CN109456960A - A kind of method of redox graphene immobilization Phenylalanine dehydrogenase - Google Patents
A kind of method of redox graphene immobilization Phenylalanine dehydrogenase Download PDFInfo
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Abstract
The invention discloses a kind of methods using redox graphene immobilization Phenylalanine dehydrogenase, belong to enzyme immobilization technology field.The stable structure of redox graphene, good biocompatibility are a kind of excellent enzyme immobilization carriers.The redox graphene surface of lamellar structure lacks the groups such as epoxy group, carbonyl, carboxyl, and load area is big and has hydrophobicity.Redox graphene immobilization Phenylalanine dehydrogenase is mainly hydrophobic effect, and in immobilization process, the reducing degree and salinity of redox graphene have influence to redox graphene immobilised enzymes.The pH stability and reuse stability of gained immobilization Phenylalanine dehydrogenase are remarkably reinforced, while activity recovery with higher.The advantage of the invention is that simple process, preparation condition are mild, fixed rate is high.
Description
Technical field
The present invention relates to a kind of methods using redox graphene immobilization Phenylalanine dehydrogenase, belong to enzyme and fix
Change field.
Background technique
Phenylalanine dehydrogenase (Phenylalanine Dehydrogenase, PheDH) can be catalyzed L-phenylalanine auxiliary
Enzyme NAD+In the presence of aoxidize de- amine and form phenylpyruvic acid, also reversible reaction catalysis phenylpyruvic acid and ammonia and coenzyme NAD H
Synthesize L-phenylalanine.It can detect phenylketonuria (PKU) using PheDH, detection process is convenient, quick, can avoid endogenous glimmering
The interference of light and antibiotic.
Since free Phenylalanine dehydrogenase stability is poor, can be improved using the method for immobilization.Immobilised enzymes
Recyclable, reuse, stability are good.The performance of immobilised enzymes depend on immobilised enzymes used in carrier material property and
Process for fixation.Enzyme immobilizatio material includes inorganic, organic polymer, gel and biomaterial etc..Enzyme immobilizatio side
There are mainly four types of methods: investment, absorption method, covalent method and cross-linking method.
Redox graphene (Chemical reduced Graphene Oxide, CRGO) is graphene
(Graphene) the important derivative of one kind, has good biocompatibility, in biological medicine, medicament slow release, packing timber
The fields such as material have broad application prospects.CRGO has two-dimensional slice structure similar with graphene, by controlling electronation
Time can regulate and control reducing degree.The CRGO of different reducing degrees is on the two-dimensional surface of carbon skeleton and edge has different numbers
The oxygen-containing groups such as-OH ,-COOH and the-O- of amount have different hydrophobic performances, therefore have good biocompatibility.At present
There is not a kind of method using redox graphene immobilization Phenylalanine dehydrogenase also.
Summary of the invention
The present invention provides a kind of methods using redox graphene immobilization Phenylalanine dehydrogenase.Utilize reduction
The temperature stability of Phenylalanine dehydrogenase can be improved as immobilization carrier immobilization Phenylalanine dehydrogenase in graphene oxide
With reuse number.The method, unlimited Phenylalanine dehydrogenase can be used in oxidoreducing enzyme.
The technical solution adopted by the present invention to solve the technical problems first is that:
A method of utilizing redox graphene immobilization Phenylalanine dehydrogenase, comprising the following steps:
1) prepared by crude enzyme liquid: the recombinant strains that can express the Phenylalanine dehydrogenase with His-tag label are inoculated with
It is cultivated into LB culture medium containing kanamycin, lactose inducement IPTG is added after cultivating a period of time；Culture gained bacterium solution,
Centrifugation obtains cell, is configured to cell suspension；Ultrasonication is simultaneously centrifuged, and collecting supernatant is to contain with His-tag label
The crude enzyme liquid of Phenylalanine dehydrogenase；
Wherein, the method that building can express the recombinant strains of the Phenylalanine dehydrogenase with His-tag label: phenylpropyl alcohol
The original series of propylhomoserin dehydrogenase gene come from Bacillus nanhaiensis, as shown in SEQ ID NO.1；According to above-mentioned original
Beginning sequence is respectively provided with the Phenylalanine dehydrogenase gene of NdeI and Xhol restriction enzyme site using 5 ' end of PCR method building and 3 ' ends；
PET28a- is obtained with the above-mentioned Phenylalanine dehydrogenase gene of NdeI and Xhol difference double digestion and pET28a plasmid, connection conversion
PheDH plasmid；Above-mentioned plasmid is converted to E.coli BL21 (DE3), obtains that the phenylalanine with His-tag label can be expressed
Recombinant strains E.coli BL21 (DE3)/pET28a of dehydrogenase.
This can be expressed to the recombinant strains E.coli BL21 of the Phenylalanine dehydrogenase with His-tag label
(DE3)/pET28a is inoculated into LB culture medium containing kanamycin and cultivates, and lactose inducement is added after cultivating a period of time
IPTG.The inoculum concentration is 1~3%, the composition of the LB culture medium are as follows: 5.0~15.0g/L of tryptone, yeast leaching
Cream 0.0~15.0g/L of 1.0~10.0g/L, NaCl adjusts pH 7.0~7.5, and addition kanamycins makes its final concentration before being inoculated with
For 50~150 μ g/mL；The condition of culture is 36~38 DEG C, and inducer IPTG is added after cultivating 1.5~6h in 150~250rpm,
Make its final concentration of 5~15mg/mL, continues to cultivate 2~12h under 150~250rpm at 25~30 DEG C.Culture gained bacterium solution,
It is centrifuged (preferably 4 DEG C, 8000rpm, 15min) acquisition cells in refrigerated centrifuge, abandons supernatant, precipitating uses phosphate buffer
(pH 7~7.5) is resuspended, and is sufficiently centrifuged after washing, repetitive operation 3 times.It is configured to phosphate buffer (pH 6.5~8.0) dense
Degree is the cell suspension of 50~150g/L.
The cell suspension of preparation is placed in ice bath, ultrasonic cell disintegration instrument probe is placed in lower 1 centimetre of liquid level, power
200W, ultrasound 3 seconds are spaced 6 seconds, ultrasound 20~60 times.Then 4 DEG C, that 12000rpm centrifugation 15min removes insoluble cell is broken
Piece, supernatant are the crude enzyme liquid for containing the Phenylalanine dehydrogenase with His-tag label.
2) preparation of pure enzyme: purify to the crude enzyme liquid that step 1) obtains using nickel column and desalination；The purification column of use
For be capable of specificity purifying with His-tagged label protein HisTrap HP column, step include balance, loading,
Balance, elution, pillar regeneration；It collects the part of elution and carries out desalination using ultra-filtration centrifuge tube, the liquid obtained after desalination is i.e.
For the pure enzyme solutions of the Phenylalanine dehydrogenase with His-tag label.
3) preparation of redox graphene solution: graphene oxide is prepared with improvement Hummers method, by graphene oxide
It is dissolved in pure water, the finely dispersed graphene oxide suspension that concentration is 0.1~10mg/mL is obtained after ultrasonic disperse, from
It is graphene oxide solution that supernatant is taken after the heart, and reconstitution concentration is the graphene oxide solution of 1mg/mL, by the anti-of 0.05g
Bad hematic acid (L-AA) powder be directly added into 10mL concentration be 1mg/mL graphene oxide solution in, at room temperature vibrate 0.5~
For 24 hours, 10000rpm be centrifuged 30min obtain reduzate, and be washed with deionized three times, with phosphate buffer (pH=7~
7.4) solution is resuspended, and obtains redox graphene (CRGO) suspension, which can stable preservation in room temperature.
4) preparation of redox graphene immobilization Phenylalanine dehydrogenase: in the reduction-oxidation graphite that step 3) obtains
Be added in alkene suspension the obtained Phenylalanine dehydrogenase with His-tag label of step 2) pure enzyme solutions (volume ratio 1:
1), and make final concentration of 0.1~10mg/mL of the Phenylalanine dehydrogenase with His-tag label, adjust NaCl concentration be 0~
5M vibrates 1~5h in 4~45 DEG C of constant temperature oscillators to get redox graphene immobilised enzymes.
Preferably, in the step 3), oscillation restores 3~18h at room temperature.
Preferably, in the step 4), adjusting NaCl concentration is 3~5M.
Invention also provides immobilization phenylpropyl alcohols prepared by the method using above-mentioned redox graphene immobilised enzymes
Propylhomoserin dehydrogenase.
Beneficial effects of the present invention are as follows:
The redox graphene of lamellar structure is a kind of excellent fixation support, and surface lacks epoxy group, carbonyl, carboxylic
The functional groups such as base, load area are big.In immobilization process, redox graphene immobilization Phenylalanine dehydrogenase master
To be hydrophobic effect, using the CRGO of different reducing degrees, and regulate and control the salinity of immobilised enzymes system, can control enzyme and CRGO
Between hydrophobic effect and Hyarogen-bonding, realize the controllable immobilization of enzyme, enhance immobilised enzymes effect.And this method is to enzyme point
The structure interference of son is less, can preferably keep enzymatic activity.The advantage of the invention is that simple process, preparation condition are mild, solid
Determine rate height.The pH stability and reuse stability of gained immobilised enzymes are remarkably reinforced, while enzymatic activity with higher
The rate of recovery.
Detailed description of the invention
Present invention will be further explained below with reference to the attached drawings and examples.
Fig. 1 is activity influence of the salinity to free phenylalanine dehydrogenase.
Fig. 2 is influence of the salinity to the enzyme activity rate of recovery of CRGO-PheDH.
Fig. 3 is the influence for the ability that salinity loads free phenylalanine dehydrogenase to CRGO.
Fig. 4 is the temperature stability of 2M-CRGO-PheDH in embodiment 3.
Fig. 5 is the scanning electron microscope characterization of immobilised enzymes in embodiment 3.(A) redox graphene (CRGO) SEM
Figure；(B) immobilised enzymes (2M-CRGO-PheDH) SEM schemes.
Fig. 6 is the temperature stability of 4M-CRGO-PheDH in embodiment 5.
Fig. 7 is influence of the reduction reaction time to the enzyme activity rate of recovery of CRGO-PheDH.
Fig. 8 is the influence for the ability for the reduction reaction time free phenylalanine dehydrogenase being loaded to CRGO.
Specific embodiment
It elaborates below by embodiment to the present invention.
Embodiment 1
(1) preparation of crude enzyme liquid:
Building can express the recombinant strains of the Phenylalanine dehydrogenase with His-tag label: Phenylalanine dehydrogenase
(PheDH) original series of gene come from Bacillus nanhaiensis, as shown in SEQ ID NO.1；According to above-mentioned original
Sequence designs the primer as shown in SEQ ID NO.2 and SEQ ID NO.3, is respectively provided with using 5 ' end of PCR method building and 3 ' ends
The Phenylalanine dehydrogenase gene of NdeI and Xhol restriction enzyme site, PCR synthesis process is by the raw work biotechnology service in Shanghai
Co., Ltd completes.For pcr amplification product after the identification of 1% agarose gel electrophoresis, glue recycles PheDH genetic fragment, uses NdeI
Double digestion is carried out with XhoI digestion enzyme, recycles digestion products, (is marked with His-tag with the pET-28a plasmid of same double digestion
Label) it is attached, the plasmid connected is transformed into e. coli bl21 (DE3), obtains pET28a-PheDH plasmid.By above-mentioned matter
Grain conversion obtains the recombinant expression bacterium that can express the Phenylalanine dehydrogenase with His-tag label to E.coli BL21 (DE3)
Strain E.coli BL21 (DE3)/pET28a.
Recombination bacillus coli E.coli BL21 (DE3)/pET28a culture: with 1% inoculum concentration, strain is accessed
In 200mL LB culture medium.The group of LB culture medium becomes 10.0g/L tryptone, 5.0g/L yeast extract, 10g/L NaCl.Training
Support condition are as follows: starting pH 7.0, liquid amount volume fraction be 10%, 37 DEG C of cultivation temperature, shaking speed 200rpm, incubation time
6 hours.Inducer IPTG is added, makes its final concentration of 10mg/mL, continuation is cultivated 12 hours under the conditions of 25 DEG C, 200rpm.
Culture terminates the fermentation liquid obtained, and (4 DEG C, 8000rpm, 15min) acquisition cells are centrifuged in refrigerated centrifuge, abandons
Supernatant, precipitating are resuspended with phosphate buffer (pH=7~7.4), are sufficiently centrifuged after washing, repetitive operation 3 times, are used phosphoric acid buffer
Liquid (pH 7~7.4) is configured to the cell suspension that concentration is 50~150g/L.
The cell suspension of preparation is placed in ice bath, cell liquid is handled using Ultrasonic Cell Disruptor, cell crushing instrument
Probe is placed in 1cm under liquid level, and broken condition is ultrasound 3 seconds, is spaced 6 seconds, ultrasound 60 times, power 200W.Then 4 DEG C, 12,
000rpm is centrifuged 15min and removes insoluble cell fragment, and supernatant is to contain the Phenylalanine dehydrogenase with His-tag label
Crude enzyme liquid.
(2) the pure enzyme preparation of Phenylalanine dehydrogenase: using the His Trap nickel column (Histrap of GE companyTMHP, 5mL) it is right
The crude enzyme liquid that step (1) obtains is isolated and purified, and carries out ultrafiltration desalination with the ultra-filtration centrifuge tube of the 10K of PALL company.Institute
The purification column that the purification process stated uses for be capable of specificity purifying with His-tagged label protein HisTrap
HP column, step include balance, loading, balance, elution, pillar regeneration；Collect elution part and using ultra-filtration centrifuge tube into
Row desalination；The liquid obtained after desalination is the pure enzyme solutions of the Phenylalanine dehydrogenase with His-tag label purified.
(3) preparation of redox graphene solution: graphene oxide is prepared with improvement Hummers method, by graphite oxide
Alkene is placed in pure water, and ultrasonic disperse (100W, 120min) obtains the finely dispersed graphite oxide that concentration is 0.1~10mg/mL
Alkene suspension, taking supernatant after centrifugation is graphene oxide solution, and reconstitution concentration is the graphene oxide solution of 1mg/mL,
Ascorbic acid (L-AA) powder of 0.05g is directly added into the graphene oxide solution that 10mL concentration is 1mg/mL, at room temperature
For 24 hours, 10000rpm centrifugation 30min obtains reduzate, and is washed with deionized three times, with phosphate buffer (pH=for oscillation
7.4) solution is resuspended, and obtains redox graphene (CRGO) suspension.
(4) detection method of enzyme activity: the determination of activity reaction system of Phenylalanine dehydrogenase includes 10 μ L, 40mM NAD+, 160 μ L, 0.2mol/L Glycine-NaOH buffer solutions (pH 9.5), 10 μ L, 40mM L-phenylalanine solution and 20
μ L enzyme solution at 37 DEG C, measures enzyme activity under 340nm wavelength.Enzyme activity is defined as under the above conditions, oxidation consumption per minute (or
Generate) 1 μm of ol NAD+Required enzyme amount is an enzyme activity unit.
Salting liquid (salinity includes 0M, 1M, 2M, 3.5M, 4M, 5M) is added in Phenylalanine dehydrogenase enzyme activity determination system,
Investigating different salinity has activation to free phenylalanine dehydrogenase enzyme activity.As a result as shown in Figure of description 1.
(5) preparation of redox graphene immobilization Phenylalanine dehydrogenase: in the oxygen reduction fossil that step (3) obtains
The pure enzyme solutions for the Phenylalanine dehydrogenase with His-tag label that step (2) obtain are added in black alkene suspension, and make band
The final concentration of 0.4116mg/mL of the Phenylalanine dehydrogenase of His-tag label, oscillation 4h is to get also in 4 DEG C of constant temperature oscillators
Former graphene oxide immobilised enzymes 0M-CRGO-PheDH, the enzyme activity rate of recovery can reach 18.39%, as shown in Figure of description 2.
Redox graphene load capacity is 0.370mg/mg, as shown in Figure of description 3.
(6) measurement of immobilised enzymes temperature stability: by resolvase and immobilised enzymes 0M-CRGO-PheDH at 40 DEG C
Constant temperature water bath 3h measures an enzyme activity every 30min in this process.Relative to resolvase, the temperature of immobilised enzymes is stablized
Property significantly improve, with the initial enzyme activity of immobilised enzymes be 100%, immobilised enzymes 0M-CRGO- is obtained after 40 DEG C of water bath with thermostatic control 3h
PheDH, opposite enzyme activity are 13.03%.
Embodiment 2
(1)~(4) the step of experimental procedure such as embodiment 1 (1)~(4).
(5) preparation of redox graphene immobilization Phenylalanine dehydrogenase: in the oxygen reduction fossil that step (3) obtains
The pure enzyme solutions for the Phenylalanine dehydrogenase with His-tag label that step (2) obtain are added in black alkene suspension, and make band
The final concentration of 0.4116mg/mL of the Phenylalanine dehydrogenase of His-tag label, adjusting NaCl concentration are 1M, 4 DEG C of constant temperature oscillations
4h is vibrated in device, obtains immobilised enzymes 1M-CRGO-PheDH, and the enzyme activity rate of recovery can reach 42.15%, such as 2 institute of Figure of description
Show.Redox graphene immobilised enzymes load capacity is 0.409mg/mg, as shown in Figure of description 3.
(6) measurement of immobilised enzymes temperature stability: by resolvase and immobilised enzymes 1M-CRGO-PheDH at 40 DEG C
Constant temperature water bath 3h measures an enzyme activity every 30min in this process.Relative to resolvase, the temperature of immobilised enzymes is stablized
Property significantly improve, with the initial enzyme activity of immobilised enzymes be 100%, immobilised enzymes 1M-CRGO- is obtained after 40 DEG C of water bath with thermostatic control 3h
PheDH, opposite enzyme activity are 89.68%.
Embodiment 3
(1)~(4) the step of experimental procedure such as embodiment 1 (1)~(4).
(5) preparation of redox graphene immobilization Phenylalanine dehydrogenase: in the oxygen reduction fossil that step (3) obtains
The pure enzyme solutions for the Phenylalanine dehydrogenase with His-tag label that step (2) obtain are added in black alkene suspension, and make band
The final concentration of 0.4116mg/mL of the Phenylalanine dehydrogenase of His-tag label, adjusting NaCl concentration are 2M, 4 DEG C of constant temperature oscillations
4h is vibrated in device, obtains immobilised enzymes 2M-CRGO-PheDH, and the enzyme activity rate of recovery can reach 46.44%, such as 2 institute of Figure of description
Show.The load capacity of redox graphene immobilised enzymes is 0.410mg/mg, as shown in Figure of description 3.
(6) measurement of immobilised enzymes temperature stability: by resolvase and immobilised enzymes 2M-CRGO-PheDH at 40 DEG C
Constant temperature water bath 3h measures an enzyme activity every 30min in this process.Relative to resolvase, the temperature of immobilised enzymes is stablized
Property significantly improve, with the initial enzyme activity of immobilised enzymes be 100%, immobilised enzymes 2M-CRGO- is obtained after 40 DEG C of water bath with thermostatic control 3h
PheDH, opposite enzyme activity is 52.29%, as shown in Figure of description 4.
(7) immobilised enzymes scanning electron microscope (SEM) characterization: with SEM observation redox graphene (CRGO),
The shape characteristic of immobilised enzymes (2M-CRGO-PheDH), as shown in Figure of description 5.
Embodiment 4
(1)~(4) the step of experimental procedure such as embodiment 1 (1)~(4).
(5) preparation of redox graphene immobilization Phenylalanine dehydrogenase: in the oxygen reduction fossil that step (3) obtains
The pure enzyme solutions for the Phenylalanine dehydrogenase with His-tag label that step (2) obtain are added in black alkene suspension, and make band
The final concentration of 0.4116mg/mL of the Phenylalanine dehydrogenase of His-tag label, adjusting NaCl concentration are 3.5M, 4 DEG C of constant temperature vibrations
It swings and vibrates 4h in device, obtain immobilised enzymes 3.5M-CRGO-PheDH, the enzyme activity rate of recovery can reach 69.02%, such as Figure of description
Shown in 2.The load capacity of redox graphene immobilised enzymes is 0.410mg/mg, as shown in Figure of description 3.
(6) measurement of immobilised enzymes temperature stability: by resolvase and immobilised enzymes in 40 DEG C of constant temperature water bath 3h,
An enzyme activity is measured every 30min during this.Relative to resolvase, the temperature stability of immobilised enzymes is significantly improved, with
The initial enzyme activity of immobilised enzymes is 100%, and immobilised enzymes 3.5M-CRGO-PheDH, phase are obtained after 40 DEG C of water bath with thermostatic control 3h
It is 75.78% to enzyme activity.
Embodiment 5
(1)~(4) the step of experimental procedure such as embodiment 1 (1)~(4).
(5) preparation of redox graphene immobilization Phenylalanine dehydrogenase: in the oxygen reduction fossil that step (3) obtains
The pure enzyme solutions for the Phenylalanine dehydrogenase with His-tag label that step (2) obtain are added in black alkene suspension, and make band
The final concentration of 0.4116mg/mL of the Phenylalanine dehydrogenase of His-tag label, adjusting NaCl concentration are 4M, 4 DEG C of constant temperature oscillations
4h is vibrated in device, obtains immobilised enzymes 4M-CRGO-PheDH, and the enzyme activity rate of recovery can reach 71.2%, such as 2 institute of Figure of description
Show.The load capacity of redox graphene immobilised enzymes is 0.408mg/mg, as shown in Figure of description 3.
(6) measurement of immobilised enzymes temperature stability: by resolvase and immobilised enzymes in 40 DEG C of constant temperature water bath 3h,
An enzyme activity is measured every 30min during this.Relative to resolvase, the temperature stability of immobilised enzymes is significantly improved, with
The initial enzyme activity of immobilised enzymes is 100%, and immobilised enzymes 4M-CRGO-PheDH is obtained after 40 DEG C of water bath with thermostatic control 3h, opposite
Enzyme activity is 28.82%, as shown in Figure of description 6.
Embodiment 6
(1)~(4) the step of experimental procedure such as embodiment 1 (1)~(4).
(5) preparation of redox graphene immobilization Phenylalanine dehydrogenase: in the oxygen reduction fossil that step (3) obtains
The pure enzyme solutions for the Phenylalanine dehydrogenase with His-tag label that step (2) obtain are added in black alkene suspension, and make band
The final concentration of 0.4116mg/mL of the Phenylalanine dehydrogenase of His-tag label, adjusting NaCl concentration are 5M, 4 DEG C of constant temperature oscillations
4h is vibrated in device, obtains immobilised enzymes 5M-CRGO-PheDH, and the enzyme activity rate of recovery can reach 70.9%, such as 2 institute of Figure of description
Show.The load capacity of redox graphene immobilised enzymes is 0.408mg/mg, as shown in Figure of description 3.
(6) measurement of immobilised enzymes temperature stability: by resolvase and immobilised enzymes in 40 DEG C of constant temperature water bath 3h,
An enzyme activity is measured every 30min during this.Relative to resolvase, the temperature stability of immobilised enzymes is significantly improved, with
The initial enzyme activity of immobilised enzymes is 100%, and immobilised enzymes 5M-CRGO-PheDH is obtained after 40 DEG C of water bath with thermostatic control 3h, opposite
Enzyme activity is 29.67%.
Embodiment 7
(1), the step of (2) experimental procedure such as embodiment 1 (1), (2).
(3) preparation of redox graphene solution: graphene oxide is prepared with improvement Hummers method, by graphite oxide
Alkene is placed in pure water, and it is suspended that ultrasonic disperse (100W, 120min) obtains the finely dispersed graphene oxide that concentration is 1mg/mL
Liquid, taking supernatant after centrifugation is graphene oxide solution, and reconstitution concentration is the graphene oxide solution of 1mg/mL, will
Ascorbic acid (L-AA) powder of 0.05g is directly added into the graphene oxide solution that 10mL concentration is 1mg/mL, is shaken at room temperature
Swing 3h, 10000rpm centrifugation 30min obtains reduzate, and be washed with deionized three times, with phosphate buffer (pH=7~
7.4) solution is resuspended, and obtains redox graphene (CRGO) suspension.
(4) detection method of enzyme activity: the determination of activity reaction system of Phenylalanine dehydrogenase includes 10 μ L, 40mM NAD+, 160 μ L, 0.2mol/L Glycine-NaOH buffer solutions (pH 9.5), 10 μ L, 40mM L-phenylalanine solution and 20
μ L enzyme solution at 37 DEG C, measures enzyme activity under 340nm wavelength.Enzyme activity is defined as under the above conditions, oxidation consumption per minute (or
Generate) 1 μm of ol NAD+Required enzyme amount is an enzyme activity unit.
(5) preparation of redox graphene immobilization Phenylalanine dehydrogenase: in the oxygen reduction fossil that step (3) obtains
The pure enzyme solutions for the Phenylalanine dehydrogenase with His-tag label that step (2) obtain are added in black alkene suspension, and make band
The final concentration of 0.4116mg/mL of the Phenylalanine dehydrogenase of His-tag label vibrates 4h in 4 DEG C of constant temperature oscillators, consolidate
Surely change enzyme 3h-CRGO-PheDH, the enzyme activity rate of recovery can reach 42.22%, as shown in Figure of description 7.Redox graphene
Immobilised enzymes load capacity is 0.402mg/mg, as shown in Figure of description 8.
Embodiment 8
(1), the step of (2) experimental procedure such as embodiment 1 (1), (2).
(3) preparation of redox graphene solution: graphene oxide is prepared with improvement Hummers method, by graphite oxide
Alkene is placed in pure water, and it is suspended that ultrasonic disperse (100W, 120min) obtains the finely dispersed graphene oxide that concentration is 1mg/mL
Liquid, taking supernatant after centrifugation is graphene oxide solution, and reconstitution concentration is the graphene oxide solution of 1mg/mL, will
Ascorbic acid (L-AA) powder of 0.05g is directly added into the graphene oxide solution that 10mL concentration is 1mg/mL, is shaken at room temperature
Swing 6h, 10000rpm centrifugation 30min obtains reduzate, and be washed with deionized three times, with phosphate buffer (pH=7~
7.4) solution is resuspended, and obtains redox graphene (CRGO) suspension.
(4) detection method of enzyme activity: the determination of activity reaction system of Phenylalanine dehydrogenase includes 10 μ L, 40mM NAD+, 160 μ L, 0.2mol/L Glycine-NaOH buffer solutions (pH 9.5), 10 μ L, 40mM L-phenylalanine solution and 20
μ L enzyme solution at 37 DEG C, measures enzyme activity under 340nm wavelength.Enzyme activity is defined as under the above conditions, oxidation consumption per minute (or
Generate) 1 μm of ol NAD+Required enzyme amount is an enzyme activity unit.
(5) preparation of redox graphene immobilization Phenylalanine dehydrogenase: in the oxygen reduction fossil that step (3) obtains
The pure enzyme solutions for the Phenylalanine dehydrogenase with His-tag label that step (2) obtain are added in black alkene suspension, and make band
The final concentration of 0.4116mg/mL of the Phenylalanine dehydrogenase of His-tag label vibrates 4h in 4 DEG C of constant temperature oscillators, consolidate
Surely change enzyme 6h-CRGO-PheDH, the enzyme activity rate of recovery can reach 45%, as shown in Figure of description 7.Redox graphene is fixed
Change enzyme load capacity is 0.411mg/mg, as shown in Figure of description 8.
Embodiment 9
(1), the step of (2) experimental procedure such as embodiment 1 (1), (2).
(3) preparation of redox graphene solution: graphene oxide is prepared with improvement Hummers method, by graphite oxide
Alkene is placed in pure water, and it is suspended that ultrasonic disperse (100W, 120min) obtains the finely dispersed graphene oxide that concentration is 1mg/mL
Liquid, taking supernatant after centrifugation is graphene oxide solution, and reconstitution concentration is the graphene oxide solution of 1mg/mL, will
Ascorbic acid (L-AA) powder of 0.05g is directly added into the graphene oxide solution that 10mL concentration is 1mg/mL, is shaken at room temperature
Swing 12h, 10000rpm centrifugation 30min obtains reduzate, and be washed with deionized three times, with phosphate buffer (pH 7~
7.4) solution is resuspended, and obtains redox graphene (CRGO) suspension.
(4) detection method of enzyme activity: the determination of activity reaction system of Phenylalanine dehydrogenase includes 10 μ L, 40mM NAD+, 160 μ L, 0.2mol/L Glycine-NaOH buffer solutions (pH 9.5), 10 μ L, 40mM L-phenylalanine solution and 20
μ L enzyme solution at 37 DEG C, measures enzyme activity under 340nm wavelength.Enzyme activity is defined as under the above conditions, oxidation consumption per minute (or
Generate) 1 μm of ol NAD+Required enzyme amount is an enzyme activity unit.
(5) preparation of redox graphene immobilization Phenylalanine dehydrogenase: in the oxygen reduction fossil that step (3) obtains
The pure enzyme solutions for the Phenylalanine dehydrogenase with His-tag label that step (2) obtain are added in black alkene suspension, and make band
The final concentration of 0.4116mg/mL of the Phenylalanine dehydrogenase of His-tag label vibrates 4h in 4 DEG C of constant temperature oscillators, consolidate
Surely change enzyme 12h-CRGO-PheDH, the enzyme activity rate of recovery can reach 46.44%, as shown in Figure of description 7.Redox graphene
Immobilised enzymes load capacity is 0.4116mg/mg, as shown in Figure of description 8.
Embodiment 10
(1), the step of (2) experimental procedure such as embodiment 1 (1), (2).
(3) preparation of redox graphene solution: graphene oxide is prepared with improvement Hummers method, by graphite oxide
Alkene is placed in pure water, and it is suspended that ultrasonic disperse (100W, 120min) obtains the finely dispersed graphene oxide that concentration is 1mg/mL
Liquid, taking supernatant after centrifugation is graphene oxide solution, and reconstitution concentration is the graphene oxide solution of 1mg/mL, will
Ascorbic acid (L-AA) powder of 0.05g is directly added into the graphene oxide solution that 10mL concentration is 1mg/mL, is shaken at room temperature
Swing 18h, 10000rpm centrifugation 30min obtains reduzate, and be washed with deionized three times, with phosphate buffer (pH=7~
7.4) solution is resuspended, and obtains redox graphene (CRGO) suspension.
(4) detection method of enzyme activity: the determination of activity reaction system of Phenylalanine dehydrogenase includes 10 μ L, 40mM NAD+, 160 μ L, 0.2mol/L Glycine-NaOH buffer solutions (pH 9.5), 10 μ L, 40mM L-phenylalanine solution and 20
μ L enzyme solution at 37 DEG C, measures enzyme activity under 340nm wavelength.Enzyme activity is defined as under the above conditions, oxidation consumption per minute (or
Generate) 1 μm of ol NAD+Required enzyme amount is an enzyme activity unit.
(5) preparation of redox graphene immobilization Phenylalanine dehydrogenase: in the oxygen reduction fossil that step (3) obtains
The pure enzyme solutions for the Phenylalanine dehydrogenase with His-tag label that step (2) obtain are added in black alkene suspension, and make band
The final concentration of 0.4116mg/mL of the Phenylalanine dehydrogenase of His-tag label vibrates 4h in 4 DEG C of constant temperature oscillators, consolidate
Surely change enzyme 18h-CRGO-PheDH, the enzyme activity rate of recovery can reach 42.08%, as shown in Figure of description 7.Redox graphene
Immobilised enzymes load capacity is 0.410mg/mg, as shown in Figure of description 8.
The above is only the preferred embodiment of the present invention, the range implemented of the present invention that therefore, it cannot be limited according to, i.e., according to
Equivalent changes and modifications made by the invention patent range and description, should still be within the scope of the present invention.
Sequence table
<110>Xiamen University
<120>a kind of method of redox graphene immobilization Phenylalanine dehydrogenase
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1101
<212> DNA
<213> Bacillus nanhaiensis
<400> 1
atgtttgaaa aaatatcaca gcatgagcaa gttgtgtttt gtaacgatcc atcaacaggt 60
ctcaaggcaa ttatcgctat acataacaca acattaggcc cagcactcgg cggatgcaga 120
atgagaccgt atggatcggt tgatgaggca cttgaggatg tgctgagatt gtcaaaaggt 180
atgacataca aatgcgctgg tgcagatgtt gacttcggtg gaggtaaatc ggtcatcatc 240
ggggatccga tgacggatcg tacaccagag ttgttccgag cattcggaca gtttgtagat 300
tcattaaacg gtcgctttta tacaggaaca gatatgggaa caacacctga tgattttatg 360
cacgcgttaa aagaaacgaa ttgtatcgtc ggggtacccg aagaatatgg cggcagcggc 420
gattcttctg ttccaacagc acaaggagtt atatacggac ttcaagctac cattcagacg 480
cttgaaggaa cagatgaact ttcaggtaag tcatactcta tacaaggttt aggaaaagta 540
ggttttaagg tcgcagagca actgctcgca gccggtacac aaatctatgt tactgatatt 600
aatgaaaaag cattaaagat gattcaagaa cgagcagaac tcctacctgg aaatgtggaa 660
gtagttgaag gaagcgacat ctacggggtg gatgctgata ttttcattcc ttgcgcactc 720
ggcggaatca ttcacgatga aacaattgaa caactaaaag taaaagcgat cgtaggaagt 780
gccaacaatc agctcttaga agataagcac ggactttatt tgcagcaaaa aggaatttta 840
tacggacccg attatatcgt gaacgcagga gggcttattc aggtagctga tgagctttat 900
ggaccgaata aagcccgtgt attaacgaaa acgagagcga tctatgacag tctgatacag 960
atttatagcg agagtacaaa gaaccagata tctacgatgg aagcagcaaa tcttttctgt 1020
gaggagaagc ttttggcccg ctcaaaacga aacagctttt tcgctcacaa ccgaagacca 1080
aaatggcagg ttagacatta a 1101
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<210> 3
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<212> DNA
<213>artificial sequence (Artificial Sequence)
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Claims (6)
1. a kind of method using redox graphene immobilization Phenylalanine dehydrogenase, it is characterised in that: including following step
It is rapid:
1) prepared by crude enzyme liquid: the recombinant strains that can express the Phenylalanine dehydrogenase with His-tag label being inoculated into and are contained
It is cultivated in the LB culture medium for having kanamycins, lactose inducement IPTG is added after cultivating a period of time；Culture gained bacterium solution, centrifugation
Cell is obtained, cell suspension is configured to；Ultrasonication is simultaneously centrifuged, and collecting supernatant is to contain the phenylpropyl alcohol with His-tag label
The crude enzyme liquid of propylhomoserin dehydrogenase；
2) preparation of pure enzyme: purify to the crude enzyme liquid that step 1) obtains using nickel column and desalination, obtains band His-tag label
Phenylalanine dehydrogenase；
3) preparation of redox graphene solution: graphene oxide is dissolved in water, is uniformly dispersed after ultrasonic disperse
Graphene oxide suspension, taking supernatant after centrifugation is graphene oxide solution；Graphite oxide is added in ascorbic acid
In alkene solution, the mass ratio of ascorbic acid and graphene oxide is 4~6:1；At room temperature oscillation reduction 0.5~for 24 hours, centrifugation obtains
Reduzate, washing several times, are resuspended with the phosphate buffer of pH 7~7.4 to get redox graphene suspension is arrived；
4) preparation of redox graphene immobilization Phenylalanine dehydrogenase: outstanding in the redox graphene that step 3) obtains
The Phenylalanine dehydrogenase with His-tag label that step 2) obtains is added in turbid, and makes the phenylpropyl alcohol ammonia with His-tag label
Final concentration of 0.1~10mg/mL of acidohydrogenase, adjusting NaCl concentration are 0~5M, oscillation 1 in 4~45 DEG C of constant temperature oscillators~
5h is to get redox graphene immobilization Phenylalanine dehydrogenase.
2. the method for utilizing redox graphene immobilization Phenylalanine dehydrogenase as described in claim 1, feature exist
In: in the step 1), the construction method of the recombinant strains of the Phenylalanine dehydrogenase with His-tag label can be expressed
Are as follows: as shown in SEQ ID NO.1,5 ' end of PCR method building and 3 ' ends are respectively provided with the original series of Phenylalanine dehydrogenase gene
The Phenylalanine dehydrogenase gene of NdeI and Xhol restriction enzyme site；The above-mentioned phenylalanine dehydrogenation of double digestion is distinguished with NdeI and Xhol
Enzyme gene and pET28a plasmid, connection conversion, obtain pET28a-PheDH plasmid；Above-mentioned plasmid is converted to E.coli BL21
(DE3), obtain can expressing the recombinant strains E.coli BL21 (DE3) of the Phenylalanine dehydrogenase with His-tag label/
pET28a。
3. the method for utilizing redox graphene immobilization Phenylalanine dehydrogenase as claimed in claim 2, feature exist
In: in the step 1), E.coli BL21 (the DE3)/pET28a is inoculated into 1~3% inoculum concentration described containing card
It is cultivated in the LB culture medium of that mycin, the LB culture medium includes: 5.0~15.0g/L of tryptone, yeast extract 1.0~
0.0~15.0g/L of 10.0g/L, NaCl, adjust pH 7.0~7.5, be inoculated with before addition kanamycins make its final concentration of 50~
150μg/mL；Condition of culture is 36~38 DEG C, and inducer IPTG is added after cultivating 1.5~6h in 150~250rpm, makes its final concentration
For 5~15mg/mL, continue to cultivate 2~12h under 150~250rpm at 25~30 DEG C；Culture gained bacterium solution, centrifugation obtains thin
Born of the same parents abandon supernatant, and precipitating is resuspended with the phosphate buffer of pH 7~7.5, is sufficiently centrifuged after washing, and repetitive operation several times, is used
The phosphate buffer of pH 6.5~8.0 is configured to the cell suspension that concentration is 50~150g/L.
4. the method for utilizing redox graphene immobilization Phenylalanine dehydrogenase as described in claim 1, feature exist
In: in the step 3), oscillation restores 3~18h at room temperature.
5. the method for utilizing redox graphene immobilization Phenylalanine dehydrogenase as described in claim 1, feature exist
In: in the step 4), adjusting NaCl concentration is 3~5M.
6. one kind is according to any one of claim 1 to 5 to utilize redox graphene immobilization phenylalanine dehydrogenation
The immobilization Phenylalanine dehydrogenase of the method preparation of enzyme.
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| CN110819693A (en) * | 2019-10-31 | 2020-02-21 | 申友基因组研究院(南京)有限公司 | Method for detecting contents of phenylalanine and tyrosine by enzyme method and application thereof |
| CN111855775A (en) * | 2020-06-15 | 2020-10-30 | 厦门大学 | Amino acid dehydrogenase electrode and preparation method and application thereof |
| CN112266905A (en) * | 2020-09-27 | 2021-01-26 | 厦门大学 | Polypeptide modified amino acid dehydrogenase and preparation and immobilization method thereof |
| CN113564154A (en) * | 2021-08-16 | 2021-10-29 | 厦门大学 | Method for coordinating and immobilizing oxidoreductase by using graphene oxide-wool keratin and metal ions |
| CN116440883A (en) * | 2023-02-20 | 2023-07-18 | 江苏恰瑞生物科技有限公司 | Preparation method of blood perfusion device filler for treating phenylketonuria |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110777188A (en) * | 2019-10-31 | 2020-02-11 | 申友基因组研究院(南京)有限公司 | Method for detecting content of glutamic acid by enzyme method and application thereof |
| CN110819693A (en) * | 2019-10-31 | 2020-02-21 | 申友基因组研究院(南京)有限公司 | Method for detecting contents of phenylalanine and tyrosine by enzyme method and application thereof |
| CN110777188B (en) * | 2019-10-31 | 2024-02-27 | 申友基因组研究院(南京)有限公司 | Method for detecting glutamic acid content by enzyme method and application thereof |
| CN111855775A (en) * | 2020-06-15 | 2020-10-30 | 厦门大学 | Amino acid dehydrogenase electrode and preparation method and application thereof |
| CN112266905A (en) * | 2020-09-27 | 2021-01-26 | 厦门大学 | Polypeptide modified amino acid dehydrogenase and preparation and immobilization method thereof |
| CN113564154A (en) * | 2021-08-16 | 2021-10-29 | 厦门大学 | Method for coordinating and immobilizing oxidoreductase by using graphene oxide-wool keratin and metal ions |
| CN116440883A (en) * | 2023-02-20 | 2023-07-18 | 江苏恰瑞生物科技有限公司 | Preparation method of blood perfusion device filler for treating phenylketonuria |
| CN116440883B (en) * | 2023-02-20 | 2023-11-07 | 江苏恰瑞生物科技有限公司 | Preparation method of filling material of blood perfusion device |
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Application publication date: 20190312 |