPASE [LPBP] (T-63)

TriglyceridesT-63 PDF download
(Diagnostic Reagent Grade) Nagase Diagnostics 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
 ①Low adsorption onto cuvette
 ②Stability 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

SubstrateRelative activity
(%)
Triolein100
Trimyristin14.8
Trilaurin34.9
Tricaprylin92.2
Tricaprin87.1
Tricaproin13.5
Tributyrin38.0
Triacetin0

 

Table 3. Effect of detergents on LPBP activity

SubstrateRelative activity
(%)
None100
Adekanol NP69591.9
Adekanol NP72096.2
Adekanol SO12086.7
Adekanol B79588.1
Triton X30591.9
Emulgen B6687.9

 

Table 2. Efect of various chemicals on LPBP activity

AdditivesConcentrationRelative activity (%)
None
-
100
NiCl1mM94
MnCl21mM90
(NH4)2SO41mM92
MgCl21mM98
ZnCl1mM100
ZnSO41mM100
Ba(CH3COO)21mM101
CaCl21mM97
MoSO41mM100
CuSO40.5mM103
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
NaF20mM4

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
  1. 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

  1. 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
  1. McIlvaine buffer pH 4.2
    Mix 0.2 M Na2HPO4 and 0.1 M citrate solution and adjust pH to 4.2 at 25℃.
  2. 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.
  3. Reaction stopper
    Mixture of ethanol and acetone (1:1)
  4. Indicator
    1% (W/V) Phenolphthalein–ethanol solution
  5. Titration solution
    50 mM NaOH solution
  6. Enzyme dilution buffer
    0.1 M KH2PO4–NaOH buffer, pH 6.0 containing
    0.1% (W/V) BSA and 0.1% (W/V) NaN3
  7. 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 10 ml. Dilute it with enzyme dilution buffer to adjust the concentration as required.
Procedure
  1. 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℃.
  2. 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.
  3. After 20 min, stop the reaction with 16 ml of reaction stopper.
  4. 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
    Titrationvolume :Vs
     blank : Vc
    △V = (Vs－Vc) ≦ 1.5 ml
Calculation
  1. 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
  1. 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
  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) Biochem. Biophys.
    Acta, 341, 195–200.
  1. Sugiura, M. and Isobe, M. (1975) Chem. Pharm. Bull., 23, 1226–1230.
  2. Horiuchi, Y., Koga, H. and Gocho, S. (1976) J. Biochem., 80, 367–370.
  3. 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
  1. 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。
  2. 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。
  3. 反応停止液
    エタノール－アセトン (1:1) 混液
  4. 指示液
    1% (W/V) フェノールフタレン－エタノール溶液
  5. 滴定液
    50mM NaOH 液
  6. 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
  7. 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
  1. 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。
  2. 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。
  3. 20 分経過後、反応停止液16.0ml 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 (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)
T-63 PDF download