CN109136212B - Malathion hydrolase prepared by cross-linked enzyme aggregate immobilization method - Google Patents
Malathion hydrolase prepared by cross-linked enzyme aggregate immobilization method Download PDFInfo
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- CN109136212B CN109136212B CN201810842842.XA CN201810842842A CN109136212B CN 109136212 B CN109136212 B CN 109136212B CN 201810842842 A CN201810842842 A CN 201810842842A CN 109136212 B CN109136212 B CN 109136212B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/342—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the enzymes used
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/38—Products with no well-defined composition, e.g. natural products
- C11D3/386—Preparations containing enzymes, e.g. protease or amylase
- C11D3/38672—Granulated or coated enzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/306—Pesticides
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/40—Organic compounds containing sulfur
Abstract
The invention relates to a malathion hydrolase prepared by a cross-linked enzyme aggregate immobilization method, which comprises the following steps: (1) adding a certain amount of ammonium sulfate into the malathion hydrolase solution, uniformly stirring, and salting out to obtain an aggregate of the malathion hydrolase; (2) adding polyether with a certain concentration into the aggregate to obtain a combination of the aggregate and the polyether; (3) and finally adding glutaraldehyde with a certain concentration for cross-linking reaction, and centrifuging or filtering after the cross-linking reaction is finished to obtain insoluble particles, namely the immobilized enzyme. Compared with the prior art, the activity of the malathion hydrolase is improved by 4 times, the stability and the tolerance of a detergent are obviously improved, the enzyme activity is basically unchanged after 10 times of repeated recycling, and the malathion hydrolase shows very good activity and stability.
Description
Technical Field
The invention relates to development and preparation of an organophosphorus enzyme immobilization method, in particular to a preparation method and application of a novel cross-linked aggregate immobilized enzyme.
Background
The organophosphate substances are artificially synthesized high-toxicity compounds, including various types such as phosphate esters, phosphate thiol esters and the like, and are widely applied to pesticide insecticides, herbicides and the like, wherein malathion is widely used for preventing and treating diseases and insect pests on crops such as grains, vegetables, tobacco, tea and the like in a plurality of countries in the world. Since the naturally evolved biocatalytic system has limited ability to degrade such artificially synthesized compounds, organophosphorus compounds pose a great threat to environmental safety and human health.
The organophosphorus hydrolase reduces the toxicity of organophosphorus compounds by hydrolyzing the phosphorus ester bond of the organophosphorus compounds, can be applied to detergents, detoxification medicaments, soil restoration agents and scavengers of harmful pesticides or nerve agents, has wide application in the field of biological detoxification, and is a reliable biological repair and detoxification method. However, the currently reported organophosphorus hydrolase suffers from low catalytic efficiency, low thermal stability or low expression level, and few organophosphorus hydrolases and mutants thereof capable of meeting application requirements are still available. Meanwhile, the free enzyme has water solubility, cannot be recycled after use, is easily inactivated by high temperature, organic solvents and the like, and does not have good storage stability and operation stability. Thus, these drawbacks limit the use of free enzymes.
The above-mentioned drawbacks of the organophosphorus hydrolase can be overcome by preparing an immobilized enzyme. After immobilization, immobilized enzymes have more advantages than free enzymes: the immobilized enzyme is easy to separate from a substrate and a product, the immobilized enzyme can be recovered by simple operations such as centrifugation or filtration after the reaction is finished, the enzyme activity is reduced less, the immobilized enzyme can be used repeatedly in multiple batches, the production cost is reduced, the general stability, the thermal stability and the like are greatly improved after the immobilized treatment, and the sensitivity to an inhibitor is reduced. The immobilized enzyme is suitable for automatic and continuous production, is easy to control the catalytic process, can not bring enzyme protein into the product to cause enzyme residue, simplifies the later purification process, and improves the utilization efficiency of the enzyme. However, immobilized enzymes also have certain drawbacks: (1) the mass transfer efficiency of enzyme and substrate is reduced; (2) the conformation of the enzyme is changed, and the activity of the enzyme is reduced; (3) the enzyme is lost during the immobilization and use process. Therefore, it is necessary to select a suitable immobilization method according to different use requirements, and optimize parameters such as carrier, pH, substrate concentration, enzyme loading amount, preparation time, and the like in the enzyme immobilization process, so as to obtain an immobilized enzyme with simple preparation method, low cost, less activity loss, and high stability.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for preparing malathion hydrolase by using a cross-linked enzyme aggregate immobilization method.
The purpose of the invention can be realized by the following technical scheme: a malathion hydrolase is prepared by a cross-linked enzyme aggregate immobilization method, which is characterized by comprising the following steps:
(1) adding a certain amount of ammonium sulfate into the malathion hydrolase solution, uniformly stirring, and salting out to obtain an aggregate of the malathion hydrolase;
(2) adding polyether with a certain concentration into the aggregate to obtain a combination of the aggregate and the polyether;
(3) and finally adding glutaraldehyde with a certain concentration for cross-linking reaction, and centrifuging or filtering after the cross-linking reaction is finished to obtain insoluble particles, namely the immobilized enzyme.
The source of malathion hydrolase described in step 1 includes, but is not limited to, Pseudomonas oleovarans DSM 50188. (purchased from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, purchasing Link: https:// www.dsmz.de/catalog-Microorganisms. html # search result)
Further, the concentration of the malathion hydrolase in the step 1 is 10-40mg/ml, preferably 20 mg/ml.
Further, in step 1, ammonium sulfate is added so that the final concentration of ammonium sulfate in the solution is 0.2 to 0.8g/ml, preferably 0.3 to 0.7g/ml, and more preferably 0.5 g/ml; salting-out temperature is 0-10 deg.C, salting-out time is 15-60min, preferably salting-out temperature is 4 deg.C, salting-out time is 30 min.
Further, the polyether in the step 2 has the average molecular weight of 8000-15000, the addition amount of 0.1-1.5 times of the mass of the malathion hydrolase, the reaction temperature of 0-10 ℃ and the reaction time of 15-60 min. The polyether is a commercially available polyether, and comprises polyether Pluronic F127, the mass ratio of the polyether to malathion hydrolase is 1:0-1:6, preferably 1:4, and the reaction condition of the crosslinking aggregate and the polyether is preferably that the crosslinking aggregate and the polyether are stirred for 30min at 4 ℃.
Further, the final concentration of glutaraldehyde in step 3 is 30-120mM, preferably 90mM, the crosslinking reaction temperature is 0-10 ℃ and the reaction time is 1-5 hours, preferably the crosslinking reaction temperature is 4 ℃ and the reaction time is 3 hours.
Further, the centrifugation in step 3 was carried out under 12000rpm for 3 min.
Further, the obtained immobilized enzyme is used for degrading malathion in industrial washing, polluted soil and water body restoration.
The invention provides a novel cross-linked aggregate immobilized enzyme prepared by the method.
The novel cross-linked aggregate immobilized enzyme is applied to detoxification of malathion.
Compared with the prior art, the invention provides the preparation method of the novel cross-linked aggregate immobilized enzyme, the novel cross-linked aggregate immobilized enzyme prepared by the method has higher immobilization rate, and the enzyme activity, the operation stability and the detergent tolerance of the co-immobilized enzyme are better. The activity of malathion hydrolase is improved by 4 times, the stability and the detergent tolerance are obviously improved, the activity of the enzyme is basically unchanged after 10 times of repeated recycling, and the malathion hydrolase shows very good activity and stability. In an actual malathion degradation test, the immobilized enzyme shows a good degradation effect under a high temperature condition, can completely hydrolyze 0.15mM of malathion within 15min at 50 ℃, can completely degrade 0.15mM of malathion within 30min at room temperature, can meet the removal requirement of the malathion in industrial washing, and can be well applied to the remediation of other biological detoxification fields such as polluted soil and water.
Drawings
FIG. 1 is a graph showing the effect of the conventional immobilization method and the novel method for immobilizing a crosslinked aggregate according to the embodiment of the present invention (CLEA: enzyme + ammonium sulfate precipitation + glutaraldehyde crosslinking, 1: enzyme + polyether + ammonium sulfate + glutaraldehyde, 2: the order of addition in the embodiment of the present invention, enzyme + ammonium sulfate + polyether + glutaraldehyde, and 3: enzyme + ammonium sulfate + glutaraldehyde + polyether).
FIG. 2 is a graph showing the effect of ammonium sulfate concentration on the activity of immobilized enzymes in an example of the present invention;
FIG. 3 is a graph showing the effect of enzyme concentration on the activity of immobilized enzymes in an example of the present invention;
FIG. 4 is a graph of the effect of polyether concentration on immobilized enzyme activity in an example of the invention;
FIG. 5 is a graph showing the effect of glutaraldehyde concentration on the activity of an immobilized enzyme in an embodiment of the present invention;
FIG. 6 shows the optimum pH of an immobilized enzyme in an example of the present invention;
FIG. 7 shows the optimum temperature of an immobilized enzyme in the example of the present invention;
FIG. 9 shows that the novel immobilized enzyme CLEA-PL127(A) and the free enzyme (B) are respectively incubated in a detergent for 120min to ensure residual enzyme activity;
FIG. 8 shows the residual enzyme activity of free enzyme, conventional immobilized enzyme, and immobilized enzyme incubated at 50 ℃ in the present example;
FIG. 10 shows the operational stability of the immobilized enzyme in the example of the present invention during 10 repeated operations;
FIG. 11 shows the organophosphorus degradation rates of the immobilized enzyme in the 0.1% detergent solution at room temperature (A) and 50 ℃ respectively in the example of the present invention.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples. It should be noted, however, that the practice of the present invention is not limited to the practice described below.
Example 1
Dissolving organophosphorus hydrolase (i.e. malathion hydrolase Pseudomonas oleodorans DSM50188) in 1mL 50mM phosphate buffer solution (pH 7.0) at a concentration of 20mg/mL, adding ammonium sulfate for aggregate precipitation, and stirring at 4 deg.C for 30min to obtain a solution with a final concentration of 0.4 g/mL; then adding polyether Pluronic F127, wherein the mass ratio of the polyether Pluronic F127 to the malathion hydrolase is 1:4, and stirring for 30min at 4 ℃; finally, glutaraldehyde is added, the concentration of the glutaraldehyde is 90mM, and crosslinking is carried out for 3 hours at 4 ℃. After the reaction, the reaction solution was collected and centrifuged at 12000rpm for 3min to separate the immobilized enzyme.
And (3) enzyme activity determination:
the immobilized enzyme is placed on a constant temperature oscillation reactor at 50 ℃ and is kept for 7 hours. A certain amount of immobilized enzyme was added to 1ml of 50mM Tris-HCl buffer (pH9.0, containing 2mM Ellman's reagent) containing malathion at a final concentration of 0.5mM per incubation for a certain period of time, and the absorbance change rate of the reaction solution was measured for 1min, respectively. The change of the activity of the enzyme within 7h was measured spectrophotometrically with no immobilized enzyme as a blank. The result shows that the residual activity of the immobilized enzyme is more than 65% after incubation for 7h at 50 ℃, the immobilized enzyme is obviously superior to the immobilized enzyme and free enzyme prepared by the conventional method, the adaptability to high temperature is very strong, and the immobilized enzyme can be completely used for degrading malathion in the environment with higher temperature.
Testing of the activity and stability of the immobilized enzymes:
the immobilized enzymes were added to 50mM Tris-HCl buffer (pH9.0, containing 2mM Ellman's reagent) containing 0.1% of a commercial detergent, respectively, and incubated for 120min, and the enzyme activity was measured every 20 min. Experimental results show that the activity and the stability of the immobilized enzyme are greatly superior to those of free enzyme, the immobilized enzyme has quite excellent performance no matter the immobilized enzyme does not contain detergent or contains detergent, the enzyme activity is all increased after the incubation is started, and the enzyme activity is increased by 2 times of the initial activity after the 120min incubation is finished. For free enzyme, the enzyme activity is reduced from the beginning of incubation, wherein SDS and APG have the most serious influence on the free enzyme, 70 percent of the enzyme activity is lost after 60min, and the activity of the free enzyme is lost by 30 percent even if the free enzyme is incubated in COD solution with the minimum influence for 60 min.
Operational stability of immobilized enzyme:
an appropriate amount of immobilized enzyme was added to 990. mu.l of 50mM Tris-HCl buffer (pH9.0, containing 2mM Ellman's reagent), 10. mu.l of 50mM malathion was added thereto, the mixture was shaken and mixed, and after 1min of reaction on a spectrophotometer, the absorbance at 412nm was measured, and the initial rate of hydrolysis reaction of the batch was calculated. And (3) after continuing to react for 10min, centrifuging at 12000rpm for 3min to collect immobilized enzyme, washing for 3 times by using a buffer solution, reacting for the next batch, and repeating the operation for 10 times. The immobilized enzyme shows good reuse stability, and the activity is still maintained at about 86% after 10 batches of the immobilized enzyme are reused.
Experiment for degrading malathion by immobilized enzyme in 0.1% detergent
A certain amount of immobilized enzyme was added to three different commercial detergents (COD, APG, SDS) at 0.1% concentration, respectively, and the reaction was carried out with 0.15mM malathion as a substrate, and absorbance values were recorded every 1min for 30min in a spectrophotometer. The experimental result shows that the malathion can be completely hydrolyzed after the immobilized enzyme reacts in the solution without the detergent and 0.1% COD for 30min at room temperature, and the malathion with the same concentration can be completely hydrolyzed after the immobilized enzyme with the same amount reacts in the solution without the detergent and 0.1% COD for 15min at 50 ℃.
Example 2
A malathion hydrolase is prepared by a cross-linked enzyme aggregate immobilization method, which comprises the following steps:
(1) adding a certain amount of ammonium sulfate into the malathion hydrolase solution, uniformly stirring, and salting out to obtain an aggregate of the malathion hydrolase;
(2) adding polyether with a certain concentration into the aggregate to obtain a combination of the aggregate and the polyether;
(3) and finally adding glutaraldehyde with a certain concentration for cross-linking reaction, and centrifuging or filtering after the cross-linking reaction is finished to obtain insoluble particles, namely the immobilized enzyme.
FIG. 1 is a graph comparing the effects of the conventional immobilization process and the novel cross-linked aggregate immobilization process, and illustrates that the specific activity of all the cross-linking processes after adding polyether Pluronic F127 is higher than that of the conventional CLEA, wherein the optimal addition mode is as follows: PoOPHM9 precipitated with ammonium sulfate to form aggregates, added polyether PL127, and finally cross-linked with glutaraldehyde, the enzyme specific activity of the immobilization method was 5 times that of conventional CLEA, which is the novel immobilization method in the examples of the present invention.
FIG. 2 is a graph showing the influence of the addition of ammonium sulfate of different concentrations on the enzyme activity of the final immobilized enzyme, and it can be seen from the graph that the enzyme activity is detected when the concentration of ammonium sulfate is 0.2-0.8g/mL, and the enzyme activity is highest when the concentration of ammonium sulfate is 0.5 g/mL;
FIG. 3 is a graph of the effect of enzyme concentration on the activity of immobilized enzymes; namely, the influence of different concentrations of malathion hydrolase Pseudomonas oleovarans DSM50188 on the activity of the immobilized enzyme can be seen from the figure, and the malathion hydrolase with the concentration of 10-40mg/ml has better enzyme activity.
FIG. 4 is a graph of the effect of polyether concentration on immobilized enzyme activity; as can be seen from the figure, when the mass ratio of the polyether to the malathion hydrolase is 1:0-1:6, the enzyme activity is the best when the ratio is 1: 4.
FIG. 5 is a graph showing the effect of glutaraldehyde concentration on the activity of immobilized enzyme, and it can be seen from the graph that the enzyme activity is good at a final concentration of 30-120mM and the enzyme activity is best at a concentration of 90 mM.
FIG. 6 shows the optimum pH of the immobilized enzyme, wherein the reactivity is uniformly good at pH8 to 10, and the reactivity is best at pH9. Compared with free enzyme, the novel immobilized enzyme in the embodiment of the invention has no obvious change, but the pH range is widened, and the activity of CLEA-PL127 is always maintained to be more than 80% in the pH range of 8.0-11.0.
FIG. 7 is a graph showing the effect of temperature difference on the reactivity of immobilized enzyme, and it can be seen that the optimum temperature of the novel immobilized enzyme in the example of the present invention is increased from 30 ℃ to 50 ℃ and the activity is maintained at 80% or more at 40-60 ℃ as compared with the free enzyme, and 27% of the activity is maintained even at a high temperature of 80 ℃ while the free enzyme is completely inactivated.
FIG. 8 shows the residual enzyme activity of free enzyme, conventional immobilized enzyme and the immobilized enzyme in the scheme incubated at 50 ℃, and it can be seen from the figure that the residual activity of the novel immobilized enzyme CLEA-PL incubated at 12750 ℃ for 7h in the embodiment of the invention is maintained at more than 65%, while the activity of the conventional immobilized enzyme CLEA is lost by 50% after incubation for 7 h. Free enzyme activity decreased rapidly after 1h incubation at 50 ℃. This indicates that the novel immobilized enzymes of the examples of the present invention have better high temperature tolerance. FIG. 9 shows that the residual enzyme activity of the immobilized enzyme CLEA-PL127(A) and the free enzyme (B) of the novel immobilized enzyme of the embodiment of the invention is 120min after incubation in a detergent, and as can be seen from the graph, the enzyme activity of the novel immobilized enzyme of the embodiment of the invention is increased to about 2 times of the initial activity after incubation for 120min in the detergent and without the detergent. And the free enzyme has low enzyme activity all the time from the beginning of incubation, which shows that the stability of the novel immobilized enzyme in the embodiment of the invention in a detergent is greatly higher than that of the free enzyme.
FIG. 10 shows the operation stability of the immobilized enzyme in the process of repeating 10 times, and it can be seen from the figure that the novel immobilized enzyme CLEA-PL127 in the embodiment of the present invention shows good reuse stability, and the activity is still maintained at about 86% after 10 batches of reuse.
FIG. 11 shows that the degradation rate of organophosphorus of immobilized enzyme in 0.1% detergent solution at room temperature (A) and 50 deg.C (B) respectively is better, and the novel immobilized enzyme in the embodiment of the invention has better degradation effect at 50 deg.C.
Claims (3)
1. A preparation method of malathion hydrolase immobilized by using a cross-linked enzyme aggregate is characterized by comprising the following steps:
(1) adding ammonium sulfate into malathion hydrolase solution, stirring to obtain final concentration of 0.4-0.7g/ml ammonium sulfate, salting out to obtain malathion hydrolase aggregate at 0-10 deg.C for 15-60 min; the malathion hydrolase is derived from pseudomonas oleovorans (A), (B)Pseudomonas oleovorans) DSM50188；
(2) Adding polyether into the aggregate to obtain a combination of the aggregate and the polyether; the average molecular weight of the polyether is 8000-15000, the mass ratio of the polyether to the malathion hydrolase is 1:1-1:6, the reaction temperature is 0-10 ℃, and the reaction time is 15-60 min;
(3) finally adding glutaraldehyde for cross-linking reaction, and centrifuging or filtering to obtain insoluble particles as immobilized enzyme, wherein the final concentration of glutaraldehyde is 60-120 mM, the cross-linking reaction temperature is 0-10 ℃, and the reaction time is 1-5 hours;
the polyether is Pluronic F127.
2. The method for preparing malathion hydrolase immobilized by using crossiinked enzyme aggregate according to claim 1, wherein the concentration of the malathion hydrolase in the step 1 is 10 to 40 mg/ml.
3. The method for preparing malathion hydrolase immobilized by using crossiinked enzyme aggregate as claimed in claim 1, wherein the centrifugation in step 3 is carried out under 12000rpm for 3 min.
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