CN102174483B

CN102174483B - Method for preparing turnip chitin binding protein through macromolecular cold-condensation method

Info

Publication number: CN102174483B
Application number: CN2011100331104A
Authority: CN
Inventors: 苏保荣; 徐凤彩; 程京燕; 胡永刚
Original assignee: Zhuhai Yupintang Biotechnology Co Ltd
Current assignee: Zhuhai Yupintang Biotechnology Co Ltd
Priority date: 2011-01-30
Filing date: 2011-01-30
Publication date: 2012-11-21
Anticipated expiration: 2031-01-30
Also published as: CN102174483A

Abstract

The invention relates to a method for preparing turnip chitin binding protein through a macromolecular cold-condensation method. The principles of PAA+R.CBP -> PAA-R.CBP and PAA-R.CBP+Ca<2+> -> PAA-Ca<2+>+R.CBP are utilized, turnip used as a material is purified to obtain R.CBP powder, wherein the purification step comprises induction, extraction, selective thermal denaturation, ultrafiltration concentration, condensation reaction and displacement. The technical production equipment has low investment, the technology is simple and easy to realize industrial production; and PAA can be recycled by adopting the reaction product PAA-Ca<2+> and using a H2SO4 solution, the cost can be reduced, and no environmental pollution is caused.

Description

A kind of polymer condensation gathers the method that legal system is equipped with radish chitin-binding protein

[technical field]

The present invention relates to a kind of polymer condensation and gather the method that legal system is equipped with radish chitin-binding protein.

[background technology]

Radish chitin-binding protein (Raphanus sativus L Chitin-binding protin; Abbreviation RCBP; Radish CBP) be a kind of can with the protein of Regitex FA specific combination; Proved already that it had germicidal action (Agricultural University Of South China's journal; 1997,18 (4): 72～75): it is wider that we have studied the proteinic antimicrobial spectrum of this kind, to Staphylococcus albus (Staphylococcus), streptococcus aureus (Staphylococcus aureus), Bacillus subtilus G such as (Bacillus Subtilis (Ehrenberg) Cohn) ⁺Bacterium and Bacillus proteus (ProteusbacillusVulgaris), intestinal bacteria (Escherichia Cdi), salmonella typhimurium (Salmonellatyphimwium), kill pasteurellaceae bacillus (Pastewella multaida), white dysentery Salmonellas (Salmnella), gas bacillus G such as (Enterobacteratrogenes) more ^-Bacterium and yeast (SaccharomyceScerevisiae), fungies such as Mucor racemosus (Mucor racemosus), Rigen are mould, black mold (Aspergillus niger), mould all have the effect extremely that disappears.Simultaneously; It is also to Chinese cabbage Bacteria erwinia (Frwinia carovorasubsp), c itrus canker germ (Citrus; Baterial lanker Disease), bacterial wilt of tomato bacterium (RalstoniaSalanacearum), Pyricularia oryzae (magnaporthe grisea), Rhizoctonia solani Kuhn (RhizoctoniasolaniAG-11A), southern corn leaf blight (Drechslera maydis), banana blight bacteria (being commonly called as the banana cancer, Fusarium oxyporumsp.cubense) etc. are planted (work) thing pathogenic bacteria all has good germicidal action.

These are different fully with present commercially available N,O-Diacetylmuramidase, present commercially available N,O-Diacetylmuramidase they only to Gram-negative (G ^-) bacterium works, and to Gram-positive (G ⁺) bacterium, fungi be all inoperative.

The molecular composition of radish CBP, structure and characteristic have following characteristics:

1. be a kind of single composition monomeric enzyme, and be a kind of inducible enzyme;

2. be the basic protein of a kind of relative molecular mass less (about 11.4KD);

3. be a kind of lyase vigor that both had, have the bifunctional enzyme albumen of chitinase again;

4. its bifunctional enzyme vigor is at 0-60 ℃, and vigor is stable in the pH2.4-10.0 scope, and at room temperature vigor is basicly stable in two years;

5. under certain condition, its zymoprotein crystallization is a rectangle.The active site includes Glu, Asp, His and Trp residue.These are different with enzyme moving, mikrobe, it is a many His and Trp residue; Also different with other plant N,O-Diacetylmuramidase, it has a Trp and His residue, does not but have the Cys residue, so fungistatic effect is different;

6. it is also different with other N,O-Diacetylmuramidase and chitinase, and it is not a gp, does not contain in any chain and interchain disulfide bond, so enzymic activity is stable, fungistatic effect is obvious;

7. its n terminal amino acid residue is WDADMVAKUM in proper order, this sequence is carried out Blastp collect, and the result finds, in the available data storehouse, has no with it to be complementary for information about.Peptide quality fingerprinting atlas analysis identifies that the result finds in existing Protein Data Bank, also to have no with it to be complementary for information about.Thereby prove that it is a kind of newfound not only had N,O-Diacetylmuramidase but also active chitin-binding protein of tool chitinase.

According to its molecular composition, character and constitutional features thereof, and sterilization effect, especially to G ^-Bacterium, particularly more strong to the fungi sterilising effect, this radish CBP is with a wide range of applications and good market outlook and potentiality.Pharmaceutically, antifungal drug is few, and microbiotic is because the resistance of mikrobe and to the toxic side effect of body has limited its application; On agricultural, corps diseases is fungus-caused more than 90%, because agricultural chemicals residual and to the pollution of environment also limited application, in the industries such as disease resistance of food, daily use chemicals, livestock culture, radish CBP purposes is extremely wide equally, remains development and use.

Radishes planted all over the country can, especially in the Lingnan other places all year round Pu species, radish chitin-binding proteins produced extensive preparation materials, inexpensive, but low levels of the enzyme, the existing preparation process is more complex, difficult to achieve industrialization.

Vestolen PP 7052 glue (polycrylic Acid PAA) is the macromolecular compound that forms with the free monomer acroleic acid polymerization:

Different with the different relative molecular masses of its polymerization degree.PAA is nontoxic, soluble in water, is the poly dielectric medium.The dispersion agent of doing commonly used in industry.

[summary of the invention]

The purpose of this invention is to provide a kind of polymer condensation and gather the method that legal system is equipped with radish chitin-binding protein, technology is simpler, is easy to realize industriallization, can lower cost and non-environmental-pollution.

Above-mentioned purpose realizes through following technical scheme:

A kind of polymer condensation gathers the method that legal system is equipped with radish chitin-binding protein, it is characterized in that, may further comprise the steps:

1) material: get radish and clean, its epidermis is chopped wound, under 16-20 ℃, induced 4-7 days, make its microbiological contamination,, increase enzyme content with synthesizing of inducible enzyme with cutter;

2) extract: use 5-10mmol/L NaHCO ₃The aqueous solution is extracting solution, gets two parts of isopyknic extracting solutions, and the volume of every part of extracting solution and the mass ratio of material are 1; Get a extracting solution and material homogenate, the homogenate of winning, centrifugal, collect first supernatant; With its residue and the homogenate of another part extracting solution, get second homogenate again, centrifugal, collect second supernatant; Merge first supernatant and second supernatant;

3) selective thermal sex change: the supernatant of above-mentioned merging is heated with stirring to 50-55 ℃ in retort, and keeps 4-6min, centrifugal then the 3rd supernatant that regathers;

4) ultra-filtration concentrates: the soluble substance of relative molecular mass below 5000 got rid of in the 3rd supernatant ultra-filtration, be concentrated into 1/3 of original volume, get liquid concentrator;

5) condensation reaction: in above-mentioned liquid concentrator; Stir to add volume and above-mentioned liquid concentrator volume than being the PAA of 0.5%-0.8%, finishing to go under 5 ℃-10 ℃ of the low temperature condensation reaction 24 hours to 48 hours; Reaction formula PAA+RCBP → PAA-RCBP ↓; Condensation product, carry out centrifugal, throw out PAA-RCBP;

6) displacement: in PAA-RCBP, under agitation add and contain 80-120mmol/L CaCl ₂With the mixing solutions of 5-10mmol/L NaHCO3, the quality of PAA-RCBP is 1 with the ratio of the volume of mixing solutions: 16-17, reaction formula are PAA-RCBP+Ca ²⁺→ PAA-Ca ²⁺↓+RCBP carries out centrifugally, collects the 4th supernatant, and lyophilize gets the enzyme powder, and throw out is PAA-Ca ²⁺

The add-on of PAA in the step 5) is: the PAA volume is 0.5% with said liquid concentrator volume ratio.

CaCl in the step 6) ₂Mass concentration be 100mmol/L.

The add-on of mixing solutions is in the step 6): the ratio of the volume of mixing solutions and the quality of PAA-RCBP is 16.78.

Also comprise step 7): in PAA-Ca ²⁺The middle 80-120mmol/L H that adds ₂SO ₄Solution, reaction formula: PAA-Ca ²⁺+ SO4 ^2-→ PAA+CaSO ₄↓, the centrifugal deposition of going out is to reclaim PAA.

Advantage of the present invention:

Explained hereafter facility investment of the present invention is few, and technology is simple, is easy to realize suitability for industrialized production;

Reaction product PAA-Ca ²⁺Can be through using H ₂SO ₄Solution has reduced cost with the PAA recycling, non-environmental-pollution.

[description of drawings]

Fig. 1 is a process flow sheet of the present invention;

Fig. 2 is the synoptic diagram of PAA add-on to the influence of enzyme activity;

Fig. 3 is Ca ²⁺Concentration is to the synoptic diagram of the influence of enzyme activity;

Fig. 4 is Ca ²⁺Liquor capacity is to the synoptic diagram of enzyme influence alive.

[embodiment]

As shown in Figure 1, get radish and clean, with cutter its epidermis is cut (1-2cm) and hinder, under 16-20 ℃, induced 5 days, the radish after getting 100kg and inducing, chopping, homogenate limit, limit adds 100L 5mmol/L NaHCO ₃Solution homogenate, the homogenate of winning, first homogenate carries out centrifugal (14000r/min, room temperature), the about 175L of the supernatant of winning with tubular-bowl centrifuge; Residue adds 100L5mmol/L NaHCO ₃Solution homogenate gets second homogenate, and second homogenate carries out centrifugal (the same), gets the second supernatant 105L; Merge first supernatant and second supernatant (about 280L), residue is done and is shone (baking) as feed; Change the supernatant (pH is about 4.5-5.0) of above-mentioned merging over to retort and be heated with stirring to 50 ℃, keep 5min, carry out centrifugal (the same), collect the 3rd supernatant; The 3rd supernatant is pumped into amicon ultra-filtration (getting rid of the soluble substance of relative molecular mass below 5000) be concentrated into original volume 1/3 (about 95L), get liquid concentrator; Change liquid concentrator over to retort, stir add down with above-mentioned liquid concentrator volume than (the PAA volume: the liquid concentrator volume) be 0.5% PAA, change fresh-keeping refrigerator-freezer over to, gather 24h in 5 ℃ of-10 ℃ of following condensations, reaction formula PAA+RCBP → PAA-RCBP ↓, condensation product; Condensation product is carried out centrifugal (the same), the about 10.4kg of collecting precipitation thing PAA-RCBP; In throw out PAA-RCBP, what under agitation add 17L contains 100mmol/L CaCl ₂With 5mmol/L NaHCO ₃Mixing solutions, carry out centrifugal (the same), collect the 4th supernatant (RCBP solution), lyophilize gets 2.3kg enzyme powder (being the RCBP powder), throw out is PAA-Ca ²⁺In throw out PAA-Ca ²⁺The middle 80-100mmol/L H that adds ₂SO ₄Solution, reaction formula: PAA-Ca ²⁺+ SO4 ^2-→ PAA+CaSO ₄↓, separate out a large amount of depositions again (for H to precipitating earlier dissolving fully ₂SO ₄The suitable consumption of solution, those skilled in the art can control), centrifugal (the same) removed deposition, to reclaim PAA.Separation and purification result such as the table one of RCBP.

Table one

Visible from table one and above-mentioned data, the 100kg radish produces enzyme 2.3kg, and productive rate is 2.3%; Enzyme by purifying 56.74 times, enzyme activity reaches 25600/g, is 272340.4U/mg albumen than vigor.

The enzyme activity determination method: with the micrococcus lysodeikticus is substrate, and a small amount of substrate of picking adds quantitative 1/15mol/L, pH value and be 6.2 phosphate buffered saline buffer in mortar; Grind broken wall; Constantly add above-mentioned damping fluid, grind, make suspension-s under the 450nm wavelength; Absorbance value reaches 0.6-0.9 photoabsorption unit, is substrate suspension; During the enzyme activity determination of liquid, under 25 ℃, be contrast, in measuring cuvette, add substrate suspension 2.5ml, add 0.1ml enzyme liquid rapidly, stir with above-mentioned phosphate buffered saline buffer, read absorbance value (A) immediately after, whenever read number one time at a distance from 30s, read 3min altogether; Obtain average light absorption value (Δ A).With the descend enzyme amount of 0.001 unit of the absorbance value under the 450nm wavelength of PM under these conditions is 1 enzyme activity unit (U); According to the computes enzyme activity unit:

During the enzyme activity determination of enzyme powder, earlier 1g enzyme powder is diluted, the diluent of getting 1ml then calculates A according to the enzyme activity determination method of liquid, again according to the computes enzyme activity unit:

Enzyme activity unit (U/g)=A (U/ml) * [number/enzyme quality (g) is accompanied in dilution].

Protein content determination is according to Bradford (1976) method, and is standard protein with the bovine serum albumin.

Than vigor=enzyme activity/protein contnt; The calculating of purifying multiple is that the ratio vigor with extracting solution is a benchmark, and the ratio of the ratio vigor of two kinds of liquid is the purifying multiple; The calculating of productive rate is to be benchmark with the extracting solution productive rate, (volume of the enzyme activity x contrast liquid of contrast liquid)/(volume of the enzyme activity x extracting solution of extracting solution).

Confirming of several technical parameters:

1, the PAA add-on is to the influence of enzyme activity

In waiting isopyknic liquid concentrator of matter, add 0.1%, 0.3%, 0.4%, 0.5%, 0.7%, 0.9% (PAA volume: the PAA liquid concentrator volume) respectively; Stir; After condensation gathered, centrifugal collection PAA-RCBP deposition was again with isopyknic 100mmol/L CaCl that contains ₂With 5mmol/L NaHCO ₃Mixing solutions dissolve PAA-RCBP fully after, centrifugal mensuration enzyme activity.

As shown in Figure 2, the PAA treatment group enzyme activity with 0.5% is the highest, so the add-on of PAA should be 0.5% of liquid concentrator volume.

2, Ca ²⁺Concentration is to the influence of enzyme activity

The CaCl that in the PAA-RCBP that waits matter, adds isopyknic 50mmol/L, 100mmol/L, 150mmol/L, 200mmol/L, 250mmol/L respectively ₂Solution (contains 5mmol/L NaHCO ₃), centrifugal, the enzyme activity of mensuration supernatant, result such as Fig. 3, visible from Fig. 3, with the CaCl of 100mmol/L ₂The treat enzyme vigor the highest.

3, Ca ²⁺The influence that liquor capacity is lived to enzyme

100mmol/LCa with 5ml, 10ml, 15ml, 20ml, 25ml ²⁺Solution, the PAA-RCBP of dissolving 0.3g, centrifugal, the enzyme activity of mensuration supernatant, result such as Fig. 4, visible from Fig. 4, with the 100mmol/L Ca of every gram PAA-CBP adding 16.78ml ²⁺Solution (contains 5mmol/L NaHCO ₃) enzymic activity is the highest.

In sum, the add-on of PAA is 0.5% (volume percent) of liquid concentrator in the present invention, adds the CaCl of the 100mmol/L of 16～17ml with every gram PAA-CBP ₂Dissolving PAA-CBP is best with the treatment effect that reclaims PAA with the enzyme that dissociates.

The present invention is not limited to the foregoing description, based on simple replacement the foregoing description, that do not make creative work, should belong to the scope that the present invention discloses.

Claims

1. a polymer condensation gathers the method that legal system is equipped with radish chitin-binding protein, it is characterized in that, may further comprise the steps:

2) extract: use 5-10mmol/L NaHCO ₃The aqueous solution is extracting solution, gets two parts of isopyknic extracting solutions, and the volume of every part of extracting solution and the mass ratio of material are 1L: 1Kg; Get a extracting solution and material homogenate, the homogenate of winning, centrifugal, collect first supernatant; With its residue and the homogenate of another part extracting solution, get second homogenate again, centrifugal, collect second supernatant; Merge first supernatant and second supernatant;

3) selective thermal sex change: the supernatant of above-mentioned merging is heated with stirring to 50-55 ℃ in retort, and keeps 4-6min, centrifugal then regather the 3rd supernatant;

6) displacement: in PAA-RCBP, under agitation add and contain 80-120mmol/L CaCl ₂With 5-10mmol/L NaHCO ₃Mixing solutions, the quality of PAA-RCBP is 1Kg: 16L-17L with the ratio of the volume of mixing solutions, reaction formula is PAA-RCBP+Ca ²⁺→ PAA-Ca ²⁺↓+RCBP carries out centrifugally, collects the 4th supernatant, and lyophilize gets the enzyme powder, and throw out is PAA-Ca ²⁺

2. method according to claim 1 is characterized in that, the add-on of the PAA in the step 5) is: the PAA volume is 0.5% with said liquid concentrator volume ratio.

3. method according to claim 1 and 2 is characterized in that, the CaCl in the step 6) ₂Mass concentration be 100mmol/L.

4. method according to claim 3 is characterized in that, the add-on of mixing solutions is in the step 6): the ratio of the volume of mixing solutions and the quality of PAA-RCBP is 16.78.

5. method according to claim 1 is characterized in that, also comprises step 7): in PAA-Ca ²⁺The middle 80-120mmol/L H that adds ₂SO ₄Solution, reaction formula: PAA-Ca ²⁺+ SO4 ^2-→ PAA+CaSO ₄↓, the centrifugal deposition of removing is to reclaim PAA.