LIPASE [LPBP]

(Diagnostic Reagent Grade)
ASAHI KASEI ENZYMES
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 Low liquid stability
Preparation and Specification
Appearance
: White to light brownish amorphous powder, lyophilized
Specific activity : More than 800 U/mg solid
Properties
Substrate specificity
Molecular weight
Isoelectric point
Optimum pH
pH stability
Optimum temperature
Thermal stability
Effect of metal ions
Low adsorption
Liquid stability
High reactivity after
long storage
Effect of various
chemicals
Effect of detergents
:
:
:
:
:
:
:
:
:
:
See Table 1
55 kDa(SDS‒PAGE)
pH 4.9±0.2
4.2
pH 3.5‒7.0(45℃, 60 min)
37℃(Phosphate buffer)
Stable at 37℃ and below(pH 7.5, 30 min)
See Table 2
See Figure 5
See Figure 6
: See Figure 7
: See Table 3
: See Table 4
Applications for Diagnostic Test
This enzyme is useful for enzymatic determination of triglyceride.
LPBP
TG + 3 H2O
Glycerol + 3 Fatty acid
GKZ
Glycerol + ATP
G−3−P + ADP
GPOSP
DHAP + H2O2
G−3−P + O2
POD
Quinoneimine dye + 4 H2O
2 H2O2 + 4−AA + Phenol
TG: Triglyceride DHAP: Dihydroxyacetonephosphate
98
Figure
Figure
Figure
Figure
1
2
3
4
T−63
Table 1. Substrate specificity
Table 3. Effect of various chemicals on LPBP activity
Substrate(13.3%) Relative activity(%)
Chemical(1mM)
Tricaproin
Tributyrin
Triacetin
60.4
EDTA
106
Table 4. Effect of detergents on LPBP activity
Detergent(0.1%)
100
91.9
96.2
86.7
88.1
91.9
87.9
Adekanol SO120
Adekanol B795
Triton X305
Emulgen B66
100
100
95.8
95.8
97.9
101
106
103
104
95.8
100
104
99.0
Fig.1 pH Optimum
Relative activity(%)
None
Adekanol NP695
Adekanol NP720
Relative activity(%)
None
NaCl
KCl
LiCl
MgCl2
CaCl2
CuCl2
MnCl2
ZnCl2
FeCl2
CoCl2
NiCl2
BaCl2
100
99.0
NaN3
NaF
Table 2. Effect of metal ions on LPBP activity
Metal ion(1mM)
Relative activity(%)
None
100
14.8
34.9
92.2
87.1
13.5
38.0
0
Triolein
Trimyristin
Trilaurin
Tricaprylin
Tricaprin
Fig.2 pH Stability
Fig.3 Optimum Temperature
Fig.4 Thermal Stability
100
100
100
100
80
60
40
20
0
80
60
40
20
2
4
8
6
10
12
0
Residual activity (%)
120
Relative activity (%)
120
Residual activity (%)
120
Relative activity (%)
120
80
60
40
20
2
4
8
6
pH
10
0
12
60
40
20
0
20
pH
: Macllvaine buffer
80
60
40
80
100
0
0
20
Temperature (℃)
40
60
80
100
Temperature (℃)
: Citrate buffer
pH 6.8
pH 7.5, 30 min.
: 3,3-Dimethylglutarate-NaOH
40 mM Phosphate buffer
40 mM Phosphate buffer
buffer
: Phosphate buffer
Fig.5 Influence on the measurement of
NEFA after TG assay demonstrating
low LPBP adsorption
100
Residual activity (%)
400
300
200
100
0
Residual activity (%)
100
500
NEFA(μEq/L)
Fig.6 Stability of reconstituted LPBP
80
5℃
60
40
20
0
LP
LPBP
Autoanalyzer : HITACHI 7150
80
60
37 ℃
40
20
0
0
10
20
Incubation time (days)
: detergent not added
: Adekanol B-795
: Adekatol SO-120
99
30
0
10
20
30
Incubation time (days)
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
0.75
0.75
0.75
Detergents
Tween 20
Adekatol
SO-120
0.25
0
Absorption (Abs.)
0.5
Absorption (Abs.)
Absorption (Abs.)
not added
0.5
0.25
0
0
10
5
10
5
0
(min)
5
10
(min)
: initial
: at 25℃,4 weeks later
Storage conditions: 250U/ml LPBP, 0.25 U/ml MGLPL
0.05% Tween 20 or Adekatol SO-120
■ 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.
Assay
■ Principle
The assay is based on the titration of fatty acids
liberated in the following reactions:
LPBP
Glycerol + 3 Fatty acid
(Titration)
Fatty acid + NaOH
0.25
0
0
(min)
Triglyceride + 3 H2O
0.5
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
1. McIlvaine buffer pH 4.2
Mix 0.2 M Na 2HPO 4 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(medical use)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
■ 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
Titration volume
sample : Vs
blank
△V =(Vs−Vc)≦ 1.5 ml
: Vc
■ Calculation
△V × F
1
1
Activity(U/mg of powder)= ―――― × 50 × ―― × ――
0.5
X
20
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.
100
4. Sugiura, M. and Isobe, M.(1975)Chem. Pharm. Bull., 23,
References
1226‒1230.
5. Horiuchi, Y., Koga, H. and Gocho, S.(1976)J. Biochem.,
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,
80, 367‒370.
6. Saiki, T., Takagi, Y. Suzuki, T., Narasaki, T., Tamura, G.
T.(1974)Agric. Biol. Chem., 38, 947‒952.
and Arima, K.(1969)Agric. Biol. Chem., 33, 414.
3. Sugiura, M. and Isobe, M.( 1974)Biochem. Biophys.
Acta, 341, 195‒200.
Ⅲ.測定操作法
1. 試験管(24mm i.d × 200mm H)に基質懸濁液 5.0ml
LPBP 活性測定法(Japanese)
と McIlvaine 緩衝液 2.0ml を正確に分注して攪拌混
Ⅰ.試薬液
和後、37℃で予備加温する。
1. McIlvaine 緩衝液 pH4.2
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。
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
※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。
濁液)
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
「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
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
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)NaN 3 を含む
0.1M KH2PO4‒NaOH 緩衝液 pH6.0
7. 試薬
オリーブ油 :「局方」
Ⅳ.計算
1
1
ΔV × F
活性(U/mg)= ――――― × 50 ×―― × ――
0.5
X
20
エタノール :「局方」
アデカトール SO‒120:ADEKA 製
BSA: Millipore 社製 Fraction V pH5.2 #81‒053
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)
Ⅱ.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。
101