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
- Publication number
- CN106011119B CN106011119B CN201610343627.6A CN201610343627A CN106011119B CN 106011119 B CN106011119 B CN 106011119B CN 201610343627 A CN201610343627 A CN 201610343627A CN 106011119 B CN106011119 B CN 106011119B
- Authority
- CN
- China
- Prior art keywords
- lactamase
- aggregate
- cross
- recombinant
- linked
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 108090000204 Dipeptidase 1 Proteins 0.000 title claims abstract description 75
- 102000006635 beta-lactamase Human genes 0.000 title claims abstract description 55
- 238000004065 wastewater treatment Methods 0.000 title claims abstract description 11
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 102000004190 Enzymes Human genes 0.000 claims abstract description 64
- 108090000790 Enzymes Proteins 0.000 claims abstract description 64
- 241000894006 Bacteria Species 0.000 claims abstract description 22
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 14
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 14
- 238000004132 cross linking Methods 0.000 claims abstract description 9
- 239000006228 supernatant Substances 0.000 claims abstract description 8
- 230000004931 aggregating effect Effects 0.000 claims abstract description 7
- 230000001588 bifunctional effect Effects 0.000 claims abstract description 7
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 7
- 241000588724 Escherichia coli Species 0.000 claims abstract description 6
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 6
- 239000007788 liquid Substances 0.000 claims abstract description 6
- 239000003223 protective agent Substances 0.000 claims abstract description 6
- 238000001556 precipitation Methods 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 27
- 239000002351 wastewater Substances 0.000 claims description 23
- 238000000034 method Methods 0.000 claims description 14
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 12
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 9
- 239000002244 precipitate Substances 0.000 claims description 8
- 239000000725 suspension Substances 0.000 claims description 6
- 230000001939 inductive effect Effects 0.000 claims description 5
- 108010093096 Immobilized Enzymes Proteins 0.000 claims description 4
- 230000006698 induction Effects 0.000 claims description 4
- 239000012716 precipitator Substances 0.000 claims description 4
- 108091008146 restriction endonucleases Proteins 0.000 claims description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 3
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 3
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 claims description 3
- 229960000723 ampicillin Drugs 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 3
- 238000010367 cloning Methods 0.000 claims description 3
- 238000000338 in vitro Methods 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 238000003259 recombinant expression Methods 0.000 claims description 3
- 108020004705 Codon Proteins 0.000 claims description 2
- 241001052560 Thallis Species 0.000 claims description 2
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 claims description 2
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 2
- 230000001580 bacterial effect Effects 0.000 claims description 2
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 2
- 229960004261 cefotaxime Drugs 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims description 2
- 239000013604 expression vector Substances 0.000 claims description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 2
- 239000000411 inducer Substances 0.000 claims description 2
- 239000002054 inoculum Substances 0.000 claims description 2
- 238000003756 stirring Methods 0.000 claims description 2
- 239000001963 growth medium Substances 0.000 claims 3
- 108700026244 Open Reading Frames Proteins 0.000 claims 1
- GPRBEKHLDVQUJE-VINNURBNSA-N cefotaxime Chemical compound N([C@@H]1C(N2C(=C(COC(C)=O)CS[C@@H]21)C(O)=O)=O)C(=O)/C(=N/OC)C1=CSC(N)=N1 GPRBEKHLDVQUJE-VINNURBNSA-N 0.000 claims 1
- 239000008055 phosphate buffer solution Substances 0.000 claims 1
- 230000002194 synthesizing effect Effects 0.000 claims 1
- 238000004220 aggregation Methods 0.000 abstract 1
- 230000002776 aggregation Effects 0.000 abstract 1
- 238000000605 extraction Methods 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 41
- 239000000126 substance Substances 0.000 description 9
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 7
- 229910052760 oxygen Inorganic materials 0.000 description 7
- 239000001301 oxygen Substances 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- 230000003115 biocidal effect Effects 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 108010003059 aggrecanase Proteins 0.000 description 5
- 230000008901 benefit Effects 0.000 description 5
- 239000000706 filtrate Substances 0.000 description 5
- 230000002255 enzymatic effect Effects 0.000 description 4
- 241000725101 Clea Species 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- NGHVIOIJCVXTGV-ALEPSDHESA-N 6-aminopenicillanic acid Chemical compound [O-]C(=O)[C@H]1C(C)(C)S[C@@H]2[C@H]([NH3+])C(=O)N21 NGHVIOIJCVXTGV-ALEPSDHESA-N 0.000 description 2
- NGHVIOIJCVXTGV-UHFFFAOYSA-N 6beta-amino-penicillanic acid Natural products OC(=O)C1C(C)(C)SC2C(N)C(=O)N21 NGHVIOIJCVXTGV-UHFFFAOYSA-N 0.000 description 2
- HSHGZXNAXBPPDL-HZGVNTEJSA-N 7beta-aminocephalosporanic acid Chemical compound S1CC(COC(=O)C)=C(C([O-])=O)N2C(=O)[C@@H]([NH3+])[C@@H]12 HSHGZXNAXBPPDL-HZGVNTEJSA-N 0.000 description 2
- 239000003782 beta lactam antibiotic agent Substances 0.000 description 2
- 230000000593 degrading effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 239000010842 industrial wastewater Substances 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 108020004256 Beta-lactamase Proteins 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 125000003460 beta-lactamyl group Chemical group 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- AZZMGZXNTDTSME-JUZDKLSSSA-M cefotaxime sodium Chemical compound [Na+].N([C@@H]1C(N2C(=C(COC(C)=O)CS[C@@H]21)C([O-])=O)=O)C(=O)\C(=N/OC)C1=CSC(N)=N1 AZZMGZXNTDTSME-JUZDKLSSSA-M 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 229920000831 ionic polymer Polymers 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 150000004965 peroxy acids Chemical class 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
Images
Classifications
-
- 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)
- C12N9/78—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)
-
- 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
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
- 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)
-
- C—CHEMISTRY; METALLURGY
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Hydrology & Water Resources (AREA)
- Environmental & Geological Engineering (AREA)
- Water Supply & Treatment (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Enzymes And Modification Thereof (AREA)
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 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610343627.6A CN106011119B (en) | 2016-05-23 | 2016-05-23 | Preparation method of crosslinked β -lactamase aggregate for pharmaceutical wastewater treatment |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610343627.6A CN106011119B (en) | 2016-05-23 | 2016-05-23 | Preparation method of crosslinked β -lactamase aggregate for pharmaceutical wastewater treatment |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106011119A CN106011119A (en) | 2016-10-12 |
| CN106011119B true CN106011119B (en) | 2020-03-24 |
Family
ID=57096632
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610343627.6A Expired - Fee Related CN106011119B (en) | 2016-05-23 | 2016-05-23 | Preparation method of crosslinked β -lactamase aggregate for pharmaceutical wastewater treatment |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106011119B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114075560A (en) * | 2020-08-18 | 2022-02-22 | 杭州俊丰生物工程有限公司 | Stable composition of beta-lactamase |
| CN115417510B (en) * | 2022-09-20 | 2023-07-04 | 南京农业大学 | Method for removing extracellular antibiotic resistance genes in water |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102492683B (en) * | 2011-12-01 | 2013-06-12 | 南宁奕德环境科技有限公司 | Preparation method of cross-linked oxalate decarboxylase aggregates (CLEAs) |
| CN104498470A (en) * | 2015-01-16 | 2015-04-08 | 齐鲁工业大学 | New type co-immobilization white rot fungi gel particle |
| CN104651344A (en) * | 2015-03-13 | 2015-05-27 | 福建师范大学 | Method for preparing immobilized enzyme preparation of perhydrolase |
| CN105200076A (en) * | 2015-09-23 | 2015-12-30 | 江南大学 | Bacillus subtilis recombined to express gamma-lactamase and immobilization and application |
| CN103710412B (en) * | 2013-12-11 | 2016-02-24 | 福州大学 | Beta-glucosidase cross-linked aggregates catalyzes and synthesizes the technique of rhodioside |
-
2016
- 2016-05-23 CN CN201610343627.6A patent/CN106011119B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102492683B (en) * | 2011-12-01 | 2013-06-12 | 南宁奕德环境科技有限公司 | Preparation method of cross-linked oxalate decarboxylase aggregates (CLEAs) |
| CN103710412B (en) * | 2013-12-11 | 2016-02-24 | 福州大学 | Beta-glucosidase cross-linked aggregates catalyzes and synthesizes the technique of rhodioside |
| CN104498470A (en) * | 2015-01-16 | 2015-04-08 | 齐鲁工业大学 | New type co-immobilization white rot fungi gel particle |
| CN104651344A (en) * | 2015-03-13 | 2015-05-27 | 福建师范大学 | Method for preparing immobilized enzyme preparation of perhydrolase |
| CN105200076A (en) * | 2015-09-23 | 2015-12-30 | 江南大学 | Bacillus subtilis recombined to express gamma-lactamase and immobilization and application |
Non-Patent Citations (2)
| Title |
|---|
| Crosslinked Aggregates of Rhizopus oryzae Lipase as Industrial Biocatalysts: Preparation, Optimization, Characterization, and Application for Enantioselective Resolution Reactions;Funda Kartal et al;《Biotechnology progress》;20121231;第28卷(第4期);第937-945页 * |
| 青霉素酶基因异源表达及青霉素酶固定化研究;赵洪坤;《中国优秀硕士学位论文全文数据库（电子期刊）》;20111215;第1-72页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106011119A (en) | 2016-10-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| López-Serrano et al. | Cross-linked enzyme aggregates with enhanced activity: application to lipases | |
| Wilson et al. | Encapsulation of crosslinked penicillin G acylase aggregates in lentikats: evaluation of a novel biocatalyst in organic media | |
| CN106011119B (en) | Preparation method of crosslinked β -lactamase aggregate for pharmaceutical wastewater treatment | |
| CN104120120A (en) | Immobilized recombinant penicillin G acylase and application thereof | |
| Sambyal et al. | Exploitation of E. coli for the production of penicillin G amidase: a tool for the synthesis of semisynthetic β-lactam antibiotics | |
| Zhang et al. | Preparation of crosslinked enzyme aggregates (CLEAs) of acid urease with urethanase activity and their application | |
| CN116083407A (en) | Thermostable cephalosporin enzyme composition for plate medium addition | |
| Ye et al. | Poly-lysine supported cross-linked enzyme aggregates of penicillin G acylase and its application in synthesis of β-lactam antibiotics | |
| Kumar et al. | Protease production by Bacillus subtilis immobilized on different matrices | |
| Lugtenberg et al. | A simple method for following the fate of alanine-containing components in murein synthesis in Escherichia coli | |
| CN102978192B (en) | Mutant cephalosporin C acylase, method for preparing same and method for converting 7-aminocephalosporin acid (ACA) | |
| CN103695405B (en) | A kind of production method of novel ss-lactam class antibiotic synthetic enzymes | |
| Zhao et al. | Purification and immobilization of α-amylase in one step by gram-positive enhancer matrix (GEM) particles from the soluble protein and the inclusion body | |
| CN111057695B (en) | Nitrilase and preparation method and application thereof | |
| CN103184182B (en) | Penicillin acylase, as well as high-yield strain and application thereof | |
| CN103374563B (en) | A kind of method improveing 7-ACA producing strains | |
| WO2022063770A1 (en) | Method for recovering a protein from a fermentation broth comprising a high degree of lysed cells | |
| CN101003798B (en) | Purified expression of recombined beta lactamase in superspectrum, and fermentation process in high density | |
| Ajamani et al. | Mathematically optimized production, purification and characterization of penicillin G acylase from soil bacterial isolates AA17A and AA17B | |
| CN112481236A (en) | Recombinant protein INP-AidH and preparation method and application thereof | |
| Fathi et al. | Partial purification, characterization and immobilization of a novel lipase from a native isolate of Lactobacillus fermentum | |
| CN113913416B (en) | Preparation process of alcaligenes faecalis penicillin G acylase | |
| CN103695447B (en) | A kind of novel lactam antibiotics synthetic enzyme and encoding gene thereof and application | |
| JP2013198427A (en) | Method for aligning protein | |
| RU2703169C1 (en) | Strain-producer of human glutamate oxaloacetate transaminase |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20200324 |