CN106011119B - Preparation method of crosslinked β -lactamase aggregate for pharmaceutical wastewater treatment - Google Patents
Preparation method of crosslinked β -lactamase aggregate for pharmaceutical wastewater treatment Download PDFInfo
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- C12N9/14—Hydrolases (3)
- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12N9/86—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in cyclic amides, e.g. penicillinase (3.5.2)
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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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- C12Y305/02—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amides (3.5.2)
- C12Y305/02006—Beta-lactamase (3.5.2.6)
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/34—Nature of the water, waste water, sewage or sludge to be treated from industrial activities not provided for in groups C02F2103/12 - C02F2103/32
- C02F2103/343—Nature of the water, waste water, sewage or sludge to be treated from industrial activities not provided for in groups C02F2103/12 - C02F2103/32 from the pharmaceutical industry, e.g. containing antibiotics
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Abstract
The invention discloses a preparation method of a cross-linked β -lactamase aggregate for pharmaceutical wastewater treatment, which comprises the steps of transferring a recombinant β -lactamase gene sequence into a host cell Escherichia coli Top10 to obtain recombinant bacteria, carrying out induced expression, ultrasonic crushing, centrifugal treatment and supernatant extraction to obtain crude enzyme liquid, adding bovine serum albumin serving as an aggregating agent and a protective agent into the crude enzyme liquid, carrying out precipitation and aggregation to form an enzyme aggregate, and carrying out cross-linking on the obtained enzyme aggregate by using a bifunctional reagent to prepare a cross-linked β -lactamase aggregate.
Description
Technical Field
The invention belongs to the technical field of enzyme engineering, and particularly relates to a preparation method of a crosslinked β -lactamase aggregate for pharmaceutical wastewater treatment.
Background
In recent years, the pharmaceutical industry in China is rapidly developed, but a large amount of toxic and harmful waste water discharged in the pharmaceutical process seriously harms the health of people. The pharmaceutical industrial wastewater has the advantages of complex components, high organic matter content, high toxicity, deep color, high salt content and poor biodegradability, has extremely strong inhibiting effect on the growth of microorganisms, and is difficult to naturally degrade. Meanwhile, the Chemical Oxygen Demand (COD) of the pharmaceutical industry wastewater can reach more than 6000mg/L, so that the treatment of the pharmaceutical industry wastewater and the guarantee of the health of people become global subjects.
At present, the national and local governments pay great attention to wastewater treatment projects. According to the existing processes and operation conditions for domestic and foreign wastewater treatment, the current pharmaceutical industry-sharing wastewater treatment technology mainly comprises the following steps: physical treatment technology, chemical treatment technology, biological treatment technology. The biological treatment technology is favored by many pharmaceutical enterprises due to the advantages of low treatment cost, good economic benefit, no secondary pollution and the like. The biological treatment technology is a method for degrading substances in the wastewater by selecting strains from the wastewater by using organic matters in the wastewater as a sole carbon source or energy source. The method has good degradation effect on micromolecular organic matters.
β -lactamase is an enzyme secreted by lactam antibiotics, and can catalyze and hydrolyze β -lactam cyclic amide bond in 6-aminopenicillanic acid (6-APA), 7-aminocephalosporanic acid (7-ACA) and N-acyl derivative molecules thereof, so that β -lactam ring is cracked and destroyed to lose antibacterial activity.
A cross-linked enzyme aggregate (CLEAs) immobilization method is a novel immobilization technology which is proposed in 2000 by Sheldon group at the university of Delft in the Netherlands on based on cross-linked enzyme crystals (CLECs). Firstly, taking an organic solvent, a non-ionic polymer or salt and the like as a precipitator to precipitate and aggregate enzyme protein to form enzyme aggregate; and further crosslinking the enzyme aggregate with a bifunctional reagent to prepare the crosslinked enzyme aggregate. The active site of the enzyme in the cross-linked enzyme aggregate is not damaged, the protein forms a supermolecular structure, and the antibiotic acts with the active site of the enzyme through the middle gap of the aggregate, so that the active site of the enzyme is prevented from directly acting with an external microenvironment, and the antibiotic is better degraded. In addition, the aggregate is formed, so that a hydrophobic group is formed in the whole group, the influence of pH value, temperature, organic solvent and the like in a microenvironment on the configuration of protein is obviously reduced, the tolerance of enzyme is improved, and the stability of the enzyme solution is obviously improved compared with that of a free enzyme solution. Compared with the traditional immobilization method, the CLEAs technology has the advantages of low requirement on enzyme purity, good stability and high activity of the obtained immobilized enzyme, no need of a carrier, low cost, easy popularization and the like.
Although the advantages of CLEAs technology are obvious, the universality is not strong, the preparation condition which is optimized to one enzyme is not suitable for another enzyme or even the same enzyme of different enzyme sources, so the research on new enzyme immobilization systems is continuously searched.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a preparation method of a crosslinked β -lactamase aggregate for pharmaceutical wastewater treatment, so as to obtain a crosslinked β -lactamase aggregate with high activity and good stability, which is used for degrading lactam antibiotics in pharmaceutical industrial wastewater and achieving the purpose of reducing the chemical oxygen demand of the pharmaceutical wastewater.
In order to achieve the above object, the present invention provides a method for preparing a cross-linked β -lactamase aggregate for use in pharmaceutical wastewater treatment, comprising the steps of:
(1) transferring the recombinant β -lactamase gene into a host cell escherichia coli E.coli Top10 to obtain a recombinant bacterium, and performing prokaryotic in-vitro recombinant expression;
(2) carrying out induction expression on the recombinant bacteria to obtain induced bacteria, carrying out centrifugal treatment on the induced bacteria to obtain thalli, then adding a buffer solution, carrying out ultrasonic crushing and centrifugal treatment, and taking a supernatant to obtain a crude enzyme solution;
(3) adding BSA (bovine serum albumin) with the mass of 1/3 crude enzyme solution into 1mL of 25mg/mL crude enzyme solution to serve as an aggregating agent and a protective agent, and stirring the mixture for 20 minutes on a magnetic stirrer under the ice-bath condition in a 25mL beaker; 5mL of 75% ammonium sulfate solution is used as a precipitator, saturated ammonium sulfate (precipitator) is added dropwise under the ice bath condition, and the mixture is stirred for 1 hour; at normal temperature (25 ℃), taking glutaraldehyde with the concentration of 0.5% as a cross-linking bifunctional reagent, dropwise adding glutaraldehyde (bifunctional reagent), and cross-linking for 2 hours; centrifuging the obtained suspension for 15 minutes at 4000rpm under the condition of 4 ℃ to obtain a precipitate, namely the solidified enzyme;
the immobilized enzyme was resuspended in 20ml of 50mM phosphate buffer (pH7.0), stirred for 30 minutes under ice-bath conditions, and this was repeated three times, and the resulting suspension was centrifuged at 4000rpm for 15 minutes at 4 ℃ to obtain a precipitate, i.e., the resulting crosslinked β -lactamase aggregate.
More preferably, the precipitation temperature is 0 ℃ and the crosslinking temperature is room temperature (25 ℃).
As a further preference, the pH value of the crosslinked β -lactamase aggregate is 7.5 when it is used in the treatment of wastewater from the pharmaceutical industry.
As a further preference, the temperature of the crosslinked β -lactamase aggregate applied to the treatment of wastewater in pharmaceutical industry is 30-50 ℃.
As a further preference, the pharmaceutical industry wastewater is preferably antibiotic wastewater.
The object of the invention is thus achieved.
The invention relates to a preparation method of a cross-linked β -lactamase aggregate for treating pharmaceutical wastewater, which comprises the steps of transferring a recombinant β -lactamase gene sequence into a host cell Escherichia coli E.coli Top10 to obtain recombinant bacteria, carrying out induced expression, ultrasonic crushing, centrifugal treatment and supernatant taking to obtain crude enzyme liquid, adding bovine serum albumin serving as an aggregating agent and a protective agent into the crude enzyme liquid, precipitating and aggregating to form an enzyme aggregate, and cross-linking the obtained enzyme aggregate with a bifunctional reagent to prepare the cross-linked β -lactamase aggregate.
Drawings
FIG. 1 is a schematic representation of the gene modification of β -lactamase of the present invention;
FIG. 2 is a schematic diagram of recombinant expression of recombinant β -lactamase gene in the present invention;
FIG. 3 is an expression diagram of crude enzyme solution obtained according to an embodiment of the present invention;
FIG. 4 is a graph of the effect of ammonium sulfate solution concentration (a) and amount (b) on the aggrecanase activity of the cross-linked β -lactamase according to one embodiment of the present invention;
FIG. 5 is a graph showing the effect of Bovine Serum Albumin (BSA) on the activity of the cross-linked β -lactamase aggrecanase according to one embodiment of the present invention;
FIG. 6 is a graph showing the effect of final glutaraldehyde concentration on the aggrecanase activity of the cross-linked β -lactamase according to one embodiment of the present invention;
FIG. 7 is a graph of the effect of different pH values on the aggrecanase activity of the cross-linked β -lactamase according to one embodiment of the present invention;
FIG. 8 is a graph of the effect of different temperatures on the aggrecanase activity of the cross-linked β -lactamase provided by one embodiment of the present invention;
FIG. 9 is a graph of the effect of the cross-linked β -lactamase aggregates on the chemical oxygen demand of antibiotic wastewater according to one embodiment of the present invention.
Detailed Description
The following description of the embodiments of the present invention is provided in order to better understand the present invention for those skilled in the art with reference to the accompanying drawings. It is to be expressly noted that in the following description, a detailed description of known functions and designs will be omitted when it may obscure the subject matter of the present invention.
First, an embodiment
1. Obtaining of recombinant β -lactamase Gene
The invention takes β -lactamase gene on GenBank AF143804.1 with drug resistance to cefotaxime as a base, restriction enzymes NcoI (sequence 5 ' -ccatgg-3 ') and EcoRI (sequence 5 ' -gattc-3 ') are respectively designed at the 5 ' end and the 3 ' end of a coding region (nucleotide sequence), 6 His tags (histidine catcaccatcaccatcac) are inserted before a stop codon taa at the 3 ' end to obtain a modified β -lactamase gene sequence, and the process of modification and recombination is shown in figure 1.
Nucleotide sequence GenBank: AF143804.1 is a β -lactamase gene sequence from the national center for Biotechnology Information, NCBI gene sequence database.
In this example, Nanjing Kinshire Bio Inc. synthesized according to the modified β -lactamase gene sequence, which is shown as sequence 1 in the sequence table, the synthesized β -lactamase gene was cloned into pBAD-TLX expression vector to obtain recombinant β -lactamase gene, as shown in FIG. 2.
In the present invention, the restriction enzymes NcoI and EcoRI were inserted instead of the 5 'end and the 3' end of the β -lactamase gene sequence because cohesive termini produced by NcoI and EcoRI were specifically bound to cohesive termini produced by the restriction enzymes NcoI and EcoRI on the expression (cloning) vector pBAD-TLX, thereby linking the β -lactamase gene fragment to the expression (cloning) vector.
2. Inducible expression of recombinant β -lactamases
In this example, recombinant β -lactamase gene was transferred into E.coli Top10 as a host cell to obtain recombinant bacteria, which were then subjected to in vitro prokaryotic inducible expression under conditions that the recombinant bacteria were monoclonally inoculated into 20mLLB (Luria-Bertani) medium with Amp (ampicillin) concentration of 100. mu.g/mL and cultured overnight in a shaker at 37 ℃ and 180rpm, and the cultured bacterial solution was inoculated into 200mL LB medium containing Amp in an inoculum size of 2% and cultured in a shaker at 37 ℃ and 180rpm to OD600About 0.7, standing in 16 deg.C incubator for 1.5 hr, adding 0.05mg/mL arabinose as inducer, adjusting induction temperature to 16 deg.C, changing the rotation speed of shaker to 100rpm, and inducing bacteria to OD600Ending at about 2.5 to obtain the induced bacteria.
3. Obtaining crude enzyme solution
In this example, the induced bacteria were centrifuged to obtain cells, and 200mM Tris-SO was added4And (3) ultrasonically crushing the buffer solution, centrifuging at 4 ℃ for 1 hour by using a centrifuge at 12000g after the crushed solution becomes transparent to obtain a supernatant and a precipitate, and taking the supernatant to obtain a crude enzyme solution. In this example, the supernatant and the precipitate were added to a 2 XSDS loading buffer, mixed well, and subjected to SDS-PAGE polyacrylamide gel electrophoresis to detect the target protein. As can be seen from FIG. 3, the target protein is about 38kDa, and the soluble target protein, i.e., crude enzyme solution, is successfully obtained
4. Preparation of crosslinked β -lactamase aggregates
In this example, the concentration of the supernatant protein was measured by the Coomassie brilliant blue method, and based on the concentration conversion, 1mL of crude enzyme solution was added with 1/3 mass BSA (bovine serum albumin) as an aggregating agent and a protective agent in a 25mL beaker under ice-bath conditions and stirred for 20 minutes in a magnetic stirrer, saturated ammonium sulfate (precipitant) was added dropwise under ice-bath conditions and stirred for 1 hour, glutaraldehyde (bifunctional reagent) was added dropwise under normal temperature (25 ℃) conditions and cross-linked for 2 hours, the resulting suspension was centrifuged at 4000rpm for 15 minutes at 4 ℃ to obtain a precipitate as a solidified enzyme, the immobilized enzyme was suspended in 20mL of 50mM phosphate buffer (pH7.0) and stirred for 30 minutes under ice-bath conditions, and this was repeated three times, and the resulting suspension was centrifuged at 4000rpm for 15 minutes at 4 ℃ to obtain a precipitate as the resulting cross-linked β -lactamase aggregate.
Second, test
1. Effect of saturated ammonium sulfate concentration on Cross-Linked β -lactamase aggregate enzymatic Activity
30 percent, 45 percent, 60 percent, 75 percent and 85 percent of saturated ammonium sulfate solutions are respectively prepared, the crude enzyme solution is immobilized, and the relative enzyme activity of the cross-linked β -lactamase aggregate is measured, and the result is shown in figure 4 (a). As can be seen from figure 4(a), when the concentration of the saturated ammonium sulfate solution is 75 percent, the relative enzyme activity of the cross-linked β -lactamase aggregate prepared by the invention can reach 57.1 percent.
2. Effect of saturated ammonium sulfate dosage on Cross-Linked β -lactamase aggregate enzymatic Activity
The crude enzyme solution was immobilized by adding different amounts of saturated ammonium sulfate, and the relative enzyme activity of the cross-linked β -lactamase aggregates was measured, and the results are shown in FIG. 4 (b). As shown in FIG. 4(b), when 5mL of 75% saturated ammonium sulfate solution was added, the relative enzyme activity of the cross-linked β -lactamase aggregates prepared by the present invention was 64%.
3. Effect of Bovine Serum Albumin (BSA) on the Activity of Cross-Linked β -lactamase Aggregatase
When bovine serum albumin with different masses serving as an aggregating agent and a protective agent is added for immobilization, the relative enzyme activity of the cross-linked β -lactamase aggregate is measured, and the result is shown in fig. 5. from fig. 5, it can be seen that when the mass ratio of the added bovine serum albumin to the crude enzyme solution is 1:3, the relative enzyme activity of the cross-linked β -lactamase aggregate prepared by the invention is 93.1%, which indicates that the bovine serum albumin is favorable for retaining the activity of the cross-linked β -lactamase aggregate.
4. Effect of the terminal concentration of glutaraldehyde on the Activity of Cross-Linked β -lactamase Agkistrolase
When glutaraldehyde solutions of different final concentrations were added as bifunctional reagents for immobilization, the cross-linked β -lactamase aggregate relative enzyme activity was measured, and the results are shown in fig. 6. from fig. 6, it can be seen that when the final concentration of glutaraldehyde was 0.5%, the cross-linked β -lactamase aggregate relative enzyme activity prepared by the present invention was 94.5%.
5. Effect of pH on enzymatic stability of Cross-Linked β -lactamase aggregates
The enzyme activity stability of the cross-linked β -lactamase aggregate is detected by placing the cross-linked β -lactamase aggregate in phosphate buffer solutions with different pH values, and the result is shown in figure 7. from figure 7, it can be seen that the cross-linked β -lactamase aggregate prepared by the invention has lower relative enzyme activity under the environment of peracid or over alkali, but has higher stability than the crude enzyme solution, and the cross-linked β -lactamase aggregate has higher relative enzyme activity between pH5.5 and pH8.5, which indicates that the cross-linked β -lactamase aggregate prepared by the invention has higher pH value stability.
6. Effect of temperature on enzymatic Activity stability of Cross-Linked β -lactamase aggregates
The enzyme activity stability of the cross-linked β -lactamase aggregate is detected by placing the cross-linked β -lactamase aggregate in beakers with different temperatures, and the result is shown in figure 8. from figure 8, the cross-linked β -lactamase aggregate prepared by the invention has high heat resistance, can still keep 70% of enzyme activity when placed at 70 ℃ for 6 hours, and is optimal at 30-50 ℃, and the crude enzyme liquid loses 60% of enzyme activity in 60 ℃ for half an hour.
7. Effect of Cross-Linked β -lactamase aggregates on COD value of wastewater
The method comprises the steps of placing filter paper in a conical funnel, filtering antibiotic wastewater to remove suspended matters and particulate matters to obtain filtrate, centrifuging the filtrate at a high speed, detecting the chemical oxygen demand of the filtrate before wastewater treatment by using a chemical oxygen demand rapid detector, placing the crosslinked β -lactamase aggregate into the filtrate, culturing the filtrate in a shaking table at the temperature of 30 ℃ at the rotating speed of 180rpm for 35 hours, centrifuging the treated wastewater, and measuring the chemical oxygen demand of the treated wastewater, wherein the result is shown in figure 9. the removal rate of the crosslinked β -lactamase aggregate prepared by the invention on COD of the wastewater can reach 72.9 percent, which shows that the crosslinked β -lactamase aggregate prepared by the invention has a good effect of reducing the chemical oxygen demand on the antibiotic wastewater, and the removal rate of the COD of pBAD bacteria on the wastewater can also reach 12 percent, and the pBAD bacteria show that E.coli Top10 host bacteria with pBAD-TLX carriers are introduced into the body, a control group, namely, no crude bacteria are added into the wastewater, and no enzyme activity loss occurs, and no enzyme activity result is obtained.
Although illustrative embodiments of the present invention have been described above to facilitate the understanding of the present invention by those skilled in the art, it should be understood that the present invention is not limited to the scope of the embodiments, and various changes may be made apparent to those skilled in the art as long as they are within the spirit and scope of the present invention as defined and defined by the appended claims, and all matters of the invention which utilize the inventive concepts are protected.
Claims (6)
1. A preparation method of a crosslinked β -lactamase aggregate for pharmaceutical wastewater treatment is characterized by comprising the following steps:
(1) transferring the recombinant β -lactamase gene into a host cell escherichia coli E.coli Top10 to obtain a recombinant bacterium, and performing prokaryotic in-vitro recombinant expression;
(2) carrying out induction expression on the recombinant bacteria to obtain induced bacteria, carrying out centrifugal treatment on the induced bacteria to obtain thalli, then adding a buffer solution, carrying out ultrasonic crushing and centrifugal treatment, and taking a supernatant to obtain a crude enzyme solution;
(3) adding bovine serum albumin with the mass of 1/3 crude enzyme solution into 1mL of 25mg/mL crude enzyme solution to serve as an aggregating agent and a protective agent, and stirring for 20 minutes on a magnetic stirrer under the ice-bath condition in a 25mL beaker; 5mL of 75% ammonium sulfate solution is used as a precipitator, saturated ammonium sulfate is added dropwise under the ice bath condition, and the mixture is stirred for 1 hour; under the condition of normal temperature, glutaraldehyde with the concentration of 0.5 percent is taken as a cross-linking bifunctional reagent, the glutaraldehyde is added dropwise, and cross-linking is carried out for 2 hours; centrifuging the obtained suspension for 15 minutes at 4000rpm under the condition of 4 ℃ to obtain a precipitate, namely the solidified enzyme;
the immobilized enzyme is suspended in 20ml 50mM phosphate buffer solution with pH7.0, stirred for 30 minutes under ice bath condition, the operation is repeated for three times, and the obtained suspension is centrifuged for 15 minutes at 4000rpm under the condition of 4 ℃, so that the precipitate is the obtained crosslinked β -lactamase aggregate.
2. The method according to claim 1, wherein the recombinant β -lactamase gene is obtained by the following steps:
based on β -lactamase gene on GenBank AF143804.1 with resistance to cefotaxime, restriction enzymes NcoI (sequence 5 ' -ccatgg-3 ') and EcoRI (sequence 5 ' -gattc-3 ') are respectively designed at the 5 ' end and the 3 ' end of a coding region, and 6 His tags (histidine catcaccatcaccatcac) are inserted in front of a stop codon taa at the 3 ' end to obtain a modified β -lactamase gene sequence;
synthesizing according to the modified β -lactamase gene sequence, cloning the synthesized β -lactamase gene into a pBAD-TLX expression vector, and obtaining the recombinant β -lactamase gene.
3. The method of claim 1, wherein the inducible expression is:
inoculating the recombinant bacterium monoclonal into 20mL LB culture medium, placing the culture medium in a shaker with 37 ℃ and 180rpm for overnight culture, wherein the concentration of ampicillin is 100 mug/mL; inoculating the cultured bacterial liquid into 200mL LB culture medium containing ampicillin at an inoculum size of 2%, and culturing in a shaker at 37 deg.C and rotation speed of 180rpm to OD600About 0.7, standing in 16 deg.C incubator for 1.5 hr, adding 0.05mg/mL arabinose as inducer, adjusting induction temperature to 16 deg.C, changing the rotation speed of shaker to 100rpm, and inducing bacteria to OD600Ending at about 2.5 to obtain the induced bacteria.
4. The method according to claim 1, wherein the precipitation temperature is 0 ℃ and the crosslinking temperature is room temperature.
5. The method of claim 1, wherein the cross-linked β -lactamase aggregate has a pH of 7.5 when used in the treatment of wastewater from pharmaceutical industry.
6. The preparation method of claim 1, wherein the temperature of the crosslinked β -lactamase aggregate used for the treatment of pharmaceutical industry wastewater is 30-50 ℃.
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