CN102433317B - Immobilization method for thermolysin - Google Patents
Immobilization method for thermolysin Download PDFInfo
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- CN102433317B CN102433317B CN 201110363336 CN201110363336A CN102433317B CN 102433317 B CN102433317 B CN 102433317B CN 201110363336 CN201110363336 CN 201110363336 CN 201110363336 A CN201110363336 A CN 201110363336A CN 102433317 B CN102433317 B CN 102433317B
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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
(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 it has highly selective, catalytic reaction condition gentleness, characteristics such as pollution-free, is widely used in industries such as food-processing, medicine and fine chemistry industry.But the natural enzyme poor stability, easily inactivation, can not reuse, and sneak into product after the reaction, purification difficult makes it be difficult to use more widely in industry.In addition, separation and enzyme purification and their disposable use have also increased its cost as catalyzer greatly.With this understanding, the concept of immobilized enzyme and technology are proposed and are developed, and become the emphasis of enzyme engineering research in recent years.
The immobilization of enzyme namely fetters enzyme or be limited in certain zone with solid material, still can carry out its distinctive catalyzed reaction, and a recyclable and reusable class technology.According to character and the purposes of enzyme, these are several can be divided into absorption method, 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, 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 synthetic of catalysis peptide bond also.Aspartame is a kind of dipeptide sweetener, and sugariness approximately is 200 times of sucrose, and it is distinguished the flavor of as 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, diabetes 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 with chemical technology, has clear superiority, 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 by 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 by the covalently bound realization of para benzoquinone linking agent and enzyme and carrier, the final immobilized enzyme product that obtains has obtained the thermolysin immobilized enzyme that performance is greatly improved than the free enzyme of thermolysin.
The technical solution used in the present invention is:
A kind of process for fixation of thermolysin, described 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 precipitation successively with 20～30% ethanol and pure water washing, precipitation after the washing is scattered in the MES-NaOH solution again, the carrier fluid that obtains activating;
(2) immobilization of enzyme: with the carrier fluid of activation, 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 under 0～7 ℃, 20～50W microwave condition, shine 2～4min in ice bath, centrifugal, get precipitation MES-NaOH solution washing, being fixed thermolysin;
Prepared amidized MCFs carrier is the amidized mesoporous foam silicon carrier of this area routine in the step (1), and its aperture is about 26nm.
The MES-NaOH solution composition is as follows 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 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 is subjected to the effect of microwave, and protein and polypeptide all are typical polar molecules, so microwave can accelerate 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, the immobilization technology of enzyme can be reached a new high.
Described thermolysin adds in the carrier fluid after disperseing with MES-NaOH solution, and described thermolysin concentration in MES-NaOH solution is 0.5～2mg/mL, and described MES-NaOH solution composition is as follows: NaCl 2～5M, ZnCl
210～30mM, MES-NaOH 0.01～0.05M, pH 7.0～7.5, and solvent is 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.
Described method is as follows:
(A) dispersion of enzyme: get thermolysin, disperse with MES-NaOH solution, place half an hour down at-4 ℃, namely get the fatty acid methyl ester sulfonate solution of thermolysin;
(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 precipitation and washs with 20% ethanol and pure water successively, and the precipitation after the washing is scattered in the MES-NaOH solution again, the carrier fluid that obtains activating; 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 carrier fluid of activation, by 40～100mg thermolysin (in free enzyme)/g MCFs-NH
2Enzyme concentration, the fatty acid methyl ester sulfonate solution that adds thermolysin obtains mixed solution, and is with mixed solution induction stirring reaction 20h or shine 3min under 0～7 ℃, 40W microwave condition in ice bath, centrifugal, get precipitation MES-NaOH solution washing, being fixed thermolysin;
The MES-NaOH solution composition is as follows described in the step (A)～(C): NaCl 3M, ZnCl
220mM, MES-NaOH 0.02M, pH 7.0, and solvent is water.
Among the present invention, described amidized mesoporous foam silicon carrier MCFs-NH
2Can prepare as follows:
1. water-heat process: (triblock copolymer: poly-(the propylene glycol)-block-of poly-(ethylene glycol)-block-poly-(ethylene glycol)) 5～10g water dissolves in batches to take by weighing P123 earlier, 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 ℃ are stirred 20～60min down, 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 uses water filtration at last, and the oven dry of gained carrier is stand-by;
2. remove template reaction: take by weighing step 1. made carrier 0.5～3.5g in autoclave, add 5～25ml concentrated nitric acid (65%～68%, w/w) and 1～10ml hydrogen peroxide (H
2O
2Concentration 20～40% v/v), places 50～150 ℃ of reactions of baking oven, 6～30h, washes filtration with water, and it is stand-by to obtain the oven dry of template carrier;
3. silanization: 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, lead to water of condensation again, 100～150 ℃ of reflux 6～30h of oil bath, question response finishes the back cooling and uses toluene and absolute ethanol washing respectively, after treating that organic solvent in the carrier volatilizees substantially, put into baking oven and dry, namely get described amidized mesoporous foam silicon carrier MCFs-NH
2
For microwave-assisted immobilization and ordinary method are compared, the invention process process for fixation under the conventional immobilization of thermolysin and the microwave condition, 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, 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 hatch 3.5h under 70 ℃ condition, the vigor of immobilization thermolysin does not reduce, and free enzyme has not just had activity after hatching 3h.When hatch 60min under 80 ℃ condition, 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.Hatch 2h under 70 ℃ in 5% tertiary amyl alcohol, 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 ethyl acetate, the immobilization thermolysin has also been showed powerful defensive ability/resistance ability.Hatch 2h under 70 ℃ in 5% ethyl acetate, when the vigor of free enzyme had reduced by 32.5%, immobilized enzyme was but keeping 92% vigor.And when hatching 2h under 70 ℃ in 80% ethyl acetate, 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 by the inventive method is greatly improved than free enzyme on performance.And, by 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 catalyzes and synthesizes the mechanism of aspartame precursor for thermolysin; 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 that para benzoquinone concentration is to the influence of 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 that microwave irradiation power is to the influence of 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 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-ethyl acetate of immobilized enzyme in 70 ℃ of water-baths; FE: the free enzyme MW-IME of thermolysin: microwave immobilization thermolysin;
(5) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1: the fatty acid methyl ester sulfonate solution of preparation thermolysin
(1) gets 2mg thermolysin (free enzyme, available from Sigma aldrich Shanghai branch office, together following), with 2ml pH 7.0,0.02M MES-NaOH solution disperse, place half an hour down at-4 ℃, both the fatty acid methyl ester sulfonate 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, place half an hour down at-4 ℃, both the fatty acid methyl ester sulfonate solution of the free enzyme of thermolysin of 1mg/ml.
(3) get the 2mg thermolysin, contain 3M NaCl with 2ml, 20mM ZnCL
2PH7.0, the MES-NaOH solution of 0.02M disperses, place half an hour down at-4 ℃, both the fatty acid methyl ester sulfonate 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 fatty acid methyl ester sulfonate solution for preparing the free enzyme of thermolysin: get the free enzyme of 2mg thermolysin, contain 3M NaCl with 2ml, 20mM ZnCl
2PH 7.0, the MES-NaOH solution of 0.02M disperses, place half an hour down at-4 ℃, both the fatty acid methyl ester sulfonate 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: poly-(the propylene glycol)-block-of poly-(ethylene glycol)-block-poly-(ethylene glycol) earlier, PEO-PPO-PEO) the 5.34g water dissolves in batches all to transfer in the container and goes, 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 stand-by;
2. remove template reaction: take by weighing top made carrier 1.0g in autoclave, adding 10ml concentrated nitric acid (65.5wt%) and 7ml hydrogen peroxide (30%, v/v), place 100 ℃ of reactions of baking oven 12h.Wash filtration then with water, dry stand-by;
3. silanization: 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), lead to water of condensation then, 110 ℃ of reflux 12h of oil bath, question response finishes the back cooling and uses toluene and absolute ethanol washing then respectively, treat that organic solvent in the carrier volatilizees substantially 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 precipitation and uses the washing of 20% (v/v) ethanol and pure water respectively, and the precipitation 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, because solid-liquid in the centrifugal process realize to separate, so think that vehicle weight remains unchanged, still be 10mg wherein.
(3) get the carrier fluid 2.6ml of the activation that step (2) obtains, the fatty acid methyl ester sulfonate damping fluid 0.4ml that adds the free enzyme of thermolysin that steps A makes again, mixing gets mixed solution, and enzyme concentration is the free enzyme of 40mg thermolysin/g MCFs-NH in the described immobilized enzyme preparation process
2. the mixed solution that (4) get step (3) 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, being fixed thermolysin.
Embodiment 3: the free enzyme of thermolysin is prepared into the immobilization thermolysin with microwave method:
A. the fatty acid methyl ester sulfonate solution for preparing the free enzyme of thermolysin: get the free enzyme of 2mg thermolysin, contain 3M NaCl with 2ml, 20mM ZnCL
2PH 7.0, the MES-NaOH solution of 0.02M disperses, place half an hour down at-4 ℃, both the fatty acid methyl ester sulfonate 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 precipitation and washs with 20% ethanol and pure water respectively, and the precipitation 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, because solid-liquid in the centrifugal process realize to separate, so think that vehicle weight remains unchanged, still be 10mg wherein.
(3) get the carrier fluid 2.6ml of the activation that step (2) obtains, the fatty acid methyl ester sulfonate solution 0.4ml that adds the free enzyme of thermolysin that steps A makes again, mixing gets mixed solution, and enzyme concentration is the free enzyme/gMCFs-NH of 40mg thermolysin in the described immobilized enzyme preparation process
2
(4) mixed solution of getting step (3) carries out the immobilization processing: at 0～7 ℃, irradiation is 3 minutes under the 40W microwave condition, and is centrifugal with mixing solutions, gets precipitation 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
Be that the para benzoquinone solution of 1.5mM and glutaraldehyde solution are respectively to the MCFs-NH of 10mg with the 3ml final concentration
2Carry out activation treatment, with the 2ml PH 7.0 of the carrier after the activated processing, the MES-NaOH solution dissolving of 0.02M.According to the mass ratio of 100mg enzyme/g carrier, the fatty acid methyl ester sulfonate 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 (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 fatty acid methyl ester sulfonate 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, being fixed thermolysin.These three immobilized enzyme are carried out vitality test, the results are shown in Table 1.
Table 1: the linking agent kind is to the influence of immobilization thermophilic bacteria protein enzymic activity
By table 1 as seen, 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 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 the 2ml PH 7.0 of the carrier after the activated processing, the MES-NaOH solution dissolving of 0.02M.Mass ratio according to 100mg enzyme/g carrier, the fatty acid methyl ester sulfonate 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, being fixed 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, the results are shown in Figure 1.
Its immobilization specific activity of enzyme was 2357.6U/mg when para benzoquinone concentration was 1.5mM as seen from Figure 1, be the optimal result of several groups of experiments, thereby the para benzoquinone final concentration was 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.
Be that the para benzoquinone solution of 1.5mM is to the MCFs-NH of 10mg with the 3ml final concentration
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, the fatty acid methyl ester sulfonate solution 1ml of the free enzyme of thermolysin of the 1mg/ml that adding embodiment 1 (1) obtains, mixing gets mixed solution, with mixed solution at 0～7 ℃, irradiation is 3 minutes under the 30W microwave condition, centrifugal, get precipitation with the used damping fluid washing of immobilization, obtain microwave immobilization thermolysin.And these several groups of immobilized enzyme are carried out vitality test, the relative vigor of different system correspondences sees Table 2.
Table 2: neutral salt is to the influence of immobilization thermophilic bacteria protein enzyme activity
By table 2 as seen, CaCl
2To the almost not influence of catalytic activity of immobilization thermolysin, the catalytic activity of the immobilization thermolysin of NaCl has huge raising, 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 reaction system with this system.
Embodiment 7: the contrast of conventional immobilization and microwave-assisted immobilization
(1) be that the para benzoquinone solution of 1.5mM is to the MCFs-NH of 10mg with the 3ml final concentration
2Carry out activation treatment, with the 2ml PH 7.0 of the carrier after the activated processing, the MES-NaOH solution dissolving of 0.02M.Mass ratio according to 100mg enzyme/g carrier, the fatty acid methyl ester sulfonate 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, being fixed thermolysin.
(2) be that the para benzoquinone solution of 1.5mM is to the MCFs-NH of 10mg with the 3ml final concentration
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 fatty acid methyl ester sulfonate 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, the MES-NaOH solution washing of 0.02M, being fixed thermolysin.
(3) be that the para benzoquinone solution of 1.5mM is to the MCFs-NH of 10mg with the 3ml final concentration
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 fatty acid methyl ester sulfonate 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, being fixed thermolysin.
(4) be that the para benzoquinone solution of 1.5mM is to the MCFs-NH of 10mg with the 3ml final concentration
2Carry out activation treatment, with the 2ml PH 7.0 of the carrier after the activated processing, the MES-NaOH solution dissolving of 0.02M.Mass ratio according to 100mg enzyme/g carrier, the fatty acid methyl ester sulfonate 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 ℃, irradiation is 3 minutes under the 40W microwave condition, and is centrifugal, gets precipitation PH 7.0,0.02M the MES-NaOH solution washing, obtain microwave immobilization thermolysin.
(5) be that the para benzoquinone solution of 1.5mM is to the MCFs-NH of 10mg with the 3ml final concentration
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.Mass ratio according to 100mg enzyme/g carrier, the fatty acid methyl ester sulfonate 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 ℃, irradiation is 3 minutes under the 40W microwave condition, centrifugal, get precipitation with containing 20mM ZnCL
2PH 7.0, and the MES-NaOH solution washing of 0.02M obtains microwave immobilization thermolysin.
(6) be that the para benzoquinone solution of 1.5mM is to the MCFs-NH of 10mg with the 3ml final concentration
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, the fatty acid methyl ester sulfonate solution 1ml of the free enzyme of thermolysin of the 1mg/ml that adding embodiment 1 (3) obtains, mixing gets mixed solution, with mixed solution at 0～7 ℃, irradiation is 3 minutes under the 40W microwave condition, centrifugal, get precipitation 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, the results are shown in Table 3.
Table 3: the comparison of conventional immobilization and microwave immobilization thermophilic bacteria protein enzyme activity
By table 3 as seen, 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
Be that the para benzoquinone solution of 1.5mM is to the MCFs-NH of 10mg with the 3ml final concentration
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, the fatty acid methyl ester sulfonate solution 0.4～1ml of the free enzyme of thermolysin of the 1mg/ml that adding embodiment 1 (3) obtains, mixing gets mixed solution, with mixed solution at 0～7 ℃, irradiation is 3 minutes under the 40W microwave condition, centrifugal, get precipitation 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 results are shown in Table 4.
Table 4: enzyme concentration is to the influence of immobilization thermophilic bacteria protein enzyme activity
By table 4 as seen, when the relative vigor of enzyme concentration immobilization thermolysin when being 40mg enzyme/g carrier be 115.4% for optimum.
Embodiment 9: microwave irradiation power is to the fixedly influence of thermolysin
Be that the para benzoquinone solution of 1.5mM is to the MCFs-NH of 10mg with the 3ml final concentration
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, the fatty acid methyl ester sulfonate solution 0.4ml of the free enzyme of thermolysin of the 1mg/ml that adding embodiment 1 (3) obtains, mixing gets mixed solution, with mixed solution at 0～7 ℃, irradiation is 3 minutes under 20～50W microwave condition, centrifugal, get precipitation 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 results are shown in Figure 4.
As seen from Figure 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) carries out following experiment:
(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 results are shown in Figure 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
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, the results are shown in Figure 6.
The result shows, the microwave immobilized enzyme is incubated after 2 hours vigor and remains unchanged even also keeping 46.3% activity in 80% tertiary amyl alcohol in 5% tertiary amyl alcohol.And free enzyme has lost 32.5% activity be incubated 2 hours in 5% tertiary amyl alcohol after, does not have active in 80% tertiary amyl alcohol substantially.
(3) anti-ethyl acetate
Free enzyme, microwave immobilized enzyme under 70 ℃ respectively in the system of different content ethyl acetate insulation measured its remaining vigor in 2 hours, and compare at the uninsulated sample of aqueous phase, estimate its anti-ethyl acetate, the results are shown in Figure 7.
The result shows, the microwave immobilized enzyme is incubated in 5% ethyl 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 ethyl 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 under different ZAPM concentration of substrate, test the catalytic activity of its enzyme.The results are shown in 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 (4)
1. the process for fixation of a thermolysin, described 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 precipitation and uses 20 ~ 30% ethanol and pure water washing successively, precipitation after the washing is scattered in the MES-NaOH solution again, the carrier fluid that obtains activating;
(2) immobilization of enzyme: with the carrier fluid of activation, by 40 ~ 100mg thermolysin/gMCFs-NH
2Enzyme concentration, add thermolysin and obtain mixed solution, with mixed solution induction stirring reaction 18 ~ 20h or under 0 ~ 7 ℃, 20 ~ 50W microwave condition, shine 2 ~ 4min in ice bath, centrifugal, get precipitation MES-NaOH solution washing, being fixed thermolysin; Described thermolysin adds in the carrier fluid after disperseing with MES-NaOH solution; The MES-NaOH solution composition is as follows described in step (1) and (2): NaCl2 ~ 5M, ZnCl
210 ~ 30mM, MES-NaOH0.01 ~ 0.05M, pH7.0 ~ 7.5, solvent is water.
2. 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.
3. 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.
4. the method for claim 1 is characterized in that described method is as follows:
(A) dispersion of enzyme: get thermolysin, disperse with MES-NaOH solution, place half an hour down at-4 ℃, namely get the fatty acid methyl ester sulfonate solution of thermolysin;
(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 precipitation and washs with 20% ethanol and pure water successively, and the precipitation after the washing is scattered in the MES-NaOH solution again, the carrier fluid that obtains activating;
(C) immobilization of enzyme: with the carrier fluid of activation, by 40 ~ 100mg thermolysin/gMCFs-NH
2Enzyme concentration, the fatty acid methyl ester sulfonate solution that adds thermolysin obtains mixed solution, and is with mixed solution induction stirring reaction 20h or shine 3min under 0 ~ 7 ℃, 40W microwave condition in ice bath, centrifugal, get precipitation MES-NaOH solution washing, being fixed thermolysin;
The MES-NaOH solution composition is as follows described in the step (A) ~ (C): NaCl3M, ZnCl
220mM, MES-NaOH0.02M, pH7.0, solvent are water.
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