CN106701699A - Biocatalyst as well as preparation method and application thereof - Google Patents
Biocatalyst as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a biocatalyst which contains NADH-dependent specific 3-quinuclidone reductase (QNR), glucose dehydrogenase (GDH) and Ni<2+>functionalized agarose microspheres. The biocatalyst has diffraction peaks when diffraction angles 2 theta are 27.5+/-0.2 degrees, 31.7+/-0.2 degrees, 45.5+/-0.2 degrees, 52.1+/-0.2 degrees, 66.4+/-0.2 degrees and 75.8+/-0.2 degrees respectively. The biocatalyst provided by the invention has the benefits that a preparation method is simple to operate, does not need special equipment and is mild in process conditions, low in cost and suitable for industrialization. The biocatalyst provided by the invention is used for bio-catalytic synthesis of (R)-3-quinuclidone, can realize carbonyl reduction and co-enzyme in-situ regeneration at the same time, and does not need to add expensive coenzymes NADH. Meanwhile, the biocatalyst provided by the invention can be recycled and has long-term operation stability.
Description
Technical field
The invention belongs to technical field of bioengineering, and in particular to a kind of biocatalyst and its preparation method and application.
Background technology
(R) -3- quinuclidinols (molecular formula C7H13NO, molecular weight is 127.18, No. CAS：25333-42-0) it is synthesis Ah Di
The key intermediate of the medicines such as bromine ammonium, Solifenacin, Revatropate and Talsaclidine.Chirality is industrially mainly used to urge at present
Agent asymmetric reduction 3- quinuclidones synthesize (R) -3- quinuclidinols, such as：XylSkewphos/PICA-Ruthenium (II) is combined
Thing or BINAP/IPHAN-Ru (II) compound etc..But the chemical synthesis process need to screen chiral ligand；The transition gold for being used
The expensive, toxicity of category is larger, be difficult to the removal from product；And the product optical purity of preparation is relatively low, need to be further purified.
Another method is that racemic splits, and the defect of the method is its theoretical yield maximum only 50%.
Relative to chemical synthesis process, there is substrate specificity with the biological synthesis process that enzyme makees catalyst；Elevated chemical is selected
Selecting property, regioselectivity and stereoselectivity；Reaction condition is gentle；Catalysis activity is high；The unique advantage such as Atom economy is good.It is raw
Object space method synthesis (R) -3- quinuclidinols are related to two processes：One is to utilize NADH or NADPH by 3- quinines by carbonyl reductase
Ketone is reduced to (R) -3- quinuclidinols；Two is by oxidized form NAD by regenerating coenzyme enzyme+Or NADP+Be converted into reduced-NAD H or
NADPH, realizes that dual-enzyme coupling asymmetric reduction synthesizes (R) -3- quinuclidinols.Detailed process is as follows：
Living things catalysis asymmetric reduction synthesizes (R) -3- quinuclidinol processes.
Either with wild-type microorganisms or recombination bacillus coli as biocatalyst, due to cell this body structure
Limitation causes substrate/product, coenzyme, and outer exchange is obstructed in the cell, causes that bioconversion time is long, catalytic efficiency is low.And dissociate
Enzyme makees catalyst, enzyme stability and the high cost caused by enzyme purification, is all the problem that enzyme must be solved as biocatalyst.
Additionally, both biocatalysts are all difficult to reclaim and recycle.
In practical application, in order to improve stability, catalysis activity and the selectivity of enzyme, can be by enzyme and specific support combination shape
Into immobilised enzymes, and immobilized enzyme biocatalyst is easy to be separated from reactant mixture, is recycled recycling and realizes continuous
Reaction (Robert Dicosimo, Joseph Mcauliffe, Ayrookaran J.Pouloseb and Gregory
Bohlmann,Industrial use of immobilized enzymes,Chem.Soc.Rev.,2013,42,6437-
6474；Joshua Britton,Colin L.Raston and Gregory A.Weiss,Rapid protein
immobilization for thin film continuous flow biocatalysis,Chem.Commun.,2016,
52,10159-10162).Conventional enzyme immobilization method includes physical absorption, chemical bond, embedding and forms cross-linked enzyme aggregate.
Physical method is simple, quick, be difficult to cause enzyme inactivation, but enzyme easily to reveal, and carrier also easy masking enzyme active sites in itself, hinder
Binding Capacity.Chemical method is conducive to increasing enzyme stability, but easily causes enzyme inactivation (Suwan Myung, Chun You and Y.-
H.Percival Zhang,Recyclable cellulose-containing magnetic nanoparticles:
immobilization of cellulose-binding module-tagged proteins and a synthetic
metabolon featuring substrate channeling,J.Mater.Chem.B,2013,1,4419-4427；Wen
Wang,Daniel I.C.Wang and Zhi Li,Facile fabrication of recyclable and active
nanobiocatalyst:purification and immobilization of enzyme in one pot with Ni-
NTA functionalized magnetic nanoparticle,Chem.Commun.,2011,47,8115–8117；Fuhua
Zhao,Hui Li,Yijun Jiang,Xicheng Wang and Xindong Mu,Co-immobilization of
multi-enzyme on control-reduced graphene oxide by non-covalent bonds:an
artificial biocatalytic system for the one-pot production of gluconic acid
from starch,Green Chem.,2014,16,2558-2565)。
The content of the invention
In order to solve the problems of the prior art, according to the first aspect of the invention, it is an object of the invention to provide one
Biocatalyst is planted, the biocatalyst stability is good, and catalysis activity is good, with long period of operation stability and recyclable profit again
With.
The object of the present invention is achieved like this：
A kind of biocatalyst, comprising specific 3- quinines ketoreductase (QNR), GDH that NADH is relied on
(GDH)、Ni2+Functionalization agarose microbeads, it is characterised in that：The θ of angle of diffraction 2 be 27.5 ± 0.2 °, 31.7 ± 0.2 °, 45.5
± 0.2 °, 52.1 ± 0.2 °, 66.4 ± 0.2 °, have diffraction maximum at 75.8 ± 0.2 °.
An embodiment of the invention, above-mentioned biocatalyst also contains sodium chloride and sodium dihydrogen phosphate.
An embodiment of the invention, above-mentioned biocatalyst has foam sample loose structure, and aperture is 10nm-
2.5μm。
An embodiment of the invention, above-mentioned biocatalyst enzyme load capacity is 5-15%.
According to the second aspect of the invention, another object of the present invention is to provide the preparation side of above-mentioned biocatalyst
Method.
An embodiment of the invention, the preparation method of above-mentioned biocatalyst, it is characterised in that using as follows
Step：
Step (1)
Ni is balanced with cushioning liquid A2+Functionalization agarose microbeads, add and contain with histidine-tagged 3- quinine ketoreductases
(His-QNR) and the cell with histidine-tagged GDH (His-GDH) cracking mixing supernatant, with containing 5-15mM
The cushioning liquid A of imidazoles is diluted to suspension；
Step (2)
Suspension prepared by step (1) is with 30~100rpm of rotating speed in 4~30 DEG C of temperature, the condition of pH5.0~10.0
Under continuously stir 1~3h；It is then centrifuged for, collects and carry enzyme agarose microbeads, is washed with the cushioning liquid A of step (1)；
Step (3)
Load enzyme agarose microbeads after washing are dispersed in the cushioning liquid A of step (1), 2~8 DEG C of refrigerators are placed in and are incubated
1~5 day；Both；
The pH value of the cushioning liquid A is 7.8-8.2.
An embodiment of the invention, above-mentioned cushioning liquid A sodium dihydrogen phosphates containing 50mM and 300mM sodium chloride.
According to the third aspect of the invention we, it is still another object of the present invention to provide above-mentioned biocatalyst synthesis (R)-
Application in 3- quinuclidinols.
According to the fourth aspect of the invention, it is still another object of the present invention to provide a kind of synthesis side of (R) -3- quinuclidinols
Method, the method carries out enzymic catalytic reaction using above-mentioned biocatalyst, synthesizes (R) -3- quinuclidinols.
An embodiment of the invention, a kind of synthetic method of (R) -3- quinuclidinols, it is characterised in that：
Biocatalyst is dispersed in cushioning liquid B, 3- quinuclidones, glucose, NAD is added+And NADH, in room temperature bar
Under part, pH is 7.5-8.0, and with 50~150rpm speed oscillations, bioconversion time is 1-12h；The pH value of the cushioning liquid B
It is 7.2-7.4.
An embodiment of the invention, the cushioning liquid B is 10mM PBSs (PBS), is contained
NaCl 137mM, KCl 2.7mM, Na2HPO410mM, KH2PO42mM。
Specifically, a kind of biocatalytic synthesis of (R) -3- quinuclidinols, comprise the following steps：
Step (1)
Ni is balanced with cushioning liquid A2+Functionalization agarose microbeads, add and contain with histidine-tagged 3- quinine ketoreductases
(His-QNR) and the cell with histidine-tagged GDH (His-GDH) cracking mixing supernatant, with containing 5-15mM
The cushioning liquid A of imidazoles is diluted to suspension；
Step (2)
Suspension prepared by step (1) is with 30~100rpm of rotating speed in 4~30 DEG C of temperature, the condition of pH5.0~10.0
Under continuously stir 1~3h；It is then centrifuged for, collects and carry enzyme agarose microbeads, is washed with the cushioning liquid A of step (1)；
Step (3)
Load enzyme agarose microbeads after washing are dispersed in the cushioning liquid A of step (1), 2~8 DEG C of refrigerators are placed in and are incubated
1~5 day；Biocatalyst is obtained；
Step (4)
Biocatalyst is dispersed in cushioning liquid B, 3- quinuclidones, glucose, NAD is added+And NADH, in room temperature bar
Under part, pH is 7.5-8.0, and with 50~150rpm speed oscillations, bioconversion time is 1-12h；
Cushioning liquid A sodium dihydrogen phosphates containing 50mM and 300mM sodium chloride, pH value is 7.8-8.2；
The cushioning liquid B be 10mM PBSs (PBS), 137mM containing NaCl, KCl 2.7mM,
Na2HPO410mM, KH2PO42mM, pH value is 7.2-7.4.
Beneficial effect：
1st, the present invention is based on Ni2+Strong affinity interaction and between polyhistidine label is directly simultaneously pure from cell pyrolysis liquid
Change and immobilization His-QNR and His-GDH, the biocatalyst of preparation the θ of angle of diffraction 2 be 27.5 ± 0.2 °, 31.7 ±
0.2 °, 45.5 ± 0.2 °, 52.1 ± 0.2 °, 66.4 ± 0.2 °, have diffraction maximum at 75.8 ± 0.2.The biocatalyst has bubble
Foam sample loose structure, aperture is 10nm-2.5 μm, and enzyme load capacity is up to 5-15%, and catalysis activity is good.
2nd, using biocatalyst of the present invention as immobilized enzyme catalysis agent, the living things catalysis for (R) -3- quinuclidinols is closed
Into carbonyl reduction and coenzyme in-situ regeneration can simultaneously being realized, without the expensive coenzyme NAD H of addition.Biology of the invention is urged simultaneously
Agent is recyclable and cycling and reutilization, and with long period of operation stability.
3rd, enzyme immobilization method of the present invention does not need prepurification target protein, can simultaneously complete the purifying of target protein and consolidate
Fixedization；While the activity of energy specific enrichment target protein；The immobilised enzymes good stability of preparation, method is simple to operate, is not required to
Special installation is wanted, process conditions are gentle, low cost is adapted to industrialization.
4th, using biocatalyst asymmetric syntheses (R) -3- quinuclidinols of the present invention, high income reaches 70-85%, and conversion ratio is
100%, enantiomer value is 100%, while can be recycled more than 15 times, economic benefit is big.Living things catalysis of the present invention is asymmetric
Synthesis (R) -3- quinuclidinols used medium is water, can be economized on resources, free from environmental pollution, meets the requirement of Green Chemistry.
Brief description of the drawings
Fig. 1 is the SDS-PAGE analyses of biocatalyst；
Fig. 2 is temperature to Ni2+The influence of functionalization agarose microbeads enzyme load capacity；
Fig. 3 is pH value to Ni2+The influence of functionalization agarose microbeads enzyme load capacity；
Fig. 4 is Ni2+Functionalization agarose microbeads and total protein quality compare the shadow of enzyme spcificity activity and activity recovery
Ring；
Fig. 5 is the scanning electron microscope analysis of biocatalyst；
Fig. 6 is the powder X-ray diffraction analysis of biocatalyst；
Fig. 7 is the thermogravimetric analysis of biocatalyst；
Fig. 8 is the infrared spectrum analysis of 3- quinuclidones and (R) -3- quinuclidinols；
Fig. 9 is the cycling and reutilization of biocatalyst；
Figure 10 is the SDS-PAGE analyses of the biocatalyst of different circulation batches.
Specific embodiment
The present invention is specifically described below by specific embodiment, it is pointed out here that following examples are served only for this hair
It is bright to be further described, it is impossible to be interpreted as limiting the scope of the invention, the person skilled in the art of this area can be with root
Some nonessential modifications and adaptations are made to the present invention according to foregoing invention content.The all raw materials of the present invention and reagent are commercially available
Product.Carbonyl reductase used of the invention is specific 3- quinines ketoreductase (QNR, the Accession No that NADH is relied on:
AB733448), regenerating coenzyme enzyme is GDH (GDH, Accession No:AY930464).
Embodiment 1
Enzyme assay
QNR enzyme assays：
Standard reaction mixed system：Cushioning liquid B (PBS, pH7.2-7.4), 3 μm of ol 3- quinuclidones, 0.3 μm of ol
NADH, appropriate enzyme QNR, cumulative volume 1mL.The mensuration absorbance value changes at λ=340nm.Unit of enzyme activity defines：25 DEG C,
Enzyme amount in 1min needed for 1 μm of ol NADH of conversion.
GDH enzyme assays：
Standard reaction mixed system：Cushioning liquid B (PBS, pH7.2-7.4), 10 μm of ol glucose, 1 μm of ol NAD+, fit
The enzyme GDH of amount, cumulative volume 1mL.The mensuration absorbance value changes at λ=340nm.Unit of enzyme activity defines：25 DEG C, turn in 1min
Change 1 μm of ol NAD+Required enzyme amount.
Embodiment 2
His-QNR and His-GDH is fixed altogether
Take 50mg Ni2+Functionalization agarose microbeads, with cushioning liquid A (sodium dihydrogen phosphate containing 50mM and 300mM sodium chloride,
PH value is 7.8-8.2) balance；It is subsequently adding 2mL ketoreductases containing quinine (His-QNR) and GDH (His-GDH)
Cell cracking mixing supernatant, be diluted to 5mL with the cushioning liquid A containing 10mM imidazoles.In 25 DEG C of temperature, pH8.0, rotating speed
50rpm, continuously stirs above-mentioned suspension 2h.By the centrifugation of above-mentioned suspension, collect and carry enzyme agarose microbeads, washed with cushioning liquid A
Wash twice.Load enzyme agarose microbeads after washing are dispersed in cushioning liquid A again, 4 DEG C of refrigerators are placed in and are incubated 4 days.
Polyacrylamide gel electrophoresis (SDS-PAGE) analysis is carried out to biocatalyst prepared by embodiment 2
SDS-PAGE analysis results are shown in accompanying drawing 1.M is standard protein molecular weight in figure；L1To express the restructuring of His-QNR
Escherichia coli and the recombination bacillus coli of expression His-GDH；L2For two kinds of cells of recombination bacillus coli crack mixing supernatant；
L3、L4、L5And L6It is the collection liquid that biocatalyst is eluted with 10mM, 250mM, 500mM and 1M.Molecular weight 35-25kDa it
Between, there are two obvious protein bands, QNR (25.74kDa) and GDH (28.25kDa), target protein purity are corresponded to respectively
More than 90%, it was demonstrated that the affine process for fixation that the present invention is provided can directly Simultaneous purification and fixation from cell pyrolysis liquid
Change His-QNR and His-GDH.
With reference to the preparation method of embodiment 2, temperature, pH value are investigated to Ni2+Functionalization agarose microbeads enzyme load capacity and enzyme
The influence of load efficiency；Investigate Ni2+Functionalization agarose microbeads and total protein quality compare enzyme spcificity activity and activity is reclaimed
The influence of rate.
Temperature is to Ni2+The influence result of functionalization agarose microbeads enzyme load capacity and load efficiency is shown in accompanying drawing 2.When temperature from
4 DEG C when rising to 25 DEG C, enzyme load capacity increases to 59.6mg/g (Ni from 27.52+Functionalization agarose microbeads), load efficiency from
51.6% increases to 89.7%.When temperature is more than 30 DEG C, enzyme load capacity and load efficiency do not increase, show immobilization
Optimum temperature is 25 DEG C.
PH is to Ni2+The influence result of functionalization agarose microbeads enzyme load capacity and load efficiency is shown in accompanying drawing 3.25 DEG C of temperature,
When pH value is 8.0, enzyme load capacity reaches maximum for 60.8mg/g (Ni2+Functionalization agarose microbeads), and enzyme load efficiency
It is 92.3% to reach maximum.Therefore, the optimal pH of immobilization is 8.0.
Ni2+Functionalization agarose microbeads and total protein quality compare enzyme spcificity activity and the influence of activity recovery is shown in attached
Fig. 4.Ni2+Functionalization agarose microbeads and total protein mass ratio from 4/1 increase to 8/1 when, the activity specific point of QNR and GDH
It is not to increase to 8.4U/mg and 5.6U/mg from 5.3U/mg to increase to 11.5U/mg, activity recovery increases from 61.2% respectively
Increase to 98.6% to 94.6% and 64.2%.But mass ratio from 8/1 increase to 10/1 when, activity specific and activity recovery
Change is little.During enzyme immobilization, Ni2+Functionalization agarose microbeads and total protein mass ratio are advisable with 8/1.
Electronic microscope photos is scanned to biocatalyst prepared by embodiment 2
Sample solution is added dropwise on clean cover glass, 40 DEG C of vacuum drying, metal spraying covering sample uses ESEM
(SEM, S-3000N type) and field emission scanning electron microscope (FE-SEM, FEI NOVA NanoSEM 400) are imaged, and analysis result is shown in attached
Fig. 5.Accompanying drawing 5a is Ni2+Functionalization agarose microbeads, surface is smooth.Accompanying drawing 5b is the later Ni of immobilized enzyme2+Functionalization agarose
Microballoon, i.e. biocatalyst, its configuration of surface are entirely different with shown in accompanying drawing 5a, yardstick occur and form is irregular and mutually tight
The bead of close connection.Biocatalyst is further found with FE-SEM analyses, biocatalyst surface is the porous knot of foam sample
Structure, aperture between 10nm-2.5 μm (accompanying drawing 5c, d), this loose structure be conducive to Local enrichment high concentration substrate/coenzyme and
Material is quickly exchanged, and can improve reaction speed can improve catalytic efficiency again.
Powder X-ray diffraction (XRD) analysis is carried out to biocatalyst prepared by embodiment 2
Use general analysis XD-2X-ray diffractometer determination sample crystal diffractions peak, test condition：Cu targets Electricity
Pressure 30kV, electric current 15mA, 2 °/min of sweep speed, 0.02 ° of step-length, 5 ° of sweep limits, 50 ° of to, analysis result is shown in accompanying drawing 6.From
It is visible in figure：Biocatalyst XRD spectrum in, 2 θ 27.5 ± 0.2 °, 31.7 ± 0.2 °, 45.5 ± 0.2 °, 52.1 ± 0.2 °,
Occurs characteristic peak at 66.4 ± 0.2 ° and 75.8 ± 0.2 °, intensity distribution is 845,1450,1005,850,800 and 830.And egg
White matter and Ni2+In the XRD spectra of functionalization agarose microbeads, corresponding characteristic peak is not occurred.
Thermogravimetric analysis (TGA) is carried out to biocatalyst prepared by embodiment 2
Use the enzyme load capacity of Mettler 1100SF system measurement biocatalysts.About 2mg samples are placed in aluminum pot,
Nitrogen is passed through by 20mL/min, firing rate is 15 DEG C/min, and sweep limits is 30-600 DEG C.TGA analysis results are shown in accompanying drawing 7.
Calculated according to weightlessness, biocatalyst enzyme load capacity is 5.94%.
Embodiment 3
Biocatalyst is catalyzed asymmetric syntheses (R) -3- quinuclidinols：
Reaction system：50mg biocatalysts, 5mM 3- quinuclidones, 9mM glucose, 0.05mM NAD+And 0.05mM
NADH, cushioning liquid B (10mM PBSs, PBS), 137mM containing NaCl, KCl 2.7mM, Na2HPO410mM,
KH2PO42mM, pH value is 7.2-7.4), cumulative volume 10mL.25 DEG C of reaction temperature, is continuously stirred with 100rpm, pH in course of reaction
Control is monitored in 7.5-8.0. with TLC reacts, and mobile phase is VDichloromethane/VMethyl alcohol=9/1.After the completion of bioconversion, reaction is mixed
Thing centrifugation biocatalyst.Supernatant high concentration NaOH is adjusted to pH value more than 12, is then concentrated under reduced pressure at 80 DEG C
The 1/4 of cumulative volume.Add isometric n-butanol to extract 3 times, collect organic phase, it is concentrated under reduced pressure to be evaporated.It is molten with toluene at 90 DEG C
Solution solid, indissoluble thing filters while hot, filtrate cooling, has white needle-like crystals to occur, and filters, obtain white solid (1H-NMR
(400MHz, DMSO)：δ 4.946 (s, 1H), δ 3.879 (q, 1H), δ 2.531-3.164 (m, 6H), δ 1.392-2.014 (m,
5H), the theoretical molecular of product is：127.18, mass spectroscopy is：127).
Infrared spectrum analysis and conformational analysis are carried out to white solid prepared by embodiment 3
Infrared spectrum analysis：Sample is prepared using KBr pressed disc methods, model is swept wavelength and retouched and encloses 4000~400cm-1, substrate and product
The infrared spectrogram of product is shown in accompanying drawing 8.Result shows 3- quinuclidones in 1747cm-1The carbonyl characteristic peak of left and right disappears, and
3103cm-1There is hydroxyl characteristic peak, show that 3- quinuclidones are reduced into 3- quinuclidinols.
Conformational analysis
With Clarus580GC systems (Perkin Elmer, USA) determine enantiomer value, chiral column for (HYDRODEX- β-
6-TBDM, 25m × 0.25mm × 0.25 μm, Macherey-Nagel), use flame ionic detector.From 60 DEG C of temperature programmings
To 180 DEG C, programming rate is 5 DEG C/min, keeps 3min. injectors and the temperature of detector to be respectively 220 DEG C and 250 DEG C.With
Standard items (R) and (S)-quinuclidinol are compareed, and it is 100%, its configuration R types to measure the enantiomer value of product.
With reference to embodiment 3, influence of the consumption of substrate and catalyst to transformation time, conversion ratio and enantiomer value is investigated,
It is specifically shown in table 1 below.
When biocatalyst consumption for substrate 6.12%, increase with amount of substrate, bioconversion time increases from 1.0h
To 4.5h, conversion ratio and enantiomer value are 100%.When amount of substrate is 204g/L, when catalyst halves, transformation time is
9.5h, catalysis activity is substantially reduced, and conversion ratio is less than 100%, and enantiomer value is still 100%.Show that the influence of substrate consumption is biological
The activity of catalyst, transformation time extension, but its stereoselectivity is not influenceed.
Embodiment 4
The recovery of biocatalyst and cycling and reutilization.
Reaction system：100mg biocatalysts, 10mM quinuclidones, 18mM glucose, 0.05mM NAD+And 0.05mM
NADH, cushioning liquid B (PBS, pH7.2-7.4), cumulative volume 10mL.25 DEG C of reaction temperature, is continuously stirred with 100rpm., reaction
During control pH 7.5-8.0.TLC monitoring reaction after the completion of, catalyst is collected by centrifugation, be directly used in and convert next time.Hair
The biocatalyst that the existing present invention is provided is recyclable 15 times, and the conversion ratio and enantiomer value of single cycle are 100%, as a result see
Accompanying drawing 9.After circulation 15 times, catalytic efficiency is substantially reduced.During biocatalyst cycling and reutilization, respectively the 2nd time,
After the completion of 4 times, 6 times ... .20 times convert, taking a small amount of catalyst carries out SDS-PAGE analyses, as a result sees accompanying drawing 10.It was found that biological
During recycling, the enzyme amount of immobilization is not significantly reduced catalyst, shows the biocatalyst tool of present invention offer
There is long period of operation stability.
Embodiment 5 (comparative example)
Processed without biocatalyst of the present invention, directly with expression His-QNR and the restructuring large intestine of expression His-GDH
Bacillus treatment experiment, to compare.Reaction system：0.1g mixing wet cells make biocatalyst, 5mM 3- quinuclidones, 9mM Portugals
Grape sugar, 0.05mM NAD+With 0.05mM NADH, cushioning liquid B (PBS, pH7.2-7.4), cumulative volume 10mL.Reaction temperature 25
DEG C, continuously stirred with 100rpm, control pH to be monitored with TLC in 7.5-8.0. in course of reaction and react, mobile phase is VDichloromethane/VMethyl alcohol
=9/1.Transformation time is obviously prolonged (5.5h), and conversion ratio is 95%, enantiomer value 100%, it is impossible to reclaims and recycles.
Claims (10)
1. a kind of biocatalyst, comprising specific 3- quinines ketoreductase (QNR), GDH that NADH is relied on
(GDH)、Ni2+Functionalization agarose microbeads, it is characterised in that：In angle of diffraction 2θFor 27.5 ± 0.2 °, 31.7 ± 0.2 °, 45.5
± 0.2 °, 52.1 ± 0.2 °, 66.4 ± 0.2 °, have diffraction maximum at 75.8 ± 0.2 °.
2. biocatalyst as claimed in claim 1, it is characterised in that：The biocatalyst also contains sodium chloride and di(2-ethylhexyl)phosphate
Hydrogen sodium.
3. biocatalyst as claimed in claim 1 or 2, it is characterised in that：The biocatalyst has the porous knot of foam sample
Structure, aperture is 10nm-2.5 μm.
4. the biocatalyst as described in claim any one of 1-3, it is characterised in that：The biocatalyst enzyme load capacity is 5-
15%。
5. as described in claim any one of 1-4 biocatalyst preparation method, it is characterised in that use following steps：
Step (1)
Ni is balanced with cushioning liquid A2+Functionalization agarose microbeads, add and contain with histidine-tagged 3- quinine ketoreductases
(His-QNR) and the cell with histidine-tagged GDH (His-GDH) cracking mixing supernatant, with containing 5-15mM
The cushioning liquid A of imidazoles is diluted to suspension；
Step (2)
By step（1）The suspension of preparation is connected with the rpm of rotating speed 30~100 under conditions of 4~30 °C of temperature, pH5.0~10.0
1~3 h of continuous stirring；It is then centrifuged for, collects and carry enzyme agarose microbeads, uses step（1）Cushioning liquid A washing；
Step (3)
Load enzyme agarose microbeads after washing are dispersed in step（1）Cushioning liquid A in, be placed in 2~8 °C of refrigerators and be incubated 1
~5 days；Both；
The pH value of the cushioning liquid A is 7.8-8.2.
6. preparation method as claimed in claim 5, it is characterised in that：The cushioning liquid A containing 50 mM sodium dihydrogen phosphates and
300 mM sodium chloride.
7. application of the biocatalyst in (R) -3- quinuclidinols are synthesized as described in claim any one of 1-4.
8. the method that one kind synthesizes (R) -3- quinuclidinols using biocatalyst described in any one of 1-4, it is characterised in that：
Biocatalyst is dispersed in cushioning liquid B, 3- quinuclidones, glucose, NAD is added+And NADH, in room temperature condition
Under, pH is 7.5-8.0, and with 50~150 rpm speed oscillations, bioconversion time is 1-12h；The pH value of the cushioning liquid B
It is 7.2-7.4.
9. method as claimed in claim 8, it is characterised in that：The cushioning liquid B is 10mM PBSs
(PBS), 137mM containing NaCl, KCl 2.7mM, Na2HPO410mM, KH2PO4 2mM。
10. a kind of biocatalytic synthesis of (R) -3- quinuclidinols, comprise the following steps：
Step (1)
Ni is balanced with cushioning liquid A2+Functionalization agarose microbeads, add and contain with histidine-tagged 3- quinine ketoreductases
(His-QNR) and the cell with histidine-tagged GDH (His-GDH) cracking mixing supernatant, with containing 5-15mM
The cushioning liquid A of imidazoles is diluted to suspension；
Step (2)
By step（1）The suspension of preparation is connected with the rpm of rotating speed 30~100 under conditions of 4~30 °C of temperature, pH5.0~10.0
1~3 h of continuous stirring；It is then centrifuged for, collects and carry enzyme agarose microbeads, uses step（1）Cushioning liquid A washing；
Step (3)
Load enzyme agarose microbeads after washing are dispersed in step（1）Cushioning liquid A in, be placed in 2~8 °C of refrigerators and be incubated 1
~5 days；Biocatalyst is obtained；
Step (4)
Biocatalyst is dispersed in cushioning liquid B, 3- quinuclidones, glucose, NAD is added+And NADH, in room temperature condition
Under, pH is 7.5-8.0, and with 50~150 rpm speed oscillations, bioconversion time is 1-12 h；
The cushioning liquid A contains 50 mM sodium dihydrogen phosphates and 300 mM sodium chloride, and pH value is 7.8-8.2；
The cushioning liquid B is 10mM PBSs (PBS), 137mM containing NaCl, KCl 2.7mM, Na2HPO4
10mM, KH2PO42mM, pH value is 7.2-7.4.
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