CN105969757A - Enzyme immobilization method and application - Google Patents
Enzyme immobilization method and application Download PDFInfo
- Publication number
- CN105969757A CN105969757A CN201610395274.4A CN201610395274A CN105969757A CN 105969757 A CN105969757 A CN 105969757A CN 201610395274 A CN201610395274 A CN 201610395274A CN 105969757 A CN105969757 A CN 105969757A
- Authority
- CN
- China
- Prior art keywords
- enzyme
- resin
- histidine
- immobilized
- liquid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/62—Carboxylic acid esters
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01184—Carbonyl reductase (NADPH) (1.1.1.184)
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
Abstract
The invention discloses a simple and convenient method for recombinase immobilization. According to the method, the characteristic that a histidine label of recombinant protein and metal ion affinity chromatography resin are combined specifically is utilized, and therefore recombinase is immobilized on the resin, and the immobilized recombinase is prepared. Immobilized carbonyl reductase is used for continuous production of chiral alcohol, and the space time yield is greatly increased.
Description
Technical field
The invention belongs to technical field of enzyme immobilization, be specifically related to a kind of method of simplicity for having histidine mark
Sign restructuring enzyme immobilizatio, and provide carbonyl reductase immobilized enzyme be applied to living things catalysis produce chiral alcohol
Method.
Background technology
Living things catalysis process has typical advantage, as high in reaction efficiency, stereo selectivity height and reaction condition
Gentleness, consumption of organic solvent are few, environmentally friendly etc., have become as the megatrend that current green bio manufactures,
And it is applied to chemicals production more and more widely.But, native enzyme often comes with some shortcomings, such as stability
Poor, form, with product etc., the mixed system being difficult to separate after biocatalytic reaction, these shortcomings are unfavorable for that enzyme exists
Industrial application.It addition, enzyme cost height is also one of key factor of its application of restriction.Utilize the fixing of enzyme
Change technology can reuse enzyme, reduces enzyme cost, increases the stability of enzyme, and product is easily separated, thus extensively
It is applied to the fields such as food industry, fine chemicals industry, medicine, waste water process.
In enzyme immobilizatio is studied, suitable fixation support is the factor of overriding concern.Except traditional consolidates
Surely carrier is changed, the process for fixation of based on Metal Ion Chromatography technology of development in recent years.The method is by solid
On body material, connection metal ion is as part, with some Specific amino acid such as histidine, the half Guang ammonia of protein
Acid, tryptophan etc. produce affinity interaction, thus are fixed on carrier by enzyme.It is applied to this of enzyme immobilization at present
A little carriers are all prepared by laboratory oneself, and technique is complex, and repeatability is difficult to ensure that, is of limited application.
Along with the development of recombinant protein purification technology, the Metal Ion Chromatography resin of commercialization is the most fast-developing, its
Specificity, protein load, tolerance reducing agent ability, stability, recycling and regeneration capacity etc. are obtained for
Huge improvement.The IMAC carrier of commercialization at present is mainly Ni-NTA and Ni-IDA two kinds, and the former is with agar
Sugar or other substrate covalently bound NTA group, NTA group forms tetravalence chelate with Ni, and leaves Ni
Two valencys are for being combined with other groups.The also IDA group (iminodiacetic acid) of same purpose, but NTA
The most resistance to reducing agent, therefore uses more.
It is directly used in commercialization nickel ion affinity chromatograph resin and there is histidine-tagged restructuring enzyme immobilizatio more
Adding simplicity, easily obtain, repeatability is high, and low cost is with efficient.This process for fixation can be by the thick enzyme of recombiant protein
Liquid is directly combined with immobilization material, and without purifying enzyme, and immobilization material can utilize with repeated regeneration, saves
Cost.
Summary of the invention
It is an object of the invention to provide method and the application of a kind of immobilized enzyme, specifically, utilize recombinase egg
There is histidine-tagged feature in vain, and metal affinity chromatography resin-bonded, thus enzyme is fixed on resin,
Prepare immobilized enzyme.Meanwhile, present invention also offers the application in living things catalysis of the carbonyl reductase immobilized enzyme.
Details are as follows for technical scheme:
To have histidine-tagged recombinase enzyme liquid and metal ion affinity chromatography mixed with resin, make enzyme and resin
Fully combine, discard enzyme liquid, obtain immobilized enzyme.
Described have histidine-tagged recombinase and be included in N-end, or at C-end or equal at these two ends
Having the enzyme of histidine, the quantity of histidine is 6～10.
Described have histidine-tagged recombinase and can be combined with metal ion affinity chromatography resin specificity, because of
This present invention uses pure enzyme or crude enzyme liquid can reach same effect.
Described metal ion affinity chromatography resin is: Ni2+、Co2+、Cu2+Hand over agarose Deng metal ion
Connection affine resin, can with there is histidine-tagged enzyme covalent bond, preferably Ni-NTA or
Ni-IDA。
Described conjugation condition is preferably 4～25 DEG C, and 50～100rpm, 0.5～5h.
Present invention also offers the application in living things catalysis of the carbonyl reductase ChKRED20 immobilized enzyme.
The living things catalysis system of carbonyl reductase ChKRED20 immobilized enzyme is:
Buffer, isopropanol, NAD+, immobilized enzyme and substrate.
Buffer is kaliumphosphate buffer or Tris-HCl buffer.Potassium phosphate (potassium dihydrogen phosphate and phosphoric acid hydrogen
Dipotassium) buffer 100mM, pH 7.0～8.0.Tris-HCl buffer is 25mM Tris, 50mM NaCl,
pH 8.0。
Isopropyl alcohol concentration 10%～80% (v/v).
NAD+Concentration 0.2～0.5g/l.
Immobilized enzyme enzyme amount, refer to containing target enzyme (carbonyl reductase ChKRED20) pure enzyme amount, concentration 1～10
g/l。
Substrate is dissolved in isopropanol, preferably final concentration 10～400g/l.
Preferred reaction conditions is 30～50 DEG C.
Beneficial effects of the present invention:
(1) it is directly used in metal ion affinity chromatography resin and there is histidine-tagged restructuring enzyme immobilizatio more
Adding simplicity, low cost is with efficient.This process for fixation can be by direct for the crude enzyme liquid of recombiant protein and immobilization material
In conjunction with, without purifying enzyme.Compared with tradition process for fixation, the immobilization material used by the method can be repeatedly
Regeneration, cost-effective.It is and Metal Ion Chromatography resin can directly buy, easily obtain, reproducible,
Use easy to spread.
(2) present invention is first with commercialization Ni-NTA or Ni-IDA for carrier fixing for carbonyl reductase
Change, and successive reaction prepares chiral alcohol, and space-time yield is greatly improved.
Accompanying drawing explanation
Accompanying drawing is continuous fixed bed reaction or continuous device schematic diagram
Detailed description of the invention
Below in conjunction with embodiment, the present invention will be further described, it should be pointed out that the present embodiment is only used for
The explanation present invention, rather than limitation of the scope of the invention.
The preparation of embodiment 1 carbonyl reductase ChKRED20 immobilized enzyme
The construction method of carbonyl reductase ChKRED20 (NCBI accession number: KC342020) recombinant bacterium,
Heterogenous expression, enzyme purification etc. are this area conventional method.Being separately added into restriction enzyme site at the two ends of this enzyme gene is
EcoR I and Sal I, be connected into the pET 28a after same double digestion (+) carrier segments, obtain recombiant plasmid pET
28a-ChKRED20, and proceed to e. coli bl21 (DE3) builds recombinant bacterium.This gene is at large intestine bar
In bacterium, the enzyme after heterogenous expression has 6 histidine at N-end, constitutes histidine-tagged, can be with metal
Affine resin-bonded.
The abduction delivering of carbonyl reductase ChKRED20: picking recombinant bacterium monoclonal to LB (containing kanamycin
50 μ g/ml) in culture medium, 37 DEG C, 200rpm, cultivate 16h as seed liquor, then the inoculation with 1%
Amount is forwarded in TB (containing kanamycin 50 μ g/ml) culture medium, 37 DEG C, 200rpm, cultivates 3h, adds
Entering IPTG induction (final concentration of 0.2mM), 30 DEG C are continued to cultivate to 24h.Centrifugal (8000rpm, 4
DEG C, 10min) collect thalline, brine (0.8%, w/v) washs 2 times, centrifugal, it is thus achieved that wet
Thalline.
The wet thallus of above-mentioned acquisition is resuspended in buffer (10mM Tris, 300mM NaCl, 10mM
Imidazoles, pH 8.0), after high pressure homogenize crusher machine, frozen centrifugation (12000rpm, 4 DEG C, 20min),
The supernatant obtained is crude enzyme liquid.Crude enzyme liquid, with nickel affinity chromatography resin purification, obtains pure enzyme.Protein purification
Process is the conventional method of this area.
Buffer used by purifying protein is as follows.
In conjunction with liquid: 10mM Tris, 300mM NaCl, 10mM imidazoles (pH8.0)
Rinsing liquid: 10mM Tris, 300mM NaCl, 20mM imidazoles (pH8.0)
Eluent: 10mM Tris, 300mM NaCl, 250mM imidazoles (pH8.0)
The ChKRED20 enzyme liquid afforded carries out 2 dialysis, and dialysis buffer liquid is for the first time: 25mM
Tris, 50mM NaCl, 2mM DTT, 1mM EDTA, pH8.0,4 DEG C, 100rpm, 12h.The
The buffer of secondary dialysis is 25mM Tris, 50mM NaCl, pH8.0,4 DEG C, 100rpm, 12h.
Pure enzyme liquid (concentration 4.25mg/ml) 50ml and the 5ml by buffer balance that will obtain after dialysis
Ni-NTA resin (Genscript, Nanjing Genscript Biotechnology Co., Ltd.) mixes, 4 DEG C, 100rpm
In conjunction with 3h.The buffer composition of the balance of resin is: 10mM Tris, 300mM NaCl, 10mM imidazoles
(pH8.0).Discard enzyme liquid afterwards, obtain immobilized enzyme.Measuring residual enzyme concentration in enzyme liquid is 0.85mg/ml,
Therefore the enzyme amount calculating combination is 170mg.
Above-mentioned immobilized enzyme 20ml aqueous isopropanol equilibrated immobilized enzyme 30min, isopropyl alcohol concentration is 20%,
V/v, is configured in buffer, and buffer is 25mM Tris, 50mM NaCl, pH 8.0.
Due to carbonyl reductase ChKRED20 have 6 histidine-tagged, can tie with Ni-NTA specificity
Close, the effect as therefore the present invention uses crude enzyme liquid can reach with Ni-NTA resin-bonded.
Embodiment 2 carbonyl reductase ChKRED20 immobilized enzyme and the comparison of pure enzymes biocatalysis efficiency
(1) reaction system of pure enzyme: in kaliumphosphate buffer, (100mM, pH 7.0) comprises following components,
10% (v/v) isopropanol, 0.2g/l NAD+, the pure enzyme of 2g/l ChKRED20 and the chloro-1-of substrate 2-(3,4-
Difluorophenyl) ethyl ketone, concentration 100g/l.40 DEG C, 200rpm.
(2) reaction system of immobilized enzyme: the preparation method of immobilized enzyme is with embodiment 1.Immobilized enzyme 2g/l
(this concentration refers to the target enzyme amount contained in immobilized enzyme), remaining condition is with pure enzyme reaction system.
Both catalytic efficiencies of initial reaction stage without significant difference (response time 1h, conversion ratio about 45%),
Prolongation over time, the catalytic efficiency of immobilized enzyme is higher than pure enzyme.When response time is 14h, immobilization
The conversion ratio of enzyme is 98%, and the conversion ratio of pure enzyme only has 94%.Therefore, immobilized enzyme can be stood higher
The substrate of concentration, its deactivation rate in living things catalysis system is slower, thus shows within the identical time more
High conversion ratio.
Product (S)-2-chloro-1-(3, the 4-difluorophenyl) ethanol that above biocatalytic reaction obtains, ee value > 99%.
The substrate of embodiment 3 carbonyl reductase ChKRED20 immobilized enzyme catalysis variable concentrations
Replacing pure enzyme liquid with crude enzyme liquid time prepared by immobilized enzyme, remaining is with embodiment 1.
Reaction system: in kaliumphosphate buffer (100mM, pH 7.0), 10% (v/v) isopropanol, 0.2g/l
NAD+, 5g/l ChKRED20 immobilized enzyme (the target enzyme amount contained), and the chloro-1-of substrate 2-(3,4-
Difluorophenyl) ethyl ketone.Reaction condition is 40 DEG C, 200rpm.
Concentration of substrate is 50g/l, and the time that converting (conversion ratio > 99%) completely needs is 6h；Concentration is 100g/l,
The time converting needs completely is 10h；Concentration of substrate is l50g/l, and the time converted completely is 24h.Space-time produces
Rate (Space time yield) is that 100g/l is the highest at concentration of substrate, for 10g/ (l.h).When concentration of substrate continues
Increasing to 200g/l, in 24h, conversion ratio is 92%.
Product (S)-2-chloro-1-(3, the 4-difluorophenyl) ethanol that above biocatalytic reaction obtains, ee value > 99%.
Embodiment 4 carbonyl reductase ChKRED20 immobilized enzyme list batch living things catalysis
The preparation method of immobilized enzyme is with embodiment 1.
(1) substrate is 3,5 bis trifluoromethyl 1-Phenylethanone .s
Reaction system: in kaliumphosphate buffer (100mM, pH 7.0), 40% (v/v) isopropanol, 0.2g/l
NAD+, 1g/l ChKRED20 immobilized enzyme (the target enzyme amount contained), and substrate 3,5 bis trifluoromethyl
1-Phenylethanone., concentration 400g/l.Reaction condition: 30 DEG C, 200rpm, 24h.
Immobilized enzyme after single reaction being terminated reclaims, and is re-used for next round biocatalytic reaction.Front 4 take turns
The conversion ratio of reaction > 95%, the 5th conversion ratio taking turns reaction is 94%；6th conversion ratio taking turns reaction is 92%.
It is (R)-3 that above-mentioned biocatalytic reaction obtains product, 5-bis trifluoromethyl phenethanol, ee value > 99.9%.
(2) substrate is the chloro-1-of 2-(3,4-difluorophenyl) ethyl ketone
Reaction system: in kaliumphosphate buffer (100mM, pH 7.0), 10% (v/v) isopropanol, 0.2g/l
of NAD+, 5g/l ChKRED20 immobilized enzyme (the target enzyme amount contained), and the chloro-1-of substrate 2-(3,
4-difluorophenyl) ethyl ketone, concentration 150g/l.Reaction condition: 40 DEG C, 200rpm, 24h.
Immobilized enzyme after single reaction being terminated reclaims, and is re-used for next round biocatalytic reaction.Front 4
In batch reaction, the response time is that 10h just can reach conversion ratio more than 99%；Immobilized enzyme reuses 8 batches,
Its vigor remains to keep more than 90%, illustrates good catalytic capability and stability, has continuous biocatalytic
Potentiality.
Product (S)-2-chloro-1-(3, the 4-difluorophenyl) ethanol that above biocatalytic reaction obtains, ee value > 99%.
Embodiment 5 carbonyl reductase ChKRED20 prepares immobilized enzyme with Ni-IDA carrier
The preparation method of carbonyl reductase ChKRED20 enzyme is with embodiment 1.The pure enzyme liquid (concentration 4.25 that will obtain
Mg/ml) 50ml mixes, 4 DEG C, 100 with the 5ml Ni-IDA resin (brand is BBI) balanced with buffer
Rpm combines 3h.The buffer composition of the balance of resin is: 10mM Tris, 300mM NaCl, 10mM
Imidazoles (pH8.0).Discard enzyme liquid afterwards, obtain immobilized enzyme.Measuring residual enzyme concentration in enzyme liquid is 0.73
Mg/ml, the enzyme amount therefore calculating combination is 176mg.
Reaction system is: in kaliumphosphate buffer (100mM, pH 7.0), 40% (v/v) isopropanol, 0.2
g/l NAD+, immobilized enzyme 2g/l (the target enzyme amount contained), and substrate 3,5 bis trifluoromethyl 1-Phenylethanone.,
Concentration 100g/l.30 DEG C, 200rpm.Response time 15h, conversion ratio 99.8%, obtain product for (R)
-3,5-bis trifluoromethyl phenethanol, ee value > 99.9%.
Embodiment 6 has 8 histidine-tagged carbonyl reductase ChKRED20 immobilized enzyme living things catalysis
Utilize the method for rite-directed mutagenesis after 6 histidine of N-end of carbonyl reductase ChKRED20 again
2 aminoacid that suddenly change are histidine, obtain 8 histidine-tagged enzyme ChKRED20-His8。
Carbonyl reductase ChKRED20-His8Heterogenous expression, immobilized enzyme preparation method with embodiment 1.
Reaction system is: in kaliumphosphate buffer (100mM, pH 7.0), 10% (v/v) isopropanol, 0.2g/l NAD+,
Immobilized enzyme 2g/l (the target enzyme amount contained), and the chloro-1-of substrate 2-(3,4-difluorophenyl) ethyl ketone,
Concentration 100g/l.40 DEG C, 200rpm, 24h.Response time 18h, conversion ratio 99%, obtain product (S)-2-
Chloro-1-(3,4-difluorophenyl) ethanol, ee value > 99%.
Embodiment 7 carbonyl reductase ChKRED20 immobilized enzyme packed bed reactor continuous biocatalytic
As shown in drawings, immobilized enzyme is placed in reactor bottom, substrate reactions liquid to the schematic diagram of packed bed reactor
Being entered from upper end by pump, product mixtures flows out from lower end.This device passes through chuck temperature control, makes bed maintain
Stationary temperature.
Substrate reactions liquid is: high concentration substrate (is dissolved in isopropanol, account for that overall reaction liquid is long-pending 70%),
NAD+(0.2g/l), buffer (Tris 25mM, NaCl 50mM, pH8.0).Reaction system adds
NiSO4, DTT etc., the stability of packed bed reactor reaction system can be improved.
In packed bed successive reaction, buffer is Tris 25mM, NaCl 50mM, pH8.0, real below
Execute example identical.
Product mixtures is: high concentration product (is dissolved in isopropanol), NAD+, buffer and the third of generation
Ketone and a small amount of substrate converted the most completely.
(1) substrate is 3,5-bis trifluoromethyl 1-Phenylethanone., and concentration is 100g/l, packed bed immobilized enzyme enzyme amount
160mg (the target enzyme amount contained), immobilized enzyme volume 4.5ml, reaction temperature 40 DEG C.Reactant liquor component
(accounting for 70% volume), NAD it is dissolved in isopropanol for substrate+(0.2g/l), buffer (accounting for 30% volume).
Space-time yield (Space Time Yield, STY) is also known as space-time yield or space-time yield.Refer to given
Under reaction condition, in the unit interval, unit volume (or quality) catalyst can obtain a certain product amount.It is
Weigh one of mark of catalyst activity size and reactor assembly production capacity.
Space-time yield=product amount/(the bed volume * time).
When flow velocity is 0.5ml/min, conversion ratio 99.7%, the space-time yield of calculating is 664g/ (l.h).Flow velocity
During for 0.8ml/min, conversion ratio 99.3%, the space-time yield of calculating is 1059g/ (l.h).Flow velocity is 0.9ml/min
Time, conversion ratio 95%, the space-time yield of calculating is 1140g/ (l.h).Spatiotemporal efficiency is free enzymes biocatalysis
100 times.
Being filled with a biocatalytic reaction, successive reaction 72h with flow velocity for 0.8ml/min, conversion ratio is protected
Hold 99%.
The product that above biocatalytic reaction obtains is (R)-3,5-bis trifluoromethyl phenethanol, ee value > 99.9%.
(2) substrate is the chloro-1-of 2-(3,4-difluorophenyl) ethyl ketone, concentration 50g/l, packed bed immobilized enzyme
Enzyme amount 160mg (the target enzyme amount contained), immobilized enzyme volume 4.5ml, reaction temperature 40 DEG C.Reaction
Liquid component is: substrate is dissolved in isopropanol (accounting for 70% volume), NAD+(0.2g/l), buffer (accounts for
30% volume).
When flow velocity is 0.08ml/min, conversion ratio 99.1%, the space-time yield of calculating is 53g/ (l.h).Flow velocity
During for 0.09ml/min, conversion ratio 99.1%, the space-time yield of calculating is 60g/ (l.h).Flow velocity is 0.10ml/min
Time, conversion ratio 90%, the space-time yield of calculating is 60g/ (l.h).Space-time yield is the 5 of free enzymes biocatalysis
More than Bei.
It is filled with a biocatalytic reaction, successive reaction 24h with flow velocity for 0.09ml/min, converts
Rate > 98%.
Product (S)-2-chloro-1-(3, the 4-difluorophenyl) ethanol that above biocatalytic reaction obtains, ee value > 99%.
(3) substrate is 4-chloroacetyl acetacetic ester, concentration 320g/l, packed bed immobilized enzyme enzyme amount 160mg
(the target enzyme amount contained), immobilized enzyme volume 4.5ml, reaction temperature 40 DEG C.
A. reactant liquor component is: substrate is dissolved in isopropanol (accounting for 70% volume), NAD+(0.2g/l), slow
Rush liquid (accounting for 30% volume).
When flow velocity is 0.6ml/min, conversion ratio 99%, ee value > 99%, the space-time yield of calculating is 2534
g/(l.h)。
B. reactant liquor component is: substrate is dissolved in isopropanol (accounting for 70% volume), NAD+(0.2g/l),
5mM NiSO4, 2mM DTT, buffer (accounts for 30% volume).
When flow velocity is 0.6ml/min, conversion ratio 99%, ee value > 99%, the space-time yield of calculating is 2534
g/(l.h).Space-time yield is freely enzymatic about 8 times.
Product (the S)-4-chloro-3-hydroxyl ethyl n-butyrate. that above biocatalytic reaction obtains, ee value > 99%.
Embodiment 8 mutant M135 immobilized enzyme packed bed reactor continuous biocatalytic
Mutant enzyme is carbonyl reductase ChKRED20 thermostability mutant M135 (Appl Microbiol
Biotechnol.2016.100 (8): 3567-3575).The preparation method of immobilized enzyme is with embodiment 1.
Substrate is the chloro-1-of 2-(3,4-difluorophenyl) ethyl ketone, concentration 50g/l, packed bed immobilized enzyme enzyme amount
130mg (the target enzyme amount contained), immobilized enzyme volume 4ml, reaction temperature 50 DEG C.Reactant liquor component
For: substrate is dissolved in isopropanol (accounting for 70% volume), NAD+(0.2g/l), buffer (accounting for 30% volume).
Buffer is Tris 25mM, NaCl 50mM, pH8.0.
When flow velocity is 0.12ml/min, conversion ratio 99%, the space-time yield of calculating is 90g/ (l.h).The most anti-
Answering 30h, conversion ratio keeps > 98%.
Product (S)-2-chloro-1-(3, the 4-difluorophenyl) ethanol that above biocatalytic reaction obtains, ee value > 99%.
Claims (8)
1. an enzyme immobilizatio method, is characterized in that utilizing recombinase albumen to have histidine-tagged feature, with
Metal affinity chromatography resin specificity combines, thus is fixed on resin by enzyme, prepares immobilized enzyme.
2. the answering in carbonyl reductase immobilized enzyme produces chiral alcohol of the enzyme immobilizatio method described in claim 1
With.
3. the enzyme immobilizatio method described in claim 1, is characterized in that the preparation process of immobilized enzyme is: restructuring
Enzyme enzyme liquid and metal ion affinity chromatography mixed with resin, make enzyme be combined after being fully contacted with resin, abandon
Remove enzyme liquid, obtain immobilized enzyme.
4. the enzyme immobilizatio method described in claim 1, is characterized in that having histidine-tagged recombinase includes
At N-end, or at C-end, or all having the enzyme of histidine at these two ends, the quantity of histidine is 6～10
Individual.
5. the enzyme immobilizatio method described in claim 1, is characterized in that metal ion affinity chromatography resin is: Ni2+、
Co2+、Cu2+Deng the affine resin of metal ion and agarose crosslinking, can with there is histidine mark
The enzyme covalent bond signed.
6. the conjugation condition described in claim 1 and 3 is 4～25 DEG C, 50～100rpm, 0.5～5h.
7. the enzyme immobilizatio method described in claim 3, is characterized in that recombinase enzyme liquid is pure enzyme or crude enzyme liquid.
8. the metal ion affinity chromatography resin described in claim 5 is Ni-NTA or Ni-IDA.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610395274.4A CN105969757A (en) | 2016-06-03 | 2016-06-03 | Enzyme immobilization method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610395274.4A CN105969757A (en) | 2016-06-03 | 2016-06-03 | Enzyme immobilization method and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105969757A true CN105969757A (en) | 2016-09-28 |
Family
ID=57010341
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610395274.4A Pending CN105969757A (en) | 2016-06-03 | 2016-06-03 | Enzyme immobilization method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105969757A (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108823196A (en) * | 2018-06-21 | 2018-11-16 | 湖州师范学院 | A kind of preparation method of high carrying capacity immobilization mandelate racemase |
| CN109439696A (en) * | 2017-09-04 | 2019-03-08 | 尚科生物医药（上海）有限公司 | A method of preparing the chloro- 1-(3,4- difluorophenyl of (R) -2-) ethyl alcohol |
| CN109929829A (en) * | 2019-02-15 | 2019-06-25 | 江苏理工学院 | A kind of process for fixation of carbonyl reductase |
| CN110028063A (en) * | 2019-05-10 | 2019-07-19 | 南京工业大学 | A kind of affine fixation support of graphene oxide and the preparation method and application thereof |
| CN110438116A (en) * | 2019-09-02 | 2019-11-12 | 成都信息工程大学 | A kind of process for fixation of laccase |
| CN111575334A (en) * | 2020-06-03 | 2020-08-25 | 湖州颐盛生物科技有限公司 | Method for preparing (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol |
| WO2021057817A1 (en) * | 2019-09-24 | 2021-04-01 | Abclonal Biotechnology Co., Ltd | His-mbp tagged dna endonuclease for facilitated enzyme removal |
| CN113943372A (en) * | 2021-11-09 | 2022-01-18 | 南京工业大学 | Method for improving stability of immobilized protein by using trivalent cobalt ions |
| CN114231522A (en) * | 2021-12-27 | 2022-03-25 | 上海合全药物研发有限公司 | Process for preparing immobilized N-deoxyribotransferase and deoxynucleoside |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1861798A (en) * | 2005-05-11 | 2006-11-15 | 中国科学院上海生命科学研究院 | Preparation of N-aceto-D-neuraminic acid by N-aceto-D-neuraminic acid aldonase immobilizing method |
| CN102851272A (en) * | 2012-09-25 | 2013-01-02 | 南京工业大学 | Gamma-glutamyltranspeptidase immobilized enzyme, preparation method and application thereof |
| EP2589587A1 (en) * | 2011-11-04 | 2013-05-08 | Chemo Ibérica, S.A. | Synthesis of nitrogen substituted cyclopropanes |
| CN104744266A (en) * | 2013-12-31 | 2015-07-01 | 上虞京新药业有限公司 | Preparation method of ticagrelor intermediate |
| CN105255844A (en) * | 2015-10-20 | 2016-01-20 | 杨仲毅 | Method for preparing recombinase through high-temperature wall breaking |
-
2016
- 2016-06-03 CN CN201610395274.4A patent/CN105969757A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1861798A (en) * | 2005-05-11 | 2006-11-15 | 中国科学院上海生命科学研究院 | Preparation of N-aceto-D-neuraminic acid by N-aceto-D-neuraminic acid aldonase immobilizing method |
| EP2589587A1 (en) * | 2011-11-04 | 2013-05-08 | Chemo Ibérica, S.A. | Synthesis of nitrogen substituted cyclopropanes |
| CN102851272A (en) * | 2012-09-25 | 2013-01-02 | 南京工业大学 | Gamma-glutamyltranspeptidase immobilized enzyme, preparation method and application thereof |
| CN104744266A (en) * | 2013-12-31 | 2015-07-01 | 上虞京新药业有限公司 | Preparation method of ticagrelor intermediate |
| CN105255844A (en) * | 2015-10-20 | 2016-01-20 | 杨仲毅 | Method for preparing recombinase through high-temperature wall breaking |
Non-Patent Citations (3)
| Title |
|---|
| 王爽: "《羰基还原酶的融合表达及固定化研究》", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》 * |
| 苏流利: "《固定化金属亲和载体的制备及应用》", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
| 赵广荣: "《不对称还原制备手性醇》", 《现代制药工艺学》 * |
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109439696A (en) * | 2017-09-04 | 2019-03-08 | 尚科生物医药（上海）有限公司 | A method of preparing the chloro- 1-(3,4- difluorophenyl of (R) -2-) ethyl alcohol |
| CN108823196B (en) * | 2018-06-21 | 2022-01-18 | 湖州师范学院 | Preparation method of high-load immobilized mandelic acid racemase |
| CN108823196A (en) * | 2018-06-21 | 2018-11-16 | 湖州师范学院 | A kind of preparation method of high carrying capacity immobilization mandelate racemase |
| CN109929829A (en) * | 2019-02-15 | 2019-06-25 | 江苏理工学院 | A kind of process for fixation of carbonyl reductase |
| CN109929829B (en) * | 2019-02-15 | 2023-03-10 | 江苏理工学院 | Immobilization method of carbonyl reductase |
| CN110028063A (en) * | 2019-05-10 | 2019-07-19 | 南京工业大学 | A kind of affine fixation support of graphene oxide and the preparation method and application thereof |
| CN110438116A (en) * | 2019-09-02 | 2019-11-12 | 成都信息工程大学 | A kind of process for fixation of laccase |
| CN110438116B (en) * | 2019-09-02 | 2021-09-21 | 成都信息工程大学 | Laccase immobilization method |
| WO2021057817A1 (en) * | 2019-09-24 | 2021-04-01 | Abclonal Biotechnology Co., Ltd | His-mbp tagged dna endonuclease for facilitated enzyme removal |
| CN113767170A (en) * | 2019-09-24 | 2021-12-07 | 武汉爱博泰克生物科技有限公司 | His-MBP tagged endodnases for facilitating enzyme removal |
| CN113767170B (en) * | 2019-09-24 | 2023-10-03 | 武汉爱博泰克生物科技有限公司 | His-MBP-labeled DNA endonucleases for facilitating enzymatic removal |
| CN111575334A (en) * | 2020-06-03 | 2020-08-25 | 湖州颐盛生物科技有限公司 | Method for preparing (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol |
| CN113943372A (en) * | 2021-11-09 | 2022-01-18 | 南京工业大学 | Method for improving stability of immobilized protein by using trivalent cobalt ions |
| CN114231522A (en) * | 2021-12-27 | 2022-03-25 | 上海合全药物研发有限公司 | Process for preparing immobilized N-deoxyribotransferase and deoxynucleoside |
| CN114231522B (en) * | 2021-12-27 | 2024-04-26 | 上海合全药物研发有限公司 | Immobilized N-deoxyribotransferase and deoxynucleoside preparation method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105969757A (en) | Enzyme immobilization method and application | |
| CN109609475B (en) | Glufosinate-ammonium dehydrogenase mutant and application thereof in synthesizing L-glufosinate-ammonium | |
| CN106754610B (en) | Recombinant engineering bacterium for surface display expression of glutamate decarboxylase and construction method and application thereof | |
| CN104152506B (en) | The thick enzyme system of recombinant bacterium of aldehyde ketone reductase catalyzes and synthesizes the method for (S)-N, N-dimethyl-3-hydroxyl-3-(2-thiophene)-1-propylamine | |
| CN105274070A (en) | Mutant of 7 beta-hydroxyl steroid dehydrogenase, application of mutant and synthesis method | |
| CN109576317A (en) | The method that enzyme process prepares R-3- aminobutyric acid | |
| CN107338258A (en) | The method for producing the engineering bacteria structure and its production beta Alanine of beta Alanine | |
| CN106367377B (en) | A kind of process for fixation of sucrose isomerase | |
| CN105624128B (en) | Immobilized monoamine oxidase and application thereof in synthesis of chiral azabicyclo compound | |
| CN101691560B (en) | Colibacillus and method for performing soluble expression of transglutaminase proenzyme thereof | |
| CN104131041A (en) | Production method for alpha-ketoglutaric acid | |
| CN107354118A (en) | A kind of genetic engineering bacterium and its construction method and application with γ terpinene synthesis capabilities | |
| CN109371070A (en) | A kind of method of high yield α-ketoisovaleric acid | |
| CN106222231A (en) | A kind of method of quick production high-optical-purity D lysine | |
| CN106011191B (en) | A kind of method of Whole Cell Biocatalysis production 5- aminovaleric acid | |
| CN109679978B (en) | Recombinant co-expression system for preparing L-2-aminobutyric acid and application thereof | |
| CN102433360B (en) | Method for preparing alpha-ketobutyric acid by using L-threonine as substrate | |
| CN103131659A (en) | Organic solvent-resistant lipase, as well as encoding gene, producing strain and application thereof | |
| CN105907742A (en) | Carboxyl magnetic bead immobilized glutamic acid decarboxylase and preparation method and application thereof | |
| CN104805112B (en) | A kind of construction method of 3- hydracrylic acids Producing Strain recombinant plasmid | |
| CN103865959A (en) | Biological synthesis method of xylosic acid | |
| CN111662899B (en) | Linker peptide mediated enzyme immobilized BaPAD catalyst and preparation method and application thereof | |
| CN112481320B (en) | Method for preparing (-) gamma-lactam with high catalytic efficiency | |
| CN104031892A (en) | Leucine dehydrogenase and gene for coding same | |
| CN110590888B (en) | Method for affinity purification of cyclic dinucleotide by using STING protein |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20160928 |
|
| WD01 | Invention patent application deemed withdrawn after publication |