CN105543256A

CN105543256A - Lyase of bacteriophage and sterilization application

Info

Publication number: CN105543256A
Application number: CN201610016733.3A
Authority: CN
Inventors: 杨洪江; 荆兆元; 何洋; 张粉姣
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2016-01-12
Filing date: 2016-01-12
Publication date: 2016-05-04

Abstract

The invention relates to lyase of bacteriophage and sterilization application. The amino acid sequence of the lyase is Seq ID NO.1, the nucleotide sequence of the lyase is Seq ID NO. 2, and hand sanitizer containing the lyase is also provided. The bacteriophage lysine (Endolysin) has the characteristics of high efficiency, a wide spectrum and the like and can be applied in medical treatment and public health, food processing and sanitary products as antibacterial active components. The enzyme preparation can be independently used or used in a compounding mode, bacteria can be specifically inactivated, and a safe enzyme preparation source which is free of toxic and side effects is provided for current bacterial infection prevention and food-derived bacterial contamination prevention.

Description

The lyase of a kind of phage and germicidal applications

Technical field

The invention belongs to bioengineering field, particularly a kind of Pseudomonas aeruginosa phage lyase (Endolysin) and as the application of fungicide active ingredient in health care, food-processing, hygienic articles.

Background technology

Pseudomonas aeruginosa (Pseudomonasaeruginosa) is the essential condition pathogenic bacterium causing nosocomial infection, is the important pathogen causing fiber lung patient, fire victim, AIDS patient and immune deficient patients's death.This bacterium is extensively present in various environment, and its adaptability is very strong, can resist the effect of sterilant and other antibacterials.In recent years, especially along with the appearance of multi-drug resistant bacterial strain, making this bacterium cause the treatment of infection to become more difficult, therefore, in the urgent need to researching and developing the antibacterials of brand-new mechanism of action, being used for controlling and the infection that causes for the treatment of Pseudomonas aeruginosa.

Phage (Bacteriophage) cry bacteriophage again, it can in specific intrusion bacterial cell, utilize the instrument of bacterium inside to synthesize composition needed for self, phase is by the lyase of its coding after infection, thus destroy the cell walls of bacterium, make bacteria lysis, finally kill bacterium.But phage comes with some shortcomings equally in practical application, such as bacterium easily produces exists bacteriotoxin etc. for the resistance of phage, phage splitting liquid.By contrast, bacterial virus catenase is utilized to show more powerful application potential.But so far, most research is about gram-positive microorganism bacterial virus catenase, and little for the lyase research of Gram-negative bacteria, this is because the latter has the adventitia of low permeability, limit lyase directly to the degraded of its peptidoglycan.Therefore, make bacterial virus catenase arrive its target position peptidoglycan after the material strengthening gram-negative bacteria cell wall outer membrane permeability can be added, thus exercise lytic activity.

Along with the raising day by day of people's living standard, the demand of people to Liquid soap is not only cleaning action, Liquid soap is also needed to have sterilization, effect of antibacterial and skin care, many disinfection hand cleansers are there is on the market now, but its sterilization component security is not high, after containing hypotoxic cleaning composition hand, when the direct contacting foodstuff of hand, sterilization component can cause certain injury to people's health, therefore its security is not high, although the security of other Liquid soap is high, but function is incomplete, preparation method is complicated, cost is high, general human consumer can not accept.It is CNIO3194338A that State Intellectual Property Office discloses a publication number on July 10th, 2013, name is called the patent of invention of " a kind of Liquid soap and preparation method thereof ", although this patent has certain cleaning action, but its sterilization, fungistatic effect are good not, and not containing skin care ingredient, without moisturizing and skin makeup effect.A kind of disinfection hand cleanser containing dioxide peroxide of patent of invention of Authorization Notice No. CN101766543B, its sterilant is dioxide peroxide.Although dioxide peroxide sterilization effect is good, there is intense stimulus, after contact, mainly cause eye and respiratory tract, suck the pulmonary edema that lethality can occur high density.It take parachlormetaxylenol as sterilant that patent CN101475879B discloses a kind of sterilizing skin-protective hand cleanser, has pungency and the stronger shortcoming of toxicity equally.So market is starved of a kind of soil removability good, sterilizing power is strong, safe and reliable Liquid soap.

Summary of the invention

The object of the invention is to overcome the deficiencies in the prior art part, a kind of lyase and the germicidal applications thereof that derive from Pseudomonas aeruginosa phage are provided, bacterial virus catenase provided by the invention is as sterilant, and it has the advantages such as safety non-toxic, good water solubility, antimicrobial spectrum be wide.

The technical scheme that the present invention realizes object is as follows:

A lyase gene of Pseudomonas aeruginosa phage K8, its nucleotides sequence is classified as SeqIDNO.2.

A lyase of Pseudomonas aeruginosa phage K8, its aminoacid sequence is SeqIDNO.1.

A kind of expression vector of the lyase gene containing phage.

And described prokaryotic expression carrier name is called pQE30, lyase gene according to claim 1 is inserted into the BamHI restriction enzyme site place of pQE30.

A kind of engineering bacteria of the lyase gene containing phage or clone.

And the Host Strains of described engineering bacteria is E.coliM15/pREP4, and described engineering bacteria is E.coliM15/pREP4/pQE30-gp57, it is containing the carrier described in claim 3.

A preparation method for the lyase of phage, adopts above-mentioned engineering bacterium fermentation to obtain.

And the step of preparation method is: at 37 DEG C, under 180rpm condition, with 0.5mmol/L isopropyl-beta D-thio galactopyranoside (IPTG) abduction delivering engineering bacteria, obtain expression product; By expression product through Ni ²⁺nTA affinitive layer purification is separated, and obtains bacterial virus catenase.

The application of lyase in preparation preparation control bacteriological infection medicine, medical and hygiene article, food-processing additive, hygienic articles of a kind of phage.

A Liquid soap for lyase containing phage, component and mass percent thereof are: glycerine 1 ~ 5%, Modulan 1 ~ 3%; tensio-active agent 8 ~ 30%, bacterial virus catenase 0.001 ~ 0.0001%, pH adjusting agent 0 ~ 3%; essence 0.01 ~ 0.03%, surplus is deionized water.

Advantage of the present invention and positively effect are:

The present invention adopts the bacterial virus catenase of engineering strain fermentative production as sterilant, and it has the advantages such as safety non-toxic, good water solubility, antimicrobial spectrum be wide.Lyase is the class peptidoglycan lytic enzyme in infection host late period by phage expression, and this enzyme finally causes host cell lysis by the connecting key in the amido linkage on hydrolytic bacteria whole cell peptidoglycan between sugar and peptide or peptide between amino-acid residue.Not only specific effect is in host for lyase, and action time is short, and action spectrum comparatively phage is wide.Lyase has efficient, special and does not produce the characteristics such as resistance, has the report of successful Application at present at food and pharmaceutical industries.

Bacterial virus catenase provided by the invention (Endolysin) is as the application of fungicide active ingredient in health care, food-processing, hygienic articles; This zymin can independent or composite use, can specifically inactivating bacterium, pollutes provide a kind of zymin source safely, to have no side effect for preventing and treating bacteriological infection and food borne bacteria at present.

Accompanying drawing explanation

Fig. 1 is clone and the checking of lyase gp57 gene carrier T of the present invention;

Fig. 2 is the Construction and identification of the expression plasmid of lyase gp57 gene of the present invention;

Fig. 3 is that the present invention recombinates the identification and analysis of lyase GP57 expression product; Swimming lane 1: the total protein of the JK1501 engineering bacteria of not inducing, swimming lane 2: after induction 4h, the total protein of JK1501 engineering bacteria, swimming lane 3: the target protein after purifying;

Fig. 4 is that the present invention recombinates the degradation capability analysis of lyase GP57 to Pseudomonas aeruginosa protoplastis.

Embodiment

The inventive method is described below by specific embodiment.Unless stated otherwise, technique means used in the present invention is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, but not limits the scope of the invention, and the spirit and scope of the invention only limited by claims.To those skilled in the art, under the prerequisite not deviating from essence of the present invention and scope, the various change carry out the material component in these embodiments and consumption or change also belong to protection scope of the present invention.

The invention provides a kind of lyase deriving from Pseudomonas aeruginosa phage, its aminoacid sequence is all or part of aminoacid sequence shown in SEQIDNo:1.The nucleotide sequence of its encoding gene is as shown in SEQIDNo:2.

Meanwhile, the present invention also provides expression vector, engineering bacteria or the clone of said gene (sequence 2), and carrier is pQE30-gp57, and described engineering bacteria is E.coliM15/pREP4/pQE30-gp57.

The preparation method of expression Pseudomonas aeruginosa phage lyase provided by the invention, adopts primer Lkes-F:5 '-CGC gGATCCaTGGCTCTAACTGAGCAAG-3 ' and primer Lkes-R:5 '-CGC gGATCCtTACTTGAAGGATTGATAGG-3 ', with Pseudomonas aeruginosa phage genomic dna for template amplification Pseudomonas aeruginosa phage lyase gene;

Concrete steps are as follows

(1) increase Pseudomonas aeruginosa phage lyase gene from Pseudomonas aeruginosa phage K8 genome;

(2) the recombinant expression vector of construction expression Pseudomonas aeruginosa phage lyase;

(3), by recombinant expression vector transformation of E. coli competent cell, screening obtains the engineering bacteria of expressing Pseudomonas aeruginosa phage lyase;

(4) isopropyl-beta D-thio galactopyranoside (IPTG) abduction delivering, obtains recombinant gene expression product;

(5) by recombinant gene expression product, through Ni ²⁺nTA affinitive layer purification is separated, and obtains the Pseudomonas aeruginosa phage lyase of recombinating;

Above-mentioned concrete steps adopt the mode of embodiment to carry out describing.

The extraction of embodiment 1, phage genome

(1) crude phage particle

(1) preparation of Host Strains: picking Pseudomonas aeruginosa (Pseudomonasaeruginosa) from solid medium ^[1]single bacterium colony, is inoculated in 5mLLB liquid nutrient medium, 37 DEG C of shaking culture 6-8h.

(2) preparation of phage pure culture liquid: the single plaque of picking, is inoculated in 5mL logarithmic phase Host Strains nutrient solution, 37 DEG C of shaking culture 4-6h, then by lysate in the centrifugal 10min of 10000 × g, supernatant liquor is phage pure culture liquid.

(3) preparation of the rough particle of phage: overnight culture be transferred in 100mL LB liquid medium, inoculum size is 1%, and amplification cultivation is to logarithmic phase (OD ₆₀₀about 0.4), add 5mL phage pure culture liquid, after 37 DEG C of shaking culture 6-8h, obtain phage splitting liquid.In lysate, adding DNaseI and RNaseA to final concentration is 5 μ g/mL, mixes rear 37 DEG C of standing 1h.Then adding NaCl to final concentration is 0.1mol/L, and the rear centrifugal 20min of ice bath 1h, 12000 × g is dissolved in mixing.Forwarded to by supernatant liquor after in another centrifuge tube, adding PEG6000 to final concentration is 10% (w/v), and fully vibrate in 4 DEG C of hold over night after dissolving, the centrifugal 20min of 12000 × g, abandons supernatant.With 500 μ LTM (0.05mol/LTris-HClpH7.5,0.2%MgSO ₄7H ₂o) damping fluid will precipitate resuspended, and with isopyknic chloroform repeatedly extracting once, the centrifugal 10min of 12000 × g, to remove the PEG6000 in re-suspension liquid, finally obtains the crude extract of phage particle.

(2) preparation of phage genome DNA

(1) in the phage particle of purifying, add DNaseI and RNaseA, final concentration is 1 μ g/mL, and 37 DEG C of standing 1h, with DNA or RNA of the Host Strains of degrade residual;

(2) then add 1mol/LEDTA (pH8.0) to final concentration 50mmol/L, stop DNaseI and RNaseA active;

(3) adding Proteinase K to final concentration is 50 μ g/mL, and adding SDS to final concentration is 0.5%, mixing, 56 DEG C of lh, digesting protein;

(4) add equal-volume phenol: chloroform: primary isoamyl alcohol (25: 24: 1) mixes, the centrifugal 10min of 12000 × g, collect supernatant;

(5) step 4 repeats 3 times; Collect supernatant, equal-volume chloroform, mixing, 12000 × g, 10min, collect supernatant;

(6) 95% ethanol of 1/10 volume 3mol/LNaAc and 3 times volume precooling is added, mixing, 12000 × g, 10min, precipitation DNA;

(7) add 70% ethanol (500 μ L) in precipitation, and put upside down by the centrifuge tube covered tightly for several times, the centrifugal 5min of 12000 × g, reclaims DNA.

(8) remove supernatant liquor, the alcohol drop on removing tube wall, by the centrifuge tube drying at room temperature 10min of opening, then uses the resuspended DNA of distilled water.

The structure of embodiment 2, recombinant plasmid

1, the acquisition of object fragment

(1) design of primers

According to Pseudomonas aeruginosa phage K8 lyase gene sequence, design primer:

LkesF：5′-CGCGGATCCATGGCTCTAACTGAGCAAG-3′

LkesR：5′-CGCGGATCCTTACTTGAAGGATTGATAGG-3′

(2) 50 μ L reaction systems:

(3) PCR reaction conditions:

2, chemical conversion experiment (structure of pGEM-Teasy recombinant vectors)

(1) be connected with pGEM-Teasy carrier by gp57 gene PCR product, linked system is:

10 μ L reaction systems:

Reaction system is mixed, 16 DEG C, connection of spending the night.

(2) escherichia coli DH5a Competent cell preparation:

A, microbial culture: the single bacterium colony of picking escherichia coli DH5a in 5mL LB liquid medium, 37 DEG C of incubated overnight.Switching overnight culture is in 200mLLB substratum, and inoculum size is 3%, continues to be cultured to logarithmic phase (OD in mid-term ₆₀₀about 0.35-0.4), nutrient solution is proceeded to ice bath in centrifuge tube, to cooling completely;

B, 0.1mol/L magnesium chloride treatment soln: by nutrient solution in 4 DEG C, the centrifugal 10min of 3500 × g.The resuspended rear ice bath 5min of bacterial sediment 20mL magnesium chloride solution, then in 4 DEG C, the centrifugal 10min of 3500 × g, abandons supernatant;

C, 0.1mol/L calcium chloride treatment soln: ice bath 20min after the resuspended precipitation of 20mL calcium chloride solution, then 4 DEG C, the centrifugal 10min of 3500 × g, abandons supernatant;

D, 0.1mol/L calcium chloride/15% glycerine is preserved: by the resuspended precipitation of 1mL mixing solutions, manage (centrifuge tube is ice bath 10min in advance) packing ,-80 DEG C of preservations the most finally after mixing with 100 μ L/;

(3) connection product and the 100 μ L escherichia coli DH5a competent cells of getting 3 μ LPCR products and plasmid vector pGEM-Teasy mix.In 42 DEG C of heat shock 30s after ice bath 30min, then in leaving standstill 2min on ice.Add the LB liquid nutrient medium of 800 μ L precoolings, after 37 DEG C of shaking culture 30min, get 100 μ L and be applied to (containing 100 μ g/mL penbritins, 50 μ g/mLIPTG) LB flat board on, 37 DEG C of incubated overnight, by the white bacterium colony screening of indigo plant, choose white colony.

Concussion of spending the night in picking white colony to the LB substratum of the penbritin containing 100 μ g/mL is cultivated, alkaline lysis method of extracting plasmid, and carry out digestion verification with restriction enzyme BamHI, as shown in Figure 1, carrier pGEM-Teasy and lyase gene gp57 has been there is at 3.0kb and 0.5kb place, size conforms to expection, show lyase gene gp57 and pGEM-Teasy construction of recombinant vector correct.

3, the structure of expression plasmid

(1) intestinal bacteria M15/pREP4 Competent cell preparation:

A, microbial culture: the single bacterium colony of picking intestinal bacteria M15/pREP4 in 5mL LB liquid medium (containing kantlex 25 μ g/mL), 37 DEG C of incubated overnight.Switching overnight culture is in 200mLLB substratum (containing kantlex 25 μ g/mL), and inoculum size is 1%, continues to cultivate logarithmic phase (OD ₆₀₀about 0.35-0.4), nutrient solution is proceeded to ice bath in centrifuge tube, to cooling completely;

B, 0.1mol/L magnesium chloride solution process: by nutrient solution in 4 DEG C, the centrifugal 10min of 3500 × g.The resuspended rear ice bath 5min of bacterial sediment 20mL magnesium chloride solution, then in 4 DEG C, the centrifugal 10min of 3500 × g;

C, 0.1mol/L calcium chloride solution process: ice bath 20min after the resuspended precipitation of 20mL calcium chloride solution, then 4 DEG C, the centrifugal 10min of 3500 × g;

D, 0.1mol/L calcium chloride/15% glycerine is preserved: by the resuspended precipitation of 2mL mixing solutions, manage (centrifuge tube is ice bath 10min in advance) packing ,-80 DEG C of preservations the most finally after mixing with 200 μ L/;

(2) have the carrier T plasmid of lyase gp57 gene to carry out enzyme with BamHI to plasmid pQE30 and clone to cut, it is as follows that enzyme cuts system:

100 μ L endonuclease reaction systems of gp57 gene:

Reaction system is mixed, 37 DEG C of water-bath 4h.

(3) use glue to reclaim test kit to reclaim pQE30 plasmid and lyase gp57 gene fragment.

(4) by pQE-30 plasmid dephosphorylation, and plasmid is reclaimed

(5) gp57 enzyme is cut back to close product to be connected with the pQE-30 plasmid vector after dephosphorylation, linked system is as follows:

Gp57 reclaims product 4 μ L

Carrier pQE-301 μ L

DNA ligase SolutionI5 μ L

Reaction system is mixed, 16 DEG C, connection of spending the night.

(6) get 5 μ L and connect product and the mixing of 100 μ L intestinal bacteria M15/pREP4 competent cells.In 42 DEG C of heat shock 30s after ice bath 30min, then in leaving standstill 2min on ice.Add the LB liquid nutrient medium of 800 μ L precoolings, in 37 DEG C, after 110rpm shaking culture 15min, culture is coated on the LB flat board containing 100 μ g/mL penbritins, 25 μ g/mL kantlex, is inverted incubated overnight for 37 DEG C.

(7) picking white colony is cultivated to containing concussion of spending the night in the penbritin of 100 μ g/mL and the LB liquid nutrient medium of 25 μ g/mL, alkaline lysis method of extracting plasmid, and carry out digestion verification respectively with restriction enzyme BamHI, EcoRI, as shown in Figure 2, the gene band that lyase gene gp57 and checking forward are connected has been there is at 567bp with 537bp place, size conforms to expection, show successful connection and for be just connected, by recombinant plasmid vector called after pQE30-gp57 correct for qualification, the engineering bacteria called after JK1501 containing positive recombinant plasmid.

Embodiment 3, the lyase high expression in intestinal bacteria

The frozen engineering bacteria JK1501 containing positive recombinant plasmid is inoculated in 20mLLB liquid nutrient medium (containing penbritin 100 μ g/mL, kantlex 25 μ g/mL), 37 DEG C, 220r/min, overnight shaking is cultivated, next day, bacterium liquid joins in fresh 1LLB liquid nutrient medium (containing penbritin 100 μ g/mL, kantlex 25 μ g/mL) by the inoculum size according to 1%, and 37 DEG C, 180rpm is cultured to OD ₆₀₀=0.6, add inductor IPTG (be 1mmol/L to final concentration).After inducing culture 4h, centrifugal, 4000r/min, 15min collect thalline.Every 1g thalline (weight in wet base) is resuspended in the broken bacterium damping fluid (Tris-HCl20mmol/L of 3mL, NaCl250mmol/L, PMSF1mmol/L, pH7.4) in, after abundant mixing, ultrasonic disruption cell, centrifugal 10000 × g, 4 DEG C of centrifugal 15min, remove insoluble cell debris, supernatant liquor 0.22 μm of sterilised membrane filter filters, and obtains crude enzyme liquid.

By crude enzyme liquid His affinity chromatography nickel post (GEHealthcare, Sweden) purifying, specifically illustrate that step is poly-by test kit and carry out.Obtaining protein designations is lyase GP57.Sequence 1 is seen after the order-checking of its protein sequence.

SDS-PAGE analytical results as shown in Figure 3, containing positive recombinant plasmid engineering bacteria JK1501 through IPTG induction after, in its supernatant about 22kDa (wherein albumen itself be about 20.1kDa and with his amalgamation and expression after size be about 22kDa) position have thicker induced protein bands, with expection size conform to.

Embodiment 4, lyase GP57 are to the cracking of Pseudomonas aeruginosa protoplastis

The PAK of incubated overnight is transferred in 50mLLB, is cultured to OD600=0.6; Culture 4000 × g, 4 DEG C, centrifugal 15min, abandons supernatant; Add the Tris damping fluid (pH=7.7) that 10mL0.05mol/L chloroform is saturated, softly shake 45min under room temperature, 4000 × g, 4 DEG C, centrifugal 15min, abandon supernatant and collect protoplastis; With phosphate buffered saline buffer (pH=7) re-suspended cell of 10mmol/L, 4000 × g, 4 DEG C, centrifugal 15min, then use the phosphate buffered saline buffer of 5mL10mmol/L (pH7) by resuspended for protoplastis to OD600=0.6-1.0, for subsequent use; Added by lyase in 96 porocyte culture plates, every hole adds lyase and protoplastis respectively, and makes the final concentration of lyase be 0.25 μ g/mL, arrange 3 parallel.With N,O-Diacetylmuramidase (5.0 μ g/mL) for positive control, equal-volume level pad are for negative control, every 3min measures absorbancy OD ₆₀₀, temperature of reaction is room temperature.

Result as shown in Figure 4, under the restructuring lyase splitting action that concentration is 0.25 μ g/mL, OD ₆₀₀rapid decline, along with the prolongation OD of time ₆₀₀finally tend to be steady state, during effect 2h, and OD ₆₀₀reduce 73.6%, this shows that the restructuring lyase Gp57 product of purifying has good lytic activity to Pseudomonas aeruginosa protoplastis.

Embodiment 5, lyase GP57 are as the fungicidal activity of the activeconstituents of Liquid soap

By each component concentration of Liquid soap mass percent:

In deionized water, add glycerine, Modulan, lauroylamidopropyl betaine, be heated to 55 ~ 60 DEG C, stir; be cooled to room temperature, adjust pH to 8 ~ 9 with citric acid, then add biological bactericide bacterial virus catenase, essence; stir, degassed, packing.

Table 1 biological antibiotic Liquid soap (1% diluent) 1 hour sterilization effect

Bacterial strain	Pseudomonas aeruginosa	Subtilis	Bacillus licheniformis	Bacillus mycoides
					Sterilizing rate (%)	99.64	87.81	99.99	90.00

Enzyme prepared by the present invention can be used for the application of the pollution of anti-bacteria or the medicine of hypertrophy or sterilant.Described bacterial contamination is to meat, or eggs, or milk preparation, or grain, or vegetables, or the solid of Combined machining between them or the pollution of liquid type food; Described application refer to preparation suppress described in P. aeruginosa fungi pollution for meat, or eggs, or milk preparation, or grain, or vegetables, or the solid of Combined machining between them or the medicine of pollution of liquid type food or the application of sterilant.

Pseudomonas aeruginosa phage lyase is in the application of preparation control bacteriological infection medicine.

The present invention also provides the application of a kind of Pseudomonas aeruginosa phage lyase in hand sanitizer formula.

Hand sanitizer formula containing bacterial virus catenase, all have killing action to gram-positive microorganism, Gram-negative bacteria; The mass percent of each component of filling a prescription is: glycerine 1 ~ 5%, Modulan 1 ~ 3%, tensio-active agent 8 ~ 30%, bacterial virus catenase 0.001 ~ 0.0001%, pH adjusting agent 0 ~ 3%, essence 0.01 ~ 0.03%, and all the other are deionized water; Described tensio-active agent is one or more mixing in sodium lauryl sulphate, sarcosyl, ammonium lauryl sulfate, polyoxyethylenated alcohol sodium sulfate, AESA, cocoamidopropyl dimethylamine amine second lactone, lauroylamidopropyl betaine, cocamidopropyl propyl amide amine oxide, TritonX-100, tween 80, polysorbas20, tetradecyl trimethyl ammonium chloride, ten alkyl trimethyl ammonium bromides.

The preparation method of above-mentioned Liquid soap is: in deionized water, add glycerine, Modulan, tensio-active agent; be heated to 55 ~ 60 DEG C; stir and dissolve; be cooled to room temperature; adjust pH to 8 ~ 9 with pH adjusting agent sodium hydroxide or sodium carbonate, then add biological bactericide bacterial virus catenase, essence, stir; degassed, packing.

More than show and describe ultimate principle of the present invention, principal character.The technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and specification sheets just illustrates principle of the present invention; do not departing under the spirit and scope of the present invention prerequisite; the present invention also has various changes and modifications, and these changes and improvements fall in the claimed scope of the invention.

Claims

1. a lyase gene of Pseudomonas aeruginosa phage K8, is characterized in that: its nucleotides sequence is classified as SeqIDNO.2.

2. a lyase of Pseudomonas aeruginosa phage K8, is characterized in that: its aminoacid sequence is SeqIDNO.1.

3. the expression vector containing the lyase gene of the phage described in claim 1.

4. carrier according to claim 3, is characterized in that: described prokaryotic expression carrier name is called pQE30, and lyase gene according to claim 1 is inserted into the BamHI restriction enzyme site place of pQE30.

5. the engineering bacteria containing the lyase gene of the phage described in claim 1 or clone.

6. the engineering bacteria of the lyase gene of phage according to claim 5, it is characterized in that the Host Strains of described engineering bacteria is E.coliM15/pREP4, described engineering bacteria is E.coliM15/pREP4/pQE30-gp57, and it is containing the carrier described in claim 3.

7. a preparation method for the lyase of phage, is characterized in that: adopt the engineering bacterium fermentation described in claim 5 or 6 to obtain.

8. the preparation method of the lyase of phage according to claim 7, it is characterized in that: step is: at 37 DEG C, under 180rpm condition, with 0.5mmol/L isopropyl-beta D-thio galactopyranoside (IPTG) abduction delivering engineering bacteria, obtain expression product; By expression product through Ni ²⁺nTA affinitive layer purification is separated, and obtains bacterial virus catenase.

9. the application of lyase in preparation preparation control bacteriological infection medicine, medical and hygiene article, food-processing additive, hygienic articles of a phage as claimed in claim 2.

10. the Liquid soap containing, for example the lyase of phage according to claim 2; it is characterized in that: component and mass percent thereof are: glycerine 1 ~ 5%; Modulan 1 ~ 3%; tensio-active agent 8 ~ 30%; bacterial virus catenase 0.001 ~ 0.0001%; pH adjusting agent 0 ~ 3%, essence 0.01 ~ 0.03%, surplus is deionized water.