CN101173261B - Separation purification technique for lysozyme in holothurian intestines - Google Patents
Separation purification technique for lysozyme in holothurian intestines Download PDFInfo
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- 239000004325 lysozyme Substances 0.000 title claims abstract description 18
- 108010014251 Muramidase Proteins 0.000 title claims abstract description 17
- 102000016943 Muramidase Human genes 0.000 title claims abstract description 17
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 title claims abstract description 17
- 235000010335 lysozyme Nutrition 0.000 title claims abstract description 16
- 241000251511 Holothuroidea Species 0.000 title abstract description 7
- 210000000936 intestine Anatomy 0.000 title abstract description 5
- 238000000746 purification Methods 0.000 title description 2
- 238000000926 separation method Methods 0.000 title 1
- 238000000502 dialysis Methods 0.000 claims abstract description 44
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- 238000001035 drying Methods 0.000 claims abstract 2
- 102000004190 Enzymes Human genes 0.000 claims description 52
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- 108010064696 N,O-diacetylmuramidase Proteins 0.000 claims description 37
- 239000007788 liquid Substances 0.000 claims description 37
- 239000000872 buffer Substances 0.000 claims description 34
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 28
- 239000002953 phosphate buffered saline Substances 0.000 claims description 28
- 238000005406 washing Methods 0.000 claims description 23
- 239000000243 solution Substances 0.000 claims description 22
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 16
- 239000003795 chemical substances by application Substances 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 12
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 9
- 238000007710 freezing Methods 0.000 claims description 9
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- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 claims description 8
- -1 polyoxyethylene Polymers 0.000 claims description 8
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 238000002523 gelfiltration Methods 0.000 claims description 7
- 239000012535 impurity Substances 0.000 claims description 7
- 241000258955 Echinodermata Species 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- IDOQDZANRZQBTP-UHFFFAOYSA-N 2-[2-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol Chemical compound CC(C)(C)CC(C)(C)C1=CC=CC=C1OCCO IDOQDZANRZQBTP-UHFFFAOYSA-N 0.000 claims description 4
- 229920004929 Triton X-114 Polymers 0.000 claims description 4
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- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention relates to a holothurian intestine lysozyme extraction method from abandoned holothurian intestine, which is obtained as the following: homogenate, lixiviation in 2% TritonX-114 solution, dialysis, absorption and elution in CM52 cellulose column, absorption and elution in Sephadex-G75 chromatography column, drying. The invention has the advantages of scale production, low cost, high yield rate, good safety and high stability, in particular to the usage of abandoned holothurian intestine, which not only avoids environmental pollution, but also produces the echinodermatous lysozyme of psychrophilic nature, oxidation resistibility and broad bacteriolytic spectrum compared with chicken protein lysozyme. The invention has the unique advantages and brilliant application prospect in medicine, food industry, biological high technology industry and other fields.
Description
Technical field
The present invention relates to a kind of echinoderms albumen enzyme purification technology, relate in particular to a kind of Intestinum Stichopi japonici and extract the N,O-Diacetylmuramidase technology.
Technical background
N,O-Diacetylmuramidase is a kind of enzyme that can optionally dissolve microorganism wall that nineteen twenty-two Alexander Fleming finds.In actual applications, N,O-Diacetylmuramidase has antibiotic, antiviral, hemostasis, detumescence, analgesia and accelerates function such as tissue repair.Therefore.Can be used for medical treatment and foodstuff additive, be used to extract the interior all kinds of materials of microorganism cells and carry out the protoplastis preparation and the fusion breeding.
N,O-Diacetylmuramidase is the ubiquitous a kind of enzyme of occurring in nature.The egg albumen N,O-Diacetylmuramidase is the highest N,O-Diacetylmuramidase of content, on average accounts in the egg proteicly 3.4%, is called c type N,O-Diacetylmuramidase.At present, 724 type Zeo-karbs adsorbing and extracting from Ovum Gallus domesticus album is mainly adopted in the production of N,O-Diacetylmuramidase, remove explained hereafter such as foreign protein and lyophilize through wash-out, ammonium sulfate precipitation, dialysis, iso-electric point, or be that pregnant solution extracts from eggshell with polyacrylic acid.
The N,O-Diacetylmuramidase biochemical property that extracts from echinoderms such as sea cucumber is different from those egg albumen N,O-Diacetylmuramidases.The N,O-Diacetylmuramidase of echinoderm can be resisted the infection of bacterium as a kind of digestive ferment.Come from the molluscan N,O-Diacetylmuramidase of Echinodermata and seldom be subjected to Temperature Influence, very wide subject range is arranged.The Echinodermata N,O-Diacetylmuramidase is than the pH value that egg albumen N,O-Diacetylmuramidase adapted to, and temperature, ionic strength are all low.Compare with egg white lysozyme, it has low temperature, the characteristics that anti-oxidant and bacteriolyze spectrum is wide had a liking for, the cell walls that can decompose Gram-negative, positive bacteria specifically forms bacteriolysis, have sterilization, effect such as anticorrosion, antiviral, unique advantage and vast market prospect are arranged in fields such as medicine, foodstuffs industry and biological new high-tech industries.
Intestinum Stichopi japonici is mainly derived from units such as the food factory, wineshop of a large amount of consumption sea cucumbers.The Intestinum Stichopi japonici of abandoning in batch will soon be putrid and deteriorated, causes environmental pollution, and the depleted Intestinum Stichopi japonici is used, and not only avoided environmental pollution but also increased economic benefit, is a job that is of great significance.
Summary of the invention
The purpose of this invention is to provide a kind of processing method of from discarded Intestinum Stichopi japonici, extracting N,O-Diacetylmuramidase.
Technical scheme of the present invention is: with fresh Intestinum Stichopi japonici is raw material, by the extracting of 2%TritonX-114 solution, cloud point extraction method (CPE) concentrate, dialysis, CM52 Mierocrystalline cellulose and two chromatography columns of Sephadex-G75 adsorb respectively, thereby obtain the Intestinum Stichopi japonici N,O-Diacetylmuramidase.
The objective of the invention is to realize by following technology:
1. the freezing intestines of sea cucumber are thawed homogenate.
With 3～8 times of volume Tris-HCl damping fluids (50mmol/L, pH 7.5, contain 0.1%～2%TritonX-114) lixiviate, 2～5 ℃ leave standstill 4～24h.4 ℃ of following 10000g 10min are centrifugal, and supernatant liquor both had been the crude enzyme liquid of lysozyme.
3. 25 ℃～37 ℃ water-bath 5～15min of crude enzyme liquid are made water and washing agent layering mutually, lower floor is the washing agent phase.25 ℃ then～37 ℃ centrifugal 5～15min of following 2000g collect the washing agent phase, are the concentrated solution of lysozyme, and 4 ℃ of preservations are standby.
4. concentrated solution is put into molecular weight cut-off and is 7000 dialysis tubing, then dialysis tubing is put into pH=6.0～7.0 that contain 5～10 times of volumes, the container of 0.02mol/L～0.05mol/L phosphate buffered saline buffer, 4 ℃ of dialysis 12～24h, enzyme liquid in the bag taking, both for lysozyme treat sample enzyme liquid, 4 ℃ of preservations are standby.
5. adorn the CM52 cellulose column, add upward sample enzyme liquid along post jamb, and with the 1.0ml/min flow velocity slowly by through pH=6.0～7.0, the CM52 Mierocrystalline cellulose chromatography post crossed of 0.02mol/L～0.05mol/L phosphate buffered saline buffer balance, with 3 times of volume pH=6.0～7.0,0.02mol/L～0.05mol/L phosphate buffered saline buffer washing chromatography column, remove and do not adsorb impurity again.Chromatography column is preferably the Sephadex-G75 gel column of 1.8cm * 60cm.
6. use respectively again by pH=6.0～7.0,0.02mol/L～0.05mol/L phosphate buffered saline buffer preparation 60,120,180,240,300,360,420,480,540mmol/LNaCl solution (containing 0.1%TritonX-114) carries out gradient elution, flow velocity 1.0ml/min, be in charge of collection, being in charge of the survey enzyme lives, be associated with the component that enzyme is lived, 4 ℃ of preservations are standby.
7. it is 7000 dialysis tubing that the enzyme liquid after will merging is put into molecular weight cut-off, dialysis tubing is put into to contain the container that molecular weight is 8000 polyoxyethylene glycol then, and 4 ℃ of dialysis, enzyme liquid is 3～5ml to the bag, it is standby to concentrate 4 ℃ of preservations of enzyme liquid in the bag taking.
On spissated enzyme liquid through pH=7.0,0.02mol/L～0.05mol/L phosphate buffered saline buffer (containing 0.1%TritonX-114) equilibrated Sephadex-G75 chromatography column, pH=7.0,0.02mol/L～0.05mol/L phosphate buffered saline buffer (containing 0.1%TritonX-114) one-step elution with 120% column volume, flow velocity 3.0ml/7min, be in charge of collection, being in charge of the survey enzyme lives, be associated with the component that enzyme is lived, 4 ℃ of preservations are standby.Chromatography column is preferably the Sephadex-G75 gel column of 1.8cm * 60cm.
9. the component after will merging at-40 ℃ of freezing 0.5h～2h, then at-70 ℃ of cold-trap 12h～24h, obtains the pure product of exsiccant N,O-Diacetylmuramidase at last.
Advantage of the present invention is as follows:
1. the traditional water formulation is combined with modern biotechnology, the utilization washing agent makes the fully stripping of N,O-Diacetylmuramidase in the Intestinum Stichopi japonici, has improved the degree of utilizing to raw material, has reduced production cost.
2. in concentration process, introduce cloud point extraction method (CPE), simplified concentration technology, improved the ratio of N,O-Diacetylmuramidase and lived, shortened concentration time, do not destroyed the biological activity of N,O-Diacetylmuramidase simultaneously.
Describe technical scheme of the present invention in detail below in conjunction with embodiment.
Embodiment 1
5 gram Intestinum Stichopi japonicis are packed in the beaker, and deionized water is cleaned, and pulls an oar with stirrer.With 3 times of volume Tris-HCl damping fluids (50mmol/L, pH 7.5, contain 0.1%Triton X-114) lixiviate, 2 ℃ left standstill 4 hours.4 ℃ 10 000g 10min are centrifugal down, and 25 ℃ of water-bath 5min of solution make water and washing agent layering mutually, and lower floor is the washing agent phase.25 ℃ of centrifugal 5min of following 2000g collect the washing agent phase then, are the concentrated solution of lysozyme.Then be 7000 4 ℃ of pH=6.0 of dialysis tubing, 0.02mol/L phosphate buffered saline buffer dialysis 12h, must be with interior liquid for treating sample enzyme liquid to 5 times of volumes with molecular weight cut-off.The dress post, liquid-transfering gun with 1ml adds upward sample enzyme liquid along post jamb, and with the 1.0mL/min flow velocity slowly by through pH=6.0, CM52 Mierocrystalline cellulose chromatography post that 0.02mol/L phosphoric acid buffer balance is good, with the 3 times of same phosphoric acid buffers washing of volume chromatography columns, removing is to adsorb impurity (albumen) again.Use respectively by the preparation of pH=6.5,0.02mol/L phosphate buffered saline buffer 60,120,180,240,300,360,420,480,540mmol/L NaCl solution (containing 0.1%TritonX-114) carries out gradient elution, be in charge of collection, survey enzyme work, be associated with the elutriant of enzyme N,O-Diacetylmuramidase alive.With molecular weight cut-off is that 4 ℃ of 7000 dialysis tubings are 4 ℃ of dialysis of polyoxyethylene glycol of 8000 to molecular weight to the bag liquid is 3mL.Enzyme liquid after the dialysis is used the gel-filtration of above-mentioned same phosphate buffered saline buffer equilibrated Sephadex-G75 chromatography column again, and the phosphate buffered saline buffer wash-out of 120% column volume is collected the component that has enzyme to live.Chromatography column is the Sephadex-G75 gel column of 1.8cm * 60cm.
Be associated with the elutriant that enzyme is lived,,, obtain the pure product of N,O-Diacetylmuramidase of 0.31mg at last then at-70 ℃ of cold-trap 12h at-40 ℃ of freezing 0.5h.
Embodiment 2
5 gram Intestinum Stichopi japonici deionized waters are cleaned homogenate.With 5 times of volume Tris-HCl damping fluids, 5 ℃ of lixiviate 12h.4 ℃ of following 10000g 10min are centrifugal, 25 ℃ of water-bath 10min of supernatant liquor, and 25 ℃ of centrifugal 10min of following 2000g collect lower floor's washing agent phase then, are the concentrated solution of lysozyme.Be 7000 4 ℃ of pH=6.0 of dialysis tubing, the 0.02mol/L phosphate buffered saline buffer 12h that dialyses then, must treat sample enzyme liquid 8 times of volumes with molecular weight cut-off.Last sample enzyme liquid carries out CM52 Mierocrystalline cellulose chromatography column chromatography with embodiment 1 same method and phosphoric acid buffer washing chromatography column serves as an absorption impurity (albumen) to remove.Chromatography column is the Sephadex-G75 gel column of 1.8cm * 60cm.
Adopt linear gradient elution method to collect the elutriant of lysozyme with embodiment 1, with molecular weight cut-off is that 4 ℃ of 7000 dialysis tubings are 4 ℃ of dialysis of polyoxyethylene glycol of 8000 to molecular weight to the bag liquid is 4ml, enzyme liquid after the dialysis is used through pH=6.0, the gel-filtration of 0.02mol/L phosphate buffered saline buffer equilibrated Sephadex-G75 chromatography column again, the phosphate buffered saline buffer wash-out of 120% column volume is collected the component that has enzyme to live.
Be associated with the elutriant that enzyme is lived,,, obtain the pure product of N,O-Diacetylmuramidase of 0.35mg at last then at-70 ℃ of cold-trap 24h at-40 ℃ of freezing 1h.
Embodiment 3
Clean, the homogenate of 5 gram Intestinum Stichopi japonicis.With 4 ℃ of lixiviate 24h of 8 times of volume Tris-HCl damping fluids.4 ℃ of following 10000g 10min are centrifugal, 25 ℃ of water-bath 15min of solution, and 25 ℃ of centrifugal 15min of following 2000g collect the concentrated solution of washing agent phase-lysozyme.Be that 7000 4 ℃ of pH=6.0 to 10 times of volumes of dialysis tubing, 0.02mol/L phosphate buffered saline buffer were dialysed 12 hours then, must go up sample enzyme liquid with molecular weight cut-off.Last sample enzyme liquid carries out the CM52 cellulose chromatography, washs to remove with the identical method of embodiment is absorption impurity, gradient elution, collect the elutriant of lysozyme, and be that 4 ℃ of 7000 dialysis tubings are 4 ℃ of dialysis of polyoxyethylene glycol of 8000 to molecular weight to the bag liquid is 5ml with molecular weight cut-off, enzyme liquid after the dialysis is used same phosphate buffered saline buffer equilibrated Sephadex-G75 chromatography column gel-filtration again, the phosphate buffered saline buffer wash-out of 120% column volume is collected the component that has enzyme to live.
Be associated with the elutriant that enzyme is lived,,, obtain the pure product of N,O-Diacetylmuramidase of 0.37mg at last then at-70 ℃ of cold-trap 24h at-40 ℃ of freezing 2h.
Embodiment 4
Clean, the homogenate of 5 gram Intestinum Stichopi japonicis.With 4 ℃ of lixiviate 24h of 5 times of volume Tris-HCl damping fluids.4 ℃ of following 10000g 10min are centrifugal, 30 ℃ of water-bath 10min of solution, and 30 ℃ of centrifugal 5min of following 2000g collect the concentrated solution of washing agent phase-lysozyme.Following molecular weight cut-off is the elutriant of 7000 dialysis, CM52 cellulose chromatography, washing, linear gradient elution method and collection lysozyme, be 7000 dialysis tubing with molecular weight cut-off to molecular weight be that 8000 polyoxyethylene glycol dialysis, the gel-filtration of Sephadex-G75 chromatography column and wash-out are collected and steps such as component that enzyme lives arranged with embodiment 1.
Be associated with the elutriant that enzyme is lived,,, obtain the pure product of N,O-Diacetylmuramidase of 0.42mg at last then at-70 ℃ of cold-trap 24h at-40 ℃ of freezing 1h.
Embodiment 5
, homogenate clean 5 grams.With 4 ℃ of lixiviate 24h of 5 times of volume Tris-HCl damping fluids (50mmol/L, pH 7.5, contain 2%Triton X-114).4 ℃ of following 10000g 10min are centrifugal, and 37 ℃ of water-bath 10min of solution make water and washing agent layering mutually, and 37 ℃ of centrifugal 10min of following 2000g collect lower floor's washing agent phase then.Then with molecular weight cut-off be 4 ℃ of 7000 dialysis tubings to 7 times of volume pH=6.5, the dialysis of 0.02mol/L phosphate buffered saline buffer 12 hours, must liquid for treating sample enzyme liquid.Liquid-transfering gun with 1mL adds upward sample enzyme liquid along post jamb, and with the 1.0mL/min flow velocity slowly by pH=6.5, CM52 Mierocrystalline cellulose chromatography post that 0.02mol/L phosphoric acid buffer balance is good, serve as to adsorb impurity with 3 times of same phosphoric acid buffers washing chromatography columns of volume to remove again.Chromatography column is the Sephadex-G75 gel column of 1.8cm * 60cm.
Employing is carried out gradient elution with embodiment 1 same method, collect the elutriant of lysozyme, with molecular weight cut-off is that 4 ℃ of 7000 dialysis tubings are 4 ℃ of dialysis of polyoxyethylene glycol of 8000 to molecular weight to the bag liquid is 3ml, enzyme liquid after the dialysis is used same phosphate buffered saline buffer equilibrated Sephadex-G75 chromatography column gel-filtration again, the phosphate buffered saline buffer wash-out of 120% column volume is collected the component that has enzyme to live.Chromatography column is the Sephadex-G75 gel column of 1.8cm * 60cm.
Be associated with the elutriant that enzyme is lived,,, obtain the pure product of N,O-Diacetylmuramidase of 0.45mg at last then at-70 ℃ of cold-trap 24h at-40 ℃ of freezing 1h.
Embodiment 6
Clean, the homogenate of 5 gram Intestinum Stichopi japonici deionized waters, with 4 ℃ of lixiviate 24h of 5 times of volume Tris-HCl damping fluids (50mmol/L, pH 7.5, contain 2%Triton X-114).4 ℃ of following 10000g 10min are centrifugal, and 37 ℃ of water-bath 10min of solution make water and washing agent layering mutually, and 37 ℃ of centrifugal 10min of following 2000g collect lower floor's washing agent phase then.Then be 7000 4 ℃ of pH=7.0 of dialysis tubing, 0.05mol/L phosphate buffered saline buffer dialysis 24h to 9 times of volumes with molecular weight cut-off, must liquid for treating sample enzyme liquid.Liquid-transfering gun with 1mL adds upward sample enzyme liquid along post jamb, and slowly pass through pH=7.0 with the 1.0mL/min flow velocity, 0.05mol/L the CM52 Mierocrystalline cellulose chromatography post that the phosphoric acid buffer balance is good, again with 3 times of phosphoric acid buffer washing chromatography columns that volume is same, remove and be absorption impurity (albumen), adopt the same gradient elution of embodiment 1, collect the elutriant of lysozyme, with molecular weight cut-off is that 4 ℃ of 7000 dialysis tubings are 4 ℃ of dialysis of polyoxyethylene glycol of 8000 to molecular weight to the bag liquid is 3ml, enzyme liquid after the dialysis is used through same phosphate buffered saline buffer equilibrated Sephadex-G75 chromatography column gel-filtration again, the phosphate buffered saline buffer wash-out of 120% column volume is collected the component that has enzyme to live.Chromatography column is the Sephadex-G75 gel column of 1.8cm * 60cm.
Be associated with the elutriant that enzyme is lived,,, obtain the pure product of N,O-Diacetylmuramidase of 0.46mg at last then at-70 ℃ of cold-trap 24h at-40 ℃ of freezing 1h.
Claims (4)
1. the preparation method of a N,O-Diacetylmuramidase is characterized in that it being the preparation method of Intestinum Stichopi japonici N,O-Diacetylmuramidase of Echinodermata N,O-Diacetylmuramidase, and its processing step is:
(1) homogenate: Intestinum Stichopi japonici is clean with deionized water, homogenate;
(2) lixiviate:, leave standstill 4～24h under 2～5 ℃ with the lixiviate of 3～8 times of volume Tris-HCl buffer solns; 4 ℃ of following 10000g 10min are centrifugal, and supernatant liquor both had been the crude enzyme liquid of lysozyme;
Described Tris-HCl damping fluid specification is: 50mmol/L, and pH 7.5, contain 0.1%～2%Triton X-114;
(3) concentrate: 25 ℃～37 ℃ water-bath 5～15min make water and washing agent layering mutually with crude enzyme liquid, and 25 ℃～37 ℃ centrifugal 5～15min of following 2000g collect the washing agent phase, are the concentrated solution of lysozyme, and 4 ℃ of preservations are standby;
(4) dialysis: it is 7000 dialysis tubing that concentrated solution is put into molecular weight cut-off, then dialysis tubing is put into pH=6.0～7.0 that contain 5～10 times of volumes, the container of 0.02mol/L～0.05mol/L phosphate buffered saline buffer, 4 ℃ of dialysis 12～24h;
(5) cellulose column absorption: the concentrated solution that adds the process dialysis along post jamb, with the 1.0ml/min flow velocity by through pH=6.0～7.0, the CM52 Mierocrystalline cellulose chromatography column chromatography crossed of 0.02～0.05mol/L phosphate buffered saline buffer balance, with 3 times of volume pH=6.0～7.0,0.02mol/L～0.05mol/L phosphate buffered saline buffer washing chromatography column, remove and do not adsorb impurity again;
(6) wash-out: use the NaCl solution that contains equal difference concentration by pH=6.0～7.0,0.02mol/L～0.05mol/L phosphate buffered saline buffer preparation respectively, adsorption column is carried out gradient elution, flow velocity 1.0mL/min collects the component that has enzyme to live respectively and merges 4 ℃ of preservations;
Described NaCl strength of solution is respectively 60,120,180,240,300,360,420,480,540mmol/L, all contains 0.1%TritonX-114 in the solution;
(7) dialysis: it is 7000 dialysis tubing that the enzyme liquid after will merging is put into molecular weight cut-off, dialysis tubing put into to contain the container that molecular weight is 8000 polyoxyethylene glycol then, and 4 ℃ of dialysis, enzyme liquid is 3～5ml to the bag, concentrated 4 ℃ of preservations of enzyme liquid are standby in the bag taking;
(8) gel column absorption and wash-out: elutriant is used through pH=6.0, the gel-filtration of 0.02mol/L phosphate buffered saline buffer equilibrated Sephadex-G75 chromatography column again, and the phosphate buffered saline buffer wash-out of 120% column volume is collected the component that has enzyme to live;
(9) drying: the elutriant lyophilize that will contain N,O-Diacetylmuramidase gets the pure product of N,O-Diacetylmuramidase.
2. the extracting method of N,O-Diacetylmuramidase according to claim 1 is characterized in that in (2) lixiviate step, and Intestinum Stichopi japonici stirrer homogenate is used the lixiviate of 5 times of volume 2%TritonX114 solution, 4 ℃ of standing over night again; 4 ℃ of following 10000g 10min are centrifugal.
3. the extracting method of N,O-Diacetylmuramidase according to claim 1 is characterized in that in absorption of (8) gel column and elution step the Sephadex-G75 gel column that described Sephadex-G75 chromatography column is 1.8cm * 60cm.
4. the extracting method of N,O-Diacetylmuramidase according to claim 1, it is characterized in that in (9) lyophilize step, being associated with the elutriant that enzyme is lived, at-40 ℃ of freezing 0.5h～2h, then, obtain the pure product of exsiccant N,O-Diacetylmuramidase at last at-70 ℃ of cold-trap 12h～24h.
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| Tor Haug et al..Antibacterial activity in Strongylocentrotus droebachiensis(Echinoidea), Cucumaria frondosa (Holothuroidea), andAsterias rubens (Asteroidea).Journal of Invertebrate Pathology81 2.2002,81(2),94-102. |
| Tor Haug et al..Antibacterial activity in Strongylocentrotus droebachiensis(Echinoidea), Cucumaria frondosa (Holothuroidea), andAsterias rubens (Asteroidea).Journal of Invertebrate Pathology81 2.2002,81(2),94-102. * |
| 杨西建等.海参i 型溶菌酶基因及其编码产物的结构特点.中国生物化学与分子生物学报23 7.2007,23(7),542-547. |
| 杨西建等.海参i 型溶菌酶基因及其编码产物的结构特点.中国生物化学与分子生物学报23 7.2007,23(7),542-547. * |
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