CN102433317A - Immobilization method for thermolysin - Google Patents

Immobilization method for thermolysin Download PDF

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CN102433317A
CN102433317A CN2011103633360A CN201110363336A CN102433317A CN 102433317 A CN102433317 A CN 102433317A CN 2011103633360 A CN2011103633360 A CN 2011103633360A CN 201110363336 A CN201110363336 A CN 201110363336A CN 102433317 A CN102433317 A CN 102433317A
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thermolysin
mes
enzyme
immobilization
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CN102433317B (en
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谢恬
陈飞飞
王安明
张方凯
杜方川
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Hangzhou Normal University
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Hangzhou Normal University
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Abstract

The invention provides an immobilization method for thermolysin. The immobilization method comprises the following steps of: (1) activating carriers: impregnating aminated mesocellular foam silicon carriers (MCFs-NH2) in 0.5 to 2.5 mM of benzoquinone solution, carrying out oscillating activation for 1 to 3 hours, centrifuging, taking precipitate out, washing the precipitate, and dispersing the washed precipitate in MES-NaOH solution again to obtain activated carrier liquid; and (2) immobilizing enzyme: adding the activated carrier liquid in to the thermolysin in an enzyme adding amount of 40 to 100mg thermolysin/g MCFs-NH2 to obtain mixed liquid, performing electromagnetic stirring reaction on the mixed liquid in ice bath for 18 to 20 hours or irradiating the mixed liquid for 2 to 4 minutes under the condition of 20 to 50 W of microwaves at the temperature of between 0 and 7 DEG C, centrifuging, taking precipitate out, and washing the precipitate by the MES-NaOH solution and thus obtaining the immobilized thermolysin. The performance of immobilized thermolysin prepared by the method is greatly improved compared with that of free enzymes, and the immobilization time is greatly shortened from 20 hours to 3 minutes and is totally shortened by 399 times, so that the cost of the enzyme immobilization is lowered.

Description

A kind of process for fixation of thermolysin
(1) technical field
The present invention relates to a kind of process for fixation of thermolysin.
(2) background technology
Enzyme is as a kind of biological catalyst, because of it has characteristics such as highly selective, catalytic reaction condition be gentle, pollution-free, is widely used in industries such as food-processing, medicine and fine chemistry industry.But the natural enzyme poor stability, be prone to inactivation, can not reuse, and sneak into product after the reaction, purification difficult makes it be difficult in industry, use more widely.In addition, separation and enzyme purification and their disposable use have also increased its cost as catalyzer greatly.With this understanding, the notion of immobilized enzyme and technology are able to propose and development, and become the emphasis of enzyme engineering research in recent years.
The immobilization of enzyme promptly fetters enzyme or be limited in certain zone with solid material, still can carry out its distinctive catalyzed reaction, and recyclable and reusable one type of technology.According to the character and the purposes of enzyme, can be divided into these several kinds of absorption methods, covalent attachment method, crosslinking, entrapping method.
Thermolysin (EC3.4.24.27) is a kind of thermotolerance neutral metal proteolytic enzyme that is present in the thermophilic molten albumen genus bacillus (Bacillus thermoproteolyticus), contains 4 calcium ions and 1 zine ion.It once was used to study the structure of ovotransferrin because thermolysin can complete digestion N the end leaf but stay most C end leaf and avoid destroying.Thermolysin not only is widely used in the hydrolysis of peptide bond, hydrolysis leucine particularly, phenylalanine(Phe); Isoleucine; The N end of amino acid whose hydrophobic or big amino side-chain such as Xie Ansuan, and its formation, particularly catalysis artificial sweetner ASPARTAME POWDER BP/USP synthetic of catalysis peptide bond also.ASPARTAME POWDER BP/USP is a kind of dipeptide sweetener, and sugariness approximately is 200 times of sucrose, and it is distinguished the flavor of like white sugar, and is oiliness not bitter; The low in calories fat-reducing; Can not make blood sugar increasing, it is edible to be suitable for obesity, mellitus and cardiovascular patient; Not by microbial fermentation, be not afraid of mouldyly, do not have the anxiety carious tooth, in more than 100 countries, be widely used in the bag and bottle now.Adopt the synthetic of this sweeting agent of enzyme catalysis, compare, have clear superiority with chemical technology; As: avoided the use of poisonous and harmful catalyzer, reduced the usage quantity of organic solvent, the gentleness that reaction conditions is become; From substituting the angle of traditional chemical synthesis technique catalyzer; This enzymatic reaction has very strong green attribute, the Green Chemistry transformation of traditional chemical technology is had significance, thereby this enzyme has broad application prospects.
Immobilization can improve the character of enzyme, makes immobilized enzyme specific ionization enzyme have better stability and higher service efficiency, and endonuclease capable is recycled, and greatly saves production cost.But (1.0～1.2mg/ml) have only its covalent immobilization of 9 amino (thermolysin is through amino covalently bound with carrier) to have very big difficulty again because the thermolysin solvability is low.
(3) summary of the invention
It is a kind of simple that the object of the invention is to provide; Fast; Thermolysin covalent immobilization method efficiently; This covalent immobilization is through the covalently bound realization of para benzoquinone linking agent and enzyme and carrier, finally obtains the immobilized enzyme product, has obtained the thermolysin immobilized enzyme that performance is greatly improved than the free enzyme of thermolysin.
The technical scheme that the present invention adopts is:
A kind of process for fixation of thermolysin, said method comprises:
(1) carrier activation: amidized mesoporous foam silicon carrier MCFs-NH2 is immersed in the para benzoquinone solution of 0.5～2.5mM; The vibration activation is 1～3 hour under 20～30 ℃, the condition of 100～200rpm; Centrifugal; Get deposition successively with 20～30% ethanol and pure water washing, the deposition after the washing is scattered in the MES-NaOH solution again, obtains the activatory carrier fluid;
(2) immobilization of enzyme: with the activatory carrier fluid, by 40～100mg thermolysin (free enzyme)/g MCFs-NH 2Enzyme concentration; Add thermolysin and obtain mixed solution, with mixed solution induction stirring reaction 18～20h or shine 2～4min down in ice bath, centrifugal at 0～7 ℃, 20～50W microwave condition; Get deposition and use the MES-NaOH solution washing, obtain the immobilization thermolysin;
Prepared amidized MCFs carrier is the conventional amidized mesoporous foam silicon carrier in this area in the step (1), and its aperture is about 26nm.
The MES-NaOH solution composition is following described in step (1) and (2): NaCl 2～5M, ZnCl 210～30mM, MES-NaOH 0.01～0.05M, pH 7.0～7.5, and solvent is a water.
The present invention adds 2～5MNaCl when immobilization can make the solvability of thermolysin improve about 10 times, thereby making thermolysin to be well dispersed in to be unlikely in the solution to flock together can be covalently bound with carrier well.
Microwave current radiation being widely used in Synthetic Organic Chemistry receives publicity, and it can add the carrying out of fast response widely and improve productive rate effectively.Microwave to the effect of material molecule be directly act on intramolecular.The material molecule dipole oscillation has similar frequency with microwave vibrations; In the microwave magnetic field of fast vibration; The dipole oscillation of molecule is complementary with the magnetic field vibration as possible; And the vibration of molecule often lags behind magnetic field, thereby material molecule absorbs electromagnetic energy with the effect between billions of vibration at high speed acceleration moleculars of per second.It is more obvious that wherein polar material receives the effect of microwave, and protein and polypeptide all are typical polar molecules, so microwave can quicken the covalently bound of thermolysin and carrier.Therefore it is significant to study neutral salt solubilising and microwave radiation reinforcement thermophilic bacteria protein enzyme immobilization, can the immobilization technology of enzyme be reached a new high.
Said thermolysin adds in the carrier fluid after disperseing with MES-NaOH solution, and said thermolysin concentration in MES-NaOH solution is 0.5～2mg/mL, and said MES-NaOH solution composition is following: NaCl 2～5M, ZnCl 210～30mM, MES-NaOH 0.01～0.05M, pH 7.0～7.5, and solvent is a water.
Para benzoquinone solution and MCFs-NH in the step (1) 2The ratio of volume mass consumption be 2～5ml: 10mg.
Used MES-NaOH solution and MCFs-NH when disperseing again in the step (1) 2The ratio of volume mass consumption be 2～5ml: 10mg.
Said method is following:
(A) dispersion of enzyme: get thermolysin, disperse,, promptly get the MES solution of thermolysin-4 ℃ of held half a hour with MES-NaOH solution;
(B) carrier activation: with amidized mesoporous foam silicon carrier MCFs-NH 2Be immersed in the para benzoquinone solution of 1.5mM, the vibration activation is 2 hours under 25 ℃, the condition of 160rpm, and is centrifugal, gets deposition and washs with 20% ethanol and pure water successively, and the deposition after the washing is scattered in the MES-NaOH solution again, obtains the activatory carrier fluid; Used para benzoquinone solution and MCFs-NH during activation 2The ratio of volume mass consumption be 3ml: 10mg; Used MES-NaOH solution and MCFs-NH when disperseing again 2The ratio of volume mass consumption be 2～2.6ml: 10mg;
(C) immobilization of enzyme: with the activatory carrier fluid, by 40～100mg thermolysin (in free enzyme)/g MCFs-NH 2Enzyme concentration; The MES solution that adds thermolysin obtains mixed solution, and is with mixed solution induction stirring reaction 20h or shine 3min down at 0～7 ℃, 40W microwave condition in ice bath, centrifugal; Get deposition and use the MES-NaOH solution washing, obtain the immobilization thermolysin;
The MES-NaOH solution composition is following described in the step (A)～(C): NaCl 3M, ZnCl 220mM, MES-NaOH 0.02M, pH 7.0, and solvent is a water.
Among the present invention, said amidized mesoporous foam silicon carrier MCFs-NH 2Can prepare as follows:
1. water-heat process: take by weighing P123 (triblock copolymer: gather (terepthaloyl moietie)-block-and gather (Ucar 35)-block-and gather (terepthaloyl moietie)) 5～10g water earlier and dissolve in batches, begin to stir after adding 50～100mg Neutral ammonium fluoride again, add 5～10ml TMB (1.3.5-trimethylbenzene) and 25～50ml hydrochloric acid (36%～38% again; W/w), 200～300rpm, 10～50 ℃ of following 20～60min that stir add 10～30ml TEOS (positive tetraethyl orthosilicate) then; After continuing to stir 12～24h; Solution is changed in the autoclave, place 100～200 ℃ in baking oven, aging 6～30h; Use water filtration at last, the oven dry of gained carrier is for use;
2. remove template reaction: take by weighing the 1. made carrier 0.5～3.5g of step in autoclave, adding 5～25ml concentrated nitric acid (65%～68%, w/w) with 1～10ml ydrogen peroxide 50 (H 2O 2Concentration 20～40% v/v), places 50～150 ℃ of reactions of baking oven, 6～30h, the water washing and filtering, and it is for use to obtain the oven dry of template carrier;
3. silylanization: what take by weighing that 2. 2～10g step make goes the template carrier in there-necked flask; Add toluene 200～300ml and amination reagent (for example silane coupling agent) 20～60ml, logical again water of condensation, 100～150 ℃ of reflux 6～30h of oil bath; Question response finishes postcooling and uses toluene and absolute ethanol washing respectively; After treating that organic solvent in the carrier volatilizees basically, put into baking oven and dry, promptly get said amidized mesoporous foam silicon carrier MCFs-NH 2
For microwave-assisted immobilization and ordinary method are compared; The present invention has implemented process for fixation under conventional immobilization and the microwave condition of thermolysin; The immobilization thermolysin that process for fixation obtains under the microwave condition is at catalysis activity; Thermotolerance; All obtained huge raising on the performance of organic solvent-resistant: the catalysis activity of (1) immobilization thermolysin is improved, and its catalysis activity is 1.6 times of free enzyme catalysis vigor, is 4.5 times of conventional immobilized enzyme catalysis vigor.(2) thermostability of immobilization thermolysin makes moderate progress.When under 70 ℃ condition, hatching 3.5h, the vigor of immobilization thermolysin does not reduce, and free enzyme has not just had activity after hatching 3h.When under 80 ℃ condition, hatching 60min, free enzyme has not had activity fully, and immobilized enzyme is keeping 74.1% catalytic activity.(3) the immobilization thermolysin performance of resisting organic solvent also is greatly improved.In 5% tertiary amyl alcohol, hatch 2h under 70 ℃, the activity of immobilized enzyme does not change, and the catalysis activity of free enzyme has reduced by 32.5%.Even in 80% tertiary amyl alcohol, hatch 2h under 70 ℃, and immobilized enzyme is also keeping 46.3% catalysis activity, and free enzyme has not had vigor fully.For ETHYLE ACETATE, the immobilization thermolysin has also been showed powerful defensive ability/resistance ability.In 5% ETHYLE ACETATE, hatch 2h under 70 ℃, when the vigor of free enzyme had reduced by 32.5%, immobilized enzyme was but keeping 92% vigor.And when in 80% ETHYLE ACETATE, hatching 2h under 70 ℃, free enzyme has had no vigor, and immobilized enzyme is but keeping 53.1% catalysis activity.
Beneficial effect of the present invention is embodied in, and the immobilization thermolysin that makes through the inventive method is greatly improved than free enzyme on performance.And, through the inventive method thermolysin is carried out immobilization and also shortened the immobilization time widely, shortened to 3min from 20h, totally shortened 399 times, this makes the cost of enzyme immobilization obtain reduction.
(4) description of drawings
Fig. 1 is the mechanism of the synthetic ASPARTAME POWDER BP/USP precursor of thermophilic bacteria protein enzyme catalysis; Wherein, ZD:N-carbobenzoxy-(Cbz)-L-aspartic acid FM:L-phenylalanine methyl ester ZDFM:N-carbobenzoxy-(Cbz)-L-aspartyl-L-phenylalanine methyl ester;
Fig. 2 is the influence of para benzoquinone concentration to immobilization thermolysin load factor and catalysis activity;
Fig. 3 is the reaction mechanism of sodium-chlor solubilising and microwave-assisted covalent immobilization thermolysin;
Fig. 4 is the influence of microwave irradiation power to immobilization thermolysin load factor and catalysis activity;
Fig. 5 is the temperature stability of free enzyme and immobilized enzyme; Wherein, ■: microwave immobilization thermolysin is hatched in 70 ℃ of water-baths; ●: microwave immobilization thermolysin is hatched in 80 ℃ of water-baths, ▲: the free enzyme of thermolysin is hatched in 70 ℃ of water-baths, ★: the free enzyme of thermolysin is hatched in 80 ℃ of water-baths;
Fig. 6 is free enzyme and the anti-tertiary amyl alcohol property of immobilized enzyme in 70 ℃ of water-baths; FE: the free enzyme MW-IME of thermolysin: microwave immobilization thermolysin;
Fig. 7 is free enzyme and the anti-ETHYLE ACETATE property of immobilized enzyme in 70 ℃ of water-baths; FE: the free enzyme MW-IME of thermolysin: microwave immobilization thermolysin;
(5) embodiment
Below in conjunction with specific embodiment the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1: the MES solution of preparation thermolysin
(1) gets 2mg thermolysin (free enzyme,, together following) available from Sigma aldrich Shanghai branch office; With 2ml pH 7.0; 0.02M MES-NaOH solution disperse ,-4 ℃ of held half a hour, both the MES solution of the free enzyme of thermolysin of 1mg/ml.
(2) get the 2mg thermolysin, contain 20mM ZnCl with 2ml 2PH 7.0, the MES-NaOH solution of 0.02M disperses ,-4 ℃ of held half a hour, both the MES solution of the free enzyme of thermolysin of 1mg/ml.
(3) get the 2mg thermolysin, contain 3M NaCl, 20mM ZnCL with 2ml 2PH7.0, the MES-NaOH solution of 0.02M disperses ,-4 ℃ of held half a hour, both the MES solution of the free enzyme of thermolysin of 1mg/ml.
Embodiment 2: the free enzyme of thermolysin is prepared into the immobilization thermolysin:
A. the MES solution for preparing the free enzyme of thermolysin: get the free enzyme of 2mg thermolysin, contain 3M NaCl, 20mM ZnCl with 2ml 2PH 7.0, the MES-NaOH solution of 0.02M disperses ,-4 ℃ of held half a hour, both the MES solution of the free enzyme of thermolysin of 1mg/ml.
B. the free enzyme of thermolysin is prepared into the immobilization thermolysin:
(1) carrier MCFs-NH 2Preparation:
1. water-heat process: take by weighing P123 (triblock copolymer: gather (terepthaloyl moietie)-block-and gather (Ucar 35)-block-and gather (terepthaloyl moietie) earlier; PEO-PPO-PEO) the 5.34g water dissolves in batches and it can all be transferred in the container go; Installation begins to stir after adding the 61.34mg Neutral ammonium fluoride again, adds 6.2ml TMB (1.3.5-trimethylbenzene) and 27.4ml hydrochloric acid (36.5wt%) again.It is 250rpm that mixing speed is set, and 40 ℃ are stirred 45min down, adds 12.6ml TEOS (positive tetraethyl orthosilicate) then, continues to stir 20h.After stirring finishes, solution is changed in the autoclave, as in the baking oven 120 ℃, aging 24h.Use water filtration at last, dry for use;
2. remove template reaction: take by weighing top made carrier 1.0g in autoclave, adding 10ml concentrated nitric acid (65.5wt%) and 7ml ydrogen peroxide 50 (30%, v/v), place 100 ℃ of reactions of baking oven 12h.The water washing and filtering is dried for use then;
3. silylanization: what take by weighing that 2. the 4.5g step make goes the template carrier in the middle of there-necked flask; Add toluene 270ml then, amination reagent 27ml (silane coupling agent JH-A112), logical then water of condensation; 110 ℃ of reflux 12h of oil bath; Question response finishes postcooling and uses toluene and absolute ethanol washing then respectively, treat that organic solvent in the carrier volatilizees basically after, put into baking oven dry MCFs-NH 2, the aperture is 26nm;
(2) carrier is carried out activation treatment: selecting for use para benzoquinone solution 3ml to make its final concentration at reaction solution is the MCFs-NH of 1.5mM and 10mg 2Vibration 2 hours under the condition of 160rpm is centrifugal in 25 ℃ water bath with thermostatic control shaking table, gets deposition and uses 20% (v/v) ethanol and pure water to wash respectively, and the deposition after the washing also is scattered in 2.6ml again with it and contains 3M NaCl, 20mM ZnCl 2PH 7.0, in the MES-NaOH solution of 0.02M,,, still be 10mg wherein so think that vehicle weight remains unchanged because of solid-liquid in the centrifugal process realize to separate.
(3) get the activatory carrier fluid 2.6ml that step (2) obtains; The MES damping fluid 0.4ml that adds the free enzyme of thermolysin that steps A makes again; Mixing gets mixed solution, and described immobilized enzyme prepares that enzyme concentration is the free enzyme of 40mg thermolysin/g MCFs-NH in the process 2. the mixed solution that step (3) is got in (4) carries out the immobilization processing: mixing solutions induction stirring reaction 20h in ice bath is centrifugal, and with containing 3M NaCl, 20mM ZnCl 2PH 7.0, the MES-NaOH solution washing of 0.02M obtains the immobilization thermolysin.
Embodiment 3: the free enzyme of thermolysin is prepared into the immobilization thermolysin with microwave method:
A. the MES solution for preparing the free enzyme of thermolysin: get the free enzyme of 2mg thermolysin, contain 3M NaCl, 20mM ZnCL with 2ml 2PH 7.0, the MES-NaOH solution of 0.02M disperses ,-4 ℃ of held half a hour, both the MES solution of the free enzyme of thermolysin of 1mg/ml.
B. the free enzyme of thermolysin is prepared into the immobilization thermolysin:
(1) preparation of carrier:
With embodiment 2.
(2) carrier is carried out activation treatment: selecting for use para benzoquinone solution 3ml to make its final concentration at reaction solution is the MCFs-NH of 1.5mM and 10mg 2Vibration is 2 hours under the condition of 25 ℃ water bath with thermostatic control shaking table 160rpm, and is centrifugal, gets deposition and washs with 20% ethanol and pure water respectively, and the deposition after the washing also is scattered in 2.6ml again with it and contains 3M NaCl, 20mM ZnCl 2PH 7.0, in the MES-NaOH solution of 0.02M,,, still be 10mg wherein so think that vehicle weight remains unchanged because of solid-liquid in the centrifugal process realize to separate.
(3) get the activatory carrier fluid 2.6ml that step (2) obtains; The MES solution 0.4ml that adds the free enzyme of thermolysin that steps A makes again; Mixing gets mixed solution, and described immobilized enzyme prepares that enzyme concentration is the free enzyme/gMCFs-NH of 40mg thermolysin in the process 2
(4) mixed solution of getting step (3) carries out the immobilization processing: at 0～7 ℃, the 40W microwave condition shone 3 minutes down, and is centrifugal with mixing solutions, gets deposition with containing 3M NaCl, 20mM ZnCl 2PH 7.0, the MES-NaOH solution washing of 0.02M obtains microwave immobilization thermolysin.
Embodiment 4: the linking agent kind of activated carrier is to the fixedly influence of thermolysin
Use the 3ml final concentration as the para benzoquinone solution of 1.5mM and glutaraldehyde solution respectively to the MCFs-NH of 10mg 2Carry out activation treatment, with 2ml PH 7.0, the MES-NaOH solution of 0.02M dissolves with the carrier after the activated processing.According to the mass ratio of 100mg enzyme/g carrier, the MES solution 1ml of the free enzyme of thermolysin of the 1mg/ml that adding embodiment 1 (1) obtains, mixing gets mixed solution.
MCFs for epoxy groupization does not need activation, and directly the MCFs (the preparation process is referring to embodiment 2, and the silane coupling agent of usefulness is JH-S1891 here) with the 10mg epoxy groupization is dissolved in 2ml PH7.0, in the 0.02M MES-NaOH solution.According to the mass ratio of 100mg enzyme/g carrier, the MES solution 1ml mixing of the free enzyme of thermolysin of the 1mg/ml that adding embodiment 1 (1) obtains gets mixed solution again.
With above three kinds of mixed solutions induction stirring reaction 20h in ice bath respectively, centrifugal, with PH 7.0, the MES-NaOH solution washing of 0.02M obtains the immobilization thermolysin.These three immobilized enzyme are carried out vitality test, and the result sees table 1.
Table 1: the linking agent kind is to the active influence of immobilization thermolysin
Figure BDA0000109004370000101
Visible by table 1, its immobilization specific activity of enzyme was 2357.6U/mg when linking agent was para benzoquinone, be the optimal result of these three groups experiments, thereby the linking agent that subsequent experimental is selected for use was a para benzoquinone.
Embodiment 5: the para benzoquinone concentration of activated carrier is to carrier MCFs-NH 2The fixedly influence of thermolysin
The para benzoquinone solution that is respectively 0.5mM, 1.0mM, 1.5mM, 2.0mM, 2.5mM with the 3ml final concentration is respectively to the MCFs-NH of 10mg 2Carry out activation treatment, with 2ml PH 7.0, the MES-NaOH solution of 0.02M dissolves with the carrier after the activated processing.Mass ratio according to 100mg enzyme/g carrier; The MES solution 1ml of the free enzyme of thermolysin of the 1mg/ml that adding embodiment 1 (1) obtains, mixing gets mixed solution, and induction stirring is reacted 20h in ice bath; Centrifugal; With PH 7.0, the MES-NaOH solution washing of 0.02M obtains the immobilization thermolysin.
Enzyme to these five para benzoquinone concentration activated carriers carries out vitality test respectively, and the corresponding ratio vigor of respectively organizing gained immobilization thermolysin is 1188.6U/mg, 1569.1U/mg, 2357.6U/mg, 1936U/mg, 1888.9U/mg, and the result sees Fig. 1.
Its immobilization specific activity of enzyme is 2357.6U/mg when being 1.5mM by the visible para benzoquinone concentration of Fig. 1, be the optimal result of several groups of experiments, thereby the para benzoquinone final concentration is chosen to be 1.5mM in the preparation of follow-up microwave immobilization thermolysin.
Embodiment 6: neutral salt is to the influence of immobilization thermophilic bacteria protein enzymatic activity.
Use the 3ml final concentration to be the para benzoquinone solution of 1.5mM MCFs-NH to 10mg 2Carry out activation treatment, the carrier after the activated processing is comprised CaCl respectively with 2ml 2ZnCl 2CaClL 2, ZnCl 2ZnCl 2, NaCl PH 7.0, the dissolving of the MES-NaOH solution of 0.02M.Mass ratio according to 100mg enzyme/g carrier; Add the MES solution 1ml of the free enzyme of thermolysin of the 1mg/ml that embodiment 1 (1) obtains, mixing gets mixed solution, with mixed solution at 0～7 ℃; The 30W microwave condition shone 3 minutes down; Centrifugal, get deposition with the used damping fluid washing of immobilization, obtain microwave immobilization thermolysin.And these several groups of immobilised enzymes are carried out vitality test, the corresponding relative vigor of different systems sees Table 2.
Table 2: neutral salt is to the influence of immobilization thermophilic bacteria protein enzyme activity
Visible by table 2, CaCl 2To the almost not influence of catalytic activity of immobilization thermolysin, NaCl has huge raising to the catalytic activity of immobilization thermolysin, though ZnCl 2Catalytic activity to the immobilization thermolysin has certain influence, but less relatively than its influence power of NaCl.20mM ZnCl 2, 3M NaCl system make that the relative vigor of immobilization thermolysin is 82.9%, so subsequent reactions is a reaction system with this system.
Embodiment 7: the contrast of conventional immobilization and microwave-assisted immobilization
(1) use the 3ml final concentration to be the para benzoquinone solution of 1.5mM MCFs-NH to 10mg 2Carry out activation treatment, with 2ml PH 7.0, the MES-NaOH solution of 0.02M dissolves with the carrier after the activated processing.Mass ratio according to 100mg enzyme/g carrier; The MES solution 1ml of the free enzyme of thermolysin of the 1mg/ml that adding embodiment 1 (1) obtains, mixing gets mixed solution, and induction stirring is reacted 20h in ice bath; Centrifugal; With PH 7.0, the MES-NaOH solution washing of 0.02M obtains the immobilization thermolysin.
(2) use the 3ml final concentration to be the para benzoquinone solution of 1.5mM MCFs-NH to 10mg 2Carry out activation treatment, the carrier after the activated processing is comprised 20mM ZnCl with 2ml 2PH 7.0, the MES-NaOH solution dissolving of 0.02M.According to the mass ratio of 100mg enzyme/g carrier, the MES solution 1ml of the free enzyme of thermolysin of the 1mg/ml that adding embodiment 1 (2) obtains, mixing gets mixed solution, and induction stirring is reacted 20h in ice bath, and is centrifugal, with comprising 20mM ZnCl 2PH 7.0, and the MES-NaOH solution washing of 0.02M obtains the immobilization thermolysin.
(3) use the 3ml final concentration to be the para benzoquinone solution of 1.5mM MCFs-NH to 10mg 2Carry out activation treatment, the carrier after the activated processing is comprised 3M NaCl with 2ml, 20mM ZnCl 2PH 7.0, the dissolving of the MES-NaOH solution of 0.02M.According to the mass ratio of 100mg enzyme/g carrier, the MES solution 1ml of the free enzyme of thermolysin of the 1mg/ml that adding embodiment 1 (3) obtains, mixing gets mixed solution; And induction stirring is reacted 20h in ice bath; Centrifugal, with comprising 3M NaCl, 20mM ZnCl 2PH 7.0, the MES-NaOH solution washing of 0.02M obtains the immobilization thermolysin.
(4) use the 3ml final concentration to be the para benzoquinone solution of 1.5mM MCFs-NH to 10mg 2Carry out activation treatment, with 2ml PH 7.0, the MES-NaOH solution of 0.02M dissolves with the carrier after the activated processing.According to the mass ratio of 100mg enzyme/g carrier, the MES solution 1ml of the free enzyme of thermolysin of the 1mg/ml that adding embodiment 1 (1) obtains, mixing gets mixed solution; With mixing solutions at 0-7 ℃; The 40W microwave condition shone 3 minutes down, and is centrifugal, gets deposition with PH 7.0; 0.02M the MES-NaOH solution washing, obtain microwave immobilization thermolysin.
(5) use the 3ml final concentration to be the para benzoquinone solution of 1.5mM MCFs-NH to 10mg 2Carry out activation treatment, the carrier after the activated processing is comprised 20mM ZnCl with 2ml 2PH 7.0, the MES-NaOH solution dissolving of 0.02M.According to the mass ratio of 100mg enzyme/g carrier, the MES solution 1ml of the free enzyme of thermolysin of the 1mg/ml that adding embodiment 1 (2) obtains, mixing gets mixed solution; With mixing solutions at 0～7 ℃; The 40W microwave condition shone 3 minutes down, and is centrifugal, gets deposition with containing 20mM ZnCL 2PH 7.0, and the MES-NaOH solution washing of 0.02M obtains microwave immobilization thermolysin.
(6) use the 3ml final concentration to be the para benzoquinone solution of 1.5mM MCFs-NH to 10mg 2Carry out activation treatment, the carrier after the activated processing is comprised 3M NaCl with 2ml, 20mM ZnCl 2PH 7.0, the dissolving of the MES-NaOH solution of 0.02M.Mass ratio according to 100mg enzyme/g carrier; Add the MES solution 1ml of the free enzyme of thermolysin of the 1mg/ml that embodiment 1 (3) obtains, mixing gets mixed solution, with mixed solution at 0～7 ℃; The 40W microwave condition shone 3 minutes down; Centrifugal, get deposition with containing 3M NaCl, 20mM ZnCl 2PH 7.0, the MES-NaOH solution washing of 0.02M obtains microwave immobilization thermolysin.
(7) the immobilization thermolysin that makes more than the general carries out the corresponding relative vigor of vigor test, and the result sees table 3.
Table 3: the comparison of conventional immobilization and microwave immobilization thermophilic bacteria protein enzyme activity
Figure BDA0000109004370000141
Visible by table 3, microwave radiation not only can make the catalytic activity of immobilization thermolysin be improved, but also makes the immobilization time shorten 399 times.
Embodiment 8: enzyme concentration is to the fixedly influence of thermolysin
Use the 3ml final concentration to be the para benzoquinone solution of 1.5mM MCFs-NH to 10mg 2Carry out activation treatment, the carrier after the activated processing is comprised 3M NaCl, 20mMZnCl with 2～2.6ml respectively 2PH 7.0, the dissolving of the MES-NaOH solution of 0.02M.Respectively according to the mass ratio of 40-100mg enzyme/g carrier; Add the MES solution 0.4～1ml of the free enzyme of thermolysin of the 1mg/ml that embodiment 1 (3) obtains, mixing gets mixed solution, with mixed solution at 0～7 ℃; The 40W microwave condition shone 3 minutes down; Centrifugal, get deposition with containing 3M NaCl, 20mM ZnCl 2PH 7.0, the MES-NaOH solution washing of 0.02M obtains microwave immobilization thermolysin.Again these 4 groups of immobilization thermolysins are carried out vitality test and obtain relative vigor, the result sees table 4.
Table 4: enzyme concentration is to the influence of immobilization thermophilic bacteria protein enzyme activity
Visible by table 4, when the relative vigor of enzyme concentration immobilization thermolysin when being 40mg enzyme/g carrier is 115.4% for optimum.
Embodiment 9: microwave irradiation power is to the fixedly influence of thermolysin
Use the 3ml final concentration to be the para benzoquinone solution of 1.5mM MCFs-NH to 10mg 2Carry out activation treatment, the carrier after the activated processing is comprised 3M NaCl with 2.6ml, 20mM ZnCl 2PH 7.0, the dissolving of the MES-NaOH solution of 0.02M.Mass ratio according to 40mg enzyme/g carrier; Add the MES solution 0.4ml of the free enzyme of thermolysin of the 1mg/ml that embodiment 1 (3) obtains, mixing gets mixed solution, with mixed solution at 0～7 ℃; 20～50W microwave condition shone 3 minutes down; Centrifugal, get deposition with containing 3M NaCl, 20mM ZnCl 2PH 7.0, the MES-NaOH solution washing of 0.02M obtains microwave immobilization thermolysin.Again these 7 groups of immobilization thermolysins are carried out the relative vigor that vitality test obtains and be respectively 82.9%, 110.8%, 115.4%, 129.2%, 156%, 85.7%, 82.5%, the result sees Fig. 4.
Visible by Fig. 4, when microwave irradiation power was 40W, the relative vigor of immobilization thermolysin was 156%, is optimal result.
Embodiment 10: the free enzyme of thermolysin, microwave immobilization thermophilic bacteria protein enzyme performance compare:
Get the free enzyme of thermolysin (being called for short free enzyme), the microwave immobilization thermolysin that embodiment 3 makes (being called for short the microwave immobilized enzyme) is tested as follows:
(1) temperature stability
In industry, adopt higher temperature of reaction, following advantages are arranged: higher speed of reaction, become molecular balance, better substrate solvability, lower reaction medium viscosity and the reduction of microbial contamination possibility etc.Therefore the enzyme of thermostability is significant for industrial application.Free enzyme, microwave immobilized enzyme are after differing temps is incubated 30 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 210 minutes respectively; Measure its remaining vigor; Compare with uninsulated sample, estimate its temperature stability, the result sees Fig. 5.
The result shows that microwave immobilized enzyme vigor after being incubated 3.5 hours under 70 ℃ of conditions remains unchanged, and after 1 hour, the remaining vigor of microwave immobilized enzyme still has 74.1% in insulation under 80 ℃ of conditions, and its temperature stability of freer enzyme improves a lot.
(2) anti-tertiary amyl alcohol property
Free enzyme, microwave immobilized enzyme under 70 ℃ respectively in the system of different content tertiary amyl alcohol insulation measured its remaining vigor in 2 hours, and compare at the uninsulated sample of aqueous phase, estimate its anti-tertiary amyl alcohol property, the result sees Fig. 6.
The result shows, the microwave immobilized enzyme is incubated after 2 hours vigor and remains unchanged even in 80% tertiary amyl alcohol, also keeping 46.3% activity in 5% tertiary amyl alcohol.And free enzyme has lost 32.5% activity after in 5% tertiary amyl alcohol, being incubated 2 hours, in 80% tertiary amyl alcohol, does not have active basically.
(3) anti-ETHYLE ACETATE property
Free enzyme, microwave immobilized enzyme under 70 ℃ respectively in the system of different content ETHYLE ACETATE insulation measured its remaining vigor in 2 hours, and compare at the uninsulated sample of aqueous phase, estimate its anti-ETHYLE ACETATE property, the result sees Fig. 7.
The result shows, the microwave immobilized enzyme is incubated in 5% ETHYLE ACETATE has only 8% vigor to lose after 2 hours, and free enzyme has been lost 32.5% vigor.After being incubated 2 hours in the ETHYLE ACETATE 80%, the microwave immobilized enzyme still has 53.1% catalytic activity, and free enzyme loses activity fully
(4) dynamic performance of immobilized enzyme
Free enzyme, microwave immobilized enzyme are reacted the catalytic activity of testing its enzyme under different ZAPM concentration of substrate.The result sees table 5.
Table 5: the relevant kinetic constant of microwave immobilization thermolysin
The result shows that the catalytic effect of microwave immobilized enzyme under low concentration of substrate can reach the catalytic effect of free enzyme under high concentration of substrate effect.

Claims (5)

1. the process for fixation of a thermolysin, said method comprises:
(1) carrier activation: with amidized mesoporous foam silicon carrier MCFs-NH 2Be immersed in the para benzoquinone solution of 0.5～2.5mM; The vibration activation is 1～3 hour under 20～30 ℃, the condition of 100～200rpm, and is centrifugal, gets deposition and washs with 20～30% ethanol and pure water successively; Deposition after the washing is scattered in the MES-NaOH solution again, obtains the activatory carrier fluid;
(2) immobilization of enzyme: with the activatory carrier fluid, by 40～100mg thermolysin/gMCFs-NH 2Enzyme concentration; Add thermolysin and obtain mixed solution, with mixed solution induction stirring reaction 18～20h or shine 2～4min down in ice bath, centrifugal at 0～7 ℃, 20～50W microwave condition; Get deposition and use the MES-NaOH solution washing, obtain the immobilization thermolysin;
The MES-NaOH solution composition is following described in step (1) and (2): NaCl2～5M, ZnCl 210～30mM, MES-NaOH 0.01～0.05M, pH 7.0～7.5, and solvent is a water.
2. the method for claim 1; It is characterized in that adding in the carrier fluid after said thermolysin disperses with MES-NaOH solution; Said thermolysin concentration in MES-NaOH solution is 0.5～2mg/mL, and said MES-NaOH solution composition is following: NaCl 2～5M, ZnCl 210～30mM, MES-NaOH 0.01～0.05M, pH7.0～7.5, solvent is a water.
3. the method for claim 1 is characterized in that para benzoquinone solution and MCFs-NH in the step (1) 2The ratio of volume mass consumption be 2～5ml: 10mg.
4. the method for claim 1, used MES-NaOH solution and MCFs-NH when it is characterized in that disperseing again in the step (1) 2The ratio of volume mass consumption be 2～5ml: 10mg.
5. the method for claim 1 is characterized in that said method is following:
(A) dispersion of enzyme: get thermolysin, disperse,, promptly get the MES solution of thermolysin-4 ℃ of held half a hour with MES-NaOH solution;
(B) carrier activation: with amidized mesoporous foam silicon carrier MCFs-NH 2Be immersed in the para benzoquinone solution of 1.5mM, the vibration activation is 2 hours under 25 ℃, the condition of 160rpm, and is centrifugal, gets deposition and washs with 20% ethanol and pure water successively, and the deposition after the washing is scattered in the MES-NaOH solution again, obtains the activatory carrier fluid;
(C) immobilization of enzyme: with the activatory carrier fluid, by 40～100mg thermolysin/gMCFs-NH 2Enzyme concentration; The MES solution that adds thermolysin obtains mixed solution, and is with mixed solution induction stirring reaction 20h or shine 3min down at 0～7 ℃, 40W microwave condition in ice bath, centrifugal; Get deposition and use the MES-NaOH solution washing, obtain the immobilization thermolysin;
The MES-NaOH solution composition is following described in the step (A)～(C): NaCl3M, ZnCl 220mM, MES-NaOH 0.02M, pH 7.0, and solvent is a water.
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CN104988132A (en) * 2015-06-19 2015-10-21 杭州师范大学 Microwave-assisted co-immobilization method of aldehyde ketone reductase and glucose dehydrogenase
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CN109234263A (en) * 2018-09-27 2019-01-18 福建海峡石墨烯产业技术研究院有限公司 A method of the crosslinked action immobilized enzyme based on adenosine monophosphate and graphene

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