CN110218718A - Immobilization Ketoreductase mutant and its preparing the application in Nebivolol chirality alcohol intermediate and the like - Google Patents
Immobilization Ketoreductase mutant and its preparing the application in Nebivolol chirality alcohol intermediate and the like Download PDFInfo
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- CN110218718A CN110218718A CN201910529412.7A CN201910529412A CN110218718A CN 110218718 A CN110218718 A CN 110218718A CN 201910529412 A CN201910529412 A CN 201910529412A CN 110218718 A CN110218718 A CN 110218718A
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
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Abstract
Immobilization Ketoreductase mutant and its preparing the application in Nebivolol chirality alcohol intermediate and the like.The present invention provides immobilization Ketoreductase mutants, which is characterized in that the immobilization Ketoreductase mutant is the immobilized cell or immobilised enzymes of Ketoreductase mutant, and immobilised enzymes is that the mutant is immobilized on solid phase support object；Immobilized cell is that the Ketoreductase mutant is expressed in the microbial cell being immobilized on solid phase support object, and the Ketoreductase mutant is selected from ET016, ET017 or ET031 ketoreductase.The present invention has the following technical effect that immobilization Ketoreductase mutant 1) of the invention, it can be used as catalyst, it is provided commonly for preparing Nebivolol chirality alcohol intermediate and the like with Cofactor Regeneration Systems, reaction conversion ratio is up to 90%, 95% or 99% or more, and the chiral purity angle value of product is up to 90%, 95% or 99% or more.2) enzyme after immobilization or cell can be recycled by simply filtering, and it can be used repeatedly 7-10 times after recycling, and while reusing is essentially identical with the reaction conversion ratio and chiral purity angle value that use for the first time.
Description
Technical field
The present invention relates to a kind of immobilization Ketoreductase mutant and its prepare Nebivolol chirality alcohol intermediate and its
Application in analog belongs to bio-enzyme engineering and microbe application field.
Background technique
Nebivolol hydrochloric acid [nebivolol hydrochloride, bis- [2- (6- fluorine benzodihydropyran -2- base) -2-
Hydroxyethyl] amine hydrochlorate] be to be researched and developed by Johnson Co., be a kind of hemangiectasis activity 1 adrenaline of selective β by
Body antagonist also can be used for the treatment of angina pectoris and congestive heart failure for the treatment of mild to moderate hypertensive patient, tool
The features such as effective in cure significant, convenient to take, adverse reaction is small.9.3 hundred million dollars of global marketing volume in 2016, global marketing in 2017
11.3 hundred million dollars of volume.
Nebivolol has 16 kinds of chiral enantiomters, and research finds, thus NB-8 best with (S, R, R, R) curative effect
The preparation of (S, S)/NB-8 (R, S)/NB-8 (R, R)/four kinds of configuration chirality alcohol intermediates of NB-8 (S, R) is that nebivolol hydrochloric acid closes
At committed step.
The synthesis of S- chirality alcohol intermediate at present mainly has two kinds of approach of chemical synthesis and enzymatic clarification.Chemical synthesis uses
Chemical resolution prepares chiral intermediate with chemical asymmetric syntheses, and both methods has problems, such as chemical resolution
It needs using a large amount of resolving agent, reaction step is long, and energy consumption is high and waste discharge amount is big etc.；Based on transition metal chiral ligand
The problems such as catalytic hydrogenation system, there is optical purity of products is not high and heavy-metal residual, cause yield too low, cost occupies high
Under not.The patent of nebivolol hydrochloric acid will expire in the year two thousand twenty, bulk pharmaceutical chemicals price can test again and again, traditional chemical route of synthesis because
Environmental protection and cost problem are by extreme shock.
The research report of enzymatic clarification chirality alcohol intermediate is less.Initially transition-metal catalyst is replaced using lipase
Come the problems such as carrying out chiral resolution, be greatly reduced although polluting, can not be utilized there is also fractionation low efficiency, another enantiomer.
Follow-up study is carried out chiral reduction using ketoreductase and prepares chiral alcohol intermediate.However it is domestic do not have also at present can be used for work
The research report for the enzyme and its mutant that industry metaplasia produces.
Enzyme or immobilized cell technique are by cell or enzyme solution fixation support material containing enzyme, by enzyme constraint, limit
System is in certain space or connects on a support material, retains its catalytic activity, and a kind of skill that is recyclable and reusing
Art.Such method can recycling uses in multiple times again after the reaction by expensive enzyme product, to reduce the use of enzyme
Cost.The fixation support material of cell or zymoprotein due to being connected to larger particle after immobilization simultaneously, it is industrially difficult to make
In the small molecules such as the enzyme of filtering and cell, it is condensed into the bulky grain for being easy to filter, thus so very big that reduce industrialization filtering
Operation difficulty and corresponding cost, solve the problems, such as industrial enzyme catalyst and product isolates and purifies difficulty.
Immobilised enzymes once uses water desmoenzyme, insoluble enzyme, immobilized enzyme, desmoenzyme, immobilized enzyme, enzyme resin and load in the literature
The titles such as body desmoenzyme.
Integrality of the catalysis reaction of enzyme dependent on its active site, therefore must be selected suitably in a certain enzyme of fixation
Condition keeps the group of its active site unaffected, and avoids the conditions such as high temperature, strong acid and highly basic, does not make protein denaturation.Enzyme
Process for fixation be mainly the following method.
(a) carrier combined techniques: the most commonly used is covalent coupling method, i.e. the nonessential group of zymoprotein passes through covalent bond and load
Body forms irreversible connection.The protein group of energy coupling includes: amino, carboxyl, cysteine under mild conditions
Sulfydryl, the imidazole radicals of histidine, the phenolic group of tyrosine, serine and threonine hydroxyl.It participates in and the covalently bound base of carrier
Group, cannot be group necessary to enzyme performance vigor.
(b) cross-linking method makes to crosslink between enzyme molecule by double-functional group reagent is aggregated into reticular structure, is allowed to insoluble
In water to form immobilised enzymes.Frequently with double-functional group reagent have glutaraldehyde, maleic anhydride etc..Zymoprotein dissociates
Amino, phenolic group, imidazole radicals and sulfydryl each may participate in cross-linking reaction.
(c) investment: enzyme is rolled in the thin grid of gel or is surrounded by semipermeable polymer film and become lattice type
With two kinds of microcapsule-type.Investment prepares immobilised enzymes also normal embedding cell in addition to embedding water-soluble enzyme, and immobilized cell is made,
Such as can with gelatin and glutaraldehyde embedding have PA ase vigor thallus, can continuous hydrolysis Ru base penicillin, industry
Produce 6-amino-penicillanic acid.
Enzyme compares the variation for being resistant to temperature and pH, optimal pH often slightly shifts, single-minded to substrate after immobilization
Property there is no any change, efficiency of actual improves tens times (such as industrial applications of 5 '-phosphodiesterases) even hundred times (such as
The industrial application of PA ase).
Immobilised enzymes it is various informative, the particle that can be made into good mechanical property dresses up enzyme column for continuous production；Or anti-
Progress batch in device is answered to be stirred to react；Enzyme membrane, enzyme pipe etc. be may be made as applied to analytical chemistry；It can be made into microcapsules enzyme again, make
It is applied to clinic for treatment enzyme.Now again someone with enzyme membrane (including cell, tissue, film made of microorganism) and electricity, light, heat etc.
Sensitive element forms a kind of device and claims biosensor, for measure organic compound and fermentation automatically control in information biography
Pass and environmental protection in harmful substance detection.The most commonly used is the biosensor of enzyme membrane and ion-selective electrode composition, examples
If urea sensor is made of immobilized urease, immobilized nitrobacteria and oxygen electrode, urea decomposes ammonification and carbon dioxide through urase,
Ammonia continues again by oxidation by nitrobacteria, and total oxygen demand then passes through the variation that oxygen electrode reflects electric current, to calculate the content of urea.
After Nelson and Griffin has found enzyme immobilizatio phenomenon within 1916, what scientist began to immobilised enzymes
Research work (J.M.Nelson, E.J.Griffin.Adsorption of invertase.J.Am.Chem.Soc.,
1916,38(5):1109-115).The research of immobilised enzymes since the fifties, the Grubhofer of nineteen fifty-three Germany and
Schleith uses poly- aminostyryl resin for the combination such as carrier and carboxypeptidase, amylase, pepsin, ribalgilase,
Immobilised enzymes is made.The later period sixties, immobilization technology develop rapidly.1969, Japanese thousand cigarettes, one Lang Shouci was in work
Production application immobilized aminoacylase realizes one in enzyme application history from DL- amino acid continuous production l-amino acid in industry
Great change.In the first time world enzyme engineering academic conference held in 1971, determine that the unified English name of immobilised enzymes is
Immobilized enzyme.With the development of immobilization technology, there is immobilized thallus.1973, Japan was for the first time in industry
On using the aspartase in immobilization coli somatic, by fumaric acid continuous production ASPARTIC ACID.?
On the basis of immobilised enzymes and immobilized thallus, there is immobilized cell technology in the later period seventies.1976, France used for the first time
Fixed yeast cell produces beer and alcohol, and Japan in 1978 produces amylase with immobilization hay bacillus, has started with solid
Surely change the precedent of cell production enzyme.Nineteen eighty-two, Japanese research for the first time produce glutamic acid with immobilized protoplast, make progress.
Obstacle of the immobilized protoplast due to relieving cell wall, is more advantageous to the secretion of intracellular organic matter, this produces skill for endocellular enzyme
The change of art route provides new direction.(Zou Zechang；The preparation of silicon oxide mesoporous foamed material and its papain are fixed
Change [D]；Beijing University of Technology；2009)
The prior art not offer prepare Nebivolol chirality alcohol intermediate and the like immobilization ketoreductase
Mutant and its application effect.
The immobilization of other ketoreductases has the following technical problems in the prior art: 1, immobilization higher cost, operation
Formality is complicated；2, it is few that number is recycled in immobilization；3, catalytic efficiency decline etc. after immobilization.
Summary of the invention
The technical problem to be solved in the present invention is to provide one kind to derive from Lactobacillus kefir species
(Lactobacillus kefir) ketoreductase Lk Kred gene, and provide and the cell of the enzyme and producing enzyme is immobilized
Method, and with the enzyme and its immobilized cell and immobilised enzymes of preparation, Nebivolol is used to prepare as biocatalyst
The method of chiral intermediate and the like.
Technical scheme is as follows:
Immobilization Ketoreductase mutant, which is characterized in that the immobilization Ketoreductase mutant is ketoreductase
The immobilized cell or immobilised enzymes of mutant, immobilised enzymes are that the mutant is immobilized on solid phase support object；It is fixed
Changing cell is that the Ketoreductase mutant is expressed in the microbial cell being immobilized on solid phase support object；
The Ketoreductase mutant is selected from ET016, ET017 or ET031 ketoreductase；
The amino acid sequence of the ET017 ketoreductase encodes the ET017 ketone also as shown in SEQ ID NO:2
The nucleotide sequence of nitroreductase gene is as shown in SEQ ID NO:1；
The amino acid sequence of the ET016 ketoreductase encodes the ET016 ketone also as shown in SEQ ID NO:4
The nucleotide sequence of nitroreductase gene is as shown in SEQ ID NO:3；
The amino acid sequence of the ET031 ketoreductase encodes the ET031 ketone also as shown in SEQ ID NO:6
The nucleotide sequence of nitroreductase gene is as shown in SEQ ID NO:5.
The gene order of three kinds of ketoreductase Lk Kred synthesizes institute by Jin Sirui Biotechnology Co., Ltd full genome
, NdeI and HindIII restriction endonuclease sites are added respectively at code area both ends.Target gene fragment passes through restricted interior
It after enzyme cutting NdeI and HindIII digestion, is attached, converts and screens with by pET21a (+) carrier of same double digestion, sieve
Obtained positive plasmid Lk Kred-pET21a (+) is selected to be transferred in BL21 (DE3) host strain, to construct the body of the ketoreductase
Outer heterogenous expression system.
According to existing common knowledge, any gene is connected into all kinds of expression vectors after operation or transformation, converts to suitable
Host cell, inducing through felicity condition can overexpression destination protein.Therefore, the carrier of its expression of ketoreductase Lk Kred
It can be pET or pCW or pUC or pPIC9k etc., expressive host can be Escherichia coli, Pichia pastoris, streptomycete etc..
The present invention also provides the Kred cells of Lk containing ketoreductase to immobilize cell, and obtains enzyme solution and be fixed
Change the method for enzyme.Immobilized cell is crosslinked cell containing enzyme and glutaraldehyde solution using chitosan, with natrium carbonicum calcinatum into
Row granulation, obtains immobilized cell after filtering.Immobilised enzymes is added enzyme solution and is connected to using the resin after glutaraldehyde cross-linking activation
On resin, finally with the active site on alanine closing resin, it is spare to obtain the enzyme being fixed on resin.The above immobilization behaviour
Make referring to Examples hereinafter.
The present invention also provides the immobilization Ketoreductase mutant as catalyst preparing Nebivolol chirality
The application of alcohol intermediate and the like, it is described the preparation method is as follows: being catalyzed Nebivolol ketone group in the presence of co-factor NADP
Intermediate [NB-7 (S)/NB-7 (R)] and Nebivolol chirality alcohol intermediate [NB-8 (S, S)/NB-8 (R, S)/NB-8 (R, R)/
NB-8 (S, R)] between stereospecificity balanced reaction, reaction equation is as follows:
React formula (I)
React formula (II)
React formula (III)
React formula (IV)
Described prepares Nebivolol chirality alcohol intermediate and the method for other analogs, including any one of following method
Kind:
(a) reaction carried out using isopropanol and Im-Lk Kred
(b) reaction carried out using glucose combination glucose dehydrogenase (being abbreviated as Gdh)
Reaction system are as follows: the immobilized cell or immobilised enzymes of ketoreductase Lk Kred, phosphate buffer, coenzyme NAD P,
Substrate, regenerating coenzyme substrate isopropanol or glucose.
Wherein, the dosage of immobilized cell or immobilised enzymes is in 2-20g/10ml, and buffer concentration is in 50-200mM, coenzyme
Concentration is 2-20mg/10ml, and concentration of substrate is adjusted in 1-2g/10ml, regenerating coenzyme concentration of substrate according to concentration of substrate.
Preferably, the dosage of the immobilized cell or immobilised enzymes is in 2g/10ml, and buffer concentration is auxiliary in 50mM
Enzyme concentration is 2mg/10ml, and the pH of the reaction is 7-11, and the reaction temperature is 35-40 DEG C.
Product is after acetonitrile extraction through HPLC by verifying after reaction, and reaction conversion ratio is up to 90% or 95% or more, product
Chiral purity angle value up to 90% or 95% or 98% or more.To which the provable enzyme mutant can be used for raw material NB-7 (S)
With the biological asymmetric reduction of NB-7 (R).
The enzyme that can carry out above-mentioned biocatalytic reaction includes other presence such as corresponding immobilized cell or immobilised enzymes
Form.
Cofactor Regeneration Systems used in the present invention are glucose-glucose dehydrogenase system or isopropanol -ol dehydrogenase body
System.The enzyme source that wherein glucose dehydrogenase system uses is in bacillus subtilis (Bacillus subtilis), huge gemma
The vitro recombination enzyme of bacillus (Bacillus megaterium) etc. preferably derives from bacillus subtilis (Bacillus
Subtilis the genetic recombination enzyme of glucose dehydrogenase (BsGDH)).Enzyme used in isopropanol -ol dehydrogenase system mainly comes
Derived from ketoreductase Lk Kred or the carrier format of its immobilization.It is specifically below the present invention unless otherwise specified, to use
Cofactor Regeneration Systems be isopropanol -one reductase enzyme Lk Kred Cofactor Regeneration Systems.
The present invention has the following technical effect that
1) immobilization Ketoreductase mutant of the invention, can be used as catalyst, be provided commonly for making with Cofactor Regeneration Systems
Standby Nebivolol chirality alcohol intermediate and the like, reaction conversion ratio is up to 90%, 95% or 99% or more, the chirality of product
Reinheitszahl is up to 90%, 95% or 99% or more.
2) enzyme after immobilization or cell can be recycled by simply filtering, it can be used repeatedly after recycling 7-10
It is secondary, and while reusing, is essentially identical with the reaction conversion ratio and chiral purity angle value that use for the first time.
Detailed description of the invention
Fig. 1 is the SFC testing result of embodiment 7.
Fig. 2 is the SFC testing result of embodiment 8.
Fig. 3 is the SFC testing result of embodiment 9.
Fig. 4 is the SFC testing result of embodiment 10.
Fig. 5 is the SFC testing result of embodiment 11.
Fig. 6 is the SFC testing result of embodiment 12.
Fig. 7 is the SFC testing result of embodiment 13.
Fig. 8 is the SFC testing result of embodiment 14.
Fig. 9 is the SFC testing result of embodiment 15.
Figure 10 is the SFC testing result of embodiment 16.
Figure 11 is the SFC testing result of embodiment 17.
Figure 12 is the SFC testing result of embodiment 18.
Figure 13 is process for fixation in the prior art.
Specific embodiment
Explain the present invention in detail with reference to embodiments.Embodiment to facilitate the understanding of the present invention, but not
Limitation of the present invention.
In embodiment, test method without specific conditions, usually routinely condition, such as " Molecular Cloning:A Laboratory guide "
(J. Pehanorm Brooker, D.W. Russell write, Huang Peitang, Wang Jiaxi, and Zhu's thickness plinth etc. is translated, the third edition, Beijing: Science Press,
2002) method described in carries out.
The building of 1 prokaryotic expression system of embodiment
Three kinds of ketoreductase Lk Kred genetic fragments are synthesized by Jin Sirui Biotechnology Co., Ltd, and are recombinated and arrived
On pET21a carrier.
Positive recombinant plasmid Lk Kred-pET21a (+) conversion expression host strain BL21 (DE3) (is purchased from Tiangeng biochemistry section
Skill (Beijing) Co., Ltd), it obtains prokaryotic expression bacterial strain Lk Kred-pET21a (+)/BL21 (DE3), it is anti-as subsequent catalyst
Answer primary bacterial strain.
Glucose dehydrogenase (BsGDH) (LOC111893255) and alcohol dehydrogenase (TbADH) for regenerating coenzyme
(LOC101068320) gene is synthesized by Jin Sirui Biotechnology Co., Ltd, the same Lk of the building of subsequent recombination expression plasmid
The building of Kred-pET21a (+) plasmid, respectively obtained after being transferred in BL21 (DE3) BsGDH-pET21a (+)/BL21 (DE3),
TbADH-pET21a (+)/BL21 (DE3) expresses bacterial strain.
It is prepared by the fermentation of 2 enzyme of embodiment
Expression bacterial strain Lk Kred-pET21a (+)/BL21 (DE3), the BsGDH-pET21a (+)/BL21 of above-mentioned building
(DE3), TbADH-pET21a (+)/BL21 (DE3) is in the 5ml LB liquid training added with final concentration of 100ug/ml ampicillin
Support base [10g/l tryptone (OXIOD), 5g/l yeast powder (OXIOD), 10g/l sodium chloride (traditional Chinese medicines reagent)] in 37 DEG C,
After 200rpm shaken cultivation is stayed overnight, it is inoculated in 1% (V/V) ratio containing final concentration of 100ug/ml ampicillin
In 500ml LB liquid medium, in 37 DEG C, 200rpm shaken cultivation.When OD600 is between 0.8-1.0, final concentration is added
For the inducer IPTG (isopropyl-β-D-thiogalactoside, IPTG) of 0.1mM, and in 25 DEG C of overnight inductions.Thallus exists
It is collected by centrifugation under the conditions of 8000rpm, it is spare.A part of thallus is taken, the ratio of 4g phosphate buffer is corresponded to 1g wet thallus, is suspended
In 50mM pH7.0 sodium phosphate buffer, ultrasonication (200W, 3s/5s, 10min), 4 DEG C, 12000rpm centrifugation 20min,
Take supernatant spare.
The catalytic activity of 3 enzyme of embodiment detects
Enzyme solution obtained above is used for substrate catalysis reaction.1g substrate (NB-7 (S) or NB-7 (R)) is dissolved in 8ml
In 50mM pH7.0 sodium phosphate buffer containing 10% isopropanol, 1.5g glucose is then added, is added after dissolution to be mixed
2mg NADP+, 2.5g GDH wet thallus are added 2ml enzyme supernatant for volume and add to 10ml.Reaction solution is placed in 37 DEG C of thermostatted waters
In bath, magnetic agitation reaction.It is sampled after reaction 10mim, carries out HPLC detection, the substrate transformation rate reaches 30%, and product is chiral
Reinheitszahl > 99%.
Conversion ratio is defined as product peak/(product peak+substrate peak) * 100% numerical value, and enzyme activity is defined as every min under 40 degree
Every ml enzyme solution reaction generates micromole's number of product.Calculating process is as follows: enzyme activity=conversion ratio * (1/221) * 1000000/10/
10.Conversion ratio after this 10min is 30%, then enzyme activity=30%* (1/221) * 1000000/10/10=13.6U/min/
ml。
4 HPLC detection method of embodiment is established
The detection of the substrate transformation rate is carried out by HPLC, and the condition of detection is as follows: instrument is Agilent HPLC1100, liquid phase
Column is that Agilent Agllent 5TC-C18 (2) 250*4.6mm, Mobile phase B uffer A are containing 0.1% formic acid water phase, mobile phase
Buffer B is containing 0.1% formic acid acetonitrile phase, and Gradient program: 0~20min80%A to 10%A, 20-25min 10%A are arrived
10%A to 80%A after 10%A, 25min.30 DEG C of column oven temperature, flow velocity 1mL/min, Detection wavelength 282nm, sample volume
10uL.The peak area of product is acquired into conversion ratio divided by the sum of product and the peak area of substrate after detection.
The detection of product chiral purity is detected by supercritical fluid chromatography (SFC).By the peak area of S type product after detection
Divided by the sum of the peak area of S type product and R type product, chiral purity is acquired.
Above-mentioned test sample is all made of after mobile phase is diluted to suitable concentration, is examined with 0.22 μm of membrane filtration into instrument
It surveys.
The preparation of 5 immobilized cell of embodiment
50g somatic cells addition 50ml water is stirred evenly, separately takes and 2~3g chitosan is added in 50ml water and 3ml is added
Glacial acetic acid is stirred well to chitosan and is completely dissolved.50ml chitosan solution is poured into the cell solution stirred evenly to fill
Divide stirring 30min, 10ml isopropanol is added to stir.It takes 50% 4~5ml of concentration glutaraldehyde to be diluted with water to 12ml and is added with upper casing
It is sufficiently stirred in glycan cell solution.30~50min of stirring crosslinking.3g natrium carbonicum calcinatum is added in 150ml water and is sufficiently stirred
Dissolution.Sodium carbonate liquor is slowly added to be sufficiently stirred in the above crosslinked chitosan-immobilized cell, is at this time neutrality.It crosses
Filter obtains immobilized cell, and filter cake is rinsed 2~3 times with 500ml water, is eluted with the 20mM phosphoric acid buffer of 200ml primary.It filters
To filter cake weighing about 80g, it is filtered dry and is put into 4 DEG C of refrigerators preservations.
Immobilized cell obtained above is used for substrate catalysis reaction.1g substrate (compound I) is dissolved in 40ml to contain
Have in the 50mM pH7.0 sodium phosphate buffer of 10% isopropanol, 800mg glucose is then added, is added after dissolution to be mixed
5mg NADP+, 2.5g GDH wet thallus are added 2g immobilized cell, and 10ml phosphoric acid buffer are added, volume is added to 50ml.
Reaction solution is placed in 40 DEG C of thermostat water baths, magnetic agitation reaction.It is sampled after reaction 10mim, carries out HPLC detection, substrate conversion
Rate reaches 19%, product chiral purity angle value > 99%.
Conversion ratio is defined as product peak/(product peak+substrate peak) * 100% numerical value, and enzyme activity is defined as every min under 40 degree
Every g immobilized cell reaction generates micromole's number of product.Calculating process is as follows: enzyme activity=conversion ratio * (1/221) *
1000000/10/2.Conversion ratio after this 10min is 19%, then enzyme activity=19%* (1/221) * 1000000/10/2=
43U/min/g。
The preparation of 6 immobilised enzymes of embodiment
2 are washed by pure water 2 times of 5 times of volumes of 100g resin, then with 4 times of volume pH8.0 25mmol phosphoric acid buffers
It is secondary.Resin is added to the glutaraldehyde of 4 times of resin volumes 6%, is stirred 6 hours at room temperature, crosslinking activation is carried out.Then it is set with 5 times
The pure water of rouge volume 2 times, then with 4 times volume pH8.0 25mmol phosphoric acid washing 2 times, activated resin is collected by filtration,
It is spare.
The enzyme solution in the embodiment 2 of 5 times of volumes is added on spare resin, 20 DEG C hereinafter, be slowly stirred overnight.Centrifugation
Collect resin.5 times of volume pH8.0 25mmol phosphoric acid buffer dissolution 15g l-Alanine are taken, the resin of previous step is added, stirs
It mixes 1 hour.Resin is collected by centrifugation, is washed 2 times with the pH8.0 25mmol phosphoric acid buffer of 3 times of resin volumes.Resin is collected by centrifugation,
Obtain immobilised enzymes about 98g, 4 DEG C of preservations.
Immobilised enzymes obtained above is used for substrate catalysis reaction.1g substrate NB-7 (S) is dissolved in 8ml and contains 10%
In the 50mM pH7.0 sodium phosphate buffer of isopropanol, 1.5g glucose is then added, 5mg NADP is added after dissolution to be mixed
+, 2.5g GDH wet thallus, 2g immobilised enzymes is added, and 2ml phosphoric acid buffer is added, volume is added into 10ml.Reaction solution is placed in
In 40 DEG C of thermostat water baths, magnetic agitation reaction.It is sampled after reaction 10mim, carries out HPLC detection, the substrate transformation rate reaches
22%, product chiral purity angle value > 98%.
Conversion ratio is defined as product peak/(product peak+substrate peak) * 100% numerical value, and enzyme activity is defined as every min at 40 DEG C
Every g immobilized cell reaction generates micromole's number of product.Calculating process is as follows: enzyme activity=conversion ratio * (1/221) *
1000000/10/10.Conversion ratio after this 10min is 12%, then enzyme activity=12%* (1/221) * 1000000/10/10=
5.4U/min/g。
The biocatalysis of 7 immobilization Lk Kred cell of embodiment
1g substrate NB-7 (S) is dissolved in the 50mM pH7.0 sodium phosphate buffer that 8ml contains 10% isopropanol, wait stir
2mg NADP+ is added after mixing dissolution, the ET016 immobilized cell of the above-mentioned preparation of 2g is added, and is added to volume with buffer
10mL.Reaction solution is placed in 40 DEG C of thermostat water baths, mechanic whirl-nett reaction.In reaction process every 2h with 0.1M NaOH by pH
It adjusts to 7.0 or so, reaction carries out SFC detection afterwards for 24 hours.
See Fig. 1.
By calculating, the substrate transformation rate > 98%, chiral purity angle value > 98% are obtained.
The biocatalysis of 8 immobilization Lk Kred cell of embodiment
1g substrate NB-7 (S) is dissolved in the 50mM pH7.0 sodium phosphate buffer that 8ml contains 10% isopropanol, wait stir
2mg NADP+ is added after mixing dissolution, the ET016 immobilization of the above-mentioned preparation of 2g is added in 1.5g glucose, 0.02g GDH freeze-dried powder
Cell, and volume is added into 10mL with buffer.Reaction solution is placed in 40 DEG C of thermostat water baths, mechanic whirl-nett reaction.Reaction
PH is adjusted to 7.0 or so with 0.1M NaOH every 2h in the process, reaction carries out SFC detection afterwards for 24 hours.As a result see Fig. 2.
By calculating, the substrate transformation rate > 98%, chiral purity angle value > 98% are obtained.
The biocatalysis of 9 immobilised enzymes Lk Kred of embodiment
1g substrate NB-7 (S) is dissolved in the 50mM pH7.0 sodium phosphate buffer that 8ml contains 10% isopropanol, wait stir
2mg NADP+ is added after mixing dissolution, the above-mentioned ET017 immobilised enzymes with resin crosslinking preparation of 2g is added, and with buffer by body
Product adds to 10mL.Reaction solution is placed in 40 DEG C of thermostat water baths, mechanic whirl-nett reaction.Every 2h 0.1M in reaction process
NaOH adjusts pH to 7.0 or so, and reaction carries out SFC detection afterwards for 24 hours.As a result see Fig. 3.
By calculating, the substrate transformation rate > 98%, chiral purity angle value > 98% are obtained.
The biocatalysis of 10 immobilised enzymes Lk Kred of embodiment
1g substrate NB-7 (S) is dissolved in the 50mM pH7.0 sodium phosphate buffer that 8ml contains 20% isopropanol, wait stir
2mg NADP+ is added after mixing dissolution, 1.5g glucose, 0.02g GDH freeze-dried powder, addition 2g is above-mentioned to be crosslinked preparation with resin
ET017 immobilised enzymes, and volume is added into 10mL with buffer.Reaction solution is placed in 40 DEG C of thermostat water baths, mechanical stirring
Reaction.PH is adjusted to 7.0 or so with 0.1M NaOH every 2h in reaction process, reaction carries out SFC detection afterwards for 24 hours.As a result it asks
See Fig. 4.
By calculating, the substrate transformation rate > 98%, chiral purity angle value > 98% are obtained.
The biocatalysis of 11 immobilization Lk Kred cell of embodiment
1.5g substrate NB-7 (S) is dissolved in the 50mM pH7.0 sodium phosphate buffer that 8ml contains 10% isopropanol, to
2mg NADP+ is added after stirring and dissolving, the ET017 immobilized cell of the above-mentioned preparation of 2g is added, and is supplemented volume with buffer
To 10mL.Reaction solution is placed in 40 DEG C of thermostat water baths, mechanic whirl-nett reaction.It will with 0.1M NaOH every 2h in reaction process
PH is adjusted to 7.0 or so, and reaction carries out SFC detection afterwards for 24 hours.SFC result is see Fig. 5.
By calculating, the substrate transformation rate > 95%, chiral purity angle value > 98% are obtained.
The biocatalysis of 12 immobilization Lk Kred cell of embodiment
1.5g substrate NB-7 (S) is dissolved in the 50mM pH7.0 sodium phosphate buffer that 8ml contains 10% isopropanol, to
2mg NADP+, 1.5g glucose, 0.02g GDH freeze-dried powder are added after stirring and dissolving, the ET017 that the above-mentioned preparation of 2g is added is fixed
Change cell, and volume is added into 10mL with buffer.PH is adjusted to 7.0 left sides with 0.1M NaOH every 2h in reaction process
The right side, reaction solution are placed in 40 DEG C of thermostat water baths, mechanic whirl-nett reaction.Reaction carries out SFC detection afterwards for 24 hours, as a result see Fig. 6.
By calculating, the substrate transformation rate > 96%, chiral purity angle value > 98% are obtained.
The biocatalysis of 13 immobilised enzymes Lk Kred of embodiment
1.5g substrate NB-7 (S) is dissolved in the 50mM pH7.0 sodium phosphate buffer that 160ml contains 10% isopropanol,
After dissolution to be mixed be added 0.02g NADP+, be added 2g it is above-mentioned with resin crosslinking preparation ET017 immobilised enzymes, and with buffer
Volume is added to 10mL by liquid.Reaction solution is placed in 35 DEG C of thermostat water baths, mechanic whirl-nett reaction.It is used in reaction process every 2h
0.1M NaOH adjusts pH to 7.0 or so, and reaction carries out SFC detection afterwards for 24 hours, as a result see Fig. 7.
By calculating, the substrate transformation rate > 95%, chiral purity angle value > 99% are obtained.
The biocatalysis of 14 immobilised enzymes Lk Kred of embodiment
1.5g substrate NB-7 (S) is dissolved in the 50mM pH7.0 sodium phosphate buffer that 8ml contains 10% isopropanol, to
2mg NADP+ is added after stirring and dissolving, the above-mentioned ET017 immobilised enzymes with resin crosslinking preparation of 2g is added, and will with buffer
Volume adds to 10mL.Reaction solution is placed in 35 DEG C of thermostat water baths, mechanic whirl-nett reaction.Every 2h 0.1M in reaction process
NaOH adjusts pH to 7.0 or so, and reaction carries out SFC detection afterwards for 24 hours, as a result see Fig. 8.
By calculating, the substrate transformation rate > 90%, chiral purity angle value > 98% are obtained.
The biocatalysis of 15 immobilization Lk Kred cell of embodiment
2g substrate NB-7 (S) is dissolved in the 50mM pH7.0 sodium phosphate buffer that 8ml contains 10% isopropanol, wait stir
2mg NADP+ is added after mixing dissolution, the ET017 immobilization of the above-mentioned preparation of 2g is added in 1.5g glucose, 0.02g GDH freeze-dried powder
Cell, and volume is added into 10mL with buffer.PH is adjusted to 7.0 or so with 0.1M NaOH every 2h in reaction process,
Reaction solution is placed in 40 DEG C of thermostat water baths, mechanic whirl-nett reaction.Reaction carries out SFC detection afterwards for 24 hours, as a result see Fig. 9.
By calculating, the substrate transformation rate > 85%, chiral purity angle value > 98% are obtained.
The biocatalysis of 16 immobilization Lk Kred cell of embodiment
2g substrate NB-7 (R) is dissolved in the 50mM pH7.0 sodium phosphate buffer that 8ml contains 10% isopropanol, wait stir
2mg NADP+ is added after mixing dissolution, the ET017 immobilization of the above-mentioned preparation of 2g is added in 1.5g glucose, 0.02g GDH freeze-dried powder
Cell, and volume is added into 10mL with buffer.PH is adjusted to 7.0 or so with 0.1M NaOH every 2h in reaction process,
Reaction solution is placed in 40 DEG C of thermostat water baths, mechanic whirl-nett reaction.Reaction carries out SFC detection afterwards for 24 hours, as a result see Figure 10.
By calculating, the substrate transformation rate > 86%, chiral purity angle value > 98% are obtained.
The Reusability of 17 immobilization Lk Kred cell biocatalysis of embodiment
1g substrate NB-7 (R) is dissolved in the 50mM pH7.0 sodium phosphate buffer that 8ml contains 10% isopropanol, wait stir
2mg NADP+ is added after mixing dissolution, the ET031 immobilization of the above-mentioned preparation of 2g is added in 1.5g glucose, 0.02g GDH freeze-dried powder
Cell, and volume is added into 10mL with buffer.PH is adjusted to 7.0 or so with 0.1M NaOH every 2h in reaction process,
Reaction solution is placed in 35 DEG C of thermostat water baths, mechanic whirl-nett reaction.Reaction carries out SFC detection afterwards for 24 hours, as a result see Figure 11.
By calculating, the substrate transformation rate > 98%, chiral purity angle value > 99% are obtained.
The precipitating of immobilization Lk Kred cell will be obtained after the filtering of above-mentioned reaction solution, these sedimentation cells are put into again
Above-mentioned system reuses 7 times repeatedly, chiral purity angle value > 98% that all products measure.
The Reusability of 18 immobilization Lk Kred cell biocatalysis of embodiment
1g substrate NB-7 (R) is dissolved in the 50mM pH7.0 sodium phosphate buffer that 8ml contains 10% isopropanol, wait stir
2mg NADP+ is added after mixing dissolution, the ET017 immobilization of the above-mentioned preparation of 2g is added in 1.5g glucose, 0.02g GDH freeze-dried powder
Cell, and volume is added into 10mL with buffer.PH is adjusted to 7.0 or so with 0.1M NaOH every 2h in reaction process,
Reaction solution is placed in 35 DEG C of thermostat water baths, mechanic whirl-nett reaction.Reaction carries out SFC detection afterwards for 24 hours, as a result see Figure 12.
By calculating, the substrate transformation rate > 98%, chiral purity angle value > 98% are obtained.
The precipitating of immobilization Lk Kred cell will be obtained after the filtering of above-mentioned reaction solution, these sedimentation cells are put into again
Above-mentioned system reuses 7 times repeatedly, chiral purity angle value > 98% that all products measure.
Sequence table
<110>Nanjing Qu Mei Biotechnology Co., Ltd
Shanghai Ren Mei Biotechnology Co., Ltd
<120>immobilization Ketoreductase mutant and its answering in Nebivolol chirality alcohol intermediate and the like is being prepared
With
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 756
<212> DNA
<213>Lactobacillus kefir (Lactobacillus kefir)
<400> 1
atgaccgacc gtctgaaggg caaagttgcg atcgtgaccg gtggcaccct gggtatcggt 60
ctggcgattg cggataagtt cgttgaggaa ggtgcgaaag tggttattac cggtcgtcac 120
gcggacgtgg gcgagaaggc ggcgaaaagc atcggtggca ccgatgttat tcgttttgtg 180
cagcacgacg cgagcgatga agcgggctgg accaagctgt tcgacaccac cgaggaagcg 240
tttggcccgg ttaccaccgt ggttaacaac gcgggtattg cggtggacgt tagcgtggag 300
gataccacca ccgaggaatg gcgtaaactg ctgagcgtta acctggatgg tgtgttcttt 360
ggcacccgtc tgggtatcca acgtatgaag aacaaaggtc tgggcgcgag catcattaac 420
atgagcagca ttgaaggtct ggttggcgac ccgaccctgg gtgcgtacaa cgcgagcaag 480
ggtgcggtgc gtatcatgag caaaagcgcg gcgctggatt gcgcgctgaa ggactatgat 540
gttcgtgtga acaccgttca cccgggcccg attaaaaccc cgctgctgga cgatctggag 600
ggtttcgagg aaatgcacag ccagcgtacc aagaccccga tgggtcacat cggcgaaccg 660
aacgacatcg cgtggatttg cgtgtacctg gcgagcgatg agagcaaatt cgcgaccggt 720
gcggaatttg tggttgacgg tggctatacc gcgcaa 756
<210> 2
<211> 252
<212> PRT
<213>Lactobacillus kefir (Lactobacillus kefir)
<400> 2
Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Asp
85 90 95
Val Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Glu Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Leu Leu Asp Asp Leu Glu Gly Phe Glu Glu Met His Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 3
<211> 756
<212> DNA
<213>Lactobacillus kefir (Lactobacillus kefir)
<400> 3
atgaccgacc gtctgaaggg caaagtggcg atcgttaccg gtggcaccct gggtatcggt 60
ctggcgattg cggataagtt cgtggaggaa ggtgcgaaag tggttattac cggccgtcac 120
gcggacgttg gcgagaaggc ggcgaaaagc atcggtggca ccgatgtgat tcgttttgtt 180
cagcacgacg cgagcgatga agcgggctgg accaagctgt tcgacaccac cgaggaagcg 240
tttggcccgg tgaccaccgt ggttaacaac gcgggtattg ctgtgagcaa gagcgttgag 300
gacaccacca ccgaggaatg gcgtaaactg ctgagcgtga acctggatgg tgttttcttt 360
ggcacccgtc tgggtatcca acgtatgaag aacaaaggtc tgggcgcgag catcattaac 420
atgagcagca ttgaaggttt cgtgggtgac ccgaccctgg gtgcgtacaa cgcgagcaag 480
ggtgcggttc gtatcatgag caaaagcgcg gcgctggatt gcgcgctgaa ggactacgat 540
gtgcgtgtta acaccgtgca cccgggctat attaaaaccc cgctggttga cgatctggag 600
ggtgcggagg aaatgatgag ccagcgtacc aagaccccga tgggtcacat cggcgaaccg 660
aacgacatcg cgtggatttg cgtttacctg gcgagcgatg agagcaaatt cgcgaccggt 720
gcggaatttg tggttgatgg tggctatacc gcgcaa 756
<210> 4
<211> 252
<212> PRT
<213>Lactobacillus kefir (Lactobacillus kefir)
<400> 4
Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Glu Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 5
<211> 756
<212> DNA
<213>Lactobacillus kefir (Lactobacillus kefir)
<400> 5
atgaccgacc gtctgaaggg caaagttgcg atcgttaccg gtggcaccct gggtattggc 60
ctggcgattg cggataagtt cgttgaggaa ggtgcgaaag tggttattac cggtcgtcat 120
gcggatgtgg gcgagaaggc ggcgaaaagc atcggtggca ccgatgttat tcgttttgtg 180
cagcacgacg cgagcgatga agcgggctgg accaagctgt tcgacaccac cgaggaagcg 240
tttggcccgg ttaccaccgt ggttaacaac gcgggtattt tcgtggacgt tagcgtggag 300
gataccacca ccgaggaatg gcgtaaactg ctgagcgtta acctggatgg tgtgttcttt 360
ggcacccgtc tgggtatcca acgtatgaag aacaaaggtc tgggcgcgag catcattaac 420
atgagcagca ttgaaggtct ggttggcgac ccgaccctgg gtgcgtacaa cgcgagcaag 480
ggtgcggtgc gtatcatgag caaaagcgcg gcgctggatt gcgcgctgaa ggactatgat 540
gttcgtgtga acaccgttca cccgggcccg attaaaaccc cgctgctgga cgatctggag 600
ggttttgagg aaatgcacag ccagcgtacc aagaccccga tgggtcacat cggcgaaccg 660
aacgacatcg cgtggatttg cgtgtacctg gcgagcgatg agagcaaatt cgcgaccggt 720
gcggaatttg tggttgacgg tggctatacc gcgcaa 756
<210> 6
<211> 252
<212> PRT
<213>Lactobacillus kefir (Lactobacillus kefir)
<400> 6
Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Phe Val Asp
85 90 95
Val Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Glu Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Leu Leu Asp Asp Leu Glu Gly Phe Glu Glu Met His Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
Claims (6)
1. immobilization Ketoreductase mutant, which is characterized in that the immobilization Ketoreductase mutant is prominent for ketoreductase
The immobilized cell or immobilised enzymes of variant, immobilised enzymes are that the mutant is immobilized on solid phase support object；Immobilization
Cell is that the Ketoreductase mutant is expressed in the microbial cell being immobilized on solid phase support object；
The Ketoreductase mutant is selected from ET016, ET017 or ET031 ketoreductase；
The amino acid sequence of the ET017 ketoreductase encodes the ET017 ketoreductase as shown in SEQ ID NO:2
The nucleotide sequence of gene is as shown in SEQ ID NO:1；
The amino acid sequence of the ET016 ketoreductase encodes the ET016 ketoreductase as shown in SEQ ID NO:4
The nucleotide sequence of gene is as shown in SEQ ID NO:3；
The amino acid sequence of the ET031 ketoreductase encodes the ET031 ketoreductase as shown in SEQ ID NO:6
The nucleotide sequence of gene is as shown in SEQ ID NO:5.
2. immobilization Ketoreductase mutant according to claim 1, which is characterized in that the side of the immobilized cell
Method is to be crosslinked using chitosan to cell containing enzyme and glutaraldehyde solution, is pelletized with natrium carbonicum calcinatum, is obtained after filtering
Immobilized cell.
3. immobilization Ketoreductase mutant according to claim 1, which is characterized in that the immobilised enzymes uses penta
Resin after dialdehyde crosslinking activation is added enzyme solution and is connected on resin, finally with the active site on alanine closing resin, obtains
The enzyme that must be fixed on resin.
4. immobilization Ketoreductase mutant as claimed in any one of claims 1-3 is preparing Nebivolol as catalyst
The application of chiral alcohol intermediate and the like, which is characterized in that described the preparation method is as follows: being urged in the presence of co-factor NADP
Change Nebivolol keto intermediates [NB-7 (S)/NB-7 (R)] and Nebivolol chirality alcohol intermediate [NB-8 (S, S)/NB-8 (R,
S)/NB-8 (R, R)/NB-8 (S, R)] between stereospecificity balanced reaction, reaction equation is as follows:
。
5. application according to claim 4, which is characterized in that it is characterized in that, described prepares Nebivolol chiral alcohol
The method of intermediate and other analogs, any one including following method:
(a) reaction carried out using isopropanol and Im-Lk Kred, reaction route are as follows:
(b) reaction carried out using glucose combination glucose dehydrogenase Gdh, reaction route are as follows:
In the conversion reaction system including ketoreductase Lk Kred immobilized cell or immobilised enzymes, phosphate buffer,
Coenzyme NAD P, substrate and regenerating coenzyme substrate isopropanol or glucose；
Wherein, the dosage of immobilized cell or immobilised enzymes is in 2-20g/10ml, and buffer concentration is in 50-200mM, coenzyme concentration
For 2-20mg/10ml, in 1-2g/10ml, regenerating coenzyme concentration of substrate is adjusted concentration of substrate according to concentration of substrate, described
The pH of reaction is 7-11, and the reaction temperature is 35-40 DEG C.
6. application according to claim 5, which is characterized in that the dosage of the immobilized cell or immobilised enzymes exists
2g/10ml, for buffer concentration in 50mM, coenzyme concentration is 2mg/10ml.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116790574A (en) * | 2023-07-06 | 2023-09-22 | 中科阿尔诺(深圳)生物科技有限公司 | Lactobacillus kefir immobilized microbial inoculum, lactobacillus kefir lyophilized powder and preparation method thereof |
| CN116790574B (en) * | 2023-07-06 | 2024-04-30 | 中科阿尔诺(深圳)生物科技有限公司 | Lactobacillus kefir immobilized microbial inoculum, lactobacillus kefir lyophilized powder and preparation method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080318295A1 (en) * | 2007-02-08 | 2008-12-25 | Codexis, Inc. | Ketoreductases and Uses Thereof |
| US20090093031A1 (en) * | 2007-08-24 | 2009-04-09 | Codexis, Inc. | Ketoreductase Polypeptides for the Production of (R)-3-Hydroxythiolane |
| US20090191605A1 (en) * | 2007-09-13 | 2009-07-30 | Codexis, Inc. | Ketoreductase Polypeptides for the Reduction of Acetophenones |
| US20130005001A1 (en) * | 2010-01-27 | 2013-01-03 | Corden Pharma International Gmbh | Method for producing nebivolol |
-
2019
- 2019-06-19 CN CN201910529412.7A patent/CN110218718A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080318295A1 (en) * | 2007-02-08 | 2008-12-25 | Codexis, Inc. | Ketoreductases and Uses Thereof |
| US20090093031A1 (en) * | 2007-08-24 | 2009-04-09 | Codexis, Inc. | Ketoreductase Polypeptides for the Production of (R)-3-Hydroxythiolane |
| US20090191605A1 (en) * | 2007-09-13 | 2009-07-30 | Codexis, Inc. | Ketoreductase Polypeptides for the Reduction of Acetophenones |
| US20130005001A1 (en) * | 2010-01-27 | 2013-01-03 | Corden Pharma International Gmbh | Method for producing nebivolol |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116790574A (en) * | 2023-07-06 | 2023-09-22 | 中科阿尔诺(深圳)生物科技有限公司 | Lactobacillus kefir immobilized microbial inoculum, lactobacillus kefir lyophilized powder and preparation method thereof |
| CN116790574B (en) * | 2023-07-06 | 2024-04-30 | 中科阿尔诺(深圳)生物科技有限公司 | Lactobacillus kefir immobilized microbial inoculum, lactobacillus kefir lyophilized powder and preparation method thereof |
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