CN1379088A - Radish chitin bindin and its preparing process - Google Patents
Radish chitin bindin and its preparing process Download PDFInfo
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- CN1379088A CN1379088A CN 02115151 CN02115151A CN1379088A CN 1379088 A CN1379088 A CN 1379088A CN 02115151 CN02115151 CN 02115151 CN 02115151 A CN02115151 A CN 02115151A CN 1379088 A CN1379088 A CN 1379088A
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Abstract
4 radish-chitin conjugated proteins M, M2, M3 and M4 are prepared from radish through chitosan affinity chromatography, ion exchange column chromatography, concentrating or freeze-drying the eluting liquids separately. For M1 or M2, their lysozyme and chitinase activities are 60000-70000 U/mg and 24-47 U/mL respectively. The chitinase activities of M3 and M4 are both 47-62 U/mL. The Asp, Glu, His and Trp are the necessary amino acids for M1 and M2 activities. The said conjugated proteins have the bacteriodical action to bacteria G- and G+ and the hydrolysis action to the cell wall of fungus, yeast and insect.
Description
The present invention relates to chitin-binding protein, relate in particular to the preparation of radish chitin-binding protein.
Chitin-binding protein be a class can with the protein of chitin specific combination, have now found that chitin-binding protein extensively is present in the animals and plants microorganism, wherein chitinase and N,O-Diacetylmuramidase occupy an important position, and they play a significant role in the plant defense system.Therefore research and comparison is many, and color 1993 of Chen Chongshun, Xu Feng find to have chitinase in 41 kinds of (mutation) plants of 21 sections, and compared their enzyme activity.The plant lysozyme is found early, since the root of nineteen twenty-two Fleming discovery plant contains lysozyme with flower, has successively found lysozyme from the different tissues of different plants, plant.But, from plant, isolate the chitin-binding protein M that not only has antalzyme activity but also have chitinase activity
1, M
2With chitin-binding protein M with chitinase activity
3, M
4Do not appear in the newspapers yet and lead.
N,O-Diacetylmuramidase generally is used for the sterilization, anticorrosion and fresh-keeping of food service industry, break in fermentation industry and bionic thalline, pharmaceutically be used for clinical treatment and diagnosis, the overseas utilization N,O-Diacetylmuramidase is made medicine, be used for the treatment of hemorrhage, blood urine, bloody sputum, chronic sinusitis and diseases such as nasal sinusitis, preventing dental caries, and the clinical diagnosis of disease such as leukemia, pulmonary tuberculosis, renal failure.The bacteriolyze application of enzymes is more and more extensive, and domestic lysozyme production can be satisfied the domestic market needs far away, and part lysozyme and preparation thereof need import.
Present commercially available N,O-Diacetylmuramidase, main is feedstock production with the Ovum Gallus domesticus album, and its technology of preparing complexity, raw materials cost height, product enzyme activity are low, and the egg source is limited, and contends over raw materials with food.The N,O-Diacetylmuramidase of producing with Ovum Gallus domesticus album is only to G
+Bacterium has killing action, and to G
-Bacterium, fungi are inoperative.
Chitinase can be hydrolyzed the chitin in fungi, yeast and the insect cell wall composition, thereby kills it.
And as the conjugated protein M of the chitin of production application
1, M
2With chitin desmin M as production application
3And M
4Report is not arranged yet.
At present no matter pharmaceutically, or the control fungi-medicine made from enzyme preparation on the crop protection is few.As paddy rice 100 multiple diseases, wherein be more than 90% by fungus-caused.Therefore, along with the development of new and high technology in food, medicine and the plant protection, it is imperative with new enzyme to seek new enzyme source.
Find that chitin-binding protein content is the highest in the radish in our research.Thereby found that (Chinese is called radish chitin-binding protein to radish chitin-binding protein, and the Chinese phonetic alphabet is Luobo ji ding zhi jie he dan bai; English Raphanuns SativusChitin-binding protein by name, abbreviation RCBP).Through purify radish chitin-binding protein, this radish chitin-binding protein contains four kinds of protein components, we cry it for M
1, M
2, M
3, M
4
The object of the invention is to seek a kind of bacteriolyze wide spectrum, good stability, preparation is easy, effect reaches applied range well zymin, has opened up new zymin source.The property feature of radish chitin-binding protein:
Radish chitin-binding protein comprises protein M
1, protein M
2, protein M
3, protein M
4, chitin-binding protein is protein M
1, M
2, M
3, M
4General name.Its common trait is: all be white powder, and nontoxic, tasteless, soluble in water.Its aqueous solution can reach electrophoresis pure (SDS-PAGE).Has protein structure and character.The ash content of solid articles: conventional method measures≤5%, moisture: constant weight method test period≤7%.M
1And M
2Both having had lysozyme activity, and had again chitinase activity, is bifunctional enzyme; M
3, M
4The chitin that has contains restriction endonuclease and excision enzyme vigor.Radish chitin-binding protein amino acid forms such as table 1.
Mass percent (%) residue number/mol protein residues M
1M
2M
1M
2Asp 9.80 10.31 23 22 Thr 7.52 5.72 20 14 Ser 7.10 6.54 22 19 Glu 10.98 9.38 23 18 Gly 8.78 9.67 41 43 Ala 7.90 8.61 30 30 Cys 0.00 0.00 00 Val 5.15 5.06 14 13 Met 1.30 1.69 33 Ile 4.21 4.65 10 10 Leu 4.52 5.50 11 12 Tyr 5.71 6.60 9 10 Lys 4.88 4.53 10 9 His 1.77 0.61 31 Arg 9.58 9.51 17 15 Pro 5.15 5.45 14 14 add up to 100 100 262 243
Table 1M
1, M
2Chemical property: M
1, M
2Relative molecular mass be respectively 26915 and 24831.The two is not a glycoprotein, does not all contain in Cys and the chain and interchain disulfide bond, is single composition, monomeric protein.Their biology catalytic activity center is made up of Glu, Asp, His and Trp residue.
Their pI value is 9.5-10.
When being substrate with the micrococcus lysodeikticus, M
1K
mValue is 0.287mg/mL, V
MaxBe 666.67U/min, M
2K
mValue is 0.1533mg/mL, V
MaxValue is 3333.33U/min.
Reaction activity M
1Be 3945J/mol; M
2Be 35786.01J/mol.
M
1The reaction temperature be 45-60 ℃, the reaction pH be 5-7; M
2Reaction
Temperature is 35-55 ℃, and the pH of reaction is 6-8.M
1, M
2All stable in 0-60 ℃, pH3.4-10.6 scope.When being substrate with the N-acetylglucosamine, M
1The PH of reaction is 3.6-5.6, M
2Be 2.4-4.4;
Storage stability: M
1, M
2Transformation period be 180 days, solid preparation year the rate of disintegration be about 10%; 0.002%NaClO and 24%H
2O
2Can make M
1, M
2Lose activity, papoid can make M
1, M
2Lose activity,
Inorganic ion is to M below the 50mmol/L in concentration
1, M
2Activity is promotion, shows as restraining effect more than 50mmol/L, wherein monovalent cation such as Na
+, K
+, NH
4The concentration of Yi Zhiing is 200mmol/L fully, divalent cation such as Ca
2+, Co
2+, Mn
2+Inhibition concentration is 50mmol/L fully, NO
3 -Can alleviate cationic restraining effect.The N-acetyl glucosamine is to M
1, M
2Be competitive inhibition, His is to M
1, M
2Be the uncompetitive inhibitor, histamine is to M
1, M
2For noncompetitive suppresses, imidazoles is to M
1, M
2Restraining effect is also arranged;
M
1, M
2Antalzyme activity be 3.0-3.5 ten thousand U/mg protein, M
1, M
2Chitinase activity is respectively 24 and 47U/mL, M
3, M
4Chitinase activity be 63 and 48U/mL;
M
1, M
2The activated centre contain Trp, His, Glu and Asp residue, these are different from other lysozyme, wherein the Trp residue has participated in the formation of enzyme-substrate complex; Through " induced-fit " and " substrate distortion ", His, Glu and Asp residue when having water to exist, the electronics in the participation catalytic process, the transmission of proton and to substrate enforcement catalytic action.
M
1, M
2, M
3, M
4Antalzyme activity is measured M
1, M
2, M
3, M
4Antalzyme activity is measured and is carried out according to the micrococcus lysodeikticus method of nineteen fifty-two Shugal.Measurement result M
1Antalzyme activity 3.0-4.0 ten thousand U/mg albumen, M
2Antalzyme activity 2.5-3.5U/mg albumen.M
3, M
4Can not survey antalzyme activity.M
1, M
2, M
3, M
4Chitinase activity is measured according to Chen Chong method suitable, that Xu Fengcai is published in plant resources and " research of 21 sections, 41 kinds of (mutation) plant leaf blades chitinase system " of environment on the 2nd the 2nd phase of volume in 1993 and is carried out.Measurement result is: M
1Chitinase activity is 22-25U/mL, M
2The chitin vigor be 40-50U/mL.M
3Chitinase activity is 55-70U/mL, M
4Chitinase activity is 40-50U/mL.M
1, M
2, M
3, M
4The measuring method of protein content measure by the Xylene Brilliant Cyanine G method of Bradford in 1976.Measurement result: sublimed M
1Protein content is 0.01-0.04mg/mL, sublimed M
2Protein content is 0.01-0.02mg/mL, sublimed M
3Protein content is 0.01-0.1mg/mL, sublimed M
4Protein content is 0.001-0.015mg/mL.M
1, M
2, M
3, M
4Amino acid whose mensuration amino acid forms the employing automatic amino acid analyzer and measures.The radish chitin-binding protein target level of product quality:
M
1Activity recovery is about 30%, about 90 times of purification, antalzyme activity 3.5 ten thousand U/mg albumen, chitinase activity 25U/mL.M
2Activity recovery is about 70%, about 70 times of purification, antalzyme activity 3.1 ten thousand U/mg albumen, chitinase activity 47U/mL.M
3Chitinase activity 62U/mL.M
4Chitinase activity 48U/mL.All reach electrophoresis purity.The preparation of radish chitin-binding protein:
(1) gets that fresh radish leaves or piece root are cleaned, airing, drop in the deactivation microbial inoculum and induced 2-3 days, clean, airing; (2) extract: the NaHCO through 4 ℃ of precoolings that in raw material, adds 1-1.5 times of volume material amount
3Homogenate is filtered, and filtrate is centrifugal, collects supernatant, is extract.(3) selective sex change: extract transfers to faintly acid with HCl, is incubated to take out low-temperature centrifugation after the flowing water cooling after reaching 45-55 ℃ to extract in water bath with thermostatic control.Supernatant liquor transfers to pH to 7.0 with NaOH, and low-temperature centrifugation is collected supernatant liquor.(4) ultra-filtration with above-mentioned supernatant liquor ultra-filtration to 1/4 of original volume.(5) affinity chromatography: add the chitosan gel rubber of 1.5 times of volumes in above-mentioned ultra-filtration to original volume in 1/4 the concentrated solution, after 4 ℃ of following whip attachment are complete, remove impurity, use 5mmol/LNaHCO again with the tap water flushing
3Adorn post after the balance and use 5mmol/LNaHCO
3Wash to A
280≤ 0.1.Use the 0.1mol/LHAc wash-out, collect eluent.(6) ultra-filtration again: with the elutriant ultra-filtration of collecting to 1/4 of original volume.(7) CMC ion-exchange chromatography: the 0.025mol/LpH5.4HAc-NaAc dialysis of above-mentioned concentrate through including 0.005mol/L KCl fully, upper CMC post, with the 0.025mol/LpH5.4HAC-NaAc wash-out that includes 0.05-0.4mol/LKCl, collect eluent, eluent is radish chitin-binding protein, 3mL/ pipe during collection, the 6min/ pipe, collected 8-16 pipe is protein M
4, the 17-28 pipe is protein M
3, the 29-34 pipe is protein M
2, the 35-45 pipe is protein M
1, measure protein content and the enzyme activity of each protein, as the quality standard of product.(8) each the component eluent that will collect concentrates respectively or freeze-drying, and concentrated is protein solution, freeze-drying be freeze-dried powder; The preparation of protein solution: each the component eluent that will collect, be concentrated into respectively original volume 1/3, adding protective agent and anticorrisive agent can be made into range protein solution in concentrate; The preparation of protein freeze-dried powder: each the component eluent vacuum freeze drying that will collect becomes the range protein freeze-dried powder.Protective material and sanitas are NaF, glycerine, ethanol, phenylformic acid.Each protein compression liquid mixes by following percentage by volume with protective agent and anticorrisive agent: each protein compression liquid is 74.825%, 0.01mol/LNaF is 0.125%, glycerine is 20%, benzoic acid is 0.05%, ethanol is 5%; Or each protein compression liquid is 79.95%, glycerine is 20%, benzoic acid is 0.05%.Advantage of the present invention: one. the present invention is a raw material with root, the leaf of radish, and raw material sources are easy, inexpensive, adapts to farming
The comprehensive development and utilization of byproduct.Two. explained hereafter facility investment of the present invention is few, and technology is simple.Three. this product M
1, M
2, M
3, M
4Relative molecular mass all little, all be single composition,
Monomeric protein all is a basic protein, and effect need not participate in by cofactor.The product stability height.
Trp, His, Glu and Asp residue are contained in the product activity center, this and other bacteriolyze
The enzyme difference.Four. fungicidal spectrum is wide, not only to G
+Bacterium has killing action, to G
-Bacterium has killing action equally,
And fungi, yeast had fabulous bacteriostatic action, insect also there is certain lethal effect.
These are also different with other N,O-Diacetylmuramidase.Five. M
1, M
2Be both to have had antalzyme activity, having chitinase activity again is bifunctional enzyme,
M
3, M
4Has chitinase activity.
Example: get that fresh Folium Raphani or piece root are cleaned, airing, drop into to soak in the deactivation microbial inoculum and took out afterwash, airing in 2-3 days, get the 5mmol/LNaHCO that 400g adds 4 ℃ of following precoolings of 400mL
3, homogenate, nylon cloth filters, 10000r/min4 ℃ of centrifugal 10min, get supernatant liquor, be extract, extract transfers to pH5.4 with 1mol/L HCl, in 65 ℃ of waters bath with thermostatic control, be incubated and take out after reaching 50 ℃ to extract, after the flowing water cooling, 4 ℃ of centrifugal 10min of 10000r/min.Supernatant liquor transfers to pH7.0 with 1mol/L NaOH, and 4 ℃ of centrifugal again 10min of 10000r/min collect supernatant liquor.With supernatant through hyperfiltration to 1/4 of original volume.The chitosan gel rubber that adds 1.5 times of volumes again in concentrated solution behind 4 ℃ of following whip attachment 1h, is removed impurity with the tap water flushing, uses 5mmol/LNaHCO
3Adorn post after the balance, and use 5mmol/LNaHCO
3Wash to A
280≤ 0.1. 0.1mol/LHAc wash-out is collected elutriant.Elutriant through ultra-filtration is concentrated into 1/4 of original volume.After concentrate is used the 0.025mol/LpH5.4HAc-NaAc dialysis 6h that includes 0.05mol/LKCl, upper CMC post.With containing 0.05-0.4mol/LKCl0.025mol/LpH5.4 Ac-NaAc wash-out, collect elutriant, elutriant is a radish chitin-binding protein.3mL/ pipe, 6min/ pipe during collection, and measure protein content and enzyme activity.Collected eluent, the 8-16 pipe is M
4, 17-28 pipe is M
3, 29-34 pipe is M
2, 35-45 pipe is M
1, each component is pure radish chitin-binding protein.
The analytical results of this product such as table 2 and table 3.Table 2 is the antalzyme activity of radish chitin-binding protein; Table 3 is the four components chitinase/antalzyme activity contrast of radish chitin-binding protein.Step, poly-cumulative volume total activity total protein reclaimed than vigor protein compression/purification vigor, (mL), (U), (mg), (U/mg) degree, (mg/mL) multiple, (%) crude enzyme liquid 800 40,000 1,033 38.71 1.29 1 100 affinity chromatographys 9.6 5.400 11 4,583 1.15 118.38 126M
19 10,980 0.312 35,197 0.035 909.23 27.45M
211.3 28,250 0.917 30,798 0.018 795.59 70.625M
311 0 0.77 0 0.07 0 0M
410.4 0 0.104 0 0.01 00
Table 2
M
1M
2M
3M
4The total chitinase activity of HEWL (U/mL) 24.66 47.31 62 47.78 10.49 circumscribed chitinase activity (U/mL) 9.81 14.03 21.69 11.53 2.34 endochitinase vigor (U/mL), 14.84 33.28 40.31 36.25 8.15 bacteriolyze specific activity of enzyme (U/mg Pro) the 73037.04 66095.89 00 4902.0 shared percentages of total chitinase (%) 100 100 100 100 100 circumscribed chitinases account for percentage (%) 39.8 29.7 35.0 24.3 22.3 endochitinases and account for percentage (%) 60.2 70.3 65.0 75.7 77.7 bacteriolyze specific activity of enzyme/total chitin specific activity of enzyme 99.97 122.38 00 521.49
Table 3 is got above-mentioned each elutriant 50mL to 0.066mol/L, the pH6.2 PBS 12h that dialyses, and ultrafiltration and concentration is got 10mL and added 2.5mL glycerine, 7ul phenylformic acid to 15mL, the Freeze Drying Equipment freeze-drying, the 0.7mg lyophilized powder, the ratio vigor of enzyme is 30,000 U/mg albumen.
Other gets the 5mL concentrated protein solution and respectively adds 8 μ L NaF (12.5mmol/L), 1.3mL glycerine, 0.3mL ethanol, 3 μ L phenylformic acid, promptly can be made into 40,000 U/mg protein solutions.
Claims (5)
1. radish chitin-binding protein is characterized in that: this kind chitin-binding protein has four kinds of protein, is respectively protein M
1, protein M
2, protein M
3, protein M
4All be white powder, nontoxic, tasteless, soluble in water, it is pure that its aqueous solution can reach electrophoresis: SDS-PAGE has protein structure and character, the ash content of solid articles: conventional method measures≤5%, moisture:
The constant weight method measures≤7%.M
1And M
2Both having had the lysozyme activity vigor, and had again chitinase activity, is bifunctional enzyme; M3, M4 have chitin restriction endonuclease and excision enzyme vigor.
M
1, M
2Chemical property:
M
1, M
2Relative molecular mass be respectively 26915 and 24831, the two is not glycoprotein, does not all contain in Cys and the chain and interchain disulfide bond, is single composition, monomeric protein, their biology catalytic activity center is made up of Glu, Asp, His and Trp amino acid residue;
M
1, M
2, M
3, M
4The pI value be 9.5-10;
During take micrococcus lysodeikticus as substrate, M
1The Km value be that 0.287mg/mL, Vmax are 666.67U/min, M
2The Km value be 0.1533mg/mL, Vmax is 3333.33U/min;
Reaction activity M
1Be 3945J/mol; M
2Be 35786.01J/mol;
M
1The reaction temperature be 45-60 ℃, the reaction pH be 5.0-7.0; M
2Reaction temperature be 35-55 ℃, the pH of reaction is 6.0-8.0, M
1, M
2All stable in 0-60 ℃, pH3.4-10.6 scope.During take N-Acetyl-D-glucosamine as substrate, M
1The pH of reaction is 3.6-5.6, M
2The pH of reaction is 2.4-4.4;
Storage stability: M
1, M
2Transformation period is 180 days, solid preparation year the rate of disintegration be about 10%;
0.002%NaClO and 24%H
2O
2Can make M
1, M
2Lose activity;
Papoid can make M
1, M
2Lose activity,
Inorganic ion under the concentration of 50mmol/L to M
1, M
2Activity is promotion, shows as restraining effect when the concentration of 50mmol/L is above, wherein monovalent cation such as Na
+, K
+, NH
4 +The concentration of Yi Zhiing is 200mmol/L fully, divalent cation such as Ca
2+, Co
2+, Mn
2+Inhibition concentration is 50mmol/L fully, NO
3 -Can alleviate cationic restraining effect, the N-acetyl glucosamine is to M
1, M
2Be competitive inhibition, histamine is to M
1, M
2For noncompetitive suppresses, His is to M
1, M
2Be the uncompetitive inhibitor, imidazoles is to M
1, M
2Restraining effect is also arranged;
M
1Antalzyme activity be that 3.0-4.0 ten thousand U/mg albumen, chitinase activity are 22-25U/mL, M
2Antalzyme activity be that 2.5-3.5 ten thousand U/mg albumen, chitinase activity are 40-50U/mL, M
3Chitinase activity be 55-70U/mL, M
4Chitinase activity be 40-50U/mL.
2. the preparation method of radish chitin-binding protein is characterized in that: (1) gets fresh Folium Raphani or the piece root is cleaned, airing, induces 2-3 days clean, airing with the deactivation microbial inoculum; (2) extract: the 5mmol/LNaHCO that in raw material, adds 1-1.5 times of volume material amount through 4 ℃ of precoolings
3Homogenate, filter, filtrate is centrifugal, collect supernatant liquor, be extract, (3) selectivity sex change: extract is transferred to slightly acidic with HCl, in water bath with thermostatic control, is incubated and takes out after reaching 45-55 ℃ to extract, flowing water cool off back 4 ℃ centrifugal down, supernatant liquor is transferred pH to 7.0 again with NaOH, 4 ℃ are down centrifugal, collect supernatant liquor, (4) ultra-filtration: with above-mentioned supernatant liquor ultra-filtration to 1/4 of original volume, (5) affinity chromatography: the chitosan gel rubber that adds 1.5 times of volumes in above-mentioned ultra-filtration to original volume in 1/4 the concentrated solution, complete in 4 ℃ of following whip attachment, remove impurity with the tap water flushing, use NaHCO
3Adorn post after the balance, and use NaHCO
3Wash to A
280≤ 0.1, use the 0.1mol/LHAc wash-out, collect elutriant, (6) ultra-filtration again: with the elutriant ultra-filtration of collecting to 1/4 of original volume, (7) CMC ion-exchange chromatography: above-mentioned concentrated solution is through complete to the 0.025mol/L pH5.4HAc-NaAc dialysis that includes 0.05mol/L KCl, last CMC post, with the 0.025mol/LpH5.4HAc-NaAc wash-out that includes 0.05-0.4mol/L KCl, collect elutriant, elutriant is a radish chitin-binding protein, 3mL/ pipe during collection, the 6min/ pipe, collected 8-16 pipe is protein M
4, the 17-28 pipe is protein M
3, the 29-34 pipe is protein M
2, the 35-45 pipe is protein M
1, each component elutriant that (8) will collect concentrates respectively or freeze-drying, and spissated is protein soln, and freeze dried is lyophilized powder.
3. according to the preparation method of the said radish chitin-binding protein of claim 2, it is characterized in that extract transfers to pH5.4 with 1mol/LHCl, take out after in 65 ℃ of warm water baths, making extract reach 50 ℃.
4. according to the preparation method of the said radish chitin-binding protein of claim 1, it is characterized in that said flowing water cools off back 4 ℃ of centrifugal down supernatant liquors that get, and transfers to pH7 with 1mol/NaOH.
5. according to the preparation method of the said radish chitin-binding protein of claim 1, it is characterized in that said protein NaHCO after chitosan gel rubber absorption
3Fill post after the balance, use NaHCO behind the dress post
3Washing, its NaHCO
3Concentration be 5mmol/L.
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CN101732747A (en) * | 2008-11-25 | 2010-06-16 | 宋秋兰 | Application of radish chitin-binding proteins in preparing air freshener |
CN101979571A (en) * | 2010-10-21 | 2011-02-23 | 国家海洋局第三海洋研究所 | Ridgeia piscesae chitin binding protein and preparation method thereof |
CN102114261A (en) * | 2010-12-31 | 2011-07-06 | 张胜勇 | Environmentally-friendly air freshener |
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CN101434936B (en) * | 2008-12-10 | 2011-09-07 | 宋秋兰 | Industrial preparation of radish dual-function enzyme with chitinase and antalzyme activity |
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CN101732747A (en) * | 2008-11-25 | 2010-06-16 | 宋秋兰 | Application of radish chitin-binding proteins in preparing air freshener |
CN101434936B (en) * | 2008-12-10 | 2011-09-07 | 宋秋兰 | Industrial preparation of radish dual-function enzyme with chitinase and antalzyme activity |
CN101979571A (en) * | 2010-10-21 | 2011-02-23 | 国家海洋局第三海洋研究所 | Ridgeia piscesae chitin binding protein and preparation method thereof |
CN102114261A (en) * | 2010-12-31 | 2011-07-06 | 张胜勇 | Environmentally-friendly air freshener |
CN102119720A (en) * | 2010-12-31 | 2011-07-13 | 张胜勇 | Carrot bifunctional enzyme-containing fruit and vegetable fresh-keeping agent |
CN102174483A (en) * | 2011-01-30 | 2011-09-07 | 珠海市御品堂生物科技有限公司 | Method for preparing turnip chitin binding protein through macromolecular cold-condensation method |
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CN102433351A (en) * | 2011-12-21 | 2012-05-02 | 河南省农业科学院 | Trichoderma viride chitinase gene Tvchi and expression product and application thereof |
CN102433351B (en) * | 2011-12-21 | 2014-02-19 | 河南省农业科学院 | Trichoderma viride chitinase gene Tvchi and expression product and application thereof |
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