CN101089178A - Beta-lactamase and its prepn process and application - Google Patents
Beta-lactamase and its prepn process and application Download PDFInfo
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- CN101089178A CN101089178A CN 200610051915 CN200610051915A CN101089178A CN 101089178 A CN101089178 A CN 101089178A CN 200610051915 CN200610051915 CN 200610051915 CN 200610051915 A CN200610051915 A CN 200610051915A CN 101089178 A CN101089178 A CN 101089178A
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Abstract
The present invention relates to antibiotic hydrolase, and is especially one kind of beta-lactamase and its preparation process and application in eliminating residual antibiotic. The beta-lactamase is obtained through one genetic engineering process and has high purity and high activity. Its preparation process includes the following steps: constituting engineering strain, fermentation, and separating and purifying. It is applied in eliminating residual antibiotic from animal products, medicinal product, food, beverage, etc. and possesses active effects of eliminating poisoning of residual beta-lactam and preventing drug resistance of bacteria.
Description
Technical field
The present invention relates to a kind of microbiotic lytic enzyme, its preparation method and application thereof, relate in particular to β-Nei Xiananmei, its preparation method and the application aspect the removing residual antibiotic thereof.
Background technology
Microbiotic (Antibiotics) is the chemical substance that other microbial process is suppressed or killed to having under lower concentration of microorganisms.It is one of greatest medical discovery of twentieth century, and is of a great variety, can be divided into ten surplus thousands of kinds of big classes, the clinical hundreds of kind that has commonly used.Since the penicillin forties in last century (beta-lactam antibiotics) comes out, microbiotic has been brought into play vital role in the treatment bacteriosis, therefore make the disease that is caused by infectation of bacteria more than 95% controlled, the life of its redemption is countless, says that microbiotic is that the good medicine of benefiting mankind is not excessive.
Saving life, ensureing the contribution of making in the health that because of microbiotic many people think that microbiotic can be guaranteed to cure all diseases by mistake.In fact, incorrect microbiotic use is pernicious.Li Fu points out in " antibiotic awkward situation " book: " many people believe that microbiotic can be guaranteed to cure all diseases, and the fact is that unnecessary taking does harm rather than good." so when seeing the microbiotic contribution, the toxic side effect of very important its existence.
Antibiotic toxic side effect has: 1. toxic reaction: this is a kind of untoward reaction of seeing at most, often because of dosage excessive or overlong time to people's neural system, internal organ, hemopoietic system and the toxigenicity effect of local injection place.Can cause the infringement of cochlea vestibular organ as Streptomycin sulphate, kantlex, gentamicin, cause dysequilibrium, hypoacusis or forfeiture; Paraxin can cause aplastic anemia; Phosphamidon class microbiotic can cause dermatitis, fash, vasodilation; A large amount of infringements of using tsiklomitsins can cause liver, child uses the growth that can influence tooth and bone etc.2. anaphylaxis: this is a kind of abnormal immune response, and almost every kind of microbiotic all has this reaction.Will cause anaphylaxis such as anaphylactic shock, drug fever, fash, vasodilation, cacemia, collagen disease as penicillin, Streptomycin sulphate, cephalosporin.Wherein dangerous maximum with delayed type allergy, often neglected and caused the harm that can't remedy.3. superinfection: claim that also flora replaces disease, refer to betide the new infection in the microbiotic application process.The pathogenic bacteria that superinfection takes place is mainly golden grape, fungi and enteron aisle Gram-positive bacillus.These pathogenic bacterias are owing to contact with common antibiotics such as penicillin, Streptomycin sulphate, paraxin repeatedly and produce resistance gradually, and protopathy greatly descends patient's resistibility, thus they spread and wreak havoc regular meeting's threat to life.4. bacterial resistance sexually revises: often use microbiotic can make interior, the external bacterium of human body produce resistance.Why wreaking havoc for a long time as staphylococcus, enteron aisle Gram-positive bacillus, tubercule bacillus, dysentery bacterium, is exactly the result that resistance changes.
It is quite serious to produce chemical sproof consequence, and it makes the microbiotic of a large amount of human and material resources exploitations of cost lose effect, and resistant organism is wreaked havoc but and pasted medical help.Penicillin is very effective medicine at first, and consumption is very little, even now use millions of units effect also not good.Resistance is owing to irrational drug use causes, and or else expert call as controlling antibiotic use, and the mankind probably get back to does not have the antibiotic epoch, and people will face the threat of many infectious diseases once more.Being that germ is walked crosswise on the one hand, but is that no medicine can be used on the one hand, and a large amount of communicable diseases is with serious harm human beings'health and life, and this paints an alarming picture of the situation just to scare the audience absolutely not.Because therefore the speed that resistance produces can not rely on the exploitation new antibiotic to solve the resistance problem merely far faster than the speed of new drug development,, do not use how long bacterium has just produced resistance as the xacin-series medicine.
Another major reason that causes resistant organism to spread unchecked is a microbiotic for animals.As prophylactic, feed factory or raiser generally add 1%～2% microbiotic in feed.Add microbiotic and help weakening harmful microorganism in the stomach and intestine; Inhibition, kill pathogenic organisms strengthen resistance against diseases, in case fowl is raiseeed the disease pest.Therefore microbiotic is the indispensable most valuable treasure of modern livestock industry, but it residually constitutes a serious threat to HUMAN HEALTH again, thus another directly be international community's bone of contention problem in recent years.
As previously mentioned, the equal toxic side effect of microbiotic, the frequent edible antibiotic food that contains, even trace, also can make the people urticaria occur or cause anaphylactic shock.The food of long-term edible antibiotic remains also can cause ariboflavinosis and the damage of purpura property, particularly paraxin, very easily damages the hemopoietic function of people's marrow, causes aplastic anemia.
Except the coup injury to human body, residual antibiotic also will cause the interior resistant organism of body to produce in a large number.In this process, microbiotic is pressed as selecting, and makes resistant organism than sensitive organism growth vigor be arranged, and causes Resistant strain popular.Generally add terramycin, tetracycline medication at present in animal-feed, as many as lasting low dosage medication helps pathogenic microorganism and obtains resistance.
Along with rapid economic development, the shared ratio of animal derived food is increasing, and the antibiotic remains in the livestock product is also more and more serious to people's harm.Because at present China lacks perfect management system to culturing industry, it is lower to culture practitioner's quality in addition, lacks hygienic knowledge, causes animal derived food hygiene quality to go from bad to worse.Residues of antibiotics has arrived instant stage in the control food.
Beta-lactam antibiotics is owing to have wide spectrum, cheap advantage, uses all morely on one's body at humans and animals, thereby its resistance residual and that cause is all very serious.If can make residual microbiotic inactivation, then can make it lose toxic action to human body, also can make it lose selective action, make resistant organism no longer have selective advantage, no longer advantage breeding and popular.
Summary of the invention
The objective of the invention is to seek the comparatively gentle method of the residual beta-lactam antibiotics of a kind of energy deactivation, but do not introduce new adverse factor.
First purpose of the present invention is to provide a kind of β-Nei Xiananmei.
β-Nei Xiananmei encoding sequence of the present invention is that its sequence is SEQ ID:NO.1 through dna segment codon optimized, chemosynthesis, and the aminoacid sequence of β-Nei Xiananmei is SEQ ID:NO.2.
Second purpose of the present invention is to provide a kind of preparation method of β-Nei Xiananmei.
The preparation method of β-Nei Xiananmei of the present invention is characterized in that: this method comprises the step of following order: make up engineering strain, set up zymotechnique, separation and purification.
The preparation method of beta-lactam antibiotics enzyme of the present invention is characterized in that: engineering bacteria is to contain gene order SEQ ID:NO.1, and expression vector is pET-30a, and inserting the site is BL21 (DE3) bacterial strain of Kpn I and Hind III.
The preparation method of β-Nei Xiananmei of the present invention is characterized in that: the temperature that engineering bacterium fermentation is cultivated is 28 ℃-40 ℃, and the concentration of inductor IPTG is 0.1-1.5mmol/L.
The preparation method of beta-lactam antibiotics enzyme of the present invention is characterized in that: the temperature that engineering bacterium fermentation is cultivated is 30 ℃-35 ℃, and the concentration of inductor IPTG is 0.4-1.0mmol/L.
The preparation method of beta-lactam antibiotics enzyme of the present invention is characterized in that: the temperature that engineering bacterium fermentation is cultivated is 30 ℃, and the concentration of inductor IPTG is 0.5mmol/L.
Preparation method of the present invention is characterized in that: the combination of a kind of in ion exchange chromatography, sieve chromatography, hydrophobic chromatography or the affinity chromatography or any two kinds, any three kinds, four kinds is adopted in separation and purification.
The 3rd purpose of the present invention is to provide β-Nei Xiananmei of the present invention in the application that is used for removing in the livestock product residual antibiotic.
β-Nei Xiananmei of the present invention is in the application that is used for removing in milk, food-drink processing residual antibiotic and the environment protection.
β-Nei Xiananmei of the present invention is in the application that is used for removing in livestock, poultry, meat, the medical treatment product residual antibiotic.
β-Nei Xiananleikangshengsu is the common drug of treatment infectation of bacteria.(β-lactamase) claim penicillinase again is the enzyme of bacterium excretory energy hydrolysis β-Nei Xiananleikangshengsu (comprising penicillin) to β-Nei Xiananmei.Therefore, β-Nei Xiananmei can be used for eliminating residual beta-lactam antibiotics.The advantage that this law has: at first, enzyme is an effective catalyst, only needs a spot of enzyme just can the considerable microbiotic of deactivation; Secondly, enzyme is a protein, can not cause secondary pollution, after digesting or nutritive substance.
The present invention obtains reorganization TEM1 fusion rotein with the fusion protein expression plasmid of recombinant DNA technology structure β-Nei Xiananmei TEM1 by efficiently expressing in intestinal bacteria, through the chromatographic separation purifying, obtain high purity, highly active enzyme.Concrete grammar of the present invention may further comprise the steps:
1. the selection of enzyme molecule
β-Nei Xiananmei has the hundreds of kind, as the TEM enzyme hundreds of is just arranged, and the SHV enzyme also has hundreds of.As the degrade residual β-Nei Xiananleikangshengsu, these enzymes all can be selected.In the present invention, preferred TEM1.
2. the acquisition of gene
Available PCR obtains the TEM1 gene from bacterial plasmid, also can be by the synthetic full-length gene of chemical method.Because in the TEM1 gene some rare codons being arranged, so the preferred chemosynthesis of the present invention, can be optimized the TEM1 gene like this.TEM1 gene order after optimizing is seen SEQ ID No.1.
3. the protein expression form is selected
Can lead peptide sequence by adding before gene, the mode that is secreted into pericentral siphon obtains product.Also can in kytoplasm, express natural albumen by before gene, adding the ATG codon.Can also express by the mode of fusion rotein, promptly merge segment mark label sequence at the N of natural enzyme end or C end, sequence label wherein can be: glutathione-S-transferase, maltose binding protein, Trx, His6, S-tag, Dsb A, Dsb C, or other fusion head and combination thereof.Consider expression amount and follow-up purifying technique, preference of the present invention is an expressed fusion protein, wherein preferably merges the His6/S-tag combination tag at the N end.
4. the structure of expression plasmid
The expression of T7 promoters driven can greatly improve the protein expression amount, thereby the expression vector of the preferential select tape T7 of the present invention promotor, as pET11-pET40.More preferably, the present invention adopts pET30a (+) expression vector.Multiple clone site on the available pET30a (+) is inserted gene of the present invention, more preferably, utilizes the Kpn I+HindIII site on the pET30a (+), has also introduced the His6/S-tag combination tag simultaneously naturally.
5. the abduction delivering of recombinant protein
In the preference of the present invention, it is characterized in that the temperature that engineering bacterium fermentation is cultivated is 28 ℃-40 ℃, the concentration of inductor IPTG is 0.1-1.5mmol/L; The temperature that the suboptimum engineering bacterium fermentation is cultivated is 30 ℃-35 ℃, and the concentration of inductor IPTG is 0.1-1.5mmol/L; The temperature that optimum engineering bacterium fermentation is cultivated is 30 ℃, and the concentration of inductor IPTG is 0.5mmol/L.
6. the separation of recombinant protein is purified
In a preference of the present invention, the separation and purification of recombinant protein comprises the steps:
I. to fermented sample by centrifugal or remove by filter substratum, obtain thalline;
Ii. lysing cell obtains broken bacterium liquid;
Iii is centrifugal, and broken bacterium liquid is removed bacterial chip, obtains broken bacterium liquid supernatant;
The iv chromatography purification, used chromatography is selected from ion exchange chromatography, sieve chromatography, hydrophobic interaction chromatography, affinity chromatography and two or more combination arbitrarily thereof;
7. the elimination of residual antibiotic
This enzyme can be used for eliminating microbiotic residual in the different field, as aspects such as livestock, poultry, livestock product, milk, meat, medical treatment product, environment and food-drink processing.But the application mode in different field is different.As being the domestic animals of final product with the meat product, live body injection enzyme solution is the mode that relatively is fit to.Enzyme is dissolved into the solution of proper concn, is injected in the body of animal, utilize the recycle system of animal that enzyme is carried to different tissues, organ, make enzyme more effectively degrade residual in the intravital microbiotic of animal.Certainly, also can soak meat and eliminate wherein residual antibiotic, but obviously this method all is not so good as the live body injection from the aspect of economy and efficient with enzyme solution.Concerning as liquid products such as milk, can be by enzyme being dissolved into the solution of proper concn, effect for some time is to eliminate wherein residual microbiotic in the adding product.Also can make enzyme reactor, product is eliminated wherein residual microbiotic by enzyme reactor with enzyme immobilization technology.For microbiotic residual in vessel or the environment, can eliminate by the form of immersion or spray.
Meaning of the present invention and advantage:
1. from narrowly, domestic some animal derived food penicillin exceeds standard seriously now, and the present invention can be people safe food is provided.
2. in a broad sense, the present invention can avoid the popular of resistant organism to a certain extent, for safeguarding health of people, avoids occurring disease and walks crosswise and but do not have medicine available situation.
3. technologically, the expression process of this invention is simple, and purifying process is simple, rate of recovery height.SEQ ID：NO.1
Length: 789bp
Type: thymus nucleic acid
Chain number: two strands
Geometry: linearity
Source: chemosynthesis
Feature: coding TEM1 albumen (sequence is through optimizing).
5’CAC CCA GAA ACG CTG GTG AAA GTA AAA GAT GCT GAA GAT CAG
TTG GGT GCA CGA GTG GGT TAC ATC GAA CTG GAT CTC AAC AGC GGT
AAG ATC CTT GAG AGT TTT CGC CCC GAA GAA CGT TTT CCA ATG ATG
AGC ACT TTT AAA GTT CTG CTA TGT GGC GCG GTA TTA TCC CGT GTT
GAC GCC GGG CAA GAG CAA CTC GGT CGC CGC CTG CAC TAT TCT CAG
AAT GAC TTG GTT GAG TAC TCA CCA GTC ACA GAA AAG CAT CTT ACG
GAT GGC ATG ACA GTA CGC GAA TTA TGC AGT GCT GCC CTG ACC ATG
AGT GAT AAC ACT GCG GCC AAC TTA CTT CTG ACA ACG ATC GGA GGA
CCG AAG GAG CTA ACC GCT TTT TTG CAC AAC ATG GGG GAT CAT GTA
ACT CGC CTT GAT CGT TGG GAA CCG GAG CTG AAT GAA GCC CTG CCA
AAC GAC GAG CGT GAC ACC ACG ATG CCT GCA GCA ATG GCA ACA ACG
TTG CGC AAA CTA TTA ACT GGC GAA CTA CTT ACT CTA GCT TCC CGG
CAA CAA TTA CTG GAC TGG ATG GAG GCG GAT AAA GTT GCA GGA CCA
CTT CTG CGC TCG GCC CTT CCG GCT GGC TGG TTT ATT GCT GAT AAA
TCT GGA GCC GGT GAG CGT GGG TCT CGC GGT ATC ATT GCA GCA CTG
GGG CCA GAT GGT AAG CCC TCC CGT ATC GTA GTT ATC TAC ACG ACG
GGG AGT CAG GCA ACT ATG GAT GAA CGA AAT CGC CAG ATC GCT GAG
CTG GGT GCC TCA CTGATTAAG CAT TGG TAA 3’
SEQ ID：NO.2
Length: 263 aa
Type: protein
Geometry: linearity
Feature: the aminoacid sequence of natural TEM1
HPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCG
AVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMS
DNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTM
PAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADK
SGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW
Description of drawings
Fig. 1 is that (1 is marker to the analysis of reorganization TEM1 Expression of Fusion Protein; 2 for before inducing; 3-8 is for after inducing; Can find out induce the back proteic expression amount account for total protein 30%).
Fig. 2 is the affinity purification chromatography collection of illustrative plates of reorganization TEM1 fusion rotein.
Fig. 3 is the anionresin purifying chromatography collection of illustrative plates of reorganization TEM1 fusion rotein.
Fig. 4 be TEM1 fusion rotein behind the purifying through electrophoresis evaluation figure, show that purity is greater than 95%.
Fig. 5 is with the microbiotic collection of illustrative plates in the TEM1 enzyme elimination skimmed milk substratum: from left to right, the 1st pipe is not for containing microbiotic, and the 2nd pipe contains microbiotic but be not enzyme-added, and all the other contain microbiotic and enzyme-added processing.The person of reddening is that antibiotic remains is qualified.
Fig. 6 is that recombinase is handled the required time of same amount penicillin under differing temps, is 100 calculating with 37 ℃ of required times.
Fig. 7 is that recombinase is handled the required time of same amount penicillin in the different pH values skimmed milk substratum, and the required time is 100 calculating during with pH7.0.
Embodiment
The invention will be further described below in conjunction with embodiment.But they are not limitation of the invention.The experimental technique of unreceipted actual conditions, the condition that typically refers to experiment condition routinely or provide by manufacturer.
The structure of embodiment one TEM1 amalgamation and expression system
Expression plasmid is pET30a (+), and the host bacterium is BL21 (DE3).
TEM1 gene after chemosynthesis is optimized adds the base sequence and the Kpn I restriction enzyme site of coding enteropeptidase recognition site at its 5 ' end, so that discharge natural TEM1 albumen in case of necessity.Add terminator codon TAA and introduce Hind III site at its 3 ' end, be cloned between the Kpn I and Hind III site of plasmid pET30a, introduce the His6/S-tag sequence label at the N of TEM1 end nature like this.
The expression plasmid that builds is directly transformed BL21 (DE3) competent cell, the strain of screening kalamycin resistance; Alkaline lysis prepares plasmid in a small amount, the screening of Kpn I+Hind III double digestion is inserted with the clone of synthetic TEM1 gene, with T7 promotor (promoter) and the two-way order-checking of terminator (terminator), confirm cloned sequence and implementation sequence (SEQ ID.NO.1) in full accord.
After this project bacterium was cultivated, after IPTG induced 4h, target protein accounted for 30% of total protein, sees Fig. 1 for details.Similarly method makes up the native protein expression plasmid, and carries out expression analysis, and expression amount is much lower than the fusion rotein system, less than 10%.
The selection of embodiment two inductive conditions
Temperature and inductor concentration are two two important factors that influence protein expression amount and expression-form (solvable or inclusion body).Therefore, be substratum with LB, the combination of temperature and inductor is analyzed the influence of TEM1 expressing fusion protein, see the following form.
| System | Temperature (℃) | Inductor concentration (mmol/L) |
| 1 | 25 | 0.1 |
| 2 | 25 | 0.5 |
| 3 | 25 | 1.0 |
| 4 | 25 | 1.5 |
| 5 | 30 | 0.1 |
| 6 | 30 | 0.5 |
| 7 | 30 | 1.0 |
| 8 | 30 | 1.5 |
| 9 | 37 | 0.1 |
| 10 | 37 | 0.5 |
| 11 | 37 | 1.0 |
| 12 | 37 | 1.5 |
| 13 | 40 | 0.1 |
| 14 | 40 | 0.5 |
| 15 | 40 | 1.0 |
| 16 | 40 | 1.5 |
The result shows, when 37 ℃ are induced with 1mmol/L IPTG (system 11), proteic expression amount is the highest, but most (＞90%) is present in the inclusion body.And when 30 ℃ are induced with 0.5mmol/L IPTG (system 6), though expression amount is low, most of albumen be that (＞80%) is soluble-expression.Thereby the condition of preferred system 6 is as inductive condition.
The purifying of embodiment three reorganization TEM1 fusion roteins
1, induces expression of recombinant proteins with reference to the system 6 of embodiment 2.4 ℃ of tunnings, centrifugal 15 minutes of 6000rpm gets thalline.
2, ultrasonic treatment cell, centrifugal 30 minutes of 13000rpm collects supernatant.
3, chromatography purification target protein
A. affinity chromatography
Medium: Ni-Chelating Sepharose FF
Solution A: 20mM Tris-Cl, 500mM NaCl, 5mM imidazoles (pH8.0)
Solution B: 20mM Tris-Cl, 250mM imidazoles (pH8.0)
The supernatant liquor of will the centrifugal back of broken bacterium collecting is with the flow velocity upper prop of 150cm/h, is washed till with solution A that electricity is led and uv-absorbing all no longer changes, and use the 100%B wash-out, the collection elution peak.The collection of illustrative plates of affinity chromatography is seen Fig. 2.
B. anion-exchange chromatography
Medium: DEAE Sepharose FF
Solution A: 20mM Tris-Cl (pH8.0)
Solution B: 20mM Tris-Cl+1M NaCl (pH8.0)
With 4 times of the affinity chromatography peak dilutions of collecting,, be washed till with solution A that electricity is led and uv-absorbing all no longer changes with A liquid, in 10 times of column volumes, solution risen to 50%B from 0%B, collect the TEM1 peak with linear gradient with the flow velocity upper prop of 150cm/h.The collection of illustrative plates of ion exchange chromatography is seen Fig. 3.
C. molecular sieve
Medium: Sephadex G-25
Solution A: 50mM PB (pH7.0)
With the TEM1 peak upper prop that ion exchange chromatography is collected, use the solution A wash-out then, collect TEM1 fusion rotein peak.
Behind inferior three purification steps, obtained purity and reached 95%, and the lower TEM1 fusion rotein of salt concn is seen Fig. 4.
Embodiment four-function reorganization TEM1 enzyme is eliminated residual microbiotic
1, the residual penicillin in the elimination skimmed milk substratum
In 10ml 12% skimmed milk substratum, add penicillin to 40IU/mL (corresponding to surpassing 10000 times of national penicillin residue criterions).To the reorganization TEM1 fusion rotein that wherein adds different amounts, the room temperature effect measured with the TTC method whether microbiotic exceeds standard in the milk by GB/T 4789.27-2003 after 15 minutes.Wherein do not add penicillin and redden, show that antibiotic remains has met national standard with the substratum of adding enzyme amount greater than 0.07 microgram.Do not redden and add pipe (comprising the not enzyme-added pipe) substratum that the enzyme amount is lower than 0.07 microgram, show that antibiotic remains exceeds standard.Therefore, according to the present invention, eliminated in the milk residual penicillin effectively with the TEM1 enzyme and seen Fig. 5.
2, during differing temps, reorganization TEM1 enzyme is eliminated antibiotic situation in the milk
Be simulation Various Seasonal and different places, under different temperature condition with the residual antibiotic in this enzyme elimination skimmed milk substratum.Method is with 1, and the enzyme amount of adding is 0.07 microgram, and used differing temps comprises 4 ℃, and 10 ℃, 20 ℃, 25 ℃, 30 ℃, 37 ℃ and 45 ℃.Making the qualified required time of antibiotic remains during with 37 ℃ is 100, and the histogram of required action time is seen Fig. 6 when making differing temps.The result shows that under different envrionment conditionss, this enzyme all can effectively be eliminated residual microbiotic, and required time was wanted corresponding prolongation when just temperature was low, or required enzyme amount is wanted corresponding increase.
3, during different pH, reorganization TEM1 eliminates antibiotic situation in the milk
For simulating the possible potential of hydrogen fluctuation of different milk, under different pH, eliminate residual microbiotic in the skimmed milk substratum with this enzyme.The pH of skimmed milk substratum is adjusted to 6.5,7.0 or 7.5, and operative temperature is room temperature (25 ℃).Making the qualified required time of antibiotic remains with 7.0 o'clock is 100, and the histogram of required action time is seen Fig. 7 when making different pH.The result shows that under different envrionment conditionss, this enzyme can be eliminated residual microbiotic in the above-mentioned potential of hydrogen skimmed milk substratum effectively.
4, eliminate residual microbiotic in the environment
We are by showing during coating penicillin is with simulated environment or solid surface residual antibiotic such as vessel at the LB flat board, the amount of coating penicillin is 40IU/cm
2Equally, get among the PB (pH7.0) that 0.07 microgram recombinase is diluted in proper volume, be coated on planar surface.Act on after 15 minutes, the coating e.colidh5 is inverted for 37 ℃ and was cultivated 14 hours.The result shows, the dull and stereotyped aseptic length of being born of uncoated enzyme, and the flat board of coating enzyme grows a lot of bacterium colonies, to such an extent as to be linked to be a slice, has grown up to lawn.Because no available inactivator and do not destroy flat board or do not influence the method for bacterial growth is not so handle enzyme-deactivating after acting on.
5, eliminate some other beta-lactam antibiotics commonly used with TEM1
We use the penbritin in the reorganization TEM1 enzyme elimination skimmed milk substratum.In 10ml 12% skimmed milk substratum, add ammonia benzyl to 100 μ g/mL.To the reorganization TEM1 fusion rotein that wherein adds different amounts, the room temperature effect measured with the TTC method whether microbiotic exceeds standard in the milk by GB/T 4789.27-2003 after 15 minutes.Wherein not ammonification benzyl and adding enzyme amount redden greater than the substratum of 0.2 μ g, show that antibiotic remains has met national standard.Do not redden and add pipe (comprising the not enzyme-added pipe) substratum that the enzyme amount is lower than 0.2 μ g, show that antibiotic remains exceeds standard.Therefore, according to the present invention, eliminated penbritin residual in the milk effectively with the TEM1 enzyme.
Sequence table
＜110〉Hangzhou Biodoor Biotechnology Co., Ltd.
＜120〉a kind of β-Nei Xiananmei, its preparation method and application thereof
<160>2
<210>1
<211>792
<212>DNA
＜213〉artificial sequence
<220>
<221>CDS
<222>(1)..(792)
<220>
<221>misc_feature
＜223〉TEM1 encoding sequence
<400>1
cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc 50
acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga 100
gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg 150
ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg 200
tcgccgcctg cactattctc agaatgactt ggttgagtac tcaccagtca 250
cagaaaagca tcttacggat ggcatgacag tacgcgaatt atgcagtgct 300
gccctgacca tgagtgataa cactgcggcc aacttacttc tgacaacgat 350
cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg 400
taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cctgccaaac 450
gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa 500
actattaact ggcgaactac ttactctagc ttcccggcaa caattactgg 550
actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt 600
ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc 650
tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg 700
tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaatcgc 750
cagatcgctg agctgggtgc ctcactgatt aagcattggt aa 792
<210>2
<211>263
<212>PRT
＜213〉intestinal bacteria (Escherichia Coli)
<400>2
His Pro Glu Thr Leu Val Lys Val Lys Asp Ala Glu Asp Gln Leu
1 5 10 15
Gly Ala Arg Val Gly Tyr Ile Glu Leu Asp Leu Asn Ser Gly Lys
20 25 30
Ile Leu Glu Ser Phe Arg Pro Glu Glu Arg Phe Pro Met Met Ser
35 40 45
Thr Phe Lys Val Leu Leu Cys Gly Ala Val Leu Ser Arg Val Asp
50 55 60
Ala Gly Gln Glu Gln Leu Gly Arg Arg Ile His Tyr Ser Gln Asn
65 70 75
Asp Leu Val Glu Tyr Ser Pro Val Thr Glu Lys His Leu Thr Asp
80 85 90
Gly Met Thr Val Arg Glu Leu Cys Ser Ala Ala Ile Thr Met Ser
95 100 105
Asp Asn Thr Ala Ala Asn Leu Leu Leu Thr Thr Ile Gly Gly Phe
110 115 120
Lys Glu Leu Thr Ala Phe Leu His Asn Met Gly Asp His Val Thr
125 130 135
Arg Leu Asp Arg Trp Glu Pro Glu Leu Asn Glu Ala Ile Pro Asn
140 145 150
Asp Glu Arg Asp Thr Thr Met Pro Ala Ala Met Ala Thr Thr Leu
155 160 165
Arg Lys Leu Leu Thr Gly Glu Leu Leu Thr Leu Ala Ser Arg Gln
170 175 180
Gln Leu Ile Asp Trp Met Glu Ala Asp Lys Val Ala Gly Pro Leu
185 190 195
Leu Arg Ser Ala Leu Pro Ala Gly Trp Phe Ile Ala Asp Lys Ser
200 205 210
Gly Ala Gly Glu Arg Gly Ser Arg Gly Ile Ile Ala Ala Leu Gly
215 220 225
Pro Asp Gly Lys Pro Ser Arg Ile Val Val Ile Tyr Thr Thr Gly
230 235 240
Ser Gln Ala Thr Met Asp Glu Arg Asn Arg Gln Ile Ala Glu Ile
245 250 255
Gly Ala Ser Leu Ile Lys His Trp
260
Claims (10)
1. β-Nei Xiananmei, it is characterized in that: the β-Nei Xiananmei encoding sequence is that its sequence is SEQ ID:NO.1 through dna segment codon optimized, chemosynthesis, and the aminoacid sequence of β-Nei Xiananmei is SEQ ID:NO.2.
2. the preparation method of β-Nei Xiananmei according to claim 1, it is characterized in that: this method comprises the step of following order: make up engineering strain, set up zymotechnique, separation and purification.
3. the preparation method of β-Nei Xiananmei according to claim 2, it is characterized in that: engineering bacteria is to contain gene order SEQ ID:NO.1, and expression vector is pET-30a, inserting the site is BL21 (DE3) bacterial strain of Kpn I and HindIII.
4. the preparation method of β-Nei Xiananmei according to claim 2 is characterized in that: the temperature that engineering bacterium fermentation is cultivated is 28 ℃-40 ℃, and the concentration of inductor IPTG is 0.1-1.5mmol/L.
5. the preparation method of β-Nei Xiananmei according to claim 4 is characterized in that: the temperature that engineering bacterium fermentation is cultivated is 30 ℃-35 ℃, and the concentration of inductor IPTG is 0.4-1.0mmol/L.
6. the preparation method of β-Nei Xiananmei according to claim 5 is characterized in that: the temperature that engineering bacterium fermentation is cultivated is 30 ℃, and the concentration of inductor IPTG is 0.5mmol/L.
7. preparation method according to claim 2 is characterized in that: the combination of a kind of in ion exchange chromatography, sieve chromatography, hydrophobic chromatography or the affinity chromatography or any two kinds, any three kinds, four kinds is adopted in separation and purification.
8. β-Nei Xiananmei according to claim 1 is in the application that is used for removing in the livestock product residual antibiotic.
9. β-Nei Xiananmei according to claim 1 is in the application that is used for removing in milk, food-drink processing residual antibiotic and the environment protection.
10. β-Nei Xiananmei according to claim 1 is in the application that is used for removing in livestock, poultry, meat, the medical treatment product residual antibiotic.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102321643A (en) * | 2011-09-22 | 2012-01-18 | 北京凯因科技股份有限公司 | , the optimization dna molecular of coding ADI and express the engineering bacteria of ADI |
| CN110157699A (en) * | 2018-02-12 | 2019-08-23 | 中国科学院微生物研究所 | Beta-lactamase and its encoding gene and their application |
| CN111197064A (en) * | 2020-02-21 | 2020-05-26 | 重庆医药高等专科学校 | Preparation method of penicillin antibiotic impurity |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1300822A (en) * | 1999-12-21 | 2001-06-27 | 复旦大学 | Polypeptide-beta-lactamase 1.5 and polynucleotide for coding this polypeptide |
| CN1361281A (en) * | 2000-12-26 | 2002-07-31 | 上海博德基因开发有限公司 | New polypeptide Beta lactamase 11.66 and polynucleotides encoding this polypeptide |
| WO2006017759A2 (en) * | 2004-08-05 | 2006-02-16 | Kirin Brewery Co., Ltd. | Tumor endothelial marker-1 (tem1) binding antibodies and uses thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102321643A (en) * | 2011-09-22 | 2012-01-18 | 北京凯因科技股份有限公司 | , the optimization dna molecular of coding ADI and express the engineering bacteria of ADI |
| CN102321643B (en) * | 2011-09-22 | 2014-12-10 | 北京凯因科技股份有限公司 | Optimized DNA (Deoxyribonucleic Acid) molecule for coding ADI (Aiginine Deiminase) and engineering bacteria for expressing ADI |
| CN110157699A (en) * | 2018-02-12 | 2019-08-23 | 中国科学院微生物研究所 | Beta-lactamase and its encoding gene and their application |
| CN111197064A (en) * | 2020-02-21 | 2020-05-26 | 重庆医药高等专科学校 | Preparation method of penicillin antibiotic impurity |
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