CN108931604A - A kind of online Screening Platform of reactive compound based on enzyme reactor and application - Google Patents
A kind of online Screening Platform of reactive compound based on enzyme reactor and application Download PDFInfo
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Abstract
The invention belongs to active ingredient screening technical field, a kind of online Screening Platform of the reactive compound based on enzyme reactor and application are disclosed.The online Screening Platform is connected and composed by acetylcholine ester enzyme reactor with identification equipment is separated；The acetylcholine ester enzyme reactor refers to the Organic Polymer Monolithic Columns for being fixed with acetylcholinesterase.Reactive compound based on enzyme reactor of the invention sieves platform with respect to conventional medicament screening technique online, it is with easy to operate, targeting is strong, high degree of automation advantage, the platform integrates affine fish of acetylcholine ester enzyme reactor and separates identification with LC-MS, can effectively improve reactive compound screening efficiency in natural products.
Description
Technical field
The invention belongs to active ingredient screening technical fields, and in particular to a kind of reactive compound based on enzyme reactor exists
Line Screening Platform and application.
Background technique
Natural products (natural product) is that nature biotechnology is retained in permanent evolutionary process through natural selection
Secondary metabolites [D.H.Williams, M.J.Stone, P.R.Hauck, S.K.Rahman, the Journal of to get off
Natural Products,1989,52:1189-1208.] comprising from the animal of nature, plant, marine organisms,
Bioactive substance [G.M.Cragg, D.J.Newman, K.M.Snader, Journal of Natural of microorganism etc.
Products,1997,60:52-60.].Natural products is ubiquitous, and the life of the mankind is also closely bound up therewith.Natural products because
Its ingredient multiplicity, therapeutic effect is significant, less toxic side effect and other advantages, is widely used in always in the prevention and treatment of various human diseases
[K.Chan,Trends in Pharmacological Sciences,1995,16:182-187.；D.Normile,Asian
medicine,Science,2003,299:188-190.；R.Yuan,Y.Lin,Pharmacology and
Therapeutics,2000,86:191-198.].As a huge resource treasure-house, natural products already becomes modern medicine
Object research and development primary study object [D.J.Newman, G.M.Cragg, Journal of Natural Products, 2007,
70:461-477.].Containing the hundreds of or even thousands of kinds of compounds with different structure and concentration in natural products, however, at present
Only a few compounds be elucidated with its pharmacological activity and/or toxic side effect [X.Huang, L.Kong, X.Li, X.Chen, M.Guo,
H.Zou,Journal of Chromatography B,2004,812:71-84.].This, which is mainly due to natural products, has height
The bio-diversity and structural complexity of degree, so that the screening operation to researcher's bioactive substance brings great challenge
[X.X.Yang,F.Xu,D.Wang,Z.W.Yang,H.R.Tan,M.Y.Shang,X.Wang,S.Q.Cai.,Journal of
Chromatography A,2015,1413:33-46.]。
The screening of reactive compound in natural products, method the most traditional are using chemical research as guiding to naturalization
It closes object and extracts separation, obtain the compound of various structure types as much as possible, and analyze and identify to its structure, after
It is continuous that determination of activity [W.wang, Y.H.Zeng et al.J.Nat.Prod.2010,73,1815-1820] is carried out to each compound.
Though the method achieves significant achievement on active ingredient screening, with biggish blindness, the test period is long and tries for it
Agent consumption is big.In recent years, the affine screening strategy of fishing based on affinity chromatography theory is concerned because of its strong targeting
[R.J.Zhuo,H.Liu,N.N.Liu,Y.Wang,Molecules,2016,21(11):1516.].Affine screening strategy of fishing
With the relevant target protease of same disease or receptor etc. for bait, accurately and rapidly fish directly from natural products to tie therewith
The ligand of conjunction, to substantially increase screening efficiency.
Currently, with it is affine fish strategy Development go out screening technique mainly have affinity ultrafiltration method [X.X.Yang, F.Xu,
D.Wang,Z.W.Yang,H.R.Tan,M.Y.Shang,X.Wang,S.Q.Cai,Journal of Chromatography A,
2015,1413:33-46.；Y.Choi,K.Jermihov,S.J.Nam,M.Sturdy,K.Maloney,X.Qiu,
L.R.Chadwick,M.Main,S.N.Chen,A.D.Mesecar,Analytical Chemistry,2011,83:1048-
1052.], magnetic microsphere is fished method [F.G.D.Almeida, K.L.Vanzolini, Q.B.Cass, Journal of
Pharmaceutical and Biomedical Analysis,2016,132:159-164.；M.C.de Moraes,
J.B.Santos,D.M.Dos Anjos,L.P.Rangel,T.C.Vieira,R.Moaddel,S.J.Da,Journal of
Chromatography A,2015,1379:1-8.；L.Yuan,P.L.Xu,Q.Zeng,Y.M.Liu,L.S.Ding,X.Liao,
Materials Science and Engineering C,2015,56:401-408.] etc..Affinity ultrafiltration method and magnetism are fished
Method can effectively be enriched with the ligand for having combination in natural products with target protein, subsequent to carry out separation mirror by LC-MS again
It is fixed, the relevant molecule information for the potential ligand fished can be obtained.But above method be off-line mode, be both needed to by incubation, separation,
This series of process is dissociated and analyzes, cumbersome, the degree of automation is low, is not able to satisfy still and lives now to rapidly and efficiently screening
The requirement of property compound.In view of this, the advantage and LC-MS technology screened in reactive compound based on affine method of fishing are in compound point
From the accurate reliability in identification, establish rapidly and efficiently integrate active in the affine natural products for fishing and separating identification
The online screening technique of compound can provide crucial technical support for drug discovery.
Summary of the invention
In place of the above shortcoming and defect of the existing technology, it is based on the primary purpose of the present invention is that providing one kind
The online Screening Platform of the reactive compound of enzyme reactor.
Another object of the present invention is to provide the above-mentioned online Screening Platforms of the reactive compound based on enzyme reactor in day
Application in right active ingredient screening.
The object of the invention is achieved through the following technical solutions：
A kind of online Screening Platform of reactive compound based on enzyme reactor, by acetylcholine ester enzyme reactor with separate mirror
Determine instrument to connect and compose；The acetylcholine ester enzyme reactor refers to that the organic polymer for being fixed with acetylcholinesterase is whole
Column.
Preferably, the Organic Polymer Monolithic Columns refer to that internal diameter (I.D.) is 400 μm, outer diameter (O.D.) is 800 μm
Large scale Organic Polymer Monolithic Columns.
Preferably, the separation identification equipment refers to LC-MS system (liquid phase-mass spectrometer system).
Further, in the online Screening Platform of the above-mentioned reactive compound based on enzyme reactor, the acetyl gallbladder of inactivation is set
Alkali Esterase reaction device is used as with acetylcholine ester enzyme reactor parallel connection and compares；The acetylcholine ester enzyme reactor of the inactivation refers to
Heating makes the acetylcholine ester enzyme reactor after enzyme inactivation.
The Organic Polymer Monolithic Columns for being fixed with acetylcholinesterase are prepared via a method which to obtain：
Function monomer, crosslinking agent, pore-foaming agent and initiator are mixed, modified quartz is circulated into after ultrasonic dissolution, degassing
In capillary, the sealing two ends of quartz capillary column are put into water-bath, pass through thermocatalytic home position polymerization reaction, end of reaction
The unreacted reactant in quartz capillary is removed afterwards, is obtained organic polymer integral material, is then filled acetylcholine ester enzyme solutions
It infuses in gained organic polymer integral material and immobilizes reaction, obtain the organic polymer for being fixed with acetylcholinesterase
Integral post.
Preferably, the quartz capillary of the modification refers to the large scale quartz wool that internal diameter is 400 μm, outer diameter is 800 μm
Tubule.
Preferably, the quartz capillary of the modification refers to using 3- (isobutene acyl-oxygen) propyl trimethoxy silicane (γ-
MAPs) modified quartz capillary.Specific modification procedure is as follows：Quartz capillary 15min is rinsed with the NaOH solution of 1mol/L,
Quartz capillary sealing two ends are placed on 100 DEG C of water-bath reaction 2h again；Then deionized water scouring stone English capillary is used, until
The liquid pH flowed out is 7.0；Then it is rinsed after quartz capillary with methanol and dries 4h with nitrogen；After drying, volume
Than being 1:1 methanol and 3- (isobutene acyl-oxygen) propyl trimethoxy silicane (γ-MAPs) mixed liquor is thrown into quartz capillary,
It sealing two ends and is placed in 60 DEG C of water-baths and reacts 12h；30min, the dry 12h of nitrogen are respectively finally rinsed with first alcohol and water respectively,
Up to modified quartz capillary.
Preferably, the function monomer be glycidyl methacrylate (Glycidyl methacrylate,
GMA), crosslinking agent is ethylene glycol dimethacrylate (Ethyleneglycol dimethacrylate, EDMA), pore-foaming agent
For 1,4- butanediol (1,4-butanediol, BDO), normal propyl alcohol (N-propanol, NPA) and water (water, H2O it) forms
Triplex mixture system, initiator are thermal initiator azodiisobutyronitrile (Azobisisobutyronitrile, AIBN).Wherein function
Energy monomer and crosslinking agent quality ratio are 3:2, pore-foaming agent BDO:NPA:H2O=24.26:68.90:6.84, the additional amount of initiator
It is the 1% of function monomer, crosslinking agent and pore-foaming agent gross mass.
Preferably, the mass ratio of the gross mass and pore-foaming agent of the function monomer and crosslinking agent is (20~40):(80~
60)。
Preferably, the thermocatalytic home position polymerization reaction, which refers to, reacts 12h in 65 DEG C of water-baths.
Preferably, the unreacted reactant removed in quartz capillary is to follow the steps below operation：By quartz wool
One end of tubule is connect with high-pressure pump, is rinsed respectively with first alcohol and water.
Preferably, the acetylcholine ester enzyme solutions refer to that the acetylcholinesterase being dissolved in ammonium acetate buffer solution is molten
Liquid, the concentration of acetylcholine ester enzyme solutions are 0.2~1.0mg/mL.
Preferably, the time of the immobilized reactant is 3~12h.
Application of the online Screening Platform of the above-mentioned reactive compound based on enzyme reactor in active skull cap components screening.
The online Screening Platform of the above-mentioned reactive compound based on enzyme reactor answering in the screening of rhizoma corydalis plant extracts
With the applying step is as follows：By acetylcholine ester enzyme reactor first use buffer solution balance a period of time, after with rhizoma corydalis
Plant extracts is sample feeding, removes non-specific binding component under buffer solution effect, then uses organic solvent first instead
Alcohol dissociates the ingredient with fixed acetylcholinesterase specific binding, carries out separation identification into separation identification equipment.
Further, the online Screening Platform of the above-mentioned reactive compound based on enzyme reactor is sieved in rhizoma corydalis plant extracts
In the application process chosen, the acetylcholine ester enzyme reactor that inactivation is arranged is in parallel with acetylcholine ester enzyme reactor, as right
According to.
The present invention is based on affinity chromatography theories, i.e. affinity interaction between protein ligand, will be with organic polymer integral material
Be acetylcholine ester enzyme reactor prepared by carrier as affinity extraction column, play its affine function of fishing, so by its
It is connected with commercial instrument component and constructs the online Screening Platform of reactive compound based on enzyme reactor.
Compared with the existing technology, the invention has the advantages that and beneficial effect：
(1) the online screening technique of the reactive compound of the invention based on enzyme reactor and existing screening technique such as activity
Tracking partition method, affinity ultrafiltration method and the magnetic bead method of fishing are compared, and integrate that reactive compound is affine to fish and separate identification,
Operation is simpler, the degree of automation is higher, improves the screening efficiency of reactive compound in natural products.
(2) the online Screening Platform of the reactive compound based on enzyme reactor that constructs of the present invention is by functional acetylcholine ester
Enzyme reactor is in parallel with the acetylcholine ester enzyme reactor of inactivation, can effectively be excluded because of compound and carrier by intuitively comparing
Material or enzyme nonactive site combine caused by non-specificity, improve the accurate of reactive compound screening in natural products
Property.
(3) acetylcholine ester enzyme reactor prepared by the present invention uses (400 μm of large-sized capillary integral material
I.D. × 800 μm O.D.) it is used as fixation support, with capillary integral material (the 100 μm of I.D. for being commonly used in immobilised enzymes
× 375 μm of O.D.) it compares, there is bigger enzyme supported quantity, affine better effect of fishing.
Detailed description of the invention
Fig. 1 is poly (GMA-co-EDMA) organic polymer integral carriers material internal pattern obtained in embodiment 2
Scanning electron microscope (SEM) photograph.
Fig. 2 is the scanning electron microscope (SEM) photograph of acetylcholinesterase inside reactor pattern obtained in embodiment 4.
Fig. 3 is the fluorescence of enzyme reactor prepared by acetylcholinesterase of the embodiment 4 with marked by fluorescein isothiocyanate
Phenogram.
Fig. 4 is the online Screening Platform schematic diagram of reactive compound based on enzyme reactor constructed by embodiment 6.
Fig. 5 is the online Screening Platform of the reactive compound based on enzyme reactor of embodiment 7 through galanthamine and vinegar fourth Lip river
The result figure of your correct mixture verifying.
Fig. 6 is that the online Screening Platform of the reactive compound based on enzyme reactor of embodiment 8 is planted in natural products rhizoma corydalis
The selection result figure in object extract.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
Embodiment 1
The preparation of the present embodiment organic polymer integral carriers material poly (GMA-co-EDMA)：
By function monomer glycidyl methacrylate (Glycidyl methacrylate, GMA), crosslinking agent second two
Alcohol dimethylacrylate (Ethyleneglycol dimethacrylate, EDMA), pore-foaming agent 1,4-butanediol (Isosorbide-5-Nitrae-
Butanediol, BDO), normal propyl alcohol (N-propanol, NPA) and water (water, H2O mixture and initiator azo two) is different
Butyronitrile (Azobisisobutyronitrile, AIBN) mixes, and ultrasonic degassing 5 minutes, pours into modified quartz capillary column
In (400 μm of I.D. × 800 μm O.D.), reaction temperature is 65 DEG C, reacts 12h, connect high-pressure pump first alcohol and water rinse to get
Integral carriers material (poly (GMA-co-EDMA)).Wherein the mass ratio of function monomer GMA and crosslinking agent EDMA is 3:2, pore
Agent BDO:NPA:H2O=24.26:68.90:6.84, the mass ratio of the quality sum and pore-foaming agent of function monomer and crosslinking agent is
30:70, the quality of initiator is the 1% of the quality sum of function monomer, crosslinking agent and pore-foaming agent.
The quartz capillary of the modification is prepared by the following method：Quartz capillary is rinsed with the NaOH solution of 1mol/L
15min, then quartz capillary sealing two ends are placed on 100 DEG C of water-bath reaction 2h；Then deionized water scouring stone English capillary is used
Pipe, until the liquid pH flowed out is 7.0；Then it is rinsed after quartz capillary with methanol and dries 4h with nitrogen；After drying,
It is 1 volume ratio:1 methanol and 3- (isobutene acyl-oxygen) propyl trimethoxy silicane (γ-MAPs) mixed liquor throws quartz wool into
In tubule, sealing two ends and it is placed in 60 DEG C of water-baths and reacts 12h；30min is respectively finally rinsed with first alcohol and water respectively, nitrogen is dry
Dry 12h is to get modified quartz capillary.
The organic polymer integral carriers material (poly being prepared in the present embodiment is observed with scanning electron microscope
(GMA-co-EDMA)) interior microscopic pattern, integral carriers material has micron-sized through-hole and micron-sized three as the result is shown
Tie up continuous skeleton.
Embodiment 2
The preparation of the present embodiment organic polymer integral carriers material poly (GMA-co-EDMA)：
By function monomer GMA, crosslinking agent EDMA, pore-foaming agent (BDO, NPA and H2The mixed system of O) and initiator A IBN it is mixed
It is even, it ultrasonic degassing 5 minutes, pours into modified quartz capillary column (400 μm of I.D. × 800 μm O.D.), reaction temperature 65
DEG C, 12h is reacted, high-pressure pump first alcohol and water is connect and rinses to get integral carriers material (poly (GMA-co-EDMA)).Wherein function
The mass ratio of energy monomer GMA and crosslinking agent EDMA is 3:2, pore-foaming agent BDO:NPA:H2O=24.26:68.90:6.84, function list
The mass ratio of the sum of body and crosslinking agent quality and pore-foaming agent is 35:65, the quality of initiator is function monomer, crosslinking agent and pore
The 1% of the quality sum of agent.
The quartz capillary of the modification is prepared by the following method：Quartz capillary is rinsed with the NaOH solution of 1mol/L
15min, then quartz capillary sealing two ends are placed on 100 DEG C of water-bath reaction 2h；Then deionized water scouring stone English capillary is used
Pipe, until the liquid pH flowed out is 7.0；Then it is rinsed after quartz capillary with methanol and dries 4h with nitrogen；After drying,
It is 1 volume ratio:1 methanol and 3- (isobutene acyl-oxygen) propyl trimethoxy silicane (γ-MAPs) mixed liquor throws quartz wool into
In tubule, sealing two ends and it is placed in 60 DEG C of water-baths and reacts 12h；30min is respectively finally rinsed with first alcohol and water respectively, nitrogen is dry
Dry 12h is to get modified quartz capillary.
Organic polymer integral carriers material (poly (the GMA-co-EDMA)) interior microscopic being prepared in the present embodiment
The scanning electron microscope (SEM) photograph (SEM) of pattern is as shown in Figure 1.The same capillary of Monolithic column of integral carriers material known to Fig. 1 result
Inner wall is combined closely, and without de- wall phenomenon, and polymer is uniformly distributed with microspheric, short texture, ensure that good permeability.
Embodiment 3
The preparation of the present embodiment acetylcholine ester enzyme reactor：
Ammonium acetate buffer solution (15mM, pH 8.0) is filled by integral carriers prepared by embodiment 2 using constant flow pump
In material poly (GMA-co-EDMA), continue 10min；Replacing ammonium acetate buffer solution (15mM, pH 8.0) is 0.2mg/mL's
Acetylcholinesterase (is dissolved in gained in ammonium acetate buffer solution (15mM, pH 8.0)) by acetylcholine ester enzyme solutions, is carried out
Enzyme immobilizatio, time 3h；Then remained in inside integral material using ammonium acetate buffer solution (15mM, pH 8.0) flushing
Unbonded acetylcholinesterase, time 10min.It is that 60 μ L/min are constant that rate of flooding is kept during this, is fixed
There are the Organic Polymer Monolithic Columns (functionalization acetylcholine ester enzyme reactor) of acetylcholinesterase.
Embodiment 4
The preparation of the present embodiment acetylcholine ester enzyme reactor：
Ammonium acetate buffer solution (15mM, pH 8.0) is filled by integral carriers prepared by embodiment 2 using constant flow pump
In material poly (GMA-co-EDMA), continue 10min；Replacing ammonium acetate buffer solution (15mM, pH 8.0) is 0.5mg/mL second
Acetylcholinesterase (is dissolved in gained in ammonium acetate buffer solution (15mM, pH 8.0)) by acetylcholinesterase solution, carries out enzyme
Immobilization, time 3h；Then remained in inside integral material not using ammonium acetate buffer solution (15mM, pH 8.0) flushing
In conjunction with acetylcholinesterase, time 10min.It is that 60 μ L/min are constant that rate of flooding is kept during this, is fixed with
The Organic Polymer Monolithic Columns (functionalization acetylcholine ester enzyme reactor) of acetylcholinesterase.
The acetylcholine ester enzyme reaction being prepared in the present embodiment based on integral carriers material poly (GMA-co-EDMA)
Prepared by the scanning electron microscope (SEM) photograph (SEM) of the interior microscopic pattern of device and the acetylcholinesterase with marked by fluorescein isothiocyanate
The Fluorescent Characterization figure difference of enzyme reactor is as shown in Figures 2 and 3.After fixing acetylcholinesterase as seen from Figure 2, integral carriers material
The inner surface of material is slightly roughening, but polymer is still uniformly distributed with microspheric, and internal micron-sized big aperture ensure that good
Permeability.From the figure 3, it may be seen that the acetylcholinesterase through marked by fluorescein isothiocyanate is successfully fixed on integral carriers material
On.
Embodiment 5
The preparation of the present embodiment inactivation acetylcholine ester enzyme reactor：
Ammonium acetate buffer solution (15mM, pH 8.0) is filled by integral carriers prepared by embodiment 2 using constant flow pump
In material poly (GMA-co-EDMA), continue 10min；Replacing ammonium acetate buffer solution (15mM, pH 8.0) is 0.5mg/mL second
Acetylcholinesterase (is dissolved in gained in ammonium acetate buffer solution (15mM, pH 8.0)) by acetylcholinesterase solution, carries out enzyme
Immobilization, time 3h；Then remained in inside integral material not using ammonium acetate buffer solution (15mM, pH 8.0) flushing
In conjunction with acetylcholinesterase, time 10min.It is that 60 μ L/min are constant that rate of flooding is kept during this, after this is made
The Organic Polymer Monolithic Columns for being fixed with acetylcholinesterase be placed in 80 DEG C of water-baths reaction 3h to get inactivation acetylcholine ester
Enzyme reactor.
Embodiment 6
The foundation of the online Screening Platform of reactive compound of the present embodiment based on enzyme reactor：
The present embodiment is by functionalization acetylcholine ester enzyme reactor and inactivation prepared by above-described embodiment 4 and embodiment 5
Acetylcholine ester enzyme reactor is connected with commercial instrument component, and reactive compound of the foundation based on enzyme reactor screens flat online
Platform, as shown in Figure 4.
Platform is established specific as follows：(1) it fishes module：Using two six-way valve A by functionalization acetylcholine ester enzyme reaction
Device and inactivation acetylcholinesterase reactor group are at parallel module, functionalization enzyme reactor and two six-way valve A valve positions 1 and 2 phases
Even；Inactivation enzyme reactor is connected with two six-way valve A valve positions 4 and 5；Another two six-way valves A valve position 3 is the same as autosampler component two
Position six-way valve valve position 3 is connected, and valve position 6 is connected with two position four-way valve valve positions 2；(2) separation module：It will be in business LC-MS instrument
Two six-way valve valve positions 4 of autosampler component are connected with binary pump 1；(3) LC-MS identifies module：It will using 290 μ L sample rings
Two six-way valve B valve positions 1 are connected with 4 in LC-MS system；Binary pump 2 is connected with two six-way valve B valve positions 5；C18 chromatographic column
One end is connected with two six-way valve B valve positions 6 in LC-MS system, and the other end is connected with above-mentioned two position four-way valves valve position 4；Two six
Port valve B valve position 3 is connected with two position four-way valve valve positions 3.
Active skull cap components screening process is specific as follows：Under Fig. 4 (A) state, One-Dimensional flows phase ammonium acetate buffer solution
(buffer solution A, 10mM, pH 7.4) is provided via pump 1, and flow velocity is 10 μ L/min, and sample injection volume is 3 μ L, which continues
15min；Under Fig. 4 (B) state, mobile phase is changed to methanol (elution by ammonium acetate buffer solution (10mM, pH 7.4) at pump 1
Liquid B), flow velocity is 20 μ L/min, which continues 13min；Under Fig. 4 (C) state, the specific binding that will be captured in sample loop
Ingredient is transferred in two-dimentional module, carries out LC-MS separation identification, at the same time, one-dimensional to be converted to by functionalization enzyme reactor flow path
Enzyme reactor flow path is inactivated, the above process is repeated.
Embodiment 7
The screening capacity verifying for the online Screening Platform of the reactive compound based on enzyme reactor that the present embodiment is established：
Using 6 reactive compound online Screening Platform of the gained based on enzyme reactor of embodiment, under Fig. 4 (A) state, with
The sample mixing of standard items galanthamine and acebutolol composition is sample through autosampler sample introduction, 3 μ L of sample volume, mobile phase acetic acid
Ammonium buffer solution (buffer solution A, 10mM, pH 7.4) is provided via pump 1, and flow velocity is 5 μ L/min, which continues 40min, at this time
Ingredient with immobilized enzyme on enzyme reactor without combination is then rinsed to waste liquid port, and has the ingredient of combination to be then retained in enzyme reactor
Place；After switch to Fig. 4 (B) state, mobile phase is changed to methanol (elution by ammonium acetate buffer solution (10mM, pH 7.4) at pump 1
Liquid B), flow velocity more 20 μ L/min, the process continues 13min, is retained in the ingredient on enzyme reactor at this time after methanol dissociates
It is stored in sample loop；Fig. 4 (C) state is then switched to again, and the specific binding member captured in sample loop is transferred to LC-
MS is identified in module, carries out separation identification (LC-MS test condition is as follows).At the same time, one-dimensional by functionalization enzyme reactor stream
Road is converted to inactivation enzyme reactor flow path, repeats the above screening process.As a result as shown in Figure 5.
LC-MS test condition：
Chromatographic column：SunFireTM C18column(4.6×250mm,5μm,Waters)；
Mobile phase (pump 2)：Methanol (mobile phase A)；0.2M potassium dihydrogen phosphate (Mobile phase B, pH 6.8, NaOH tune)；
Flow velocity (pump 2)：1mL/min；
It is isocratic：45%A, 55%B；
Column temperature：25℃；
Detection wavelength：290nm.
Embodiment 8
The online Screening Platform of the reactive compound based on enzyme reactor the answering in natural products that the present embodiment is established
With：
Using 6 reactive compound online Screening Platform of the gained based on enzyme reactor of embodiment, under Fig. 4 (A) state, with
Rhizoma corydalis plant extracts is sample through autosampler sample introduction, 3 μ L of sample volume, mobile phase ammonium acetate buffer solution (buffer
A, 10mM, pH 7.4) it is provided via pump 1, flow velocity is 5 μ L/min, which continues 40min, is fixed at this time on enzyme reactor
Ingredient of the enzyme without combination is then rinsed to waste liquid port, and has the ingredient of combination to be then retained at enzyme reactor；After switch to Fig. 4
(B) state pumps mobile phase at 1 and is changed to methanol (eluent B) by ammonium acetate buffer solution (10mM, pH 7.4), and flow velocity is more
20 μ L/min, the process continue 13min, and the ingredient being retained on enzyme reactor at this time is stored in sample loop after methanol dissociates
In；Fig. 4 (C) state is then switched to again, and the specific binding member captured in sample loop is transferred in LC-MS identification module,
Carry out separation identification (LC-MS test condition is as follows).At the same time, one-dimensional that fermentoid is converted to by functionalization enzyme reactor flow path
Reactor flow path repeats the above screening process.As a result as shown in Figure 6.
LC-MS test condition：
Liquid-phase condition：
Chromatographic column：XTerra MS C18column(250mm×4.6mm,5μm,Waters)；
Mobile phase (pump 2)：Acetonitrile (mobile phase A)；0.2% acetic acid aqueous solution (Mobile phase B, pH 5.0, ammonium hydroxide tune)；
Flow velocity (pump 2)：1mL/min；
Gradient：20~80%A, 80~20%B；40min；
Column temperature：25℃；
Detection wavelength：280nm.
Mass Spectrometry Conditions：Positive ion mode, full scan range are 100-600m/z, capillary voltage 4000V, atomization air pressure
Power is 40psi, and dryer temperature is 350 DEG C；Dry gas stream speed is 10.0L/min；Fragment voltage is 100V.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (10)
1. a kind of online Screening Platform of reactive compound based on enzyme reactor, it is characterised in that：The online Screening Platform by
Acetylcholine ester enzyme reactor is connected and composed with identification equipment is separated；The acetylcholine ester enzyme reactor, which refers to, is fixed with acetyl
The Organic Polymer Monolithic Columns of cholinesterase.
2. the online Screening Platform of a kind of reactive compound based on enzyme reactor according to claim 1, it is characterised in that：
The separation identification equipment refers to LC-MS system.
3. the online Screening Platform of a kind of reactive compound based on enzyme reactor according to claim 1, it is characterised in that：
The Organic Polymer Monolithic Columns refer to the large scale Organic Polymer Monolithic Columns that internal diameter is 400 μm, outer diameter is 800 μm.
4. the online Screening Platform of a kind of reactive compound based on enzyme reactor according to claim 1, it is characterised in that：
The acetylcholine ester enzyme reactor that inactivation is arranged is used as with acetylcholine ester enzyme reactor parallel connection to be compareed；The acetyl gallbladder of the inactivation
Alkali Esterase reaction device refers to that heating makes the acetylcholine ester enzyme reactor after enzyme inactivation.
5. the online Screening Platform of a kind of reactive compound based on enzyme reactor according to any one of claims 1 to 4,
It is characterized in that：The Organic Polymer Monolithic Columns for being fixed with acetylcholinesterase are prepared via a method which to obtain：
Function monomer, crosslinking agent, pore-foaming agent and initiator are mixed, modified quartzy capillary is circulated into after ultrasonic dissolution, degassing
In pipe, the sealing two ends of quartz capillary column are put into water-bath, by thermocatalytic home position polymerization reaction, are removed after completion of the reaction
The unreacted reactant in quartz capillary is removed, organic polymer integral material is obtained, then arrives acetylcholinesterase infusion
Reaction is immobilized in gained organic polymer integral material, the organic polymer for obtaining being fixed with acetylcholinesterase is whole
Column.
6. the online Screening Platform of a kind of reactive compound based on enzyme reactor according to claim 5, it is characterised in that：
The quartz capillary of the modification refers to the quartz capillary modified using 3- (isobutene acyl-oxygen) propyl trimethoxy silicane；Institute
The function monomer stated is glycidyl methacrylate, and crosslinking agent is ethylene glycol dimethacrylate, and pore-foaming agent is Isosorbide-5-Nitrae-
The triplex mixture system of butanediol, normal propyl alcohol and water composition, initiator are thermal initiator azodiisobutyronitrile.
7. the online Screening Platform of a kind of reactive compound based on enzyme reactor according to claim 6, it is characterised in that：
The function monomer and crosslinking agent quality ratio are 3:2, pore-foaming agent 1,4-butanediol:Normal propyl alcohol:Water=24.26:68.90:6.84
The additional amount of initiator be function monomer, crosslinking agent and pore-foaming agent gross mass 1%, the gross mass of function monomer and crosslinking agent with
The mass ratio of pore-foaming agent is (20~40):(80~60).
8. the online Screening Platform of a kind of reactive compound based on enzyme reactor according to claim 5, it is characterised in that：
The acetylcholine ester enzyme solutions refer to the acetylcholine ester enzyme solutions being dissolved in ammonium acetate buffer solution, acetylcholinesterase
The concentration of solution is 0.2~1.0mg/mL；The time of the immobilized reactant is 3~12h.
9. the online Screening Platform of a kind of reactive compound based on enzyme reactor according to any one of claims 1 to 8 is natural
Application in active ingredient screening.
10. the online Screening Platform of a kind of reactive compound based on enzyme reactor according to any one of claims 1 to 8 is prolonging recklessly
Application in the screening of rope plant extracts, it is characterised in that the applying step is as follows：Acetylcholine ester enzyme reactor is first adopted
A period of time is balanced with buffer solution, then using rhizoma corydalis plant extracts as sample feeding, is removed under buffer solution effect
Non-specific binding component, then use instead organic solvent methanol dissociate with fixed acetylcholinesterase specific binding at
Point, separation identification is carried out into separation identification equipment.
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