CN104698108A - Method for screening I type 17 beta-hydroxysteroid dehydrogenase inhibitor through immobilized enzyme - Google Patents
Method for screening I type 17 beta-hydroxysteroid dehydrogenase inhibitor through immobilized enzyme Download PDFInfo
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Abstract
The invention provides a method for screening a I type 17 beta-hydroxysteroid dehydrogenase inhibitor through an immobilized enzyme, and belongs to the technical field of enzymology and enzyme engineering. I type 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD1) has the important function in treating hormone-dependent diseases. At present, a substrate radiolabelling method is mainly adopted in studying the activity of 17 beta-HSD1, due to the fact that the free enzyme of 17 beta-HSD1 is likely to be inactivated, 17 beta-HSD1 is hard to prepare, a fresh placenta is needed for preparing an enzyme source in a new medicine screening experiment of every time, raw materials are hard to obtain, the price is high, and difficulty is brought to the screening work of new medicine. According to the method, an amino-modified silicon ball is adopted as a carrier, a glutaraldehyde crosslinking method is utilized, 17 beta-HSD1 extracted from the placenta is fixed, an external immobilization 17 beta-HSD1 enzyme model is built, androstenedione is adopted as a substrate, a high performance liquid chromatography method is used for detecting products, and the potential 17 beta-HSD1 inhibitor is screened. According to the method, instability of the free enzyme is overcome, operation is easy, the manufacturing cost is low, and repeated using is achieved.
Description
Technical field
The present invention relates to a kind of method utilizing immobilised enzymes to screen I type 17 beta hydroxysteroid dehydrogenase inhibitors, especially one utilizes immobilised enzymes, sets up the method for external drug screening, belongs to zymetology and technical field of enzyme engineering.
Background technology
I type 17 11-beta-hydroxysteroid dehydrogenase type (17 β-HSD1) is the enzyme that a class has oxidation restoring function, participate in the key enzyme of estrogen and male hormone metabolism, regulating and controlling effect is played to the redox reaction between the ketone group on its C17 position and alcohol radical, mutual conversion between catalysis high activity hormone and low activity hormone, the conversion namely between testosterone, estradiol and androstenedione, oestrone.17 β-HSD1 are catalyzing enzymes of final step in sex hormone building-up process, in close relations with breast cancer, carcinoma of endometrium, endometriosis etc., therefore play a significant role in treatment hormone-dependent diseases.(Design and validation of specific inhibitors of 17b-hydroxysteroid dehydrogenases for therapeutic application in breast and prostate cancer，and in endometriosis[J].Day JM，Tutill HJ，Purohit A，Reed MJ，Endocr Relat Cancer，2008，15：665-692.)
17 β-HSD1 are widely distributed, are mainly present in the tissues such as placenta, liver, testis, prostate.Nineteen fifty-seven Langer and Engel (Human placental estradiol-17beta dehydrogenase 1concentration, characterization and assay [J] .Langer LJ, Engel LL, JBiol Chem, 1958,233:583-588.) part abstraction and purification placenta 17 β-HSD1.1962, (Purification of a 17 β-hydroxysteroid dehydrogenase of human placenta and studies on its transhydrogenase function [J] the .Jarabak J such as Jarabak, Adams JA, Williams-Ashman HG, Talalay P, JBiol Chem, 1962,237:345-357.) be separated from human placenta, purifying 17 β-HSD1, and the biological characteristics of this enzyme to be described in detail.This is the 17 β-HSD1 found the earliest.17 β-HSD1 can affect estrogenic metabolism, reductase is expressed too much in the tissue, or Oxidase Expression is very few, all can cause generating too much estrogen in tissue, cause the hyperplasia of local organization, cause the generation of disease, especially the disease of estrogen-dependent, breast cancer, carcinoma of endometrium etc. as higher in the current incidence of disease.If effectively suppress 17 β-HSD1, not only can regulate and control estrogenic synthesis, level before acceptor can also block the effect of stimulin, therefore, the research of 17 beta-HSD 1 inhibitors, the therapeutic potential for estrogen-dependent diseases is great.At present, for 17 β-HSD1, oneself filters out some specific inhibitor, such as flavonoids, coumarin kind compounds etc., and in some specific animal disease model its curative effect of preliminary identification.
At present, measure the method that 17 β-HSD1 enzymes are lived, normal employing substrate radioactive label method (Insulin-like growth factor type I and insulin-like growth factor type II stimulate oestradiol-17 β hydroxysteroid dehydrogenase (reductive) activity in breast cancer cells [J] .Singh A, Reed MJ, JEndocrinol, 1991,129:R5-R8.), but this method employs radioactive material, bring hidden danger to aftertreatment and personnel protection.In addition to the above drawbacks, with free aromatizing enzyme for model also exists following open defect: (1) enzyme source is hard-earned, owing to there is no commercial aromatizing enzyme supply, each shaker test all needs to find Freshman placenta to prepare the enzyme source of microsome as aromatizing enzyme, and starting material obtain difficulty; (2) the artificial resolvase preservation condition extracted is harsh, easy in inactivation; (3) free aromatizing enzyme does not dissolve in organic solvent, and many micromolecular compounds to be screened water insoluble, organic solvent or emulsifying agent must be added, thus affect enzymatic activity; (4) free aromatizing enzyme is easily assembled in course of reaction, and enzyme molecule can not fully be contacted with substrate molecule, and catalytic efficiency is low, causes shaker test failure; (5) free activity of aromatizing enzyme is unstable, easily by environmental factor as temperature, the impacts such as potential of hydrogen and inactivation.(6) reacted enzyme and product separation difficulty, cannot recycling.Due to above reason, bring challenges usually to the screening study of 173-HSD1 potential inhibitor.
Enzymic catalytic reaction has the advantages such as efficient, single-minded and reaction conditions is gentle, but the practical application of enzymic catalytic reaction is still subject to multifactorial restriction perhaps, and as unstable in enzyme, easy inactivation, is difficult to repeat to recycle.For overcoming above shortcoming, people have developed the method for immobilised enzymes (Immobilized enzyme, IE).At present, enzyme immobilization technology is own is used widely in industries such as food industry, fine chemicals industry, medicine, its theory of development is work in cell based on endocellular enzyme, be similar to the immobilised enzymes made with embedding method, therefore immobilised enzymes can be thought to a certain extent in order to make enzyme react under closer to its interior state.Immobilised enzymes, compared with resolvase, has many advantages: 1) very easily with substrate, product separation; 2) can carry out Reusability in a long time, cost reduces; 3) stability of enzyme can be improved; 4) can improve (Developments in immobilized-enzyme technology [J] .Powell LW, Biotechnol Genet Eng Rev, 1984,2:409-438.) such as catalytic efficiencies.Up to the present, there is not yet both at home and abroad about the immobilized research report of 17 β-HSD1.
Enzyme immobilizatio method is often divided into absorption method, e, cross-linking method, investment and microencapsulation etc. according to mechanism difference.The present invention is with amido modified silicon ball for carrier, and adopt Euplotes woodruffi to fix 17 β-HSD1, the immobilised enzymes stability obtained improves, and can reuse 5 times.The present invention, by immobilization 17 β-HSD1, simulated in vivo environment, effective 17 beta-HSD 1 inhibitors of in-vitro screening, effectively simple to operate.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of method utilizing immobilised enzymes to screen 117 beta-HSD 1 inhibitors.Mainly with amido modified silicon ball for carrier, the 17 β-HSD1 utilizing glutaraldehyde cross-linking to fix to extract from placenta.
To achieve these goals, a kind of method utilizing immobilised enzymes to screen 117 beta-HSD 1 inhibitors of the present invention, comprises the steps:
1) extraction of 17 β-HSD1
Institute all carries out in steps on 0 ~ 4 DEG C of ice cube.Placenta removing chorion and trunk, with 50mM PBS (pH=7.4 includes 1%KCl), shred with scissors after cleaning, carry out homogenate with 50mM PBS (including 0.25M sucrose).Centrifugal 2500 × g30min, discards precipitation, and the centrifugal 60000 × g 60min of supernatant is with precipitation line plastochondria composition.Again discard precipitation, after adding the ammonium sulfate solids of a certain amount of recrystallization in supernatant, precipitation accumulation, centrifugal rear supernatant discarded, by pellet resuspended in 50mMPBS (including 1mM EDTA and 1mM cysteine hydrochloride), and in-80 DEG C of preservations.
2) preparation of amido modified silicon ball
Get 200mg silica gel, add the hydrochloric acid that 10 ~ 30mL concentration is 5% ~ 10%, in 90 DEG C of backflow 10 ~ 12h, washing is to neutral, and 100 DEG C of dried overnight, obtain activated silica gel.Get 50mg activated silica gel, add dry toluene and 3-aminopropyl triethoxysilane (APTES), under nitrogen protection, 90 DEG C of backflow 18 ~ 24h.The silica gel toluene of APTES on bonding and acetone are washed successively, in 105 DEG C of dried overnight.
3) preparation of immobilised enzymes
The silicon ball that 100mg amination is modified is placed in original place flask, and adding 10 ~ 20mL concentration is 1% ~ 2.5% glutaraldehyde solution, stirs 5 ~ 12h, and washing is extremely without glutaraldehyde smell, and dried overnight at 50 ~ 70 DEG C, is stored in refrigerator stand-by.Get 50mg and be cross-linked silicon ball, add the 17 β-HSD1 extracts that PBS damping fluid and concentration are 4.8mg/mL, at 4 ~ 25 DEG C, stir 8 ~ 12h, washing to not containing resolvase, dried overnight at 25 ~ 35 DEG C, and being stored in 0 ~ 4 DEG C.
4) mensuration of activity of the immobilized enzyme
Get described immobilised enzymes temperature and incubate substrates androstenedione 30min ~ 24h, centrifuging and taking supernatant, utilize the content of high effective liquid chromatography for measuring each time point product testosterone, prepare enzymatic curve, calculate enzyme kinetics coefficient, as negative control.
5) screening of 17 beta-HSD 1 inhibitors
Get described immobilised enzymes, add compound to be screened, temperature incubates substrates androstenedione 30min ~ 24h, centrifuging and taking supernatant, utilizes the content of high effective liquid chromatography for measuring each time point product testosterone, prepares enzymatic curve, calculate enzyme kinetics coefficient, and compare with negative control.Calculate and add inhibiting rate under variable concentrations compound and IC
50value, evaluates inhibiting effect.
Wherein, step 1) described in, 50mM phosphate buffer PBS (pH=7.4) component is: the content of sodium hydrogen phosphate is 19.332g/L, and the content of sodium dihydrogen phosphate is 1.976g/L, and all the other are water.
Step 2) described in, 10 ~ 30mL concentration is the hydrochloric acid of 5% ~ 10%, and preferred 20mL concentration is the hydrochloric acid of 10%.
Step 2) described in, silicon spherolite footpath is 5 ~ 50 μm.
Step 2) described in, to add toluene be 3 ~ 10mL, APTES is 20 ~ 30 μ L, preferred 5mL toluene, 25 μ LAPTES.
Step 3) described in, glutaraldehyde cross-linking reaction system pH is 7.4, because glutaraldehyde is shown in that light decomposes, and lucifuge when reaction is stirred.
Step 3) described in, adding 10 ~ 20mL concentration is 1% ~ 2.5% glutaraldehyde solution, and preferred 10mL concentration is the glutaraldehyde solution of 2.5%.
Step 3) described in, adding PBS is 0.5 ~ 1mL, and 17 β-HSD1 extracts are 50 ~ 100 μ L, preferred PBS is 1mL, and 17 β-HSD1 are 100 μ L.
Step 3) described in, enzyme immobilization reaction stirs 8 ~ 12h at 4 ~ 25 DEG C, preferably stirs 12h at 25 DEG C.
Step 4) and step 5) described in, the temperature condition of incubating of enzyme is: pH=8.0, and temperature is 37 ~ 45 DEG C, and androstenedione concentration is 30 ~ 40 μMs, and adding volume is 100 ~ 150 μ L, and NAPDH concentration is 20 ~ 25mg/mL, and adding volume is 10 ~ 20 μ L.
Step 4) and step 5) described in, high-efficient liquid phase chromatogram determining condition: Stationary liquid is octadecylsilane chemically bonded silica, mobile phase is acetonitrile: 0.1% aqueous formic acid (70: 30), determined wavelength is 240nm, column temperature is 25 ~ 35 DEG C, flow velocity is 1.0mL/min, and sampling volume is 20 μ L.
Step 4) and step 5) described in, the blank temperature for not adding NAPDH incubates system.
Step 5) described in, inhibiting rate computing formula is: I (%)=(A
1-A
2)/A1 × 100%, A
1the peak area of product during for not adding inhibitor, A2 is the peak area adding inhibitor afterproduct.Take inhibitor concentration as horizontal ordinate, inhibiting rate is ordinate, formulates regression curve, calculates IC50 value.
The invention provides a kind of method that external temperature incubates screening 17 beta-HSD 1 inhibitors, abandoned the method for carrying out active medicine screening with traditional resolvase, the 17 β-HSD1 extracted are fixed on amido modified silicon ball, the tolerance of the enzyme of raising in placenta.
Because enzyme is fixed in carrier, in pressed powder state, be easily separated with substrate, reaction mixture system, the change of high performance liquid chromatography direct-detection substrate and product assay can be passed through, and immobilised enzymes be by centrifugal recycling.
Embodiment
Embodiment 1: the preparation of immobilised enzymes
1) extraction of 17 β-HSD1
Institute all carries out in steps on 0 ~ 4 DEG C of ice cube.Placenta removing chorion and trunk, with 50mM PBS (pH=7.4 includes 1%KCl), shred with scissors after cleaning, carry out homogenate with 50mM PBS (including 0.25M sucrose).Centrifugal 2500 × g 30min, discards precipitation, and the centrifugal 60000 × g 60min of supernatant is with precipitation line plastochondria composition.Again discard precipitation, after adding the ammonium sulfate solids of a certain amount of recrystallization in supernatant, precipitation accumulation, centrifugal rear supernatant discarded, by pellet resuspended in 50mM PBS (including 1mM EDTA and 1mM cysteine hydrochloride), and in-80 DEG C of preservations.
2) preparation of amido modified silicon ball
Get the silica gel 200mg that particle diameter is 5 μm, add the hydrochloric acid that 20mL concentration is 10%, in 90 DEG C of backflow 12h, washing is to neutral, and 100 DEG C of dried overnight, obtain activated silica gel.Get 50mg activated silica gel, add 5mL dry toluene and 25 μ L3-aminopropyl triethoxysilanes (APTES), under nitrogen protection, 90 DEG C of backflow 24h.The silica gel toluene of APTES on bonding and acetone are washed 3 times successively, in 105 DEG C of dried overnight.
3) enzyme immobilizatio
The silicon ball that 100mg amination is modified, be placed in round-bottomed flask, adding 10mL concentration is 2.5% glutaraldehyde solution, and under the condition of pH=7.4, lucifuge stirs 12h, and washing is extremely without glutaraldehyde smell, and dried overnight at 60 DEG C, is stored in refrigerator stand-by.Get 50mg and be cross-linked silicon ball, add the 17 β-HSD1 extract 100 μ L that 1mL PBS damping fluid and concentration are 4.8mg/mL, at 25 DEG C, stir 12h, washing to not containing resolvase, dried overnight at 30 DEG C, and being stored in 0 ~ 4 DEG C.
Example 2: the zymologic property of immobilised enzymes
1) enzymatic reaction optimum reaction conditions
Get appropriate immobilised enzymes and resolvase, add the androstenedione of 150 μ L concentration 35 μMs respectively, the NAPDH of 20 μ L concentration 25mg/mL, incubates 8h in 37 DEG C of temperature, measures the content of product testosterone.Its optimal reaction pH is 8.0, and optimal reactive temperature is 40 DEG C.Reuse after 5 times, immobilised enzymes inactivation.
2) enzyme kinetics research
Get described immobilised enzymes 20mg, add the NAPDH of 200 μ L concentration 25mg/mL successively, the androstenedione of 1500 μ L concentration 35 μMs, after concussion mixing, average mark dresses up 10 pipes, and under the phosphate buffer of pH=8.0,37 DEG C of temperature incubate 0min, 15min, 30min, 1h, 2h, 4h, 6h, 10h, 12h, 24h, centrifuging and taking supernatant, utilize the content of each time point product of high effective liquid chromatography for measuring, prepare enzymatic curve, calculate enzyme kinetics coefficient Vm and Km.
High-efficient liquid phase chromatogram determining condition: Stationary liquid is octadecylsilane chemically bonded silica, mobile phase is acetonitrile: 0.1% aqueous formic acid (70:30), determined wavelength is 240nm, column temperature is 30 DEG C, flow velocity is 1.0mL/min, and sampling volume is 20 μ L, and detection time is 6min, the retention time of androstenedione is 3.85min, and the retention time of testosterone is 3.15min.
Example 3: apiolin is to the research of immobilised enzymes inhibition
1) apiolin enzymatic suppresses curve
Get described immobilised enzymes 20mg, add the NAPDH of 200 μ L concentration 25mg/mL successively, the androstenedione of 1500 μ L concentration 35 μMs, the apiolin of 100 μ L concentration 400nM, after concussion mixing, average mark dresses up 10 pipes, under the phosphate buffer of pH=8.0,37 DEG C of temperature incubate 0min, 15min, 30min, 1h, 2h, 4h, 6h, 10h, 12h, 24h, centrifuging and taking supernatant, utilizes the content of high effective liquid chromatography for measuring each time point product (deducting corresponding blank), prepare enzymatic curve, calculate enzyme kinetics coefficient V
mand K
m.
2) apiolin IC
50the mensuration of value
Get described immobilised enzymes 160mg, add the NAPDH of 160 μ L concentration 25mg/mL successively, the androstenedione of 1200 μ L concentration 35 μMs, after concussion mixing, average mark dresses up 8 pipes, label 0 ~ 7 respectively, No. 0 pipe adds 20 μ LPBS damping fluids, and it is 5 μMs that 1 ~ No. 7 pipe adds 20 μ L concentration respectively, 2 μMs, 1 μM, 500nM, 250nM, 100nM, the apiolin of 50nM, again after concussion mixing, incubate 8h in 37 DEG C of temperature, measure the content of product testosterone, calculate the inhibiting rate of variable concentrations apiolin, its computing formula is: I (%)=(A
1-A
2)/A
1× 100%, A
1the peak area of product during for not adding inhibitor, A
2for adding the peak area of inhibitor afterproduct.Take inhibitor concentration as horizontal ordinate, inhibiting rate is ordinate, formulates regression curve, calculates IC
50value.
Example 4: archen is to the research of immobilised enzymes inhibition
1) archen enzymatic suppresses curve
Get described immobilised enzymes 20mg, add the NAPDH of 200 μ L concentration 25mg/mL successively, the androstenedione of 1500 μ L concentration 35 μMs, the archen of 100 μ L concentration 500 μMs, after concussion mixing, average mark dresses up 10 pipes, under the phosphate buffer of pH=8.0,37 DEG C of temperature incubate 0min, 15min, 30min, 1h, 2h, 4h, 6h, 10h, 12h, 24h, centrifuging and taking supernatant, utilizes the content of high effective liquid chromatography for measuring each time point product (deducting corresponding blank), prepare enzymatic curve, calculate enzyme kinetics coefficient V
mand K
m.
2) archen IC
50the mensuration of value
Get described immobilised enzymes 160mg, add the NAPDH of 160 μ L concentration 25mg/mL successively, the androstenedione of 1200 μ L concentration 35 μMs, after concussion mixing, average mark dresses up 8 pipes, label 0 ~ 7 respectively, No. 0 pipe adds 20 μ LPBS damping fluids, and it is 5mM, 2mM that 1 ~ No. 7 pipe adds 20 μ L concentration respectively, 1mM, 500 μMs, 250 μMs, 100 μMs, the archen of 50 μMs, again after concussion mixing, incubate 8h in 37 DEG C of temperature, measure the content of product testosterone, calculate the inhibiting rate of variable concentrations archen, its computing formula is: I (%)=(A
1-A
2)/A
1× 100%, A
1the peak area of product during for not adding inhibitor, A
2for adding the peak area of inhibitor afterproduct.Take inhibitor concentration as horizontal ordinate, inhibiting rate is ordinate, formulates regression curve, calculates IC
50value.
The present invention is with by preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, what therefore protection scope of the present invention should define with claims is as the criterion.
Claims (6)
1. utilize immobilised enzymes to screen a method for I type 17 beta hydroxysteroid dehydrogenase inhibitors, it is characterized in that, comprise the steps:
Step one: the extraction of 17 β-HSD1: institute all carries out in steps on 0 ~ 4 DEG C of ice cube.Placenta removing chorion and trunk, with 50mM PBS (pH=7.4 includes 1%KCl), shred with scissors after cleaning, carry out homogenate with 50mM PBS (including 0.25M sucrose).Centrifugal 2500 × g 30min, discards precipitation, and the centrifugal 60000 × g 60min of supernatant is with precipitation line plastochondria composition.Again discard precipitation, after adding the ammonium sulfate solids of a certain amount of recrystallization in supernatant, precipitation accumulation, centrifugal rear supernatant discarded, by pellet resuspended in 50mM PBS (including 1mM EDTA and 1mM cysteine hydrochloride), and in-80 DEG C of preservations.
Step 2: the preparation of amido modified silicon ball: get 200mg silica gel, adds the hydrochloric acid that 10 ~ 30mL concentration is 5% ~ 10%, and in 90 DEG C of backflow 10 ~ 12h, washing is to neutral, and 100 DEG C of dried overnight, obtain activated silica gel.Get 50mg activated silica gel, add dry toluene and 3-aminopropyl triethoxysilane (APTES), under nitrogen protection, 90 DEG C of backflow 18 ~ 24h.The silica gel toluene of APTES on bonding and acetone are washed successively, in 105 DEG C of dried overnight.
Step 3: the preparation of immobilised enzymes: the silicon ball that 100mg amination is modified is placed in original place flask, adding 10 ~ 20mL concentration is 1% ~ 2.5% glutaraldehyde solution, stirs 5 ~ 12h, and washing is extremely without glutaraldehyde smell, dried overnight at 50 ~ 70 DEG C, is stored in refrigerator stand-by.Get 50mg and be cross-linked silicon ball, add the 17 β-HSD1 extracts that PBS damping fluid and concentration are 4.8mg/mL, at 4 ~ 25 DEG C, stir 8 ~ 12h, washing to not containing resolvase, dried overnight at 25 ~ 35 DEG C, and being stored in 0 ~ 4 DEG C.
Step 4: the mensuration of activity of the immobilized enzyme: get described immobilised enzymes temperature and incubate substrates androstenedione 30min ~ 24h, centrifuging and taking supernatant, utilize the content (deducting corresponding blank) of high effective liquid chromatography for measuring each time point product testosterone, prepare enzymatic curve, calculate enzyme kinetics coefficient, as negative control.
Step 5: the screening of 17 beta-HSD 1 inhibitors: get described immobilised enzymes, add compound to be screened, temperature incubates substrates androstenedione 30min ~ 24h, centrifuging and taking supernatant, utilize the content (deducting corresponding blank) of high effective liquid chromatography for measuring each time point product testosterone, prepare enzymatic curve, calculate enzyme kinetics coefficient, and compare with negative control.Calculate and add inhibiting rate under variable concentrations compound and IC
50value, evaluates inhibiting effect.
2. described in requiring according to right 1, utilize immobilised enzymes to screen the method for I type 17 beta hydroxysteroid dehydrogenase inhibitors, it is characterized in that, in step 2, silicon spherolite footpath is 5 ~ 50 μm, and to add toluene be 3 ~ 10mL, APTES is 20 ~ 30 μ L.
3., described in requiring according to right 1, utilize immobilised enzymes to screen the method for I type 17 beta hydroxysteroid dehydrogenase inhibitors, it is characterized in that, in step 3, glutaraldehyde cross-linking reaction system pH is 7.4, because glutaraldehyde is shown in that light decomposes, and lucifuge when reaction is stirred.
4., described in requiring according to right 1, utilize immobilised enzymes to screen the method for I type 17 beta hydroxysteroid dehydrogenase inhibitors, it is characterized in that, in step 3, adding PBS is 0.5 ~ 1mL, and 17 β-HSD1 extracts are 50 ~ 100 μ L.
5. described in requiring according to right 1, immobilised enzymes is utilized to screen the method for I type 17 beta hydroxysteroid dehydrogenase inhibitors, it is characterized in that, in step 4 and step 5, the condition that enzyme temperature is incubated: pH=8.0, temperature is 37 ~ 45 DEG C, androstenedione concentration is 30 ~ 40 μMs, adding volume is 100 ~ 150 μ L, and NAPDH concentration is 20 ~ 25mg/mL, and adding volume is 10 ~ 20 μ L.
6. described in requiring according to right 1, immobilised enzymes is utilized to screen the method for I type 17 beta hydroxysteroid dehydrogenase inhibitors, it is characterized in that, in step 4 and step 5, high-efficient liquid phase chromatogram determining condition: Stationary liquid is octadecylsilane chemically bonded silica, mobile phase is acetonitrile: 0.1% aqueous formic acid (70: 30), determined wavelength is 240nm, column temperature is 25 ~ 35 DEG C, and flow velocity is 1.0mL/min, and sampling volume is 20 μ L.
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| US11485958B2 (en) | 2017-01-23 | 2022-11-01 | Regeneron Pharmaceuticals, Inc. | HSD17B13 variants and uses thereof |
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| US11702700B2 (en) | 2017-10-11 | 2023-07-18 | Regeneron Pharmaceuticals, Inc. | Inhibition of HSD17B13 in the treatment of liver disease in patients expressing the PNPLA3 I148M variation |
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| CN108753911B (en) * | 2018-05-18 | 2022-04-01 | 山东省科学院海洋仪器仪表研究所 | Immobilized enzyme-based method for rapidly evaluating activity inhibition of marine natural product cyclooxygenase-2 |
| CN114088898A (en) * | 2021-11-02 | 2022-02-25 | 大连理工大学 | Method for preparing drug metabolizing enzyme-porous material compound and application |
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