CN107475211A - A kind of preparation of interface self assembly carbonyl reductase and the application in the synthesis of S licarbazepines - Google Patents
A kind of preparation of interface self assembly carbonyl reductase and the application in the synthesis of S licarbazepines Download PDFInfo
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Abstract
Application the invention discloses a kind of preparation of interface self assembly carbonyl reductase and in the synthesis of S licarbazepines, the interface self assembly carbonyl reductase are prepared as follows：Carbonyl reductase is added in buffer solution, add the toluene solution dissolved with polystyrene, under the conditions of dark, 80r/min, 30 DEG C, concussion reaction 2 hours in shaking table, centrifugation, acquisition upper strata is organic phase, and interface self assembly carbonyl reductase is distributed in oil-water interfaces, lower floor is the reaction system of aqueous phase, obtains interface self assembly carbonyl reductase；Self assembly carbonyl reductase in interface has preferable catalysis in oil-water two-phase interfaces, and Oxcarbazepine can be converted into S licarbazepines, and the Enantiomer excess value of S licarbazepines is more than 98%.In aqueous phase, coenzyme NAD H can smoothly realize in-situ regeneration, be advantageous to improve the conversion ratio of substrate.Self assembly enzyme in interface, which can be realized, to be used repeatedly, and this is also the incomparable advantage of resolvase.
Description
(1) technical field
The present invention relates to a kind of preparation of microbe-derived interface self assembly carbonyl reductase and its in antiepileptic vinegar
Application in sour Ai Si licarbazepines key chiral intermediate (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide synthesis.
(2) background technology
Interface self assembly enzyme is polymer-multienzyme complex, is that enzyme is combined to the enzyme for making to be modified with macromolecule polyalcohol to have
There is amphipathic structure, structure is similar to the surfactant based on protein, both containing hydrophilic protein head also
Containing hydrophobic polymer tail, when being placed in oil-water system, the polymer moieties in polymer-enzyme are stretched over oil phase, and
Enzyme is partly embedded in aqueous phase, and enzyme is fixed on to the chemical reaction being catalyzed on oil-water interface in water-oil phase.By carbonyl reductase
Interface self assembly carbonyl reductase is combined to form in suitable oil-water interfaces with polymer, and is applied to oil water two phase system
The asymmetric reduction of catalysis of carbonyl compound prepares chirality pharmaceutical intermediate compound for improving oil water two phase system living things catalysis efficiency
With positive meaning.
Selective carbonyl reductase can prepare chipal compounds with asymmetric reduction carbonyls.Carbonyl reduction
Enzyme is widely present in microbial body, directly using the latent chiral carbonyl chemical combination of the microorganism asymmetric reduction containing carbonyl reductase
Thing can prepare chiral alcohol.Microbial cell can generally play higher activity in aqueous, and organic substrates are water-soluble
It is relatively low, therefore that water-soluble relatively low substrate conversion efficiency is catalyzed in aqueous phase is relatively low for microbial cell.Using water/organic phase two-phase
The water-soluble relatively low substrate of system conversion is expected to improve transformation efficiency.The carbonyl reductase separation that will be present in microbial cell
Extract and be prepared into interface self assembly enzyme and be advantageously implemented the recycling of enzyme, and rapid conversion is realized in oil-water interfaces,
Be advantageous to improve the biotransformation efficiency of water insoluble substrates.
(S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide, English name：(S)-10-hydroxy-10,11-dihydro-5H-dibenz[b,f]azepine-
5-
Carboxamide, CAS accession number：104746-04-5, molecular formula C15H14N2O2, molecular weight 254.284.S- profit cards
Xiping is the synthesis precursor of acetic acid Ai Si licarbazepines, and the major active metabolite product of Oxcarbazepine.With Oxcarbazepine phase
Than, the tolerance of eslicarbazepine acetate is more preferable, available for or without Secondary cases generalized seizure adult epilepsy portion
Divide the treatment of property breaking-out, be a kind of novel voltage gated sodium channel inhibitor, by blocking the conduction of action potential, to suppress brain
The paradoxical discharge repeatedly of neuron, control epileptic attack.
Using Oxcarbazepine as substrate, using interface self assembly carbonyl reductase as catalyst, urged in oil/water two-phase system
(S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide can be converted into by Oxcarbazepine by changing reaction.The hydrophilic segment of interface self assembly enzyme is distributed in aqueous phase, hydrophobic portion
It is distributed in organic phase, interface self assembly enzyme is anchored at oil-water interfaces.Substrate Oxcarbazepine and product (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide point
In oil phase, substrate contacts and realizes catalytic reaction cloth with the interface self assembly enzyme of oil-water interfaces.Reduction reaction needs coenzyme NAD H
Hydrogen is provided for course of reaction, therefore, NADH is added in aqueous phase, while adds alcohol dehydrogenase and ethanol realizes NADH again
It is raw, endlessly hydrogen donor is provided for reduction reaction, reaction mechanism is as shown in Figure 1.
(3) content of the invention
It is an object of the present invention to provide a kind of interface self assembly carbonyl reductase, and using the interface self assembly enzyme in oil/water
Asymmetric reduction Oxcarbazepine prepares (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide in two-phase system, and new way is provided for the biosynthesis of (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide,
This method is completed to be advantageous to the separation and Extraction of substrate and product in oil/water two-phase system；Reaction condition is gentle；Interface self assembly
Enzyme can realize recycling, improve the biotransformation efficiency of substrate.
The technical solution adopted by the present invention is：
The present invention provides a kind of interface self assembly carbonyl reductase, and the interface self assembly carbonyl reductase is as follows
Prepare：Carbonyl reductase is added in pH 6.5,0.02M Tirs-HCl buffer solutions, add the first dissolved with polystyrene
Benzole soln, under the conditions of dark, 80r/min, 30 DEG C, concussion reaction 2 hours in shaking table, centrifuge (preferably by reaction solution
10000r/min centrifuges 10min), acquisition upper strata is organic phase, and interface self assembly carbonyl reductase is distributed in oil-water interfaces (in i.e.
Interbed is interface self assembly carbonyl reductase), lower floor is the reaction system of aqueous phase, will be distributed in the interface self assembly of oil-water interfaces
Enzyme is washed 3 times respectively with toluene and pH 7.0,0.02M Tris-HCl buffer solutions, obtains interface self assembly carbonyl reductase；Institute
State carbonyl reductase addition and 20~200U/ml (preferably 144U/ml), the toluene of the polystyrene are calculated as with buffer solution volume
Solution concentration is 1.0~4.0g/L (preferably 3.75g/L), and toluene solution and the buffer solution volume ratio of the polystyrene are 1-3:
1, preferably 1.6:1.
Further, the carbonyl reductase is prepared as follows：(1) by Bacillus anthracis CGMCC
No.12337 is scattered in pH 6.5,0.02M Tris-HCl buffer solutions, is placed in 0~4 DEG C of ice-water bath, in power 400W, is surpassed
Ultrasonication 20min under conditions of sound 2s, interval 4s, mixed liquor 8000r/min centrifugation 10min are crushed, supernatant is obtained, is
Crude enzyme liquid I；(2) crude enzyme liquid I is placed in ice-water bath and is cooled to 0~4 DEG C in advance, be slowly added to ammonium sulfate powder to sulphur while stirring
Sour ammonium quality saturation degree is 40%, and after ammonium sulfate is completely dissolved, ice-water bath is slowly stirred 1h, and 10min is centrifuged in 10000r/min,
Supernatant continues to add ammonium sulfate until quality saturation degree is 60%, and ice-water bath is slowly stirred 1h, is centrifuged in 10000r/min
10min must be precipitated, and gained precipitation is redissolved in the pH 6.5 of 1~2 times of precipitation volume, 0.02M Tris-HCl buffer solutions
In, obtain crude enzyme liquid II；(3) crude enzyme liquid II is added in 10kD Milipore ultra-filtration centrifuge tubes, in 3500r/min, 4 DEG C of bar
Centrifuge 20min under part, the concentrate in sleeve pipe is carbonyl reductase enzyme liquid, enzyme activity 180U/mg.
The present invention also provides a kind of interface self assembly carbonyl reductase and prepares S- profits in asymmetric reduction Oxcarbazepine
Application in oxcarbazepine.
Further, the application process is：Using interface self assembly carbonyl reductase as catalyst, using Oxcarbazepine the bottom of as
Thing, using NADH as coenzyme, using ethanol as cosubstrate, the in-situ regeneration that alcohol dehydrogenase is used for coenzyme NAD H is added, with pH3.0
The Tris-HCl/ toluene two-phase system that~8.0 Tris-HCl buffer solutions are formed with toluene is reaction medium, 20~40 DEG C,
React in 80r/min shaking table, after reaction completely, reaction solution is isolated and purified, obtain described (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide；The catalysis
Agent dosage is calculated as 5~150U/mL (preferably 36U/mL) with buffer solution volume, and substrate dosage is calculated as 1.98 with buffer solution volume~
9.52mmol/L (preferably 3.97mmol/L), the ethanol volumetric usage are 1~10% (preferably respectively in terms of buffer solution volume
6%)；The NADH dosages are calculated as 0.02~0.1mol/L (preferably 0.08mol/L), the alcohol dehydrogenase with buffer solution volume
(preferably 300U/mg) dosage is calculated as 0.01~0.4g/L, or 3000-120000U/L (preferably 6000U/ with buffer solution volume
L), the toluene and buffer solution volume ratio are 2.5-20:10 (preferably 12.5:10).
Further, after reaction terminates, reaction solution 10000r/min is centrifuged into 10min, can be obtained containing substrate after centrifugation
The organic phase of Oxcarbazepine and (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide, intermediate layer (self assembly enzyme in interface, which can be realized, to be used repeatedly) and aqueous phase,
The Tris-HCl buffer solutions obtained after centrifugation are extracted three times using equivalent ethyl acetate, and combining extraction liquid, rotary evaporation falls big portion
Merge after dividing ethyl acetate with organic phase, analysis detection is carried out using liquid chromatogram, further determines that pair of conversion ratio and product
Reflect the superfluous value of body.
Carbonyl reductase of the present invention comes from (Bacillus anthracis) CGMCC No.12337, and the strain is protected
China Committee for Culture Collection of Microorganisms's common micro-organisms collection is hidden in, positioned at Datun Road, Chaoyang District, Beijing City China
In institute of microbiology of the academy of sciences, preserving number CGMCC No.12337, preservation date on April 21st, 2016, in patent application
(CN2016105800977) disclosed in.
Compared with prior art, beneficial effect of the present invention is mainly reflected in：
Self assembly carbonyl reductase in interface has preferable catalysis in oil-water two-phase interfaces, can be by O'Casey flat turn
(S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide is turned to, the Enantiomer excess value of (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide is more than 98%.In aqueous phase, coenzyme NAD H can be realized smoothly
In-situ regeneration, be advantageous to improve the conversion ratio of substrate.Substrate and product are dissolved in organic phase, and catalytic reaction is entered in oil-water interfaces
OK, the later separation extraction of product is advantageous to.With the catalytic phase ratio of traditional resolvase in a two-phase system, it is possible to reduce enzyme activity is damaged
Lose, improve the catalytic efficiency of enzyme.Self assembly enzyme in interface, which can be realized, to be used repeatedly, and this is also incomparable excellent of resolvase
Gesture.Self assembly carbonyl reductase in interface plays catalysis in oil-water interfaces and provides new way for the synthesis of (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide.
(4) illustrate
Fig. 1 is reaction mechanism schematic diagram of the present invention.
(5) embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This：
Embodiment 1：The preparation of carbonyl reductase
(1) by Bacillus anthracis CGMCC No.12337 be scattered in 50ml Tris-HCl (0.02M,
PH6.5) in cushioning liquid, cell concentration is 2g/L (dry cell weight/buffer solution volume), is placed in ice-water bath (0~4 DEG C) and uses
Ultrasonic wave carries out clasmatosis.Ultrasonic probe is positioned over 1~1.5cm under bacteria suspension liquid level, power 400W, ultrasonic 2s, interval
4s, ultrasonication 20min.Sample after broken centrifuges 10min in 8000r/min centrifuge and obtains supernatant, is thick
Enzyme liquid I.
(2) crude enzyme liquid I is subjected to ammonium sulfate precipitation.Crude enzyme liquid I is placed in ice-water bath and is cooled to 0~4 DEG C in advance, gently
It is slowly added to be ground into the ammonium sulfate of fine powder while stirring, is 40% to ammonium sulfate quality saturation degree, ammonium sulfate is completely molten
Xie Hou, ice-water bath are slowly stirred 1h, in centrifuge 10000r/min centrifuge 10min, supernatant continue add ammonium sulfate until
Quality saturation degree is 60%, and ice-water bath is slowly stirred 1h, and 10000r/min centrifuges 10min and must precipitated in centrifuge.By gained
Precipitation is redissolved in Tris-HCl (0.02M, pH6.5) cushioning liquid of 1~2 times of precipitation volume, obtains crude enzyme liquid II.
(3) crude enzyme liquid II is concentrated by ultrafiltration.Crude enzyme liquid II is added in 10kD Milipore ultra-filtration centrifuge tubes,
3500r/min, centrifuges 20min under conditions of 4 DEG C, and the concentrate in sleeve pipe is carbonyl reduction enzyme liquid, enzyme activity 180U/mg.
The assay method of carbonyl reduction protein content of the present invention is：Using Bradford methods, with cow's serum
Albumin is as standard protein.
The assay method of carbonyl reductase enzyme activity of the present invention is：Enzyme activity be defined as 25 DEG C under the conditions of 1 point
Catalysis 1umol NADH generations NAD in clock+The amount of required carbonyl reductase.100 μ L enzyme is added to containing 0.25mmol/
It is anti-under the conditions of 25 DEG C in the 5ml of L NADH and 0.25mmol/L acetophenones, pH7.9,50mmol/L Tris-HCl buffer solution
Answer 5min.Detect absorbance value of the sample at 340nm.Enzyme activity uses formula (1), is carried out with respect to enzyme activity using formula (2)
Calculate.
Activity (U)=E × V × 103/(6220×1) (1)
Specific activity of enzyme (U/mg)=Activity/Protein content (2)
Activity represents the vigor of enzyme.E represents the change of 1min absorbances at 340nm.V represents the body of reaction solution
Product.6220 represent molar absorption coefficient (Lmol-1·cm-1).1 represents light path (cm).
The preparation and application of the interface self assembly carbonyl reductase of embodiment 2
(1) preparation of interface self assembly carbonyl reductase
5mg carbonyl reductases enzyme liquid (180U/mg) prepared by embodiment 1 adds 6.25ml, pH 7.0,0.02M
0.8g/L enzyme liquid is configured in Tris-HCl cushioning liquid, it is molten to add 3.75g/L polystyrene (Mw 10000Da) toluene
Liquid 10ml, reacted 1 hour under the conditions of dark, shaking speed 80r/min, 30 DEG C.1200r/min is centrifuged after the completion of reaction
10min, interface self assembly enzyme be distributed in water and organic phase two-phase interface (i.e. upper strata is toluene, and centre is interface self assembly enzyme,
Lower floor is Tris-HCl cushioning liquid).Buffer solution and organic solvent are removed, continuously uses toluene and pH 7.0,0.02M Tris-
HCl cushioning liquid washs two-phase interface 3 times respectively, removes free carbonyl reductase and polystyrene, obtains interface self assembly
Carbonyl reductase 2mg, surface area 16.6cm2, Rate activity 150U/mg.
The assay method of self assembly carbonyl reduction protein content in interface of the present invention is：Interface self assembly carbonyl reduction
The assay method of the protein content of enzyme is calculated using the method for material balance.Free carbonyl reduction for interface self assembly
The difference remained in after the protein content of enzyme and interface self assembly between the protein content in phosphate buffer is by interface
The protein content of self assembly carbonyl reductase.Protein content is measured using Bradford methods.
The assay method of self assembly carbonyl reduction enzyme activity in interface of the present invention is：Interface self assembly carbonyl reductase
It is added to the Tris-HCl bufferings of 5mL, pH7.9,50mmol/L containing 0.25mmol/L NADH and 0.25mmol/L acetophenones
In liquid, light absorbs of the detection sample at 340nm.Enzyme activity is calculated using the formula of embodiment 1 (1) and (2).
(2) asymmetric reduction of Oxcarbazepine
It is separately added into six 100ml conical flasks with buffer solution stereometer 0.08mol/L NADH, 0.2mg alcohol dehydrogenases
It is prepared by enzyme (enzyme activity 300U/mg), 10ml Tris-HCl (0.02mol/L, pH7.0) buffer solution, 0.6ml ethanol and step (1)
Interface self assembly carbonyl reductase 2mg (180U/mg).Be separately added into above-mentioned six conical flasks 2.5ml, 5ml, 10ml,
12.5ml, 15ml and 20ml toluene, then be separately added into buffer solution stereometer 3.97mmol/L Oxcarbazepines, in 30 DEG C, 80r/
6h is reacted in min shaking table.After reaction terminates, reaction solution 10000r/min is centrifuged into 10min, is divided into three layers, upper strata is organic
Phase (toluene), intermediate layer are interface self assembly enzyme, and lower floor is buffer solution, and toluene phase and Tris-HCl buffering liquid phases can be realized very
Good separation.The organic phase containing substrate Oxcarbazepine and (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide can be obtained after centrifugation.The Tris- obtained after centrifugation
HCl buffer solutions are extracted three times using equivalent ethyl acetate, combining extraction liquid, rotary evaporation fall after ethyl acetate by sample with it is organic
Mutually merge, further analyzed using HPLC and merge the content of Oxcarbazepine and (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide in sample, determine conversion ratio and S-
The Enantiomer excess value of licarbazepine.
HPLC testing conditions：Chiral chromatographic column OD-H (4.6mm × 25cm × 5 μm；Daicel,Japan).It is by content
0.05%THF n-hexane and isopropanol by volume 98:2 ratio prepares mobile phase.Detection wavelength is 210nm, mobile phase
Flow velocity is 0.8ml/min, and column temperature is 25 DEG C, and sample size is 10 μ L.
The toluene of table 1 compares the influence of reduction reaction with Tris-HCl volume of buffer solution
The volume ratio of organic solvent and buffer solution affects the biological transformation ratio of Oxcarbazepine in biotransformation.Table 1
Show, when organic solvent and buffer solution volume ratio are less than 12.5:When 10, the mapping of the conversion ratio and (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide of reaction
Body surplus value improves with the increase of volume ratio.When the volume ratio of optimal organic solvent and buffer solution is 12.5:When 10, instead
The conversion ratio and the Enantiomer excess value of (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide answered are respectively 95.6% and 99.6%.
Embodiment 3：
(1) preparation of interface self assembly enzyme：Method is same as Example 1.
(2) asymmetric reduction of Oxcarbazepine：It is separately added into five 100ml conical flasks with buffer solution stereometer concentration
Respectively 0.02,0.04,0.06,0.08 and 0.10mol/L NADH, then 0.2mg ethanol is separately added into each conical flask
Dehydrogenase (enzyme activity 300U/mg), 10ml Tris-HCl (0.02mol/L, pH7.0) buffer solution, 0.6ml ethanol and step (1)
The interface self assembly carbonyl reductase 2mg (180U/mg) of preparation, 12.5ml toluene, using buffer solution stereometer concentration as
3.97mmol/L Oxcarbazepines carry out bioconversion reaction.6h is reacted in 30 DEG C, 80r/min shaking table., will after reaction terminates
Reaction solution 10000r/min centrifuges 10min, and toluene phase and Tris-HCl buffering liquid phases can realize good separation.Can after centrifugation
To obtain the organic phase containing substrate Oxcarbazepine and (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide.The Tris-HCl buffer solutions obtained after centrifugation use equivalent
Ethyl acetate extracts three times, and combining extraction liquid, rotary evaporation merges sample with organic phase after falling most of ethyl acetate.Using
HPLC, which is further analyzed, merges the content of Oxcarbazepine and (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide in sample, determines pair of conversion ratio and (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide
Reflect the superfluous value of body.
Influence of the table 2NADH addition to reduction reaction
NADH addition influences the biological transformation ratio of Oxcarbazepine.With the increase of NADH additions, conversion ratio improves.
Preferable NADH additions are 0.08mol/L.NADH additions number the Enantiomer excess value of (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide is influenceed not
Greatly, the Enantiomer excess value of (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide is maintained at more than 99.6%.
Embodiment 4：
(1) preparation of interface self assembly enzyme：Method is same as Example 1.
(2) asymmetric reduction of Oxcarbazepine：It is separately added into six 100ml conical flasks with buffer solution stereometer concentration
For 0.08mol/L NADH, 0.2mg alcohol dehydrogenases (enzyme activity 300U/mg), 10ml Tris-HCl (0.02mol/L, pH7.0)
Interface self assembly carbonyl reductase 2mg (180U/mg) prepared by buffer solution and step (1), 12.5ml toluene, with buffer solution volume
Meter concentration is 3.97mmol/L Oxcarbazepines.To be separately added into six conical flasks with buffer solution stereometer concentration be respectively 2%,
3%th, 4%, 5%, 6% and 7% ethanol, 6h is reacted in 30 DEG C, 80r/min shaking table.After reaction terminates, by reaction solution
10000r/min centrifuges 10min, and toluene phase and Tris-HCl buffering liquid phases can realize good separation.It can be obtained after centrifugation
Organic phase containing substrate Oxcarbazepine and (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide.The Tris-HCl buffer solutions obtained after centrifugation use equivalent acetic acid second
Ester extracts three times, and combining extraction liquid, rotary evaporation merges sample with organic phase after falling most of ethyl acetate.Entered using HPLC
The analysis of one step merges the content of Oxcarbazepine and (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide in sample, determines the enantiomer mistake of conversion ratio and (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide
Surplus value.
Influence of the addition of the ethanol of table 3 to reduction reaction
Ethanol is converted into acetaldehyde in the presence of alcohol dehydrogenase, and the NADH of generation is provided endlessly for reduction reaction
Coenzyme, realize the regeneration of coenzyme.Suitable ethanol addition is favorably improved conversion ratio.Optimal ethanol addition is 6%.
In the case of the various additions of ethanol, the Enantiomer excess value of (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide keeps stable, is maintained at 99% or so.
Embodiment 5：
(1) preparation of interface self assembly enzyme：Method is same as Example 1.
(2) asymmetric reduction of Oxcarbazepine：It is separately added into five 100ml conical flasks with buffer solution stereometer concentration
For 0.08mol/L NADH, 0.2mg alcohol dehydrogenases (enzyme activity 300U/mg), 10ml Tris-HCl (0.02mol/L pH7.0)
Interface self assembly carbonyl reductase 2mg (180U/mg) prepared by buffer solution and step (1), using buffer solution stereometer concentration as 6%
Ethanol, 12.5ml toluene.Be separately added into six conical flasks with buffer solution stereometer concentration be respectively 1.98mmol/L,
2.38mmol/L, 3.97mmol/L, 6.35mmol/L and 7.93mmol/L Oxcarbazepine carry out bioconversion reaction.30 DEG C,
6h is reacted in 80r/min shaking table.After reaction terminates, reaction solution 10000r/min is centrifuged into 10min, toluene phase and Tris-HCl
Buffering liquid phase can realize good separation.It can be obtained after centrifugation organic containing substrate Oxcarbazepine and (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide
Phase.The Tris-HCl buffer solutions obtained after centrifugation are extracted three times using equivalent ethyl acetate, and combining extraction liquid, rotary evaporation falls greatly
Sample is merged with organic phase after portion of ethyl acetate.Further analyzed using HPLC and merge Oxcarbazepine and S- profit cards in sample
The content in Xiping, determine the Enantiomer excess value of conversion ratio and (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide.
Influence of the addition of the Oxcarbazepine of table 4 to reduction reaction
With the increase of substrate Oxcarbazepine addition, conversion ratio gradually reduces.Increased mainly due to concentration of substrate, bottom
Thing there may be inhibitory action to enzymatic activity.Optimal substrate addition is 3.97mmol/L.Substrate addition is to product S- profits
The influence of oxcarbazepine Enantiomer excess value is little, and in the case of various substrate additions, the enantiomer of (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide is superfluous
Value is held in more than 99%.
Embodiment 6：
(1) preparation of interface self assembly enzyme：Method is same as Example 1.
(2) asymmetric reduction of Oxcarbazepine：It is separately added into five 100ml conical flasks with buffer solution stereometer concentration
For 0.08mol/L NADH, 0.2mg alcohol dehydrogenases (enzyme activity 300U/mg), 10ml Tris-HCl (0.02mol/L, pH7.0)
Interface self assembly carbonyl reductase 2mg (180U/mg) prepared by buffer solution and step (1), using buffer solution stereometer concentration as 6%
Ethanol, 12.5ml toluene, using buffer solution stereometer concentration as 3.97mmol/L Oxcarbazepines, five conical flasks are individually positioned in
20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C and 40 DEG C, 6h is reacted in 80r/min shaking table.After reaction terminates, by reaction solution 10000r/min
10min is centrifuged, toluene phase and Tris-HCl buffering liquid phases can realize good separation.It can be obtained after centrifugation difficult to understand containing substrate
The organic phase of oxcarbazepine and (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide.The Tris-HCl buffer solutions obtained after centrifugation are using equivalent ethyl acetate extraction three
Secondary, combining extraction liquid, rotary evaporation merges sample with organic phase after falling most of ethyl acetate.Further analyzed using HPLC
Merge the content of Oxcarbazepine and (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide in sample, determine the Enantiomer excess value of conversion ratio and (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide.
Influence of the reaction temperature of table 5 to reduction reaction
Temperature has a great influence to the conversion ratio of reduction reaction.Temperature not only affects the stability and activity of enzyme, has an effect on
The balance of reaction.After temperature is more than 30 DEG C, the Enantiomer excess value of conversion ratio and (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide declines.It is most suitable
Temperature is 30 DEG C.
Embodiment 7：
(1) preparation of interface self assembly enzyme：Method is same as Example 1.
(2) asymmetric reduction of Oxcarbazepine：It is separately added into six 100ml conical flasks with buffer solution stereometer concentration
For 0.08mol/L NADH, 0.2mg alcohol dehydrogenases (enzyme activity 300U/mg), 10ml Tris-HCl (0.02mol/L pH7.0)
Interface self assembly carbonyl reductase 2mg (180U/mg) prepared by buffer solution and step (1), using buffer solution stereometer concentration as 6%
Ethanol, 12.5ml toluene, using buffer solution stereometer concentration as 3.97mmol/L Oxcarbazepines, six conical flasks are placed on 30
DEG C, react 3h, 4h, 5h, 6h, 8h and 10h respectively in 80r/min shaking table.Reaction terminate after, by reaction solution 10000r/min from
Heart 10min, toluene phase and Tris-HCl buffering liquid phases can realize good separation.It can be obtained containing substrate oka after centrifugation
Xiping and the organic phase of (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide.The Tris-HCl buffer solutions obtained after centrifugation are extracted three times using equivalent ethyl acetate,
Combining extraction liquid, rotary evaporation merge sample with organic phase after falling most of ethyl acetate.Conjunction is further analyzed using HPLC
And the content of Oxcarbazepine and (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide in sample, determine the Enantiomer excess value of conversion ratio and (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide.
Influence of the reaction time of table 6 to reduction reaction
As a result show, with the extension in reaction time, biological transformation ratio gradually steps up, when reacted between be more than 6 hours
Conversion ratio is improved less, it is thus determined that optimum reacting time is 6h.Shadow of the reaction time to (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide Enantiomer excess value
Ring less, be held in more than 99%.
Embodiment 8：
(1) preparation of interface self assembly enzyme：Method is same as Example 1.
(2) asymmetric reduction of Oxcarbazepine：It is separately added into a 100ml conical flask with buffer solution stereometer concentration
For 0.08mol/L NADH, 0.2mg alcohol dehydrogenases (enzyme activity 300U/mg), 10ml Tris-HCl (0.02mol/L pH7.0)
Interface self assembly carbonyl reductase 2mg (180U/mg) prepared by buffer solution and step (1), using buffer solution stereometer concentration as 6%
Ethanol, 12.5ml toluene, using buffer solution stereometer concentration as 3.97mmol/L Oxcarbazepines, it is placed on 30 DEG C, 80r/min shakes
6h is reacted respectively in bed.After reaction terminates, reaction solution 10000r/min is centrifuged into 10min, toluene phase and Tris-HCl buffer solutions
Good separation mutually can be realized.The interface self assembly enzyme regained is dispersed under above-mentioned reaction condition again to be reused
Interface self assembly enzyme 9 times.Terminate per secondary response, can be obtained after centrifugation organic containing substrate Oxcarbazepine and (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide
Phase.The Tris-HCl buffer solutions obtained after centrifugation are extracted three times using equivalent ethyl acetate, and combining extraction liquid, rotary evaporation falls greatly
Sample is merged with organic phase after portion of ethyl acetate.Further analyzed using HPLC and merge Oxcarbazepine and S- profit cards in sample
The content in Xiping, determine the Enantiomer excess value of conversion ratio and (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide.
Influence of the reaction time of table 7 to reduction reaction
As a result show, self assembly enzyme in interface reuses 5 times and still maintains good vigor.The process of recycling
In, the Enantiomer excess value of product (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide is maintained at 99.6%.
Claims (7)
1. a kind of interface self assembly carbonyl reductase, it is characterised in that the interface self assembly carbonyl reductase is made as follows
It is standby：Carbonyl reductase is added in pH 6.5,0.02M Tirs-HCl buffer solutions, add the toluene dissolved with polystyrene
Solution, under the conditions of dark, 80r/min, 30 DEG C in shaking table concussion reaction 2 hours, reaction solution is transferred in centrifuge tube
1000rpm centrifuges 20min, forms the body that upper strata is organic phase, intermediate layer is interface self assembly carbonyl reductase, lower floor is aqueous phase
System, aqueous phase and organic phase are removed, obtain interface self assembly carbonyl reductase；The carbonyl reductase addition is with buffer solution volume
20~200U/ml is calculated as, the toluene solution concentration of the polystyrene is 1.0~4.0g/L, and the toluene of the polystyrene is molten
Liquid and buffer solution volume ratio are 1-3:1.
2. self assembly carbonyl reductase in interface as claimed in claim 1, it is characterised in that the carbonyl reductase enzyme activity is 180U/
mg。
3. self assembly carbonyl reductase in interface as claimed in claim 1, it is characterised in that the carbonyl reductase is as follows
Prepare：(1) Bacillus anthracis CGMCC No.12337 are scattered in pH 6.5,0.02M Tris-HCl buffer solutions
In, it is placed in 0~4 DEG C of ice-water bath, the ultrasonication 20min under conditions of power 400W, ultrasonic 2s, interval 4s, crushes mixing
Liquid 8000r/min centrifuges 10min, obtains supernatant, as crude enzyme liquid I；(2) crude enzyme liquid I is placed in ice-water bath be cooled to 0 in advance~
4 DEG C, it is 40% that ammonium sulfate powder to ammonium sulfate quality saturation degree is slowly added to while stirring, after ammonium sulfate is completely dissolved, ice
Water-bath is slowly stirred 1h, centrifuges 10min in 10000r/min, supernatant continues to add ammonium sulfate until quality saturation degree is
60%, ice-water bath is slowly stirred 1h, must be precipitated in 10000r/min centrifugations 10min, and gained precipitation is redissolved in into 1~2 times
In the pH 6.5 of precipitation volume, 0.02M Tris-HCl buffer solutions, crude enzyme liquid II is obtained；(3) crude enzyme liquid II is added into 10kD
In Milipore ultra-filtration centrifuge tubes, 20min is centrifuged under conditions of 3500r/min, 4 DEG C, the concentrate in sleeve pipe is carbonyl
Reductase enzyme liquid.
4. self assembly carbonyl reductase in interface as claimed in claim 1, it is characterised in that the carbonyl reductase addition is with slow
Fliud flushing volume is calculated as 144U/ml, and the toluene solution concentration of the polystyrene is 3.75g/L, and the toluene of the polystyrene is molten
Liquid is 1.6 with buffer solution volume ratio:1.
5. self assembly carbonyl reductase in interface prepares (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide in asymmetric reduction Oxcarbazepine described in a kind of claim 1
In application.
6. application as claimed in claim 5, it is characterised in that the application process is：Using interface self assembly carbonyl reductase as
Catalyst, using Oxcarbazepine as substrate, using NADH as coenzyme, using ethanol as cosubstrate, add alcohol dehydrogenase and be used for coenzyme
NADH in-situ regeneration, the Tris-HCl/ toluene two-phase systems formed with the Tris-HCl buffer solutions of pH3.0~8.0 and toluene
For reaction medium, reacted in 20~40 DEG C, 80r/min shaking table, after reaction completely, reaction solution is isolated and purified, obtains institute
The (S)-(+)-10,11-dihydro-10-hydroxy-5H dibenz/b,f/azepine-5-carboxamide stated；The catalyst amount is calculated as 5~150U/mL with buffer solution volume, and substrate dosage is with buffer solution volume
1.98~9.52mmol/L is calculated as, the ethanol volumetric usage is 1~10% respectively in terms of buffer solution volume；The NADH dosages
0.02~0.1mol/L is calculated as with buffer solution volume, the alcohol dehydrogenase enzyme dosage is calculated as 3000- with buffer solution volume
120000U/L, the toluene and buffer solution volume ratio are 2.5-20:10.
7. application as claimed in claim 6, it is characterised in that the catalyst amount is calculated as 36U/mL, bottom with buffer solution volume
Thing dosage is calculated as 3.97mmol/L with buffer solution volume, and the ethanol volumetric usage is calculated as 6% with buffer solution volume；The NADH
Dosage is calculated as 0.08mol/L with buffer solution volume, and the alcohol dehydrogenase enzyme dosage is calculated as 6000U/L with buffer solution volume, described
Toluene is to select 12.5 with buffer solution volume ratio:10.
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| CN108998430A (en) * | 2018-06-29 | 2018-12-14 | 浙江工业大学 | A kind of interface self assembly carbonyl reductase and the application in the synthesis of (R) -3- hydroxyl -3- phenylpropionate |
| CN114958937A (en) * | 2022-05-12 | 2022-08-30 | 黄冈人福药业有限责任公司 | Synthesis process of eslicarbazepine acetate and intermediate thereof |
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| CN106190910A (en) * | 2016-07-19 | 2016-12-07 | 浙江工业大学 | Bacillus anthracis and the application in preparing S licarbazepine thereof |
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| CN106190910A (en) * | 2016-07-19 | 2016-12-07 | 浙江工业大学 | Bacillus anthracis and the application in preparing S licarbazepine thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108998430A (en) * | 2018-06-29 | 2018-12-14 | 浙江工业大学 | A kind of interface self assembly carbonyl reductase and the application in the synthesis of (R) -3- hydroxyl -3- phenylpropionate |
| CN108998430B (en) * | 2018-06-29 | 2020-11-13 | 浙江工业大学 | Interface self-assembly carbonyl reductase and application thereof in synthesis of (R) -3-hydroxy-3-ethyl phenylpropionate |
| CN114958937A (en) * | 2022-05-12 | 2022-08-30 | 黄冈人福药业有限责任公司 | Synthesis process of eslicarbazepine acetate and intermediate thereof |
| CN114958937B (en) * | 2022-05-12 | 2023-12-29 | 黄冈人福药业有限责任公司 | Eslicarbazepine acetate and synthesis process of intermediate thereof |
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