CN102199550A - Recombinant strain expressing organophosphorus hydrolase, complex enzyme preparation technology, complex enzyme preparation and fruit and vegetable detergent - Google Patents
Recombinant strain expressing organophosphorus hydrolase, complex enzyme preparation technology, complex enzyme preparation and fruit and vegetable detergent Download PDFInfo
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- CN102199550A CN102199550A CN 201110071973 CN201110071973A CN102199550A CN 102199550 A CN102199550 A CN 102199550A CN 201110071973 CN201110071973 CN 201110071973 CN 201110071973 A CN201110071973 A CN 201110071973A CN 102199550 A CN102199550 A CN 102199550A
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Abstract
The invention relates to a recombinant strain expressing organophosphorus hydrolase, a complex enzyme preparation technology, a complex enzyme preparation and a fruit and vegetable detergent, wherein the recombinant strain comprises a controllable lyase gene. The complex enzyme preparation technology comprises the following steps: culturing the recombinant strain expressing organophosphorus hydrolase, adding proper start-up condition induced expression lyase; and using the obtained culture solution to prepare the complex enzyme preparation. The recombinant strain expressing organophosphorus hydrolase is cultured and the expression of lyase lysed cells is induced, thus the possible influences of complicated mechanical and chemical pyrolysis methods on the expression of enzyme activity can be avoided and the cheap industrialized production is convenient to realize. The complex enzyme preparation produced on the basis of the preparation technology has organophosphorus hydrolase and cell lyase simultaneously, has the double functions of pesticide degradation and sterilization and can be used in the development of the new fruit and vegetable detergent.
Description
Technical field
The present invention relates to biological technical field, more particularly, relate to a kind of recombinant bacterial strain, prozyme preparation technology, compound enzymic preparation and fruits and vegetables washing composition of expressing organophosphor hydrolytic enzyme.
Background technology
Agricultural chemicals is the production means necessary in the agriculture production, and global chemical pesticide kind is approximately kind more than 14000, and annual production is to reach 3,000,000 tons with bulk drug.China is the big producing country of riskiest pesticide, wherein the use of organophosphorus pesticide accounts for 57% of whole agricultural chemicals, the residual meeting of agricultural chemicals causes the severe contamination of environment and food, and pollute wide, longer duration, have a strong impact on the foreign exchange earning of people's healthy and agricultural-food, caused the extensive concern of more and more societies.
How to handle the pesticide residue problem effectively, guarantee that garden stuff pesticide residue meets food hygienic standard, relate to every citizen's life and health.Garden stuff pesticide residue improvement technology is the crucial supporting technology of food safety production, and main method has the clear water washing to soak or the washing composition cleaning at present.But general agricultural chemicals all is fat-soluble, and the solubleness in water is very little, so the clear water washing generally can not obtain satisfied washing effect.Though and general soapless soap can reduce pesticide residue to a certain extent, but can bring secondary pollution, can cause destruction to a certain degree simultaneously to the nutritive ingredient of fruits and vegetables, so exploitation is efficient, economical, safe pesticide residue treatment process has become the research direction with important economy and social effect that needs to be resolved hurrily.
Biodegradable enzyme is a kind of lytic enzyme, very easily water-soluble, this biological enzyme is some microorganisms that come from the soil, a kind of enzyme that is produced as bacterium, fungi and actinomycetes etc., it is the phosphoric acid ester bond on the hydrolysis organophosphorus pesticide molecule specifically, organophosphorus pesticide is decomposed, and the organophosphorus pesticide after the decomposition has not been a phosphate compounds, thereby has eliminated the toxicity of agricultural chemicals fully.Because enzyme itself is exactly a protein, be widely used in foodstuff additive and wine brewing etc., can as the chemosynthesis washing composition, also can not cause harmful secondary pollution as improper use.The biological degradation enzyme process is not only efficient but also very safe, and no side effects does not have a bit influence to the taste and the nutritive value of fruits and vegetables yet, can be directly applied for various fruit, vegetables, tealeaves, cereal, beans, egg milk and meat.
At present, scientist separates the multiple degradation of pesticide enzyme that comes from microorganism, purifying, and it is carried out deep physiology and Biochemical Research, the existing research and development of just being devoted to purify the practice and the suitability for industrialized production of pesticide residual contamination.But, be difficult to realize the cheap production of industrialization because the content of degradation of pesticide enzyme in natural protoenzyme pearl is too low.
Summary of the invention
The technical problem to be solved in the present invention is that the deficiency at existing organophosphor hydrolytic enzyme technology provides a kind of recombinant bacterial strain, prozyme preparation technology, compound enzymic preparation and fruits and vegetables washing composition of expressing organophosphor hydrolytic enzyme.
The technical solution adopted for the present invention to solve the technical problems is:
Construct a kind of recombinant bacterial strain of expressing organophosphor hydrolytic enzyme, wherein, also contain controllable cracking enzyme gene in the described recombinant bacterial strain.
Recombinant bacterial strain of the present invention, wherein, described lyase is bacterial virus catenase or N,O-Diacetylmuramidase.
Recombinant bacterial strain of the present invention, wherein, adopt following method to obtain described recombinant bacterial strain:
The organophosphor hydrolytic enzyme expression plasmid is transformed into the host bacterium of controllable cracking;
The expression plasmid conversion host bacterium that perhaps, will contain organophosphor hydrolytic enzyme gene and controllable cracking enzyme Gene Double cistron;
Perhaps, with organophosphor hydrolytic enzyme expression plasmid and controllable cracking expression of enzymes plasmid cotransformation host bacterium.
Recombinant bacterial strain of the present invention, wherein, described host bacterium is bacterium or fungi, and described bacterium comprises intestinal bacteria at least, and described fungi comprises yeast at least.
The present invention also provides a kind of prozyme preparation technology of each described recombinant bacterial strain of application of aforementioned, may further comprise the steps:
A, the recombinant bacterial strain of expressing organophosphor hydrolytic enzyme is cultivated, added suitable entry condition abduction delivering lyase;
B, described medium centrifugal is directly got supernatant, obtain composite enzyme solution;
Perhaps, with resuspended with damping fluid behind the described medium centrifugal collecting cell, centrifuging and taking supernatant after freeze thawing obtains composite enzyme solution again;
Perhaps, with using the resuspended processing of lysis buffer behind the described medium centrifugal collecting cell,, obtain composite enzyme solution again through the centrifuging and taking supernatant.
Prozyme preparation technology of the present invention, wherein, in the described steps A, described suitable entry condition is meant:
Elevated temperature to 37～45 ℃ processing 1～6 hour, or adding was induced agent treated 1～6 hour;
The described reagent of inducing is pectinose or isopropylthiogalactoside.
The present invention also provides a kind of compound enzymic preparation that adopts aforementioned prozyme preparation technology preparation, wherein, comprises organophosphor hydrolytic enzyme and lysis enzyme.
Compound enzymic preparation of the present invention wherein, also comprises one or more combinations in anion surfactant, glycine, sodium bicarbonate, Tris alkali, ethylenediamine tetraacetic acid (EDTA), sodium-chlor and the carboxymethyl cellulose;
Described compound enzymic preparation is with lyophilized powder, effervescent tablet or liquid form packing.
Compound enzymic preparation of the present invention, wherein, the per-cent that described organophosphor hydrolytic enzyme accounts for the compound enzymic preparation gross weight is 0.00001%～80%, the per-cent that described lysis enzyme accounts for the compound enzymic preparation gross weight is 0.00001%～80%.
The present invention also provides a kind of fruits and vegetables washing composition, wherein, comprises aforementioned each described compound enzymic preparation.
Beneficial effect of the present invention is: by the reorganization organophosphor hydrolytic enzyme expression strain that is obtained is cultivated, the cracking of abduction delivering lyase, this technological operation is simple, avoided complicated mechanical and chemical cracking method issuable to expressing the influence of enzymic activity, reduce production cost simultaneously, be convenient to realize the cheap production of industrialization.Compound enzymic preparation based on this explained hereafter contains organophosphor hydrolytic enzyme and lysis enzyme simultaneously, has degradation of pesticide and germ-resistant dual-use function, can be used for development of new vegetable and fruit washing composition.
Description of drawings
The invention will be further described below in conjunction with drawings and Examples, in the accompanying drawing:
Fig. 1 is the OD that the pectinose of the embodiment of the invention 5 is induced the different time nutrient solution
600The result;
Fig. 2 is that the pectinose of the embodiment of the invention 5 induces the different time supernatant to measure enzyme shared ratio alive.
Embodiment
The recombinant bacterial strain of the expression organophosphor hydrolytic enzyme of preferred embodiment of the present invention contains controllable cracking enzyme gene.Adopt following method to obtain this recombinant bacterial strain: the host bacterium that the organophosphor hydrolytic enzyme expression plasmid is transformed into controllable cracking; The expression plasmid conversion host bacterium that perhaps, will contain organophosphor hydrolytic enzyme gene and controllable cracking enzyme Gene Double cistron; Perhaps, with organophosphor hydrolytic enzyme expression plasmid and controllable cracking expression of enzymes plasmid cotransformation host bacterium.Wherein, the host bacterium is bacterium or fungi, and bacterium comprises intestinal bacteria at least, and fungi comprises yeast at least.Lyase comprises bacterial virus catenase (peptidoglycan lytic enzyme, peptidoglycan hydrolase) or N,O-Diacetylmuramidase (lysozyme), is preferably the lambda particles phage lyase.
Adopt above-mentioned recombinant bacterial strain to make to be used for the compound enzymic preparation of fruits and vegetables washing, concrete technology is as follows: 1), the recombinant bacterial strain of expressing organophosphor hydrolytic enzyme is cultivated, add suitable entry condition abduction delivering lyase; 2), described medium centrifugal is directly got supernatant, the acquisition composite enzyme solution; Perhaps, with resuspended with damping fluid behind the described medium centrifugal collecting cell, centrifuging and taking supernatant after freeze thawing obtains composite enzyme solution again; Perhaps, with using the resuspended processing of lysis buffer behind the described medium centrifugal collecting cell,, obtain composite enzyme solution again through the centrifuging and taking supernatant.Wherein, suitable entry condition is meant: elevated temperature was handled 1～6 hour to 37-45 ℃, or added and induced agent treated 1～6 hour, and inducing reagent is pectinose or isopropylthiogalactoside (IPTG).Can avoid the issuable influence of complicated mechanical and chemical cracking method like this, reduce production cost simultaneously, be convenient to realize the cheap production of industrialization the expression enzymic activity.Compound enzymic preparation based on this explained hereafter contains organophosphor hydrolytic enzyme and lysis enzyme simultaneously, has degradation of pesticide and germ-resistant dual-use function, can be used for development of new vegetable and fruit washing composition.
In the various embodiments of the present invention, the pET15 that is adopted, pET22, pET28, pEAS, restriction endonuclease (Restriction Enzyme) BamHI, Bg1II, NcoI, NdeI, SacI, XbaI and XhoI, host bacterium, pectinose, isopropylthiogalactoside materials such as (IPTG) are commercial.Wherein, the host bacterium is bacterium or fungi, and bacterium comprises intestinal bacteria at least, and fungi comprises yeast at least.Intestinal bacteria comprise commercial BL21, BL21 (DE3), BL21XJb (DE3) (Autolysis
TM), BL21NTC4828, AD494, XL1-Blue and other intestinal bacteria.
In another embodiment of the present invention, a kind of compound enzymic preparation that adopts above-mentioned prepared also is provided, comprise organophosphor hydrolytic enzyme and lysis enzyme, for example N,O-Diacetylmuramidase.Effectively degrading organic phosphor pesticides is residual for this compound enzymic preparation, effectively sterilization.Wherein, the per-cent that organophosphor hydrolytic enzyme accounts for the compound enzymic preparation gross weight is 0.00001%～80%, is preferably 0.1%～10%; The per-cent that the lysis enzyme accounts for the compound enzymic preparation gross weight is 0.00001%～80%, is preferably 0.1%～10%.
In a further embodiment, above-mentioned compound enzymic preparation also comprises one or more combinations in anion surfactant, glycine, sodium bicarbonate, Tris alkali, ethylenediamine tetraacetic acid (EDTA) (EDTA), sodium-chlor and the carboxymethyl cellulose.Wherein anion surfactant comprises TritonX100, linear alkylbenzene sulphonic acid or sodium lauryl sulphate, and its weight part proportioning is 0.01%～10%, is preferably 1～5%; Glycine weight part proportioning is 0.1%～5%, is preferably 0.5～1%; Sodium bicarbonate weight part proportioning is 5%-50%, is preferably 10～30%; Carboxymethyl cellulose weight part proportioning is 0.1%～1%, is preferably 0.4～0.6%; Tris alkali weight part proportioning is 0.1%～10%, is preferably 6%; EDTA weight part proportioning is 0.05%～5%, is preferably 4%; Weight sodium chloride part proportioning is 0.1%～10%, is preferably 6%.
Compound enzymic preparation in the foregoing description is with lyophilized powder, effervescent tablet, foam spraying agent or other liquid form packing.
Below by a plurality of specific embodiments prozyme preparation technology of the present invention and prepared compound enzymic preparation are described in detail.
The prozyme preparation technology of preferred embodiment of the present invention may further comprise the steps: a), make up reorganization organophosphor hydrolytic enzyme pET28-OPH, be the applying gene recombinant technology with the organophosphor hydrolytic enzyme gene clone to the pET28 carrier, construction expression plasmid pET28-OPH, this plasmid has the T7 promotor, can be under the inducing of IPTG express recombinant organophosphor hydrolytic enzyme albumen; B), the reorganization organophosphor hydrolytic enzyme expression strain of preparation controllable cracking; C), the reorganization organophosphor hydrolytic enzyme expression strain that is obtained is carried out the engineering fermentation culture, induce pET28-OPH to express lyase; D), the fermented liquid that obtains in the step c) is prepared compound enzymic preparation.Like this, by the reorganization organophosphor hydrolytic enzyme expression strain that is obtained is carried out the engineering fermentation culture, induce pET28-OPH to express the lyase lysing cell, avoided complicated mechanical and chemical cracking method issuable, be convenient to realize the cheap production of industrialization expressing the influence of enzymic activity.
Further, above-mentioned processing step a) specifically may further comprise the steps:
A1), pcr amplification organophosphor hydrolytic enzyme OPH gene;
According to the encoding sequence of organophosphor hydrolytic enzyme, design specific oligonucleotide primer, sequence is
P1?5-AAACCCCCATGGATGAGCATTGGAACCGGCGATCGTA-3，
P2?5-CCCAAACTCGAGGCTTGCCCGAAGAGTCGGGCTCAGA-3，
NcoI and XhoI restriction enzyme site are contained in its two ends.
A2), adopt restriction endonuclease (Restriction Enzyme) NcoI and XhoI that the pET28 carrier is carried out double digestion, be connected with the OPH amplified production that same enzyme is cut;
Further, above-mentioned processing step b) comprising: will connect product pET28-OPH and transform autothermic cracking host bacterium BL21 XJb (the DE3) (Autolysis that is subjected to the responsive promotor of pectinose (pBAD promoter) regulation and control
TM), screening positive clone on 50mg/L kantlex flat board.
Further, above-mentioned processing step c) may further comprise the steps:
C1), bacterial screening: comprise that--bacterium is used in lab scale expression--amplification cultivation---production in the test tube cultivation, be specially, choose and clone that (peptone 10g+ yeast powder 5g+NaCL10g moisturizing is to 1000ml in the LB of 5ml liquid nutrient medium, with NaOH or HCL PH is transferred to 7.2-7.4) in reorganization organophosphor hydrolytic enzyme expression strain, add 1mM inductor (sec.-propyl-β-D-sulfo-galactopyranoside for example, IPTG) induced 16～24 hours in 28 ℃, adding the 3mM pectinose again induced 1～4 hour, make nutrient solution become gradually clearly, with nutrient solution under the 6000rpm condition centrifugal 5～15 minutes, collecting cell, resuspended with 50mMTris-HCL damping fluid (pH 8.0), place and be transferred to room temperature freeze thawing half an hour after-20 ℃ of freezing half an hour, under the 6000rpm condition centrifugal 10 minutes subsequently, collect supernatant liquor; Adopt the 1mM parathion-methyl to carry out enzyme measurement alive, measuring solution is 50mMTris-HCL damping fluid (pH 8.0), and reaction product 405nm surveys absorption value.Get active higher clone's amplification cultivation bacterial classification and use bacterium as producing, packing is frozen.
C2), seed liquor cultivates: get frozen bacterial classification LB plate line, cultivated 16～24 hours for 37 ℃, choose single colony inoculation in liquid LB substratum, cultivated 10～15 hours in 37 ℃, the shaking table of 150-200rpm;
C3), fermentor cultivation: fermentor tank at first 121 ℃ the sterilization 30 minutes, then the seed liquor 5% that obtains among the step c2 is inserted, control oxygen dissolving value by rotating speed and air flow, when jar internal absorbance value (OD value) when reaching 3, add inductor and induced 16～24 hours, add suitable entry condition subsequently and induce the host bacterium to express lyase; Wherein, suitable entry condition is meant that adding the 3mM pectinose handled 2～4 hours.
Further, at above-mentioned processing step d) in, with step c3) in the fermentation culture that obtains under the 6000rpm condition centrifugal 5～25 minutes, collecting cell, resuspended with 50mMTris-HCL (pH 8.0) damping fluid, place and be transferred to room temperature freeze thawing half an hour after-20 ℃ of freezing half an hour, under the 6000rpm condition centrifugal 10 minutes subsequently, the supernatant lyophilize is added anion surfactant, glycine, sodium bicarbonate, Tris alkali, ethylenediamine tetraacetic acid (EDTA) (EDTA), one or more combinations in sodium-chlor and the carboxymethyl cellulose, the dry powder compound enzymic preparation that contains organophosphor hydrolytic enzyme and lysis enzyme is made in packing.Wherein anion surfactant comprises TritonX100, linear alkylbenzene sulphonic acid or sodium lauryl sulphate, and its weight part proportioning is 0.01%～10%, is preferably 1～5%; Glycine weight part proportioning is 0.1%～5%, is preferably 0.5～1%; Sodium bicarbonate weight part proportioning is 5%-50%, is preferably 10～30%; Carboxymethyl cellulose weight part proportioning is 0.1%～1%, is preferably 0.4～0.6%; Tris alkali weight part proportioning is 0.1%～10%, is preferably 6%; EDTA weight part proportioning is 0.05%～5%, is preferably 4%; Weight sodium chloride part proportioning is 0.1%～10%, is preferably 6%.
Adopt pcr amplification organophosphor hydrolytic enzyme OPH gene earlier, again according to the encoding sequence of organophosphor hydrolytic enzyme, design specific oligonucleotide primer, sequence is P3 5-AAACCCCATATGATGAGCATTGGAACCGGCGATCGTA-3, P45-CCCAAAGGATCCGCTTGCCCGAAGAGTCGGGCTCAGA-3, adopt restriction endonuclease (Restriction Enzyme) NdeI and BamHI that the pET15 carrier is carried out double digestion, be connected with the OPH amplified production that same enzyme is cut; To connect the responsive autothermic cracking host of product invert point bacterium BL21 NTC4828, screening positive clone on 100mg/L penbritin flat board.
Choose single colony inoculation in liquid LB substratum, in 30 ℃, the shaking table of 150-200rpm, cultivated 10～15 hours; With 121 ℃ of sterilizations of fermentor tank 30 minutes, then the seed liquor 5% that obtains is inserted, control oxygen dissolving value by rotating speed and air flow, when the OD value reaches 3 in the jar, add IPTG and induced 16 hours, subsequent fermenting jar temperature is increased to 42 ℃ of processing and induced the escherichia coli expression lyase in 3 hours.At last, with the fermentation culture that obtains under the 6000rpm condition centrifugal 5 minutes, collecting cell, with lysis buffer (50mM Tris-Cl, 10mM EDTA, 100mM NaCl, 0.5% (v/v) TritonX-100, pH8.0) resuspended, placed after half an hour under the 6000rpm condition centrifugal 10 minutes, with one or more combinations in supernatant lyophilize adding anion surfactant, glycine, sodium bicarbonate and the carboxymethyl cellulose, the dry powder compound enzymic preparation that contains organophosphor hydrolytic enzyme and lysis enzyme is made in packing.
Embodiment 3
The structure of one .pET28-OPH
At first according to the encoding sequence of organophosphor hydrolytic enzyme, design the specific oligonucleotide primer, NcoI and XhoI restriction enzyme site are contained in two ends, pcr amplification OPH gene.Primer sequence is as follows:
P1?5-AAACCCCCATGGATGAGCATTGGAACCGGCGATCGTA-3
P2?5-CCCAAACTCGAGGCTTGCCCGAAGAGTCGGGCTCAGA-3，
With Restriction Enzyme the pET28 carrier is carried out double digestion, be connected, connect product transformed into escherichia coli BL21 (DE3), screening positive clone on 50mg/L kantlex flat board with the OPH amplified production that same enzyme is cut.
The structure of two .pET22-λ SR
1. synthetic pectinose pBAD promotor
According to the nucleotide sequence synthetic pBAD promotor of pectinose pBAD promotor, and add Bg1II and NcoI restriction enzyme site respectively at its two ends.Shown in sequence is constructed as follows:
2.PCR amplification lambda particles phage lyase gene
According to lambda particles phage lyase gene SR sequences Design Auele Specific Primer, two ends are added NcoI and XhoI restriction enzyme site respectively.Primer sequence is as follows:
P5?5-AAACCCCCATGGGCCACTGTCTGTCCTGAATTC-3
P6?5-AAACCCCTCGAGTAGGCATTTATACTCCGCTGGA-3，
Extract the genomic dna of lambda particles phage, and as template, amplification SR gene.
3. expression vector establishment
Carrier pET22 is carried out double digestion with restriction enzyme Bg1II and XhoI, SR gene to pcr amplification carries out double digestion with NcoI and XhoI, two kinds of double digestion products are connected with synthetic pectinose pBAD promotor, connect product transformed into escherichia coli BL21 (DE3), screening positive clone on 100mg/L penbritin flat board.
Three. two plasmid cotransformation intestinal bacteria
Extract pET28-OPH and pET22-λ SR plasmid, get and wait mole transformed into escherichia coli BL21 (DE3), screening positive clone on the flat board that contains 50mg/L kantlex and 100mg/L penbritin.
Choose single colony inoculation in liquid LB substratum, in 37 ℃, the shaking table of 150-200rpm, cultivated 10～15 hours; With 121 ℃ of sterilizations of fermentor tank 30 minutes, then the seed liquor 5% that obtains is inserted, control oxygen dissolving value by rotating speed and air flow, when the OD value reaches 3 in the jar, add IPTG and induced 16 hours, add the processing of 3mM pectinose subsequently and induced the escherichia coli expression lyase in 2～4 hours.At last, with the fermentation culture that obtains under 6000r pm condition centrifugal 5 minutes, collecting cell, with lysis buffer (50mM Tris-Cl, 10mM EDTA, 100mM NaCl, 0.5% (v/v) TritonX-100, pH8.0) resuspended, placed after half an hour under the 6000rpm condition centrifugal 10 minutes, with one or more combinations in supernatant lyophilize adding anion surfactant, glycine, sodium bicarbonate and the carboxymethyl cellulose, the dry powder compound enzymic preparation that contains organophosphor hydrolytic enzyme and lysis enzyme is made in packing.
Embodiment 4
Adopt pcr amplification organophosphor hydrolytic enzyme OPH gene earlier, again according to the encoding sequence of organophosphor hydrolytic enzyme, design specific oligonucleotide primer, sequence is P75-AAACCCGAGCTCAATGAGCATTGGAACCGGCGATCGTA-3, P85-CCCAAATCTAGATTAGCTTGCCCGAAGAGTCGGGCTCAGA-3
Adopt restriction endonuclease (Restriction Enzyme) SacI and XbaI that the pEAS carrier is carried out double digestion, be connected with the OPH amplified production that same enzyme is cut; Product be will connect and host bacterium XL1-Blue, screening positive clone on 100mg/L penbritin flat board transformed.
Choose single colony inoculation in liquid LB substratum, in 30 ℃, the shaking table of 150-200rpm, cultivated 10～15 hours; With 121 ℃ of sterilizations of fermentor tank 30 minutes, then the seed liquor 5% that obtains is inserted, control oxygen dissolving value by rotating speed and air flow, when the OD value reaches 3 in the jar, add IPTG and induced 16 hours, subsequent fermenting jar temperature is increased to 43 ℃ of processing and induced escherichia coli expression lyase lysing cell in 3 hours.
At last, with the fermentation culture that obtains under the 6000rpm condition centrifugal 5 minutes, with one or more combinations in supernatant lyophilize adding anion surfactant, glycine, sodium bicarbonate, Tris alkali, ethylenediamine tetraacetic acid (EDTA) (EDTA), sodium-chlor and the carboxymethyl cellulose, the dry powder compound enzymic preparation that contains organophosphor hydrolytic enzyme and lysis enzyme is made in packing.
Embodiment 5
Adopt pcr amplification organophosphor hydrolytic enzyme OPH gene earlier, again according to the encoding sequence of organophosphor hydrolytic enzyme, design specific oligonucleotide primer, sequence is P75-AAACCCGAGCTCAATGAGCATTGGAACCGGCGATCGTA-3, P85-CCCAAATCTAGATTAGCTTGCCCGAAGAGTCGGGCTCAGA-3
Adopt restriction endonuclease (Restriction Enzyme) SacI and XbaI that the pEAS carrier is carried out double digestion, be connected with the OPH amplified production that same enzyme is cut; Product be will connect and host bacterium BL21, screening positive clone on 100mg/L penbritin flat board transformed.
Choose and clone that (peptone 10g+ yeast powder 5g+NaCL10g moisturizing is to 1000ml in the LB of 5ml liquid nutrient medium, with NaOH or HCL PH is transferred to 7.2～7.4) in reorganization organophosphor hydrolytic enzyme expression strain, adding 1mM sec.-propyl-β-D-sulfo-galactopyranoside IPTG induced 16 hours in 28 ℃, subsequent fermenting jar temperature is increased to 42 ℃ and handled 4 hours, make nutrient solution become gradually clearly, as shown in Figure 1;
With nutrient solution under the 6000rpm condition centrifugal 5 minutes, collect supernatant liquor; Adopt the 1mM parathion-methyl to carry out enzyme measurement alive, measuring solution is 50mMTris-HCL damping fluid (pH 8.0), and reaction product 405nm surveys absorption value.Induced 1 hour, 2 hours and the enzyme of 4 hours supernatants is lived and improved gradually, as shown in Figure 2.
In the foregoing description 1 to embodiment 5, describe five kinds of different treatment process respectively, adopted the technology in the foregoing description, can prepare and contain organophosphor hydrolytic enzyme and lysis enzyme simultaneously, perhaps also comprised the compound enzymic preparation of other compositions.Wherein the component concentration of compound enzymic preparation is as shown in table 1 below.
Table 1 compound enzymic preparation component concentration (weight percent %)
Because length is limit, the compound enzymic preparation of other component ratios 16 groups of experimental group enumerating in above-mentioned table 1 is as long as contained each composition in aforesaid scope, can be thought to be not described in detail in this in the application's protection domain.
In another embodiment of the present invention, a kind of fruits and vegetables washing composition also is provided, comprising the described compound enzymic preparation of the arbitrary embodiment in front is arranged, also can comprise other ancillary components.Through a large amount of evidences, adopt this fruits and vegetables washing composition, can effectively remove residues of banned pesticides and bacterium on the vegetable and fruit.
Need to prove, in the foregoing description, organophosphor hydrolytic enzyme (organophosphorusHydrolase, OPH) can also adopt Methyl Parathion Hydrolase (methyl parathion hydrolase, MPH) or organophosphorus acid anhydrides enzyme (Organophosphorous Acid Anhydrase, OPAA) replace, can reach the effect of above-mentioned each group experiment equally.Therefore, adopt Methyl Parathion Hydrolase or organophosphorus acid anhydrides enzyme to substitute and finish above-mentioned each test, should be understood in the application's protection domain.
In sum, the present invention is by carrying out the engineering fermentation culture to the reorganization organophosphor hydrolytic enzyme expression strain that is obtained, abduction delivering lyase lysing cell, this technological operation is simple, avoided complicated mechanical and chemical cracking method issuable to expressing the influence of enzymic activity, reduce production cost simultaneously, be convenient to realize the cheap production of industrialization.Compound enzymic preparation based on this explained hereafter contains organophosphor hydrolytic enzyme and lysis enzyme simultaneously, has degradation of pesticide and germ-resistant dual-use function, can be used for development of new vegetable and fruit washing composition.
Should be understood that, for those of ordinary skills, can be improved according to the above description or conversion, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.
Claims (10)
1. a recombinant bacterial strain of expressing organophosphor hydrolytic enzyme is characterized in that, also contains controllable cracking enzyme gene in the described recombinant bacterial strain.
2. recombinant bacterial strain according to claim 1 is characterized in that, described lyase is bacterial virus catenase or N,O-Diacetylmuramidase.
3. recombinant bacterial strain according to claim 1 is characterized in that, adopts following method to obtain described recombinant bacterial strain:
The organophosphor hydrolytic enzyme expression plasmid is transformed into the host bacterium of controllable cracking;
The expression plasmid conversion host bacterium that perhaps, will contain organophosphor hydrolytic enzyme gene and controllable cracking enzyme Gene Double cistron;
Perhaps, with organophosphor hydrolytic enzyme expression plasmid and controllable cracking expression of enzymes plasmid cotransformation host bacterium.
4. recombinant bacterial strain according to claim 3 is characterized in that, described host bacterium is bacterium or fungi, and described bacterium comprises intestinal bacteria at least, and described fungi comprises yeast at least.
5. the prozyme preparation technology of each described recombinant bacterial strain among application such as the claim 1-4 is characterized in that, may further comprise the steps:
A, the recombinant bacterial strain of expressing organophosphor hydrolytic enzyme is cultivated, added suitable entry condition abduction delivering lyase;
B, described medium centrifugal is directly got supernatant, obtain composite enzyme solution;
Perhaps, with resuspended with damping fluid behind the described medium centrifugal collecting cell, centrifuging and taking supernatant after freeze thawing obtains composite enzyme solution again;
Perhaps, with using the resuspended processing of lysis buffer behind the described medium centrifugal collecting cell,, obtain composite enzyme solution again through the centrifuging and taking supernatant.
6. prozyme preparation technology according to claim 5 is characterized in that, in the described steps A, described suitable entry condition is meant:
Elevated temperature to 37～45 ℃ processing 1～6 hour, or adding was induced agent treated 1～6 hour;
The described reagent of inducing is pectinose or isopropylthiogalactoside.
7. the compound enzymic preparation of each described prozyme prepared in employing such as the claim 5～6 is characterized in that, comprises organophosphor hydrolytic enzyme and lysis enzyme.
8. compound enzymic preparation according to claim 7 is characterized in that, also comprises one or more combinations in anion surfactant, glycine, sodium bicarbonate, Tris alkali, ethylenediamine tetraacetic acid (EDTA), sodium-chlor and the carboxymethyl cellulose;
Described compound enzymic preparation is with lyophilized powder, effervescent tablet or liquid form packing.
9. compound enzymic preparation according to claim 7, it is characterized in that, the per-cent that described organophosphor hydrolytic enzyme accounts for the compound enzymic preparation gross weight is 0.00001%～80%, and the per-cent that described lysis enzyme accounts for the compound enzymic preparation gross weight is 0.00001%～80%.
10. a fruits and vegetables washing composition is characterized in that, comprises each described compound enzymic preparation among the claim 7-9.
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Cited By (2)
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|---|---|---|---|---|
| CN105062906A (en) * | 2015-04-02 | 2015-11-18 | 湖北大学 | Optimized organic phosphorus hydrolase yeast engineering bacteria and organic phosphorus hydrolase production |
| CN108355294A (en) * | 2018-01-25 | 2018-08-03 | 北京森根比亚生物工程技术有限公司 | Special pulvis and its preparation method and application is disposed in the degradation decontamination of organophosphor biochemical weapon agent |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1880460A (en) * | 2006-04-28 | 2006-12-20 | 清华大学 | Escherichia coli self-cracking method and its dedicated carrier and application |
| CN101161806A (en) * | 2007-10-03 | 2008-04-16 | 胡银安 | Detergent for removing heavy metal pollution on surface of fruits and vegetables |
| CN101732819A (en) * | 2008-11-14 | 2010-06-16 | 中国科学院沈阳应用生态研究所 | Organic phosphorus degrading enzyme preparation and preparation method thereof |
-
2011
- 2011-03-24 CN CN 201110071973 patent/CN102199550A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1880460A (en) * | 2006-04-28 | 2006-12-20 | 清华大学 | Escherichia coli self-cracking method and its dedicated carrier and application |
| CN101161806A (en) * | 2007-10-03 | 2008-04-16 | 胡银安 | Detergent for removing heavy metal pollution on surface of fruits and vegetables |
| CN101732819A (en) * | 2008-11-14 | 2010-06-16 | 中国科学院沈阳应用生态研究所 | Organic phosphorus degrading enzyme preparation and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 《Biotechnology Journal》 20090218 Zhanlin lin, et al. Cell lysis methods for high-throughput screening or miniaturized assays 210-215 1-10 第4卷, 第2期 * |
| 《Genbank》 19991111 Ohshiro K, et al. Genbank accession number: AB007293 全文 1-6 , * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105062906A (en) * | 2015-04-02 | 2015-11-18 | 湖北大学 | Optimized organic phosphorus hydrolase yeast engineering bacteria and organic phosphorus hydrolase production |
| CN105062906B (en) * | 2015-04-02 | 2018-11-16 | 湖北大学 | A kind of production method optimizing organophosphor hydrolytic enzyme Yeast engineering bacteria and its enzyme |
| CN108355294A (en) * | 2018-01-25 | 2018-08-03 | 北京森根比亚生物工程技术有限公司 | Special pulvis and its preparation method and application is disposed in the degradation decontamination of organophosphor biochemical weapon agent |
| CN108355294B (en) * | 2018-01-25 | 2021-02-26 | 北京森根比亚生物工程技术有限公司 | Special powder for degradation and decontamination treatment of organophosphorus biochemical toxicant, and preparation method and application thereof |
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