CN107893065A - A kind of preparation method of immobilised enzymes - Google Patents
A kind of preparation method of immobilised enzymes Download PDFInfo
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- CN107893065A CN107893065A CN201711186695.7A CN201711186695A CN107893065A CN 107893065 A CN107893065 A CN 107893065A CN 201711186695 A CN201711186695 A CN 201711186695A CN 107893065 A CN107893065 A CN 107893065A
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- C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
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Abstract
The invention discloses a kind of preparation method of absorption-overlay film coimmobilization enzyme, its preparation comprises the following steps：(1) enzyme is dissolved in buffer solution and enzyme solutions is made, and mixed with sorbing material, vibration absorption certain time, filtered out, obtain adsorption of immobilization enzyme；(2) overlay film protective agent is dissolved in buffer solution and protection agent solution is made, adsorption of immobilization enzyme is placed in protection agent solution, oscillation treatment certain time, filters out and drains, obtain the treated adsorption of immobilization enzyme of protective agent；(3) film covering solution is made in covering material dissolving in a solvent, the adsorption of immobilization enzyme that protective agent treats is mixed with film covering solution, vibrated overlay film certain time, filter out drying；(4) finally, with-overlay film coimmobilization enzyme must be adsorbed after deionized water or wash buffer.Present invention absorption, coating process mild condition, the enzyme activity rate of recovery is high, and the stability of immobilised enzymes is high, simple to operate, cost is low.
Description
Technical field
The invention belongs to biological technical field, and in particular to a kind of preparation method of immobilised enzymes.
Background technology
In the last few years, the modern biotechnology using genetic engineering, cell engineering, enzyme engineering, Fermentation Engineering as representative developed
Rapidly, and increasingly influence and change production and the life style of people.Enzyme engineering is the important component of biotechnology, is
Using biocatalytic Activity possessed by enzyme, corresponding raw material is changed into utility and applied to society by engineering means
The a science technology of life.It includes the preparation of enzyme preparation, enzyme immobilizatio, the side such as the modification of enzyme and transformation and enzyme reactor
Face content.
For enzyme as a kind of biocatalyst, its catalytic reaction has high efficiency, selectivity, selectivity, and reaction condition temperature
With, pharmacy, food, environmental protection, brewage, the numerous areas such as the energy is obtained for and is widely applied.But the chemistry of most of enzymes
Be in the nature protein, thus enzyme when free state uses easily by adverse effect environmental condition (such as high temperature, high pressure, strong acid,
Highly basic, heavy metal ion, organic solvent etc.) influence cause structure change reduce activity in addition inactivate.In addition, resolvase uses
When mixed with substrate, product, reaction terminate after be not easily recycled, also isolated and purified to product and cause difficulty.These are all big
Limit application of the enzyme in numerous industrial biocatalytic fields greatly.
Enzyme immobilizatio is that enzyme is fettered or is limited in certain area with immobilization material, can still provide for its and distinctive urges
Change reaction and recyclable and recycling a kind of technology.Compared with resolvase, immobilised enzymes is keeping it efficiently single-minded and warm
While the catalytic reaction characteristic of sum, the weak point of resolvase is overcome again, and presentation stability is high, separation and recovery is easy, can
Repeatedly use, operate the series of advantages such as continuous controllable, simple process.The research of immobilised enzymes starts from 1910, formally
Study in the 1960s, generally having carried out in the whole world the seventies.Current immobilised enzymes is not only in chemistry, biology and life
The research Showed Very Brisk of the ambits such as thing engineering, medical science and life science, is developed rapidly and is widely applied, Er Qieyin
To meet the strategic requirement of sustainable development with the energy, the Impacts on ecology and environment for reducing or preventing and remedying pollution with saving resource.
The preparation method of immobilised enzymes has Physical and the major class of chemical method two.Physical method includes physisorphtion, embedding
Method, the former is that enzyme or the absorption of thalline containing enzyme are made into the method for enzyme immobilization on its surface using various solid absorbents, the latter
It is that enzyme or thalline containing enzyme are embedded in the method for making enzyme immobilization in porous carrier.The advantages of Physical immobilised enzymes, is to fix
Change mild condition, enzyme does not participate in chemical reaction, and overall structure keeps constant, and the catalytic activity of enzyme is retained very well, and enzyme activity is returned
High income.But the adhesion between absorption method enzyme and carrier is weaker, enzyme is easy to that catalysis is come off and polluted from carrier
Reaction product；Easily there is the problems such as seepage of enzyme and substrate product diffusion limitation in investment.Chemical method is to pass through carrier surface
Activity functional groups and enzyme molecule on nonessential group formed chemical covalent bonds, or using some multi-functional cross-linking reagents such as
Glutaraldehyde, chemical covalent bonds are formed between enzyme molecule or between enzyme molecule and carrier molecule, so as to realize enzyme and the irreversible knot of carrier
The enzyme immobilization method of conjunction.Chemical method immobilised enzymes has the advantages of being firmly combined with, stability is high, but immobilization process reaction ratio
It is fiercer, the inactivation of enzyme is easily caused, the enzyme activity rate of recovery is low.
With deepening continuously for enzyme immobilization technology research, many new immobilization means continue to bring out, but on being all
The improvement or combination of method are stated, is still difficult to take into account the high enzyme activity rate of recovery and high stability.Therefore, new enzyme is developed to fix
Change method is still one of important research content of enzyme engineering.
The content of the invention
It is an object of the invention to provide a kind of preparation method of absorption-overlay film coimmobilization enzyme, the immobilised enzymes system
Preparation Method is simple, and the immobilization enzyme activity rate of recovery is high, stability is high, repeat performance is good.
A kind of preparation method of absorption provided by the invention-overlay film coimmobilization enzyme, comprises the following steps：
(1) enzyme is dissolved in buffer solution and enzyme solutions is made, and mixed with sorbing material, vibration absorption certain time, filter
Go out, obtain adsorption of immobilization enzyme；
(2) overlay film protective agent is dissolved in buffer solution and protection agent solution is made, adsorption of immobilization enzyme is placed in protective agent
In solution, oscillation treatment certain time, filter out and drain, obtain the treated adsorption of immobilization enzyme of protective agent；
(3) by covering material dissolving film covering solution is made in a solvent, by protective agent treat adsorption of immobilization enzyme with
Film covering solution mixes, and vibrates overlay film certain time, filters out drying；
(4) finally, with-overlay film coimmobilization enzyme must be adsorbed after deionized water or wash buffer.
According to immobilised enzymes preparation method of the present invention, enzyme described in step (1) for hydrolase (such as amylase,
Protease, lipase, phosphatase, glycosidase etc.), redox enzymes (such as laccase, horseradish peroxidase, polyphenol oxidase
Enzyme, superoxide dismutase, catalase, fatty acid oxidase, ketoreductase, alcohol dehydrogenase etc.), transferase (such as
Transmethylase, aminopherase, acyltransferase etc.), lyases (such as dehydratase, decarboxylase, carbonic anhydrase, aldehyde contracting
Enzyme etc.), isomerase (such as isomerase, epimerase, racemase), synthetic enzyme (such as glutamine synthelase, DNA connect
Connect enzyme, carboxylase, phosphorylase etc.) in one or more, it is preferable that for one kind, or it is a variety of have synergy enzyme,
Or a variety of enzymes for being catalyzed cascade reaction.
According to immobilised enzymes preparation method of the present invention, the sorbing material described in step (1) is attapulgite, diatom
Soil, kaolin, bentonite, atlapulgite, zeolite, montmorillonite, expanded perlite, biological ceramic particle, gross porosity microsphere silica gel, porous silicon
One or more in glue bead, activated carbon, activated alumina, volcanic rock filtrate, mesoporous material, macroreticular resin, corncob, preferably
Ground, for one kind, or the sorbing material that various appearances form is consistent.Sorbing material classification, granular size should according to be fixedization enzyme
Flexibly selected with field, technique for applying.
According to immobilised enzymes preparation method of the present invention, zymoprotein and sorbing material in enzyme solutions in step (1)
Ratio is 3-300mg/g, and the mg in unit mg/g refers to the quality of zymoprotein, and g refers to the quality of sorbing material, it is preferable that according to suction
The difference of enclosure material, the throwing amount of zymoprotein are less than the 30% of sorbing material maximal absorptive capacity.
According to immobilised enzymes preparation method of the present invention, the overlay film protective agent described in step (2) is glycerine, sorb
One or more in alcohol, xylitol, polyethylene glycol, sucrose, lactose, trehalose, glucose, xylose, maltose, skimmed milk；
The protectant concentration of overlay film is 0.25~15% (w/v), it is therefore preferable to 0.5~6.0% (w/v)；Adsorption of immobilization enzyme and overlay film
The ratio of protection agent solution be 0.05-0.5g/mL, and the g in unit g/mL refers to the quality of adsorption of immobilization enzyme, and mL refers to overlay film and protected
The volume of agent solution.
According to immobilised enzymes preparation method of the present invention, the covering material described in step (3) is polyvinyl alcohol, card
The cellulose derivative such as ripple nurse and cellulose acetate, cellulose diacetate, methylcellulose, ethyl cellulose, it is preferable that Gu
Surely change enzyme and be applied to aqueous catalysis, using water such as cellulose acetate, cellulose diacetate, methylcellulose, ethyl celluloses not
Cellulose of solubleness analog derivative is as covering material；Immobilised enzymes is catalyzed applied to nonaqueous phase, uses polyvinyl alcohol, carbomer
As covering material.
According to immobilised enzymes preparation method of the present invention, the covering material solvent described in step (3) is water, acetone,
Ethyl acetate, methanol, chloroform, pyridine, dimethyl sulfoxide (DMSO), dimethylformamide, dichloromethane, MEK, it is preferable that polyethylene
Alcohols, the solvent of carbomer are water, and the solvent of cellulose family is acetone, ethyl acetate, methanol, chloroform, pyridine, dimethyl Asia
One or more in sulfone, dimethylformamide, dichloromethane, MEK.Covering material is dissolved in film covering solution made of solvent
Concentration is 0.1~3.0% (w/v), it is therefore preferable to 0.25~1.5% (w/v).
According to immobilised enzymes preparation method of the present invention, the absorption that the protective agent described in step (3) treats is fixed
The ratio for changing the mixing of enzyme and film covering solution is 0.05-0.5g/mL, and the g in unit g/mL, which refers to the absorption that protective agent treats, to be fixed
Change the quality of enzyme, mL refers to the volume of film covering solution.
According to immobilised enzymes preparation method of the present invention, buffer solution described in step (1), (3) and (4) for 10~
200mmoL/LpH 2.0~10.0 buffer solution, it is preferable that be adapted to the optimal pH buffer solution of the enzyme of to be fixedization.
According to immobilised enzymes preparation method of the present invention, vibration absorption in step (1), (2), (3), oscillation treatment,
The temperature for vibrating overlay film is 4~35 DEG C, it is therefore preferable to which 15~25 DEG C, vibration rotating speed is 50~300rpm, it is therefore preferable to 100~
200rpm, vibration adsorption time are 2~24 hours, it is therefore preferable to which 6~12 hours, the time of oscillation treatment was 2~24 hours, excellent
Selection of land is 4~8 hours, and the time for vibrating overlay film is 2~60 minutes, it is therefore preferable to 5~20 minutes.
According to immobilised enzymes preparation method of the present invention, the immobilised enzymes drying temperature in step (3) after overlay film is 0
~40 DEG C, it is therefore preferable to which 4~10 DEG C, drying time is 0~24 hour, it is therefore preferable to 6-12 hours.
Beneficial effects of the present invention：
(1) absorption, coating process are physical processes in the present invention, mild condition, absorption-overlay film coimmobilization enzyme
The enzyme activity rate of recovery is high；
(2) stability of absorption of the present invention-overlay film coimmobilization enzyme is high, avoids traditional single adsorption or single covers
The shortcomings that film immobilization enzyme stability difference；
(3) present invention can flexibly select the class of sorbing material according to the application field of final immobilised enzymes, technique for applying
Other and granular size；
(4) present invention can according to the molecular size of the enzyme of to be fixedization, catalysis substrate or (and) molecular size of product
The temperature and time flexibly dried after the concentration of change film covering solution, overlay film, to reach optimal overlay film effect, makes enzyme molecule not
Easily leakage, substrate product mass transfer are easy；
(5) the reaction system difference that the present invention can apply according to final immobilised enzymes flexibly selects covering material, applies
Water-insoluble covering material is selected in aqueous catalysis, water-soluble covering material is selected applied to nonaqueous phase catalysis, so as to avoid covering
Swelling rupture after membrane material film forming in multiple batches of catalytic reaction；
(6) the protectant use of overlay film of the present invention effectively prevent cellulose family covering material dissolution solvent in overlay film pair
The enzyme activity adverse effect of the enzyme of to be fixedization；
(7) present invention can be applicable the immobilization of all enzymes；
(8) present invention is simple to operate, cost is low, is easy to industrial applications.
Brief description of the drawings
Fig. 1 is the optimal pH of absorption-overlay film coimmobilization papain.
Fig. 2 is the optimum temperature of absorption-overlay film coimmobilization papain.
Fig. 3 is the operational stability of absorption-overlay film coimmobilization papain.
Fig. 4 is the pH stability of absorption-overlay film coimmobilization papain.
Fig. 5 is the heat endurance of absorption-overlay film coimmobilization papain.
Fig. 6 is the optimal pH of absorption-overlay film coimmobilization laccase.
Fig. 7 is the optimum temperature of absorption-overlay film coimmobilization laccase.
Fig. 8 is the operational stability of absorption-overlay film coimmobilization laccase.
Fig. 9 is the pH stability of absorption-overlay film coimmobilization laccase.
Figure 10 is the heat endurance of absorption-overlay film coimmobilization laccase.
Embodiment
Specific embodiment is for the present invention is furture elucidated, it should be understood that these embodiments are merely to illustrate this hair
Bright rather than limitation the scope of the present invention, the equivalent change or modification that all Spirit Essences according to the present invention are made, should all contain
Lid is within the scope of the present invention.
The measure of the enzyme solutions protein concentration of embodiment 1
Protein concentration uses Bradford methods, and Coomassie brilliant G-250 dyestuff is in pale red in acid ethanol solution, with
The blue solution that protein is formed after combining has maximum light absorption at 595nm.Absorbance is directly proportional to protein concentration, therefore
The size for detecting solution absorbance value at 595nm calculates the content of albumen.
(1) Bradford stores liquid：Weigh 350mg Coomassie brilliant G-250s and be dissolved in the ethanol of 100mL 95%, add
200mL85% phosphoric acid, it is placed in 4 DEG C of refrigerators and preserves；
(2) Bradford working solutions：In 425mL distilled waters add the ethanol of 15mL 95%, the phosphoric acid of 30mL 85% and
30mL Bradford store liquid, are placed in 4 DEG C of refrigerators and preserve after mixing.Every time before use, being used after being filtered with filter paper；
(3) standard bovine serum albumin (BSA)：50mg standard BSA are weighed, 0.5%NaCl solution is dissolved in and is settled to
100mL, it is configured to 500ug/mL standard liquid.
The making of standard curve：Be separately added into some centrifuge tubes 500ug/mL BSA standard liquids 0uL, 5uL,
10uL, 20uL, 30uL, 40uL, 50uL, 60uL and 70uL supply 200uL with ultra-pure water；Added in each pipe
2.0mLBradford working solutions, vibration mix, and room temperature places 5min, light absorption value are determined at 595nm, according under each group concentration
Light absorption value and protein concentration relation, draw standard curve.
The measure of enzyme solutions protein concentration：30uL enzyme solutions to be measured are taken, 200uL is supplied with ultra-pure water, adds 2.0mL
Bradford working solutions, room temperature places 5min after shaken well, the measure light absorption value 595nm at, each sample do three it is parallel.
Enzyme solutions protein concentration is calculated according to the standard curve of drafting and the 595nm light absorption values measured.
The vitality test of the enzyme solutions of embodiment 2 and immobilised enzymes
The vigor of enzyme is also referred to as the activity of enzyme, refers to the ability of a certain chemical reaction of enzymatic.The unit of activity of enzyme is U,
Represent under certain conditions, the enzyme amount in the unit interval required for 1 micromole's product of 1 micromole substrate of conversion or generation is one
Individual unit of activity (U).Certain condition is usually the optimum temperature of enzyme, optimal pH condition.Taken during actual enzyme activity determination certain
The enzyme solutions of volume or the immobilised enzymes of certain mass are catalyzed certain density substrate and chemically reacted under certain condition.Enzyme
The vigor of solution is expressed as U/mL, and the vigor of immobilised enzymes is expressed as U/g.
The enzyme immobilizatio rate of embodiment 3 and the immobilised enzymes enzyme activity rate of recovery
The enzyme immobilizatio rate=(albumen before immobilization after protein content-immobilization of enzyme solutions in immobilization solution supernatant
Amount) protein content × 100% of enzyme solutions before/immobilization.The enzyme activity of the immobilised enzymes enzyme activity rate of recovery=immobilised enzymes/(input enzyme
The enzyme activity of immobilization solution supernatant after enzyme activity-immobilization of solution) × 100%.
Embodiment 4 adsorbs-overlay film coimmobilization enzyme preparation
(1) 50g papain enzyme powder is taken, is dissolved in appropriate pH7.0 100mmol/L phosphate buffers.Treat
After papain fully dissolves, regulation pH value is settled to 500mL, obtains papain solution to 7.0；
(2) take 10g diatomite to be placed in 250mL conical flasks, add 100mL papain solutions, in 15 DEG C,
100rpm concussions absorption 12 hours, then filters out, obtains adsorption of immobilization papain；
(3) take 0.5g Macrogol 4000 overlay film protective agents to be dissolved in pH7.0 100mmol/L phosphate buffers to be made
0.5% (w/v) Macrogol 4000 solution；
(4) polyethylene glycol of adsorption of immobilization papain prepared by 5g steps (2) and 20mL 0.5% (w/v) is taken
4000 solution mix, and are handled 8 hours in 15 DEG C, 100rpm concussions, then filter out and drain, and obtain the treated absorption of protective agent and fix
Change papain；
(5) 0.25g methylcellulose (40000~50000) is taken to be dissolved in the overlay film for obtaining 0.25% (w/v) in 100mL acetone
Solution；
(6) the treated adsorption of immobilization papain of the protective agent for obtaining step (4) is prepared with 20mL steps (5)
Film covering solution mixing, 15 DEG C, 100rpm concussion overlay films 20 minutes, filter out and dried 12 hours in 4 DEG C；
(7) immobilised enzymes that step (6) is dried is rinsed 3 times with pH7.0 100mmol/L phosphate buffers, must inhaled
Attached-overlay film coimmobilization papain.
The optimal pH of the absorption-overlay film coimmobilization papain prepared by above-mentioned steps is shown in Fig. 1.
Embodiment 5 adsorbs-overlay film coimmobilization enzyme preparation
(1) papain solution is prepared with the step of embodiment 4 (1)；
(2) take 10g diatomite to be placed in 500mL conical flasks, add 200mL papain solutions, in 20 DEG C,
150rpm concussions absorption 8 hours, then filters out, obtains adsorption of immobilization papain；
(3) take 3.0g sorbierite overlay film protective agents to be dissolved in pH 7.0 100mmol/L phosphate buffers and be made 3.0%
(w/v) sorbitol solution；
(4) sorbitol solution of adsorption of immobilization papain prepared by 5g steps (2) and 20mL 3.0% (w/v) is taken
Mixing, handled 6 hours in 20 DEG C, 150rpm concussions, then filter out and drain, obtain the treated adsorption of immobilization pawpaw egg of protective agent
White enzyme；
(5) take 0.75g methylcellulose (12000~13000) to be dissolved in 100mL ethyl acetate and obtain 0.75% (w/v's)
Film covering solution；
(6) the treated adsorption of immobilization papain of the protective agent for obtaining step (4) is prepared with 20mL steps (5)
Film covering solution mixing, 20 DEG C, 150rpm concussion overlay films 10 minutes, filter out and dried 8 hours in 6 DEG C；
(7) immobilised enzymes that step (6) is dried is rinsed 3 times with pH7.0 100mmol/L phosphate buffers, must inhaled
Attached-overlay film coimmobilization papain.
The optimum temperature of the absorption-overlay film coimmobilization papain prepared by above-mentioned steps is shown in Fig. 2.
Embodiment 6 adsorbs-overlay film coimmobilization enzyme preparation
(1) papain solution is prepared with the step of embodiment 4 (1)；
(2) take 10g diatomite to be placed in 500mL conical flasks, add 300mL papain solutions, in 25 DEG C,
200rpm concussions absorption 6 hours, then filters out, obtains adsorption of immobilization papain；
(3) take 6.0g maltose overlay film protective agents to be dissolved in pH 7.0 100mmol/L phosphate buffers and be made 6.0%
(w/v) maltose solution；
(4) maltose solution of adsorption of immobilization papain prepared by 5g steps (2) and 20mL 6.0% (w/v) is taken
Mixing, handled 4 hours in 25 DEG C, 200rpm concussions, then filter out and drain, obtain the treated adsorption of immobilization pawpaw egg of protective agent
White enzyme；
(5) 1.5g cellulose diacetates are taken to be dissolved in the film covering solution for obtaining 1.5% (w/v) in 100mL chloroforms；
(6) the treated adsorption of immobilization papain of the protective agent for obtaining step (4) is prepared with 20mL steps (5)
Film covering solution mixing, 25 DEG C, 200rpm concussion overlay films 5 minutes, filter out and dried 6 hours in 10 DEG C；
(7) immobilised enzymes that step (6) is dried is rinsed 3 times with pH7.0 100mmol/L phosphate buffers, must inhaled
Attached-overlay film coimmobilization papain.
The operational stability of the absorption-overlay film coimmobilization papain prepared by above-mentioned steps is shown in Fig. 3.
Embodiment 7 adsorbs-overlay film coimmobilization enzyme preparation
(1) papain solution is prepared with the step of embodiment 4 (1)；
(2) a kind of 10g macroreticular resins HPD-417 (macropore trees of Cangzhou Bon Adsorption Material Science and Technology Co., Ltd's production are taken
Fat) it is placed in 250mL conical flasks, 100mL papain solutions are added, shake absorption 15 hours in 10 DEG C, 150rpm, so
After filter out, obtain adsorption of immobilization papain；
(3) take 1.0g Macrogol 4000 overlay film protective agents to be dissolved in pH7.0 100mmol/L phosphate buffers to be made
1.0% (w/v) Macrogol 4000 solution；
(4) polyethylene glycol of adsorption of immobilization papain prepared by 5g steps (1) and 20mL 1.0% (w/v) is taken
4000 solution mix, and are handled 12 hours in 10 DEG C, 150rpm concussions, then filter out and drain, and obtain the treated absorption of protective agent and consolidate
Surely papain is changed；
(5) 0.75g methylcellulose (40000~50000) is taken to be dissolved in the overlay film for obtaining 0.75% (w/v) in 100mL acetone
Solution；
(6) the treated adsorption of immobilization papain of the protective agent for obtaining step (4) is prepared with 20mL steps (5)
Film covering solution mixing, 10 DEG C, 150rpm concussion overlay films 15 minutes, filter out and dried 15 hours in 4 DEG C；
(7) immobilised enzymes that step (6) is dried is rinsed 3 times with pH7.0 100mmol/L phosphate buffers, must inhaled
Attached-overlay film coimmobilization papain.
The optimal pH of the absorption-overlay film coimmobilization papain prepared by above-mentioned steps is shown in Fig. 1, and pH stability is shown in
Fig. 4.
Embodiment 8 adsorbs-overlay film coimmobilization enzyme preparation
(1) papain solution is prepared with the step of embodiment 4 (1)；
(2) take 10g macroreticular resins HPD-417 to be placed in 500mL conical flasks, add 200mL papain solutions, in
15 DEG C, 200rpm concussion absorption 10 hours, then filter out, obtain adsorption of immobilization papain；
(3) take 3.0g sorbierite overlay film protective agents to be dissolved in pH7.0 100mmol/L phosphate buffers and be made 3.0%
(w/v) sorbitol solution；
(4) sorbitol solution of adsorption of immobilization papain prepared by 5g steps (1) and 20mL 3.0% (w/v) is taken
Mixing, handled 8 hours in 15 DEG C, 200rpm concussions, then filter out and drain, obtain the treated adsorption of immobilization pawpaw egg of protective agent
White enzyme；
(5) take 1.0g methylcellulose (12000~13000) to be dissolved in 100mL ethyl acetate and obtain covering for 1.0% (w/v)
Coating solution；
(6) the treated adsorption of immobilization papain of the protective agent for obtaining step (4) is prepared with 20mL steps (5)
Film covering solution mixing, 15 DEG C, 200rpm concussion overlay films 10 minutes, filter out and dried 10 hours in 6 DEG C；
(7) immobilised enzymes that step (6) is dried is rinsed 3 times with pH7.0 100mmol/L phosphate buffers, must inhaled
Attached-overlay film coimmobilization papain.
The optimum temperature of the absorption-overlay film coimmobilization papain prepared by above-mentioned steps is shown in Fig. 2, heat endurance
See Fig. 5.
Embodiment 9 adsorbs-overlay film coimmobilization enzyme preparation
(1) papain solution is prepared with the step of embodiment 4 (1)；
(2) take 10g macroreticular resins HPD-417 to be placed in 500mL conical flasks, add 300mL papain solutions, in
20 DEG C, 250rpm concussion absorption 5 hours, then filter out, obtain adsorption of immobilization papain；
(3) take 5.0g maltose overlay film protective agents to be dissolved in pH 7.0 100mmol/L phosphate buffers and be made 5.0%
(w/v) maltose solution；
(4) maltose solution of adsorption of immobilization papain prepared by 5g steps (1) and 20mL 5.0% (w/v) is taken
Mixing, handled 5 hours in 20 DEG C, 250rpm concussions, then filter out and drain, obtain the treated adsorption of immobilization pawpaw egg of protective agent
White enzyme；
(5) 2.0g cellulose diacetates are taken to be dissolved in the film covering solution for obtaining 2.0% (w/v) in 100mL chloroforms；
(6) the treated adsorption of immobilization papain of the protective agent for obtaining step (4) is prepared with 20mL steps (5)
Film covering solution mixing, 20 DEG C, 250rpm concussion overlay films 5 minutes, filter out and dried 5 hours in 10 DEG C；
(7) immobilised enzymes that step (6) is dried is rinsed 3 times with pH 7.0 100mmol/L phosphate buffers, must inhaled
Attached-overlay film coimmobilization papain.
The operational stability of the absorption-overlay film coimmobilization papain prepared by above-mentioned steps is shown in Fig. 3.
Embodiment 10 adsorbs-overlay film coimmobilization enzyme preparation
(1) 50g laccase enzyme powder is taken, is dissolved in appropriate pH 4.5 100mmol/L phosphate buffers.Treat that laccase fills
After dividing dissolving, regulation pH value is settled to 500mL, obtains laccase solution to 4.5；
(2) take 10g diatomite to be placed in 250mL conical flasks, add 100mL laccase solutions, shaken in 15 DEG C, 100rpm
Absorption 12 hours, then filters out, obtains adsorption of immobilization laccase；
(3) take 0.5g Macrogol 4000 overlay film protective agents to be dissolved in pH 4.5 100mmol/L phosphate buffers to make
Into 0.5% (w/v) Macrogol 4000 solution；
(4) the Macrogol 4000 solution of adsorption of immobilization laccase prepared by 5g steps (2) and 20mL 0.5% (w/v) is taken
Mixing, handled 8 hours in 15 DEG C, 100rpm concussions, then filter out and drain, obtain the treated adsorption of immobilization laccase of protective agent；
(5) 0.25g methylcellulose (40000~50000) is taken to be dissolved in the overlay film for obtaining 0.25% (w/v) in 100mL acetone
Solution.
(6) overlay film that the treated adsorption of immobilization laccase of the protective agent for obtaining step (4) is prepared with 20mL steps (5)
Solution mixes, and 15 DEG C, 100rpm concussion overlay films 20 minutes, filters out and is dried 12 hours in 4 DEG C；
(7) immobilised enzymes that step (6) is dried is rinsed 3 times with pH 4.5 100mmol/L phosphate buffers, must inhaled
Attached-overlay film coimmobilization laccase.
The optimal pH of the absorption-overlay film coimmobilization laccase prepared by above-mentioned steps is shown in Fig. 6.
Embodiment 11 adsorbs-overlay film coimmobilization enzyme preparation
(1) laccase solution is prepared with the step of embodiment 10 (1)；
(2) take 10g diatomite to be placed in 500mL conical flasks, add 200mL laccase solutions, shaken in 20 DEG C, 150rpm
Absorption 8 hours, then filters out, obtains adsorption of immobilization laccase；
(3) take 3.0g sorbierite overlay film protective agents to be dissolved in pH 4.5 100mmol/L phosphate buffers and be made 3.0%
(w/v) sorbitol solution；
(4) the adsorption of immobilization laccase for taking 5g steps (2) to prepare mixes with 20mL 3.0% (w/v) sorbitol solution,
Handled 6 hours in 20 DEG C, 150rpm concussions, then filter out and drain, obtain the treated adsorption of immobilization laccase of protective agent；
(5) take 0.75g methylcellulose (12000~13000) to be dissolved in 100mL ethyl acetate and obtain 0.75% (w/v's)
Film covering solution；
(6) overlay film that the treated adsorption of immobilization laccase of the protective agent for obtaining step (4) is prepared with 20mL steps (5)
Solution mixes, and 20 DEG C, 150rpm concussion overlay films 10 minutes, filters out and is dried 8 hours in 6 DEG C；
(7) immobilised enzymes that step (6) is dried is rinsed 3 times with pH 4.5 100mmol/L phosphate buffers, must inhaled
Attached-overlay film coimmobilization laccase.
The optimum temperature of the absorption-overlay film coimmobilization laccase prepared by above-mentioned steps is shown in Fig. 7.
Embodiment 12 adsorbs-overlay film coimmobilization enzyme preparation
(1) laccase solution is prepared with the step of embodiment 10 (1)；
(2) take 10g diatomite to be placed in 500mL conical flasks, add 300mL laccase solutions, shaken in 25 DEG C, 200rpm
Absorption 6 hours, then filters out, obtains adsorption of immobilization laccase；
(3) take 6.0g maltose overlay film protective agents to be dissolved in pH 4.5 100mmol/L phosphate buffers and be made 6.0%
(w/v) maltose solution；
(4) the adsorption of immobilization laccase for taking 5g steps (2) to prepare mixes with 20mL 6.0% (w/v) maltose solution,
Handled 4 hours in 25 DEG C, 200rpm concussions, then filter out and drain, obtain the treated adsorption of immobilization laccase of protective agent；
(5) 1.5g cellulose diacetates are taken to be dissolved in the film covering solution for obtaining 1.5% (w/v) in 100mL chloroforms；
(6) overlay film that the treated adsorption of immobilization laccase of the protective agent for obtaining step (4) is prepared with 20mL steps (5)
Solution mixes, and 25 DEG C, 200rpm concussion overlay films 5 minutes, filters out and is dried 6 hours in 10 DEG C；
(7) immobilised enzymes that step (6) is dried is rinsed 3 times with pH 4.5 100mmol/L phosphate buffers, must inhaled
Attached-overlay film coimmobilization laccase.
The operational stability of the absorption-overlay film coimmobilization laccase prepared by above-mentioned steps is shown in Fig. 8.
Embodiment 13 adsorbs-overlay film coimmobilization enzyme preparation
(1) laccase solution is prepared with the step of embodiment 10 (1)；
(2) take 10g macroreticular resins HPD-417 to be placed in 250mL conical flasks, add 100mL laccase solutions, in 10 DEG C,
150rpm concussions absorption 15 hours, then filters out, obtains adsorption of immobilization laccase；
(3) take 1.0g Macrogol 4000 overlay film protective agents to be dissolved in pH 4.5 100mmol/L phosphate buffers to make
Into 1.0% (w/v) Macrogol 4000 solution；
(4) the Macrogol 4000 solution of adsorption of immobilization laccase prepared by 5g steps (1) and 20mL 1.0% (w/v) is taken
Mixing, handled 12 hours in 10 DEG C, 150rpm concussions, then filter out and drain, obtain the treated adsorption of immobilization laccase of protective agent；
(5) 0.75g methylcellulose (40000~50000) is taken to be dissolved in the overlay film for obtaining 0.75% (w/v) in 100mL acetone
Solution；
(6) overlay film that the treated adsorption of immobilization laccase of the protective agent for obtaining step (4) is prepared with 20mL steps (5)
Solution mixes, and 10 DEG C, 150rpm concussion overlay films 15 minutes, filters out and is dried 15 hours in 4 DEG C；
(7) immobilised enzymes that step (6) is dried is rinsed 3 times with pH 4.5 100mmol/L phosphate buffers, must inhaled
Attached-overlay film coimmobilization laccase.
The optimal pH of the absorption-overlay film coimmobilization laccase prepared by above-mentioned steps is shown in Fig. 6, and pH stability is shown in Fig. 9.
Embodiment 14 adsorbs-overlay film coimmobilization enzyme preparation
(1) laccase solution is prepared with the step of embodiment 10 (1)；
(2) take 10g macroreticular resins HPD-417 to be placed in 500mL conical flasks, add 200mL laccase solutions, in 15 DEG C,
200rpm concussions absorption 10 hours, then filters out, obtains adsorption of immobilization laccase；
(3) take 3.0g sorbierite overlay film protective agents to be dissolved in pH 4.5 100mmol/L phosphate buffers and be made 3.0%
(w/v) sorbitol solution；
(4) the adsorption of immobilization laccase for taking 5g steps (1) to prepare mixes with 20mL 3.0% (w/v) sorbitol solution,
Handled 8 hours in 15 DEG C, 200rpm concussions, then filter out and drain, obtain the treated adsorption of immobilization laccase of protective agent；
(5) take 1.0g methylcellulose (12000~13000) to be dissolved in 100mL ethyl acetate and obtain covering for 1.0% (w/v)
Coating solution；
(6) overlay film that the treated adsorption of immobilization laccase of the protective agent for obtaining step (4) is prepared with 20mL steps (5)
Solution mixes, and 15 DEG C, 200rpm concussion overlay films 10 minutes, filters out and is dried 10 hours in 6 DEG C；
(7) immobilised enzymes that step (6) is dried is rinsed 3 times with pH 4.5 100mmol/L phosphate buffers, must inhaled
Attached-overlay film coimmobilization laccase.
The optimum temperature of the absorption-overlay film coimmobilization laccase prepared by above-mentioned steps is shown in Fig. 7, and heat endurance is shown in Fig. 9.
Embodiment 15 adsorbs-overlay film coimmobilization enzyme preparation
(1) laccase solution is prepared with the step of embodiment 10 (1)；
(2) take 10g macroreticular resins HPD-417 to be placed in 500mL conical flasks, add 300mL laccase solutions, in 20 DEG C,
250rpm concussions absorption 5 hours, then filters out, obtains adsorption of immobilization laccase；
(3) take 5.0g maltose overlay film protective agents to be dissolved in pH 4.5 100mmol/L phosphate buffers and be made 5.0%
(w/v) maltose solution；
(4) the adsorption of immobilization laccase for taking 5g steps (1) to prepare mixes with 20mL 5.0% (w/v) maltose solution,
Handled 5 hours in 20 DEG C, 250rpm concussions, then filter out and drain, obtain the treated adsorption of immobilization laccase of protective agent；
(5) 2.0g cellulose diacetates are taken to be dissolved in the film covering solution for obtaining 2.0% (w/v) in 100mL chloroforms；
(6) overlay film that the treated adsorption of immobilization laccase of the protective agent for obtaining step (4) is prepared with 20mL steps (5)
Solution mixes, and 20 DEG C, 250rpm concussion overlay films 5 minutes, filters out and is dried 5 hours in 10 DEG C；
(7) immobilised enzymes that step (6) is dried is rinsed 3 times with pH 4.5 100mmol/L phosphate buffers, must inhaled
Attached-overlay film coimmobilization laccase.
The operational stability of the absorption-overlay film coimmobilization laccase prepared by above-mentioned steps is shown in Fig. 8.
It is above-mentioned that the method for absorption-overlay film coimmobilization enzyme is described with reference to embodiment, be it is illustrative without
It is restricted, several embodiments can be included according to limited scope, therefore in the case where not departing from overall thought of the present invention
It is any to change and modifications, it should belong within protection scope of the present invention.
Claims (10)
1. a kind of preparation method of immobilised enzymes, it is characterised in that comprise the following steps：
(1) enzyme is dissolved in buffer solution and enzyme solutions is made, and mixed with sorbing material, vibration absorption certain time, filtered out, obtain
To adsorption of immobilization enzyme；
(2) overlay film protective agent is dissolved in buffer solution and protection agent solution is made, adsorption of immobilization enzyme is placed in protection agent solution
In, oscillation treatment certain time, filter out and drain, obtain the treated adsorption of immobilization enzyme of protective agent；
(3) film covering solution is made in covering material dissolving in a solvent, the adsorption of immobilization enzyme and overlay film that protective agent is treated
Solution mixes, and vibrates overlay film certain time, filters out drying；
(4) finally, with obtaining absorption-overlay film coimmobilization enzyme after deionized water or wash buffer.
A kind of 2. preparation method of immobilised enzymes according to claim 1, it is characterised in that：Enzyme described in step (1) is
Hydrolase (such as amylase, protease, lipase, phosphatase, glycosidase), redox enzymes (such as laccase, horseradish mistake
Oxide enzyme, polyphenol oxidase, superoxide dismutase, glucose oxidase, catalase, fatty acid oxidase, ketone are also
Protoenzyme, alcohol dehydrogenase etc.), transferase (such as transmethylase, aminopherase, acyltransferase), lyases
(such as dehydratase, decarboxylase, carbonic anhydrase, aldolase), isomerase (such as isomerase, epimerase, racemase), close
One or more into enzyme (such as glutamine synthelase, DNA ligase, carboxylase, phosphorylase).
A kind of 3. preparation method of immobilised enzymes according to claim 1, it is characterised in that：Absorption described in step (1)
Material be attapulgite, diatomite, kaolin, bentonite, atlapulgite, zeolite, montmorillonite, expanded perlite, biological ceramic particle,
Gross porosity microsphere silica gel, Porasil, activated carbon, activated alumina, volcanic rock filtrate, mesoporous material, macroreticular resin, corncob
In one or more.
A kind of 4. preparation method of immobilised enzymes according to claim 1, it is characterised in that：In step (1) in enzyme solutions
The ratio of zymoprotein and sorbing material is 3-300mg/g, and the mg in unit mg/g refers to the quality of zymoprotein, and g refers to sorbing material
Quality.
A kind of 5. preparation method of immobilised enzymes according to claim 1, it is characterised in that：Overlay film described in step (2)
Protective agent is glycerine, sorbierite, xylitol, polyethylene glycol, sucrose, lactose, trehalose, glucose, xylose, maltose, degreasing
One or more in breast；The protectant concentration of overlay film is 0.25~15% (w/v)；Adsorption of immobilization enzyme and overlay film protective agent are molten
The ratio of liquid is 0.05-0.5g/mL, and the g in unit g/mL refers to the quality of adsorption of immobilization enzyme, and mL refers to overlay film protection agent solution
Volume.
A kind of 6. preparation method of immobilised enzymes according to claim 1, it is characterised in that：Overlay film described in step (3)
Material is the fibres such as polyvinyl alcohol, carbomer and cellulose acetate, cellulose diacetate, methylcellulose, ethyl cellulose
Tie up plain derivative；Covering material solvent is water, acetone, ethyl acetate, methanol, chloroform, pyridine, dimethyl sulfoxide (DMSO), dimethyl methyl
One or more in acid amides, dichloromethane, MEK；Covering material be dissolved in film covering solution concentration made of solvent for 0.1~
3.0% (w/v).
A kind of 7. preparation method of immobilised enzymes according to claim 1, it is characterised in that：Protection described in step (3)
The ratio that the treated adsorption of immobilization enzyme of agent mixes with film covering solution is 0.05-0.5g/mL, and the g in unit g/mL refers to protection
The quality of the treated adsorption of immobilization enzyme of agent, mL refer to the volume of film covering solution.
A kind of 8. preparation method of immobilised enzymes according to claim 1, it is characterised in that：Step (1), (3) and (4) institute
The buffer solution stated is 10~200mmoL/LpH 2.0~10.0 buffer solution.
A kind of 9. preparation method of immobilised enzymes according to claim 1, it is characterised in that：In step (1), (2), (3)
Vibration absorption, oscillation treatment, the temperature of vibration overlay film are 4~35 DEG C, and vibration rotating speed is 50~300rpm, and vibration adsorption time is
2~24 hours, the time of oscillation treatment was 2~24 hours, and the time for vibrating overlay film is 2~60 minutes.
A kind of 10. preparation method of immobilised enzymes according to claim 1, it is characterised in that：In step (3) after overlay film
Immobilised enzymes drying temperature is 0~40 DEG C, and drying time is 0~24 hour.
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