CN107589193A - A kind of method that protein inhibitor is screened using micro-reaction device - Google Patents
A kind of method that protein inhibitor is screened using micro-reaction device Download PDFInfo
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- CN107589193A CN107589193A CN201711004907.5A CN201711004907A CN107589193A CN 107589193 A CN107589193 A CN 107589193A CN 201711004907 A CN201711004907 A CN 201711004907A CN 107589193 A CN107589193 A CN 107589193A
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Abstract
The invention discloses a kind of method that protein inhibitor is screened using micro-reaction device, step are as follows：First solution and the second solution are pumped into the micro-mixer of micro-reaction device simultaneously respectively, the first microreactor that micro-reaction device is passed through after being sufficiently mixed carries out reaction and forms composition libraries；The second microreactor for being passed into micro-reaction device of first microreactor is subjected to the screening of protein inhibitor, the nano particle of the second microreactor filling area load albumen；The discharging of first microreactor and the discharging of the second microreactor are subjected to Mass Spectrometer Method and contrasting detection result, screening obtains protein inhibitor.The screening technique of protein inhibitor provided by the invention, it is more by the component in the composition libraries of micro-reaction device structure, the screening of inhibitor can be completed by continuous reaction；It is not required to that single inhibitor and protein, substrate are mixed and screened respectively, saves reactions steps, also improves the degree of accuracy of screening, it is simple to operate controllable.
Description
Technical field
The present invention relates to the screening of protein inhibitor, and in particular to a kind of to screen protein inhibitor using micro-reaction device
Method.
Background technology
It is well known that drug development process need to expend substantial amounts of money and time, and risk is larger.Current research table
Bright, the discovery of a new drug will consume 1.8 hundred million dollars.In addition, the medicine that can finally introduce to the market only has 10%, nearly
50% new drug has just been counted out in clinical three phases.Therefore, among the new drug of research and development, only 1/3rd can withdraw throwing
Provide cost.Although many newtype drugs due to lacking the effect of certain to human body in the clinical second phase trial stage because declare to lose
Lose, but the most important problem of failure is still toxicology, pharmacokinetics and security.And these characteristics are by dividing
The son chemical constitution of itself determines that this shows that the discovery of lead compound determines a potential medicine in drug development process
The success or failure of thing molecule.Therefore, it is how easy, fast and efficiently obtain lead compound and just show at medicament research and development initial stage
Obtain particularly important.
As a kind of method that can effectively identify biological targets part, dynamic combinatorial chemistry (DCC) by chemical synthesis with
Active ligand screening combines, and by the self assembly of balance controlled, synthesizes largely using simple chemical raw material
Complex compound, and the screening of reactive compound is realized simultaneously.
In most cases, once adding target spot, one or more storehouse components will be in combination, thus may change
Reaction system balances, and is obtained with more efficient ligands in theory.At present, it is reversible to be widely used in some by DCC
In reaction, for example the metathesis reaction of alkene, the synthesis of imines and hydrazone, ester exchange reaction, the exchange of mercapto alcohol-disulphide are anti-
Should, transketalation reaction etc..Initially, this method is only used for sieving using organic cation and anion as the reactive compound of target spot
Choosing.Recently, in DCC research, many molecules include ammonium ion, barbiturate, polypeptide, even nucleotides, richness
Happy alkene has all been used as target spot, carries out reactive compound screening.
The great attention of combinatorial compound has caused chemical research person, because it can be with a large amount of novel compounds of Fast back-projection algorithm
Thing, thus suffer from wide application prospect in biology, chemistry and pharmaceutical field.Due to the diffusion in micro flow field reactor away from
It is high from shorter and mass transfer heat exchange efficiency, thus the reaction in micro flow field reactor is carried out comparatively fast, and reagent and solvent-oil ratio
It is less.Therefore, micro flow field reactor has been widely used in organic synthesis, the field such as biomaterial and nano particle.Utilize group
The advantage of combination and micro flow field reactor, researcher be combined in micro flow field reaction system synthesis achieve it is a series of
Progress.Under hydrodynamics control, using single channel glass microractor, pass through 1,3- dicarbonyl compounds and hydrazine
Knorr reacts, and can synthesize the compound library using pyrazoles as parent nucleus.It is parallel organic by 2 × 2 in single glass microchip
Synthetic system, using acid amides synthetic reaction as model reaction, amides compound storehouse can be prepared.
In the medicament research and development stage, the screening of toxicity trial and drug candidate is carried out usually using enzyme level experiment.
In addition, enzyme level experiment is also used to screening and the inhibitor of the closely related enzyme of disease, such as hiv protease and monoamine oxidation
Enzyme.On microtiter plate, by high flux screening (HTS) technology, the extensive compound library for drug target can be carried out
Screening analysis.Recently, HTS development trend is by the way that screening test device is miniaturized so that the reagent and sample of consumption
Less, R＆D costs are reduced.It is miniaturized in chip system, only several nanoliters of samples need to be used in each test cell, thus often puts down
1000 samples can be screened in square centimetre of test zone.
Microchannel analysis system a series of enzyme levels experiment in be widely used, from microchannel as enzyme-substrate-
The reaction vessel of inhibitor, but the component in this method reaction system is too many, and complicated course of reaction can inactivate enzyme, will lead
Cause the selection result degree of accuracy of enzyme inhibitor not high；And be only capable of adding a kind of inhibitor per secondary response, filter out suitable suppression
Agent needs to carry out more secondary responses.
The content of the invention
Goal of the invention：The invention provides a kind of new method that protein inhibitor is screened using micro-reaction device.
Technical scheme：A kind of method that protein inhibitor is screened using micro-reaction device of the present invention, including following step
Suddenly：
(1) the first solution and the second solution are pumped into the micro-mixer in micro-reaction device simultaneously respectively, are sufficiently mixed
The first microreactor being passed through afterwards in micro-reaction device carries out reaction and forms compound library；
(2) by the second microreactor being passed into micro-reaction device of the first microreactor, second micro- reaction
The nano particle of filling area load albumen in device；
(3) discharging of the first microreactor and the discharging of the second microreactor detect simultaneously contrasting detection result, the
The material that the discharging of two microreactors disappears relative to the discharging of the first microreactor is protein inhibitor.
The present invention lays special stress on protecting the general screening technique of protein inhibitor, and the compound library formed in the step (1) takes
The species of target protein certainly in step (2), a kind of compound of inhibitory action, ability can be theoretically produced to target protein
Solute in the solution of compound library reasonable selection first and the second solution that field technique personnel can build as needed.
Preferably, Mass Spectrometer Method or gas chromatographic detection are detected as in the step (3).
The albumen is lysozyme, and first solution is dissolved in volume fraction by aminated compounds and aldehyde compound and is
Prepare and obtain in 5~15% DMSO aqueous solution, second solution by sodium cyanoborohydride be dissolved in volume fraction for 5~
Prepare and obtain in the 15% DMSO aqueous solution.Wherein, aminated compounds is the combination of a kind of compound or multiple compounds；Wherein
Aldehyde compound is the combination of a kind of compound or multiple compounds.Preferably, the solvent of first solution and the second solution
Middle DMSO volume fraction is identical.
Preferably, aminated compounds is cyclopropylamine, cyclohexylamine, aniline and 2-Acetamido-2-deoxy-D-glucose in first solution
In any one or a few, the concentration of any of which component is 10~25mM；The aldehyde compound be propionic aldehyde, isopropyl aldehyde,
In benzaldehyde, p-bromobenzaldehyde, P-methoxybenzal-dehyde, p-tolyl aldehyde and m-methoxybenzaldehyde any one or it is several
Kind, the concentration of any of which component is 5~15mM；Aminated compounds and aldehyde compound are individually prepared, and then pass through pump respectively
Aminated compounds and aldehyde compound are formed into the first solution, by the flow velocity of controlling pump, the total moles of the aminated compounds
The ratio between integral molar quantity of amount and the aldehyde compound is 1~1.5:1；The concentration of sodium cyanoborohydride is in second solution
50~150mM；Sodium cyanoborohydride rubs in the integral molar quantity of aldehyde compound and second solution in first solution
The ratio between your amount is 1:1~1.5.
It is highly preferred that the molar concentration of aminated compounds any component is identical in first solution, the aldehydes chemical combination
The molar concentration of thing any component is identical.
Reaction temperature in first microreactor is room temperature, and reaction time is 1~2h；Second micro- reaction
Reaction temperature in device is 15~35 DEG C, and reaction time is 30~60min.
The albumen is vascular endothelial cell growth factor R-2, and first solution is dissolved in body by aldehyde compound
Fraction is to prepare to obtain in 5~15% DMSO aqueous solution, wherein described aldehyde compound is any one in chemical compounds I
Kind is several；Second solution is dissolved in the DMSO aqueous solution that volume fraction is 5~15% by aminated compounds with obtained
Arrive, wherein described aminated compounds is any one or a few in compound ii 1~II 27；
Preferably, DMSO volume fraction is identical in the solvent of first solution and the second solution.
Preferably, the concentration of any component is 5~15mM in the aldehyde compound；Any group in the aminated compounds
The concentration divided is 5~15mM；The ratio between the integral molar quantity of the aldehyde compound and the integral molar quantity of aminated compounds are 1:1~
1.5, it is highly preferred that the molar concentration of any component is identical in first solution, any component rubs in second solution
Your concentration is identical.
The structural formula of the chemical compounds I of table 1
The structural formula of the compound ii of table 2
Reaction temperature in first microreactor is room temperature, and reaction time is 1~3h, second micro- reaction
Reaction temperature in device is 15~35 DEG C, and reaction time is 30~60min.
The albumen is bovine serum albumin(BSA), and first solution is dissolved in organic solvent A by fatty acid compound
Preparation obtains；Second solution is dissolved in prepare in organic solvent B by alcohol compound and obtained, and the organic solvent A and is had
Solvent B is identical, is n-hexane, pentane or normal heptane；First microreactor fills lipase.Wherein fatty acid
Compound is the combination of a kind of compound or multiple compounds, and alcohol compound is the combination of a kind of compound or multiple compounds.
Preferably, fatty acid compound is appointing in palmitic acid, oleic acid, stearic acid and linoleic acid in first solution
Meaning is one or more of, and the concentration of any of which component is 5~15mM；Alcohol compound is ethanol and positive third in second solution
Any one in alcohol or two kinds, the concentration of any of which component is 20~30mM；Fatty acid chemical combination in first solution
The ratio between integral molar quantity of alcohol compound is 1 in the integral molar quantity of thing and second solution:1~6.It is highly preferred that described
The molar concentration of any component is identical in one solution, and the molar concentration of any component is identical in second solution.
Reaction temperature in first microreactor is 25~60 DEG C, and reaction time is 10~30min；Described
Reaction temperature in two microreactors is 25~50 DEG C, and reaction time is 10~30min.
Described lipase is that Novi believes lipase 435, the lipase from antarctic candida, dwelt from thermophilic
The lipase of hot bacterium, the lipase from Mycotoruloides or the lipase from Pseudomonas fluorescens.
The preferred high resolution mass spectrum detection of the Mass Spectrometer Method, by comparing going out for the first microreactor and the second microreactor
The mass spectral results of material, the material to be disappeared from the discharging of the second microreactor is protein inhibitor.
The nano particle is α-Fe2O3、γ-Fe2O3、Fe3O4、TiO2Or SiO2Nano particle, preferably α-Fe2O3、γ-
Fe2O3Or Fe3O4Nano particle.
The micro-reaction device include be sequentially connected in series by connecting tube micro-mixer, the first microreactor and second it is micro- instead
Device is answered, the charging aperture of the micro-mixer is connected with the first liquor inlet and the second liquor inlet；First microreactor and
The capillary inner diameter of second microreactor is 1~3mm, and capillary volume is 1~5mL.
Preferably, the capillary inner diameter is 2~3mm.
Beneficial effect：(1) screening technique of protein inhibitor provided by the invention, the combination built by micro-reaction device
Component in thing storehouse is more, and the screening of inhibitor can be completed by continuous reaction；Need not be by single inhibitor and albumen
Matter, substrate mix to be screened respectively, not only saves reactions steps, also improves the degree of accuracy of screening, operation is more
Add simple；(2) screening technique of protein inhibitor provided by the invention, need not be carried out after the discharging of the second micro passage reaction
Separation, the differential screening of the Mass Spectrometer Method result to be discharged by comparing the first micro passage reaction and the second micro passage reaction press down
Preparation, save screening step；(3) present invention is continuous process using the screening of micro-reaction device progress protein inhibitor, work
Skill easy operation control, sample size is small and composition libraries component is more, and reaction time is short, and screening efficiency is high；(4) enzyme is filled in
In microreactor, the inactivation of enzyme can be avoided, improves the recycling performance of enzyme.
Brief description of the drawings
Fig. 1 is the process flow diagram that protein inhibitor is screened using micro-reaction device；
First microreactor discharging mass spectrogram during Fig. 2 BSA inhibitor screenings；
Second microreactor discharging mass spectrogram during Fig. 3 BSA inhibitor screenings.
Embodiment
See Fig. 1, the micro-reaction device includes micro-mixer 1, the and of the first microreactor 2 being sequentially connected in series by connecting tube
Second microreactor 3, the charging aperture of the micro-mixer are connected with the first liquor inlet 4 and the second liquor inlet 5.
Embodiment 1
Palmitic acid, oleic acid, stearic acid and linoleic acid are dissolved in n-hexane first, are designated as the first solution；By ethanol and just
Propyl alcohol is dissolved in n-hexane, is designated as the second solution.After Y type micro-mixers, injection is filled with promise for first solution and the second solution
In the first filling type micro-reactor (internal diameter 1mm, 3mL) of dimension letter lipase 435,15min is reacted at 30 DEG C, carries out esters
The combinatorial compound of compound；Then, α-Fe of the first filling type micro-reactor discharging injection filled with area load BSA2O3Nanometer
In the second filling type micro-reactor (internal diameter 1mm, 3mL) of grain, 10min is reacted at 25 DEG C.Wherein, palmitic acid, oleic acid, tristearin
Sour and linoleic concentration is 10mM, and the concentration of ethanol and normal propyl alcohol is 25mM, by controlling the first solution and second molten
The flow velocity of liquid so that the ratio between the integral molar quantity of aliphatic acid and the integral molar quantity of alcohol are 1:1.5.First filling type micro-reactor and
The discharging of two filling type micro-reactors carries out high resolution mass spectrum detection respectively, contrasts the discharging mass spectrum of two filling type micro-reactors
As a result (Fig. 2 and Fig. 3), the inhibitor for analyzing BSA are ethyl palmitate.
Embodiment 2
Method is with embodiment 1, the difference is that the concentration of fatty acid compound any component is 5mM in the first solution, the
The concentration of alcohol compound any component is 20mM in two solution, and the ratio between the integral molar quantity of aliphatic acid and the integral molar quantity of alcohol are 1:
3.5；Reaction temperature in first microreactor is 25 DEG C, reaction time 30min；Reaction temperature in second microreactor
Spend that for 25 DEG C, reaction time 30min, to analyze BSA inhibitor be ethyl palmitate.
Embodiment 3
Method is with embodiment 2, the difference is that the concentration of fatty acid compound any component is 15mM in the first solution, the
The concentration of alcohol compound any component is 30mM in two solution, and the ratio between the integral molar quantity of aliphatic acid and the integral molar quantity of alcohol are 1:
6；Reaction temperature in first microreactor is 60 DEG C, reaction time 10min；Reaction temperature in second microreactor
For 50 DEG C, reaction time 10min, the nano particle for loading BSA is γ-Fe2O3, the inhibitor for analyzing BSA is palm fibre
Ethyl gallate.
Embodiment 4
Method is with embodiment 2, the difference is that load BSA nano particle is Fe3O4, the inhibitor for analyzing BSA is palm
Acetoacetic ester.
Embodiment 5
Method is with embodiment 4, the difference is that the capillary inner diameter of the first microreactor and the second microreactor is 2mm, hair
Tubule volume is 5mL, and the inhibitor for analyzing BSA is ethyl palmitate.
Embodiment 6
Method is with embodiment 4, the difference is that the capillary inner diameter of the first microreactor and the second microreactor is 3mm, hair
Tubule volume is 1mL, and the inhibitor for analyzing BSA is ethyl palmitate.
Embodiment 7
Method, the difference is that lipase is the lipase from antarctic candida, analyzes BSA's with embodiment 6
Inhibitor is ethyl palmitate.
Embodiment 8
First by aminated compounds cyclopropylamine, cyclohexylamine, aniline and 2-Acetamido-2-deoxy-D-glucose and aldehyde compound third
Aldehyde, isopropyl aldehyde, benzaldehyde, p-bromobenzaldehyde, P-methoxybenzal-dehyde, p-tolyl aldehyde and m-methoxybenzaldehyde solution product
Fraction is in the 10% DMSO aqueous solution, is designated as the first solution；The concentration of each component is 25mM wherein in aminated compounds, aldehydes
The concentration of each component is 10mM in compound, the ratio between integral molar quantity of the integral molar quantity of aminated compounds and the aldehyde compound
For 1.5:1.Sodium cyanoborohydride is dissolved in prepare in the DMSO aqueous solution that volume fraction is 10% and obtains the second solution, cyano group
The concentration of sodium borohydride is 100mM.After Y type micro-mixers, the filling of injection first declines anti-for first solution and the second solution
Answer in device (internal diameter 1mm, 3mL), react 1h at room temperature, carry out the combinatorial compound of compound；Then, first the reaction that declines is filled
α-Fe of the device discharging injection filled with area load lysozyme2O3Nano particle the second filling type micro-reactor (internal diameter 1mm,
In 3mL), 45min is reacted at 25 DEG C.Pass through the flow velocity for controlling the first solution and the second solution so that aldehyde compound it is total
The ratio between mole and the mole of sodium cyanoborohydride are 1：1.2.First filling type micro-reactor and second fills the reaction that declines
The discharging of device carries out high resolution mass spectrum detection respectively, contrasts the discharging mass spectral results of two filling type micro-reactors, analyzes molten
The inhibitor of bacterium enzyme is compound III-compound V.
Embodiment 9
Method with embodiment 8, unlike in the first solution the concentration of aminated compounds each component be 10mM, aldehydes chemical combination
The concentration of thing each component is 15mM, and the concentration of sodium cyanoborohydride is 150mM in second solution, and aminated compounds always rubs
It is 1 that you, which measure the ratio between integral molar quantity with the aldehyde compound,:1, by the flow velocity for controlling the first solution and the second solution so that
The ratio between the integral molar quantity of aldehyde compound and the mole of sodium cyanoborohydride are 1:1.5；Reaction in first microreactor stops
It is 2h to stay the time；Reaction temperature in second microreactor is 35 DEG C, reaction time 30min, analyzes lysozyme
Inhibitor is the same as embodiment 8.
Embodiment 10
Method with embodiment 8, unlike in the first solution the concentration of aminated compounds each component be 10mM, aldehydes chemical combination
The concentration of thing each component is 5mM, and the concentration of sodium cyanoborohydride is 50mM in second solution, by control the first solution and
The flow velocity of second solution so that the ratio between the integral molar quantity of aldehyde compound and the mole of sodium cyanoborohydride are 1:1；First is micro-
Reaction time in reactor is 2h；Reaction temperature in second microreactor is 15 DEG C, and reaction time is
60min, the inhibitor of lysozyme is analyzed with embodiment 8.
Embodiment 11
The aldehyde compound shown in table 3 is dissolved in the DMSO aqueous solution that volume fraction is 10% first, it is molten to be designated as first
Liquid, the concentration of each component is 10mM；Aminated compounds shown in table 4 is dissolved in the DMSO aqueous solution that volume fraction is 10%,
The second solution is designated as, the concentration of each component is 10mM.First solution and the second solution are after Y type micro-mixers, injection first
In filling type micro-reactor (internal diameter 1mm, 3mL), 1h is reacted at room temperature, carries out the combinatorial compound of compound；Then, first fill out
Fill α-Fe of the reactor discharging injection filled with area load vascular endothelial cell growth factor R-2 that decline2O3Nano particle
In second filling type micro-reactor (internal diameter 1mm, 3mL), 45min is reacted at 25 DEG C.It is molten by the first solution of control and second
The flow velocity of liquid so that the ratio between the integral molar quantity of aldehyde compound and the integral molar quantity of aminated compounds are 1:1.5.First filled type
The discharging of microreactor and the second filling type micro-reactor carries out high resolution mass spectrum detection respectively, contrasts two and fills the reaction that declines
The discharging mass spectral results of device, analyze the inhibitor of vascular endothelial cell growth factor R-2 as compound VI (I -3 II -
2), compound VII (I -4 II -21), compound VIII (I -3 II -1), compound Ⅸ (I -6 II -27).
The structural formula of the chemical compounds I of table 3
The structural formula of the compound ii of table 4
Embodiment 12
Method with embodiment 11, unlike in the first solution the concentration of aldehyde compound each component be 5mM, the second solution
The concentration of middle aminated compounds each component is 5mM, by the flow velocity for controlling the first solution and the second solution so that aldehyde compound
Integral molar quantity and the ratio between the integral molar quantity of aminated compounds be 1:1.The reaction time of first microreactor is 3h, second
Reaction temperature in microreactor is 15 DEG C, reaction time 60min.Analyze vascular endothelial growth factor receptor
2 inhibitor is the same as embodiment 11.
Embodiment 13
Method is with embodiment 11, the difference is that the concentration of aldehyde compound each component is 15mM in the first solution, second is molten
The concentration of aminated compounds each component is 15mM in liquid, by the flow velocity for controlling the first solution and the second solution so that aldehydes
The ratio between the integral molar quantity of compound and the integral molar quantity of aminated compounds are 1:1.The reaction time of first microreactor is 3h,
Reaction temperature in second microreactor is 35 DEG C, reaction time 30min.Analyze vascular endothelial growth factor
The inhibitor of acceptor 2 is the same as embodiment 11.
Claims (10)
- A kind of 1. method that protein inhibitor is screened using micro-reaction device, it is characterised in that comprise the following steps：(1) the first solution and the second solution are pumped into the micro-mixer in micro-reaction device simultaneously respectively, led to after being sufficiently mixed Enter the first microreactor in micro-reaction device and carry out reaction to form compound library；(2) by the second microreactor being passed into micro-reaction device of the first microreactor, in second microreactor Fill the nano particle of area load albumen；(3) discharging of the first microreactor and the discharging of the second microreactor detect simultaneously contrasting detection result, second is micro- The material that the discharging of reactor disappears relative to the discharging of the first microreactor is the inhibitor of albumen described in step (2).
- 2. according to the method for claim 1, it is characterised in that the albumen is lysozyme, and first solution is by amine Compound and aldehyde compound are dissolved in prepare in the DMSO aqueous solution that volume fraction is 5~15% and obtained, second solution It is dissolved in prepare in the DMSO aqueous solution that volume fraction is 5~15% by sodium cyanoborohydride and is obtained.
- 3. according to the method for claim 2, it is characterised in that aminated compounds is cyclopropylamine, ring in first solution Any one or a few in hexylamine, aniline and 2-Acetamido-2-deoxy-D-glucose, the concentration of any of which component is 10~25mM；Institute It is propionic aldehyde, isopropyl aldehyde, benzaldehyde, p-bromobenzaldehyde, P-methoxybenzal-dehyde, p-tolyl aldehyde and a first to state aldehyde compound Any one or a few in epoxide benzaldehyde, the concentration of any of which component is 5~15mM；The aminated compounds always rubs It is 1~1.5 that you, which measure the ratio between integral molar quantity with the aldehyde compound,:1；The concentration of sodium cyanoborohydride in second solution For 50~150mM；Sodium cyanoborohydride in the integral molar quantity of aldehyde compound and second solution in first solution The ratio between mole is 1:1~1.5.
- 4. according to the method for claim 2, it is characterised in that the reaction temperature in first microreactor is room temperature, Reaction time is 1~2h；Reaction temperature in second microreactor is 15~35 DEG C, reaction time is 30~ 60min。
- 5. according to the method for claim 1, it is characterised in that the albumen is vascular endothelial cell growth factor R-2, First solution is dissolved in prepare in the DMSO aqueous solution that volume fraction is 5~15% by aldehyde compound and obtained, wherein institute The aldehyde compound stated is any one or a few in chemical compounds I；Second solution is dissolved in volume by aminated compounds Fraction is to prepare to obtain in 5~15% DMSO aqueous solution, wherein described aminated compounds is in compound ii 1~II 27 Any one or a few；Wherein Ar is Ar1~Ar11In any one or a few；
- 6. according to the method for claim 5, it is characterised in that in the aldehyde compound concentration of any component be 5~ 15mM；The concentration of any component is 5~15mM in the aminated compounds；The integral molar quantity and amine of the aldehyde compound The ratio between integral molar quantity of compound is 1:1~1.5.
- 7. according to the method for claim 5, it is characterised in that the reaction temperature in first microreactor is room temperature, Reaction time is 1~3h, and the reaction temperature in second microreactor is 15~35 DEG C, reaction time is 30~ 60min。
- 8. according to the method for claim 1, it is characterised in that the albumen is bovine serum albumin(BSA), first solution It is dissolved in prepare in organic solvent A by fatty acid compound and is obtained；Second solution is dissolved in organic by alcohol compound Prepare and obtain in solvent B, the organic solvent A is identical with organic solvent B；First microreactor fills lipase.
- 9. according to the method for claim 8, it is characterised in that the organic solvent A is n-hexane, pentane or positive heptan Alkane；Fatty acid compound is any one in palmitic acid, oleic acid, stearic acid and linoleic acid or several in first solution Kind, the concentration of any of which component is 5~15mM；Alcohol compound is any in ethanol and normal propyl alcohol in second solution One or two, the concentration of any of which component is 20~30mM；The total moles of fatty acid compound in first solution The ratio between integral molar quantity of alcohol compound is 1 in amount and second solution:1~6.
- 10. according to the method for claim 8, it is characterised in that reaction temperature in first microreactor for 25~ 60 DEG C, reaction time is 10~30min；Reaction temperature in second microreactor is 25~50 DEG C, and reaction stops Time is 10~30min.
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