CN106011119A - Preparation method of cross-linked beta-lactamase aggregate for treating pharmaceutical wastewater - Google Patents
Preparation method of cross-linked beta-lactamase aggregate for treating pharmaceutical wastewater Download PDFInfo
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- CN106011119A CN106011119A CN201610343627.6A CN201610343627A CN106011119A CN 106011119 A CN106011119 A CN 106011119A CN 201610343627 A CN201610343627 A CN 201610343627A CN 106011119 A CN106011119 A CN 106011119A
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- enzyme
- lactamase
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- 108090000204 Dipeptidase 1 Proteins 0.000 title claims abstract description 38
- 239000002351 wastewater Substances 0.000 title claims abstract description 31
- 102000006635 beta-lactamase Human genes 0.000 title claims abstract description 19
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 102000004190 Enzymes Human genes 0.000 claims abstract description 109
- 108090000790 Enzymes Proteins 0.000 claims abstract description 109
- 239000007788 liquid Substances 0.000 claims abstract description 25
- 241000894006 Bacteria Species 0.000 claims abstract description 19
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 14
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 14
- 238000004220 aggregation Methods 0.000 claims abstract description 10
- 230000002776 aggregation Effects 0.000 claims abstract description 10
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 9
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 8
- 241000588724 Escherichia coli Species 0.000 claims abstract description 6
- 239000006228 supernatant Substances 0.000 claims abstract description 6
- 230000004931 aggregating effect Effects 0.000 claims abstract description 5
- 238000004132 cross linking Methods 0.000 claims description 52
- 150000003952 β-lactams Chemical class 0.000 claims description 42
- 238000000034 method Methods 0.000 claims description 18
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 12
- 239000003814 drug Substances 0.000 claims description 10
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 9
- 230000008569 process Effects 0.000 claims description 9
- 238000003756 stirring Methods 0.000 claims description 9
- 238000004065 wastewater treatment Methods 0.000 claims description 8
- 230000001588 bifunctional effect Effects 0.000 claims description 6
- 230000001939 inductive effect Effects 0.000 claims description 6
- 239000000725 suspension Substances 0.000 claims description 6
- 239000003223 protective agent Substances 0.000 claims description 5
- 230000009466 transformation Effects 0.000 claims description 5
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 4
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 4
- 230000006698 induction Effects 0.000 claims description 4
- 239000008363 phosphate buffer Substances 0.000 claims description 4
- 239000002244 precipitate Substances 0.000 claims description 4
- 238000001556 precipitation Methods 0.000 claims description 4
- 238000002525 ultrasonication Methods 0.000 claims description 4
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 3
- 238000013461 design Methods 0.000 claims description 3
- 238000003760 magnetic stirring Methods 0.000 claims description 3
- 239000002773 nucleotide Substances 0.000 claims description 3
- 125000003729 nucleotide group Chemical group 0.000 claims description 3
- 238000012545 processing Methods 0.000 claims description 3
- 238000007711 solidification Methods 0.000 claims description 3
- 230000008023 solidification Effects 0.000 claims description 3
- 108091026890 Coding region Proteins 0.000 claims description 2
- 206010059866 Drug resistance Diseases 0.000 claims description 2
- 241001597008 Nomeidae 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
- 230000015572 biosynthetic process Effects 0.000 claims description 2
- 229960004261 cefotaxime Drugs 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
- 238000000338 in vitro Methods 0.000 claims description 2
- 239000002054 inoculum Substances 0.000 claims description 2
- 238000005215 recombination Methods 0.000 claims description 2
- 230000006798 recombination Effects 0.000 claims description 2
- 238000003786 synthesis reaction Methods 0.000 claims description 2
- 229910021529 ammonia Inorganic materials 0.000 claims 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 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
- 230000000694 effects Effects 0.000 abstract description 15
- 239000000126 substance Substances 0.000 abstract description 7
- 230000003115 biocidal effect Effects 0.000 abstract description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 abstract description 5
- 229910052760 oxygen Inorganic materials 0.000 abstract description 5
- 239000001301 oxygen Substances 0.000 abstract description 5
- 238000012360 testing method Methods 0.000 abstract description 3
- 230000002457 bidirectional effect Effects 0.000 abstract 1
- 230000001376 precipitating effect Effects 0.000 abstract 1
- 239000011814 protection agent Substances 0.000 abstract 1
- 230000002255 enzymatic effect Effects 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 6
- 230000000844 anti-bacterial effect Effects 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 239000000706 filtrate Substances 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 241000725101 Clea Species 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 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
- 230000008485 antagonism Effects 0.000 description 2
- 239000003782 beta lactam antibiotic agent Substances 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 230000000593 degrading effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241000228143 Penicillium Species 0.000 description 1
- 102000018120 Recombinases Human genes 0.000 description 1
- 108010091086 Recombinases Proteins 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
- 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 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 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
- 238000005336 cracking Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 239000010842 industrial wastewater Substances 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000013618 particulate matter Substances 0.000 description 1
- 150000004965 peroxy acids Chemical class 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 150000005846 sugar alcohols Polymers 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
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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)
- C12N9/78—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
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- Life Sciences & Earth Sciences (AREA)
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- 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 beta-lactamase aggregate for treating pharmaceutical wastewater. The preparation method comprises the following steps: transferring a recombinant beta-lactamase gene sequence into a host cell E.coli Top10 to obtain recombined bacteria; carrying out induced expression, ultrasonic crushing and centrifugal treatment and taking supernatant to obtain rough enzyme liquid; adding bovine serum albumin into the rough enzyme liquid as an aggregation agent and a protection agent; precipitating and aggregating to form an enzyme aggregate; crossly linking the obtained enzyme aggregate through a bidirectional reagent so as to prepare the cross-linked beta-lactamase aggregate. After a test, the cross-linked beta-lactamase aggregate prepared by the invention is high in enzyme activity and good in stability, can be repeatedly utilized and is used for treating antibiotic wastewater; the chemical oxygen demand is reduced by 72.9 percent; the cross-linked beta-lactamase aggregate has an important application value in the treatment of the antibiotic wastewater in a pharmaceutical industry.
Description
Technical field
The invention belongs to technical field of enzyme engineering, more specifically, relate to a kind of for pharmacy wastewater treatment
Crosslinking beta-lactam enzyme aggregate preparation method.
Background technology
In recent years, China's pharmaceuticals industry has obtained fast development, but the having in a large number of discharge in pharmacy procedure
The healthy of people in poison harmful waste water then serious harm.Because pharmaceuticals industry waste component is complicated, Organic substance
Content is high, toxicity is big, color is deep, salinity is high, biodegradability is poor, has extremely strong pressing down to growth of microorganism
Make and use, it is difficult to natural degradation.Meanwhile, COD (the Chemical Oxygen of pharmaceuticals industry waste water
Demand, COD) may be up to more than 6000mg/L, therefore, administer pharmaceuticals industry waste water, ensure people
Healthy become global task.
At present, country and local government pay much attention to waste water treatment engineering.Process according to domestic and international waste water
The technique used and ruuning situation, current pharmacy technology for treating industrial waste water altogether is broadly divided into: physical treatment
Technology, chemical treatment method, biologic treating technique.Biologic treating technique is because of its processing cost effect low, economic
Benefit is good and the advantage such as non-secondary pollution, receives the favor of many pharmacy corporations.Biologic treating technique is to utilize
Organic substance in waste water is as only carbon source or the energy, and breeding species from waste water, to the material in waste water
Carry out a kind of method degraded.The method especially has good degradation effect to small organic molecule.
Beta-lactamase is the class of enzymes for lactam antibiotics secretion, can catalyzing hydrolysis 6-amino penicillium sp
Beta-lactam nucleus in alkanoic acid (6-APA) and 7-amino-cephalosporanic acid (7-ACA) and N-acyl derivative molecule thereof
Amido link, makes beta-lactam nucleus cracking be destroyed, loses antibacterial activity.
Cross-linked enzyme aggregate (CLEAs) process for fixation, be 2000 by Holland Delft university Sheldon little
A kind of novel immobilization technology that group proposes on the basis of crosslinked enzyme crystal (CLECs).It first with
Organic solvent, non-ionic polyalcohol or salt etc. make pheron precipitate as precipitant and assemble, and form enzyme and gather
Collective；Further enzyme aggregate bifunctional reagent is cross-linked, thus prepare cross-linking enzyme and assemble
Body.Wherein in cross-linked enzyme aggregate, the avtive spot of enzyme is not destroyed, and protein defines supramolecular structured
Structure, antibiotic is by the avtive spot effect of the intermediate gap of aggregation with enzyme, thus the avtive spot of enzyme is kept away
Exempt from directly to act on extraneous microenvironment, antibiotic of preferably degrading.Additionally aggregation is formed such that whole
Individual group defines a hydrophobic group, and pH value in such microenvironment, temperature, organic solvent etc. are to albumen
The configuration impact of matter significantly reduces, thus improves the toleration of enzyme, and relatively resolvase stability of solution significantly carries
High.Compared to tradition process for fixation, CLEAs technology has enzyme purity requirement low, it is thus achieved that immobilization
Enzyme stability height good, active, without carrier, with low cost, it is easy to the advantages such as popularization.
Although CLEAs technical advantage is obvious, but its versatility is not strong, generally prepares bar to what a kind of enzyme optimized
Part may be the most inapplicable to the same enzyme in another kind of enzyme even difference enzyme sources, so studying the fixing of new enzyme
Change system also constantly to be groped.
Summary of the invention
It is an object of the invention to overcome the deficiencies in the prior art, it is provided that a kind of friendship for pharmacy wastewater treatment
The preparation method of connection beta-lactam enzyme aggregate, high to obtain activity, the crosslinking beta-lactamase of good stability
Aggregation, for degrading the lactam antibiotics in pharmaceuticals industry waste water, reduces pharmacy waste water chemistry
The purpose of oxygen demand.
For achieving the above object, the present invention is for the crosslinking beta-lactam enzyme aggregate of pharmacy wastewater treatment
Preparation method, it is characterised in that comprise the following steps:
(1), by recombinant beta-lactamase gene proceed in host cell E. coli E.coli Top10,
To recombinant bacterium, carry out protokaryon in-vitro recombination expression；
(2), recombinant bacterium is carried out abduction delivering, obtain inducing bacterium, be centrifuged processing to induction bacterium,
To thalline, it is subsequently adding buffer, and carries out ultrasonication, centrifugal treating, take supernatant, obtain crude enzyme liquid；
(3), take 25mg/mL crude enzyme liquid 1mL and add the BSA (bovine serum albumin) of 1/3 crude enzyme liquid quality,
As aggregating agent and protective agent, put in 25mL beaker and on magnetic stirring apparatus, stir 20 points under condition of ice bath
Clock；The ammonium sulfate of 5mL 75% is as precipitant, under condition of ice bath, is added dropwise over saturated ammonium sulfate (heavy
Shallow lake agent), stir 1 hour；Under the conditions of room temperature (25 DEG C), the glutaraldehyde of concentration 0.5% is as double merits of crosslinking
Energy reagent, is added dropwise over glutaraldehyde (bifunctional reagent), cross-links 2 hours；Under the conditions of gained suspension 4 DEG C,
4000rpm is centrifuged 15 minutes, must precipitate the enzyme after being solidification；
Enzyme after immobilization is resuspended in the 50mM phosphate buffer (pH7.0) of 20ml, ice bath bar
Stirring 30 minutes under part, be so iteratively repeated three times, under the conditions of gained suspension 4 DEG C, 4000rpm is centrifuged 15
Minute, the crosslinking beta-lactam enzyme aggregate being gained must be precipitated.
As the most preferably, precipitation temperature is 0 DEG C, and crosslinking temperature is room temperature (25 DEG C).
As the most preferred, when crosslinking beta-lactam enzyme aggregate is applied to the process of pharmaceuticals industry waste water,
PH value is 7.5.
As the most preferred, when crosslinking beta-lactam enzyme aggregate is applied to the process of pharmaceuticals industry waste water,
Temperature is 30 DEG C～50 DEG C.
As the most preferred, described pharmaceuticals industry waste water preferred antibiotics waste water.
The object of the present invention is achieved like this.
The present invention for pharmacy wastewater treatment crosslinking beta-lactam enzyme aggregate preparation method, by recombinant beta-
Lactamase gene sequence proceeds to, in host cell E. coli E.coli Top10, obtain recombinant bacterium, then pass through
Abduction delivering, ultrasonication, centrifugal treating, take supernatant and obtain crude enzyme liquid；Add bovine serum albumin as poly-
Collection agent and protective agent, in crude enzyme liquid, precipitate and assemble, and form enzyme aggregate, will obtain enzyme aggregate
Cross-link with bifunctional reagent, thus prepare crosslinking beta-lactam enzyme aggregate.The present invention is prepared into
The crosslinking beta-lactam enzyme aggregate arrived, enzyme is lived high after tested, good stability, and repeatable utilization is used for resisting
Raw element waste water treatment, its COD reduces 72.9%, and this crosslinking beta-lactam enzyme aggregate is in pharmacy
Industrial treatment antibiotic waste water demonstrates important using value.
Accompanying drawing explanation
Fig. 1 is beta-lactamase genetic modification schematic diagram in the present invention；
Fig. 2 is the recombinant expressed schematic diagram of recombinant beta-lactamase gene in the present invention；
Fig. 3 is the expression figure obtaining crude enzyme liquid under a kind of detailed description of the invention of the present invention；
Fig. 4 is ammonium sulfate concentration (a) and the content (b) of a kind of detailed description of the invention of present invention offer
On described crosslinking beta-lactam enzyme aggregate enzymatic activity impact figure；
Fig. 5 be a kind of detailed description of the invention of the present invention provide bovine serum albumin (BSA) to described crosslinking β-
Lactamase aggregation enzymatic activity impact figure；
Fig. 6 is that the glutaraldehyde final concentration of a kind of detailed description of the invention of present invention offer is to described crosslinking beta-lactam
Enzyme aggregate enzymatic activity impact figure；
Fig. 7 is that the different pH value of a kind of detailed description of the invention of present invention offer are to described crosslinking beta-lactamase
Aggregation enzymatic activity impact figure；
Fig. 8 is that described crosslinking beta-lactamase is gathered by the different temperatures that a kind of detailed description of the invention of the present invention provides
Collective's enzymatic activity impact figure；
Fig. 9 is that the described crosslinking beta-lactam enzyme aggregate antagonism that a kind of detailed description of the invention of the present invention provides is raw
Element chemical oxygen demand of waste water affect figure.
Detailed description of the invention
Below in conjunction with the accompanying drawings the detailed description of the invention of the present invention is described, in order to those skilled in the art
It is more fully understood that the present invention.Requiring particular attention is that, in the following description, when known function and
Perhaps, when the detailed description of design can desalinate the main contents of the present invention, these are described in and will be left in the basket here.
One, embodiment
1, the acquisition of recombinant beta-lactamase gene
The present invention has the beta-lactamase on the GenBank:AF143804.1 of drug resistance with Cefotaxime
Based on gene, 5 ' ends and 3 ' ends at its coding region (nucleotide sequence) separately design restricted enzyme NcoI
I.e. sequence 5 '-ccatgg-3 ' and EcoRI that is 5 '-gaattc-3 ', inserts 6 His before 3 ' end termination codoies taa
Label i.e. histidine catcaccatcaccatcac, obtains the beta-lactamase gene order of transformation, transformation restructuring
Process is as shown in Figure 1.
Nucleotide sequence GenBank:AF143804.1 comes from American National Biotechnology Information center
One in (National Center for Biotechnology Information, NCBI) geneseq database
Beta-lactamase gene order.
In the present embodiment, Nanjing Jin Sirui biology company limited is according to the beta-lactamase gene order of transformation
Synthesizing, its gene order is shown in the sequence 1 in sequence table.The beta-lactamase gene of synthesis is cloned into
In pBAD-TLX expression vector, obtain recombinant beta-lactamase gene, the most as shown in Figure 2.
In the present invention, why the upper restricted enzyme NcoI of replacement are held in the 5 ' of beta-lactamase gene order
Inserting restricted enzyme EcoRI with 3 ' ends, the cohesive end being because NcoI and EcoRI generation can be with expression
The cohesive end that restricted enzyme NcoI and EcoRI on (clone) carrier pBAD-TLX produces carries out spy
Anisogamy, thus beta-lactamase genetic fragment is connected with expressing (clone) carrier.
2, the abduction delivering of restructuring beta-lactamase
In the present embodiment, recombinant beta-lactamase gene is proceeded to host cell E. coli E.coli Top10
In, obtain recombinant bacterium, carry out the outer abduction delivering of prokaryote subsequently.Abduction delivering condition is: by recombinant bacterium list
Clone is inoculated in 20mL LB (Luria-Bertani) culture medium, and Amp (ampicillin) concentration is
100 μ g/mL, be put in 37 DEG C, rotating speed be 180rpm shaking table in incubated overnight.By cultured bacterium solution by 2%
Inoculum concentration be inoculated in the LB culture medium that 200mL contains Amp, be put in 37 DEG C, rotating speed be shaking of 180rpm
Cultivate in Chuan to OD600It is about 0.7, after then standing 1.5 hours in 16 DEG C of incubators, adds
0.05mg/mL arabinose is as derivant, and inducing temperature is adjusted to 16 DEG C, and shaking speed is changed to 100rpm, lures
Lead antibacterial to OD600It is about 2.5 end, obtains inducing bacterium.
3, crude enzyme liquid is obtained
In the present embodiment, induction bacterium is centrifuged to obtain thalline, adds 200mM Tris-SO4Buffer ultrasonication,
Liquid to be broken becomes transparence, 4 DEG C of centrifuges, and 12000g is centrifuged 1 hour, obtains cleer and peaceful precipitation, take supernatant,
Obtain crude enzyme liquid.In the present embodiment, by upper cleer and peaceful precipitation addition 2 × SDS sample-loading buffer, mixing, add
Enter in SDS-PAGE polyacrylamide gel electrophoresis testing goal albumen.Destination protein as can be seen from Figure 3
At about 38kDa, have successfully been obtained solubility destination protein i.e. crude enzyme liquid
4, the preparation of beta-lactam enzyme aggregate is cross-linked
In the present embodiment, survey supernatant protein concentration by Coomassie light blue method, according to concentration conversion, take 1mL
Crude enzyme liquid crude enzyme liquid adds the BSA (bovine serum albumin) of 1/3 crude enzyme liquid quality, as aggregating agent and protective agent,
Put in 25mL beaker and stir 20 minutes on magnetic stirring apparatus under condition of ice bath.Under condition of ice bath, dropwise add
Enter saturated ammonium sulfate (precipitant), stir 1 hour；Under the conditions of room temperature (25 DEG C), it is added dropwise over glutaraldehyde
(bifunctional reagent), cross-links 2 hours；Under the conditions of gained suspension 4 DEG C, 4000rpm is centrifuged 15 minutes, and it is heavy to obtain
Form sediment the enzyme after being solidification.Enzyme after immobilization is resuspended in the 50mM phosphate buffer (pH7.0) of 20ml
In, stir 30 minutes under condition of ice bath, be so iteratively repeated three times, under the conditions of gained suspension 4 DEG C, 4000rpm
Centrifugal 15 minutes, the crosslinking beta-lactam enzyme aggregate being gained must be precipitated.
Two, test
1, saturated ammonium sulfate concentration is on crosslinking beta-lactam enzyme aggregate enzymatic activity impact
Prepare the saturated ammonium sulfate solution of 30%, 45%, 60%, 75% and 85% respectively, crude enzyme liquid is carried out
Immobilization, measures crosslinking beta-lactam enzyme aggregate and lives relative to enzyme, shown in result such as Fig. 4 (a).From Fig. 4
(a) it can be seen that when saturated ammonium sulfate solution concentration is 75%, crosslinking beta-lactam prepared by the present invention
Enzyme aggregate relative activity is up to 57.1%.
2, the saturated ammonium sulfate consumption impact on crosslinking beta-lactam enzyme aggregate enzymatic activity
Add the most commensurability saturated ammonium sulfate to being fixed of crude enzyme liquid, measurement crosslinking beta-lactam enzyme aggregate
Relative activity, shown in result such as Fig. 4 (b).From Fig. 4 (b) it can be seen that work as the saturated of addition 75%
When ammonium sulfate is 5mL, crosslinking beta-lactam enzyme aggregate relative activity prepared by the present invention up to
64%.
3, the bovine serum albumin (BSA) impact on crosslinking beta-lactam enzyme aggregate enzymatic activity
When the bovine serum albumin of addition different quality serves as aggregating agent and being fixed of protective agent, measure crosslinking
Beta-lactam enzyme aggregate relative activity, result is as shown in Figure 5.From fig. 5, it can be seen that when the cattle added
When serum albumin and crude enzyme liquid mass ratio are 1:3, crosslinking beta-lactam enzyme aggregate prepared by the present invention is relative to enzyme
Activity 93.1%, illustrates that bovine serum albumin is conducive to retaining connection beta-lactam enzyme aggregate enzymatic activity.
4, the glutaraldehyde final concentration impact on crosslinking beta-lactam enzyme aggregate enzymatic activity
When the glutaraldehyde solution of the different final concentration of addition is as being fixed of bifunctional reagent, measurement crosslinking β-
Lactamase aggregation relative activity, result is as shown in Figure 6.From fig. 6, it can be seen that when glutaraldehyde is whole
When concentration is 0.5%, crosslinking beta-lactam enzyme aggregate relative activity prepared by the present invention is 94.5%.
5, the impact of pH stability alive on crosslinking beta-lactam enzyme aggregate enzyme
Crosslinking beta-lactam enzyme aggregate is put in the phosphate buffer of different pH, detects its enzyme and live stable
Property, result is as shown in Figure 7.It can be seen from figure 7 that crosslinking beta-lactamase prepared by the present invention is assembled
Body relative activity in the environment of peracid or mistake alkali is relatively low, but higher than crude enzyme liquid stability.?
Between pH5.5-pH8.5, crosslinking beta-lactam enzyme aggregate relative activity is higher.Illustrate that the present invention prepares
Crosslinking beta-lactam enzyme aggregate there is higher pH stability.
6, the impact of temperature stability alive on crosslinking beta-lactam enzyme aggregate enzyme
Crosslinking beta-lactam enzyme aggregate is put in the beaker of different temperatures, detects its enzyme stability alive, knot
Fruit is as shown in Figure 8.From figure 8, it is seen that crosslinking beta-lactam enzyme aggregate prepared by the present invention has the highest
Thermostability, when putting 6 in 70 DEG C of temperature and being little, it remains to keep the enzyme of 70% to live, and at 30 DEG C～50 DEG C
For most preferably；And crude enzyme liquid half an hour in 60 DEG C of environment, just loses 60% enzyme and lives.
7, the crosslinking beta-lactam enzyme aggregate impact on COD value of waste water
Filter paper is put in conical hopper, described antibiotic waste water is filtered, remove oil removal and particulate matter,
Obtain filtrate；By filtrate high speed centrifugation, the crosslinking β prepared by the chemical oxygen demand rapid measurement instrument detection present invention-
Lactamase aggregation processes the COD of front filtrate to waste water；Described crosslinking beta-lactamase is gathered
Collective puts in filtrate, is put in 30 DEG C of shaking tables, and rotating speed 180rpm cultivates 35 hours, takes processed waste water
Centrifugal, measure the COD of processed waste water.Result is as shown in Figure 9.From fig. 9, it can be seen that this
The crosslinking beta-lactam enzyme aggregate of invention preparation is to waste water COD clearance up to 72.9%, and this illustrates this
The crosslinking beta-lactam enzyme aggregate antagonism raw element waste water of bright preparation has the effect of good fall COD
Really.From Fig. 7 it can also be seen that pBAD antibacterial to waste water COD clearance also up to 12%, described pBAD
Antibacterial represents the internal E.coli Top10 Host Strains having imported pBAD-TLX carrier, and matched group is not the most appointed
The waste water of what bacterial treatment.Crude enzyme liquid joins to live less than enzyme half an hour in waste water just to be lost, therefore the thickest
Enzyme liquid result.
Although detailed description of the invention illustrative to the present invention is described above, in order to the art
Artisans understand that the present invention, it should be apparent that the invention is not restricted to the scope of detailed description of the invention, right
From the point of view of those skilled in the art, as long as various change limits in appended claim and determines
The spirit and scope of the present invention in, these changes are apparent from, all utilize present inventive concept send out
Bright creation is all at the row of protection.
Claims (6)
1., for the preparation method cross-linking beta-lactam enzyme aggregate of pharmacy wastewater treatment, its feature exists
In, comprise the following steps:
(1), by recombinant beta-lactamase gene proceed in host cell E. coli E.coli Top10,
To recombinant bacterium, carry out protokaryon in-vitro recombination expression；
(2), recombinant bacterium is carried out abduction delivering, obtain inducing bacterium, be centrifuged processing to induction bacterium,
To thalline, it is subsequently adding buffer, and carries out ultrasonication, centrifugal treating, take supernatant, obtain crude enzyme liquid；
(3), take 25mg/mL crude enzyme liquid 1mL and add the BSA (bovine serum albumin) of 1/3 crude enzyme liquid quality,
As aggregating agent and protective agent, put in 25mL beaker and on magnetic stirring apparatus, stir 20 points under condition of ice bath
Clock；The ammonium sulfate of 5mL 75% is as precipitant, under condition of ice bath, is added dropwise over saturated ammonium sulfate (heavy
Shallow lake agent), stir 1 hour；Under the conditions of room temperature (25 DEG C), the glutaraldehyde of concentration 0.5% is as double merits of crosslinking
Energy reagent, is added dropwise over glutaraldehyde (bifunctional reagent), cross-links 2 hours；Under the conditions of gained suspension 4 DEG C,
4000rpm is centrifuged 15 minutes, must precipitate the enzyme after being solidification；
Enzyme after immobilization is resuspended in the 50mM phosphate buffer (pH7.0) of 20ml, ice bath bar
Stirring 30 minutes under part, be so iteratively repeated three times, under the conditions of gained suspension 4 DEG C, 4000rpm is centrifuged 15
Minute, the crosslinking beta-lactam enzyme aggregate being gained must be precipitated.
Preparation method the most according to claim 1, it is characterised in that described restructuring beta-lactamase
Gene uses following steps to obtain:
With the beta-lactamase gene that Cefotaxime has on the GenBank:AF143804.1 of drug resistance it is
Basis, 5 ' ends and 3 ' ends at its coding region (nucleotide sequence) separately design restricted enzyme NcoI i.e. sequence
Row 5 '-ccatgg-3 ' and EcoRI that is 5 '-gaattc-3 ', inserts 6 His labels i.e. before 3 ' end termination codoies taa
Histidine catcaccatcaccatcac, obtains the beta-lactamase gene order of transformation；
Synthesize according to the beta-lactamase gene order of transformation, the beta-lactamase gene gram that synthesis obtains
Grand in pBAD-TLX expression vector, obtain recombinant beta-lactamase gene.
Preparation method the most according to claim 1, it is characterised in that described abduction delivering is:
Being inoculated into by recombinant bacterium monoclonal in 20mL LB (Luria-Bertani) culture medium, (ammonia benzyl is blue or green for Amp
Mycin) concentration is 100 μ g/mL, be put in 37 DEG C, rotating speed be 180rpm shaking table in incubated overnight.Will training
The bacterium solution supported is inoculated in the LB culture medium that 200mL contains Amp by the inoculum concentration of 2%, be put in 37 DEG C,
Rotating speed be 180rpm shaking table in cultivate to OD600It is about 0.7, in 16 DEG C of incubators, then stands 1.5
After hour, addition 0.05mg/mL arabinose is as derivant, and inducing temperature is adjusted to 16 DEG C, shaking speed
It is changed to 100rpm, Induction of bacterial to OD600It is about 2.5 end, obtains inducing bacterium.
Preparation method the most according to claim 1, it is characterised in that described precipitation temperature is 0 DEG C,
Crosslinking temperature is room temperature (25 DEG C).
Preparation method the most according to claim 1, it is characterised in that described crosslinking beta-lactamase
When aggregation is applied to the process of pharmaceuticals industry waste water, pH value is 7.5.
Preparation method the most according to claim 1, it is characterised in that described crosslinking beta-lactamase
When aggregation is applied to the process of pharmaceuticals industry waste water, temperature is 30 DEG C～50 DEG C.
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