MONOGLYCERIDE LIPASE [MGLPⅡ] (T-117)

(Diagnostic Reagent Grade) ASAHI KASEI ENZYMES T-117REACH compliant product

MONOGLYCERIDE LIPASE [MGLPⅡ]

from Bacillus sp.
(Glycerol–ester hydrolase, EC 3.1.1.23)

Monoacylglycerol + H2O → Glycerol + Fatty Acid

Preparation and Specification

Appearance
: White amorphous powder, lyophilized
Specific activity
: More than 20 U/mg solid
Contaminants
:  
Catalase
Less than 0.5% (U/U)

Properties

Substrate specificity
: See Table 1
Molecular weight
: 20 kDa (gel filtration)
Isoelectric point
: pH 4.8±0.2
Michaelis constants
: Monolaurine 1.8 × 10-4M
Optimum pH
: 6.0–8.0Figure 1
pH stability
: 6.0–8.0 (65℃, 10 min) Figure 2
Optimum temperature
: 65℃ (PIPES buffer) Figure3
Thermal stability
: Stable at 65℃ and below (pH 8.0, 10 min) Figure4
Effect of metal ions
: See Table 2
Effect of detergents
: See Table 3

Applications for Diagnostic Test

This enzyme is useful for enzymatic determination of triglyceride.

  LP + MGLP Ⅱ
TG + 3 H2O Glycerol + 3 FFA

TG: Triglyceride
FFA: Free fatty acid

 

Table 1. Substrate specificity

Substrate Relative activity
(%)
1–Monocaprylin 81.8
1–Monolaurin 100
1–Monomyristin 96.3
1–Monopalmitin 66.3
1–Monostearin 31.0
1–Monoolein 62.3
1–Monolinolein 110
Triolein 0

 

Table 2. Effect of metal ions on MGLP Ⅱ activity

Metal ion
(10mM)
Relative activity
(%)
None 100
NaCl 83.0
KCl 79.0
LiCl 78.0
MgCl2 77.0
MnCl2 77.0
CaCl2 78.0

 

Table 3. Effect of detergents on MGLP Ⅱ activity

Detergent
(0.1%)
Relative activity
(%)
None 100
Triton X–100 67.0
Triton X–114 67.0
Adekanol 795 64.0
Emulgen B–66 67.0
Emulgen 911 65.0
Emulgen 810 66.0
Emulgen 460 61.0
Rheodol TWL–106 67.0

Fig.1 pH Optimum


〇: Acetate buffer
●: Phosphate buffer
□: Tris-HCI buffer
■: Glycine-NaOH

Fig.2 pH Stability


65℃, 10min
〇: 3,3-Dimethylglutarate-NaOH buffer
●: PIPES buffer
□: Tris-HCI buffer
■: Glycine-NaOH buffer

Fig.3 Optimum Temperature


pH 7.3
50 mM PIPES buffer

Fig.4 Thermal Stability


pH 8.0, 10 min.
50 mM PIPES buffer

Assay

Principle
  1. The assay is based on the increase in absorbance at 550 nm as the formation of quinoneimine dye proceeds in the following reactions:

  MGLP Ⅱ
Monolaurin+H2O Glycerol+Lauric acid
  GK
Glycerol+ATP Glycerol–3–P+ADP
  GPOSP
Glycerol–3–P+O2 Dihydroxyacetone phosphate+H2O2
  POD
2 H2O2+4–AA+TOOS Quinoneimine dye+4 H2O

ATP : Adenosine triphosphate
GK : Glycerol kinase
GPOSP: Glycerophosphate oxidase
TOOS: Ethyl–N– (2–hydroxy–3–sulfopropyl) –m–toluidine sodium salt,
Unit definition
  1. One unit is defined as the amount of enzyme which liberates 1 μmole of monoglyceride per minute at 37℃ under the conditions specified in the assay procedure.

Reagents
  1. Reaction mixture
    0.2 M PIPES–NaOH buffer pH 7.3
    0.10 ml
    15mM 4–AA solution
    0.05 ml
    0.3% (W/V) TOOS solution
    0.05 ml
    100 U/ml POD solution 1)
    0.025 ml
    100 mM MgCl2 solution
    0.005 ml
    50 mM ATP solution pH7.0
    0.01 ml
    25 U/ml GK solution 2) 0.01 ml
  1. 150 U/ml GPOSP solution 3) 0.10 ml
    Distilled water 0.05 ml
    1) : 100 U/ml POD solution
    Dissolve 1,000 U (PPU) of POD with 10 ml of distilled water.
    2) : 25 U/ml GK solution
    Dissolve 250 U of GK with 10 ml of distilled water.
    3) :
    150 U/ml GPOSP solution
    1,500 U of GPOSP with 10 ml of distilled water.
  2. Substrate solution
    (1) Substrate preparation buffer
    5 mM MES–NaOH buffer pH 5.5 containing 0.5% (W/V) Triton X–100 MES [2– (N–monophoryno) ethanesulfonic acid monohydrate]
    (2) Substrate solution (for stock)
    0.5M Monolaurine–ethanol solution
    (3)
    Substrate solution (milky colored)
    Mix 0.2 ml of substrate solution (for stock) and 9.8 ml of substrate preparation buffer
  3. Reaction stopper
    0.5% (W/V) SDS solution
    SDS: Sodium dodecyl sulfate
  4. Enzyme dilution buffer
    10 mM PIPES–NaOH buffer pH 7.3 containing 0.1% (W/V) BSA
  5. Reagents
    PIPES [Piperazine–1,4–bis (2–ethanesulfonic acid) ]:
    Dojindo Laboratories #345–02225
    TOOS: Dojindo Laboratories #OC13
    MES: Dojindo Laboratories #349–01623
    BSA: Millipore Fraction V pH5.2 #81–053
    Monolaurin: Tokyo Kasei Kogyo Co., Ltd #G0081
    GK: Asahi Kasei Pharma Corporation #T–09
    GPOSP: Asahi Kasei Pharma Corporation #T–60
    4–AA: NACALAI TESQUE, INC. Special grade
    #01907–52
    Triton X–100: The Dow Chemical Company
    SDS: NACALAI TESQUE, INC. #316–06
    POD: Sigma Chemical Co. Type Ⅱ #P–8250
Enzyme solution
  1. Accurately weigh about 20 mg of the sample and add enzyme dilution buffer to make a total of 20 ml. Dilute it with enzyme dilution buffer to adjust the concentration as required.

Procedure
  1. Pipette accurately 0.40 ml of reaction mixture and 50 μl of substrate solution into a small test tube and preincubate at 37℃.
  2. After 3 min, add exactly 20 μl of enzyme solution and mix to start the reaction at 37℃.
    In the case of a test blank, add 20 μl of enzyme dilution buffer in place of enzyme solution.
  3. At 10 minutes after starting the reaction, add 2.0 ml of the reaction stopper to stop the reaction.
  4. Measure the absorbance at 550 nm.
  5. Absorbance sample : As
    blank : Ab
    △A = (As−Ab) ≦ 0.700Abs
Calculation
  1. Activity (U/mg of powder) = {(△ A/10)/(15.6)} × 2.47/0.02 × 1/x
    15.6 : millimolar extinction coefficient of quinoneimine dye
    at 550 nm (cm2/ μmole)
    10 : reaction time (min)
    2.47 : final volume (ml)
    0.02 : 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.

References
  1. Imamura, S., and Kitaura, S (. 2000) J. Biochem. ( Tokyo) , 127, 419–425.

MGLP Ⅱ活性測定法 (Japanese)

試薬液
  1. 反応試薬混合液
    0.2M PIPES–NaOH 緩衝液 pH7.3
    0.10 ml
    15mM 4–AA 溶液
    0.05 ml
    0.3% (W/V) TOOS 溶液
    0.05 ml
    100U/ml POD 溶液 1) 0.025 ml
    100mM 塩化マグネシウム溶液
    0.005 ml
    50mM ATP 溶液 pH7.0
    0.01 ml
    25U/ml GK 溶液 2) 0.01 ml
    150U/ml GPOSP 溶液 3) 0.10 ml
    精製水 0.05 ml
  1. 1) : 100U/ml POD 溶液
    POD 1,000 単位 (PPU) を精製水10ml で解する。
    2) :

    25U/ml GK 溶液
    GK 250 単位 (U) を精製水10ml で溶解する。
    3) :
    150U/ml GPOSP 溶液
    GPOSP 1,500 単位 (U) を精製水10ml で溶解する。
  2. 基質溶液
    基質調製用液
    0.5% (W/V) トリトンX–100 を含む5mM
    MES–NaOH 緩衝液 pH5.5


    保存基質溶液
    0.5M モノラウリンーエタノール溶液
  1. 基質溶液
    保存基質溶液0.2ml と基質調製用液9.8ml を混合 (白濁する) して基質溶液とする。
  2. 反応停止液
    0.5% (W/V) SDS 溶液
  3. 酵素溶解希釈用液
    0.1% (W/V) BSA を含む10mM PIPES–NaOH 緩衝液 pH7.3
  4. 試薬
    PIPES[ピペラジン–1,4–ビス (2–エタンスルホン酸) ]:同仁化学製 #345–02225
    TOOS[エチル–N– (2–ヒドロキシ–3–スルフォプロピル) –m–トルイジンナトリウム塩]:同仁化学製
    #OC13
    MES[2– (N–モルフォリノ) エタンスルフォン酸モノヒドレート]:同仁化学製 #349–01623
    ATP (アデノシン三リン酸・2Na・2H2O) :
    協和発酵製
    BSA : Millipore 製 Fraction V pH5.2 #81–053
    モノラウリン (Monolaurin) :
    東京化成工業製 #G0081
    GK (グリセロールキナーゼ) :旭化成ファーマ製
    #T–09
    GPOSP (グリセロリン酸オキシダーゼ) :
    旭化成ファーマ製 #T–60
    4–AA:ナカライテスク製 特級 #01907–52
    トリトンX–100:Dow Chemical 製
    SDS (ドデシル硫酸ナトリウム) :
    ナカライテスク製 #316–06
    POD:シグマ製 Type Ⅱ #P–8250
酵素試料液
  1. 検品約20mg を精密に量り、酵素溶解希釈用液で溶解して全容20ml 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. 小試験管に反応試薬混合液0.40ml と基質溶液50 μlを正確に分注し、37℃で予備加温する。
  2. 3 分経過後、酵素試料液20 μl を正確に加えて混和し、37℃で反応を開始する。
    盲検は酵素試料液の代りに酵素溶解希釈用液20 μlを加える。
  3. 10 分経過後、反応停止液2.0ml を加えて混和し、反応を停止する。
  4. 550nm における吸光度を測定する。
    求められた吸光度変化を試料液はAs、盲検液はAbとする。
    ΔA = (As−Ab) ≦ 0.700Abs
計算
活性 (U/mg) = {(ΔA/10)/(15.6)} × 2.47/0.02 × 1/x
15.6 : キノン色素の550nm におけるミリモル分子吸光係数
(cm2 /μmole)
10 : 反応時間 (min)
2.47 : 反応総液量 (ml)
0.02 : 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)