CN103397060A - Preparation method of phosphorylated peptidoglycan - Google Patents
Preparation method of phosphorylated peptidoglycan Download PDFInfo
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- CN103397060A CN103397060A CN2013103014177A CN201310301417A CN103397060A CN 103397060 A CN103397060 A CN 103397060A CN 2013103014177 A CN2013103014177 A CN 2013103014177A CN 201310301417 A CN201310301417 A CN 201310301417A CN 103397060 A CN103397060 A CN 103397060A
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Abstract
The invention discloses a preparation method of phosphorylated peptidoglycan. The method is characterized by comprising the following steps of: inoculating Lactobacillus acidophilus on a culture medium for cultivation, centrifuging to collect thallus, repeatedly washing the thallus, suspending the thallus in hydrofluoric acid solution for one night at 4 DEG C, centrifugally collecting cell wall sediment, and washing the sediment to become neutral; dissolving the sediment into Trypsin chymotrypsin phosphate buffer solution, inactivating in boiling water, centrifuging the liquid supernatant, washing the sediment; dissolving the sediment in diethyl ether, stirring and centrifuging to collect sediment, and washing the sediment with water until completely removing the smell of diethyl ether, then obtaining the peptidoglycan; dissolving the peptidoglycan in Lysostaphin phosphate buffer solution for performing enzymolysis and centrifuging, taking the liquid supernatant, centrifuging the liquid supernatant, and centrifugally washing the sediment with water to get micromolecule peptidoglycan; and finally, dissolving the micromolecule peptidoglycan in mixed phosphate solution, precipitating with alcohol after reaction, treating the alcohol precipitated peptidoglycan by freeze drying and redissolving, and adding the redissolved solution in a dialysis bag, performing dialysis concentration, and then, performing freeze drying to get the phosphorylated peptidoglycan. The method has the advantages of increasing the solubility remarkably and improving the immunological effect to a certain extent.
Description
Technical field
The present invention relates to a kind of preparation method of peptidoglycan, especially relate to a kind of preparation method of phosphorylated peptide glycan.
Background technology
Milk-acid bacteria, as a kind of important beneficial bacteria of intestinal tract, has vital role in the diseases such as regulating intestinal canal microorganism species, enhancing body immunizing power and prophylaxis of tumours.The bifidus bacillus that recent domestic studies for a long period of time except concern, Bacillus subtilus, start again target lock-on in another important member---Lactobacterium acidophilum.This bacterioid utilizes distinctive peptidoglycan composition to induce the release of various cytokines and then the immunologic function of regulating the host.
Peptidoglycan is one of chief component composition of bacteria cell wall.Its monomer is to consist of a tetrapeptide tail and a disaccharide unit.This microbial polysaccharide is because of the singularity of its peptide segment structure, the length of carbohydrate polymeric chain, and has different immune effectiveness.Just take the immuno-potentiation of mouse as model research lactobacillus peptide glycan, result shows that peptidoglycan can improve the ND Vaccine immune effect as the people such as Yao Guangguo of " microbiology circular " 34 the 1st phases of volume in 2007; The people such as Zhang Yue size and immune relation to the peptidoglycan molecular weight in 13 the 4th phases of volume in 2006 " Chinese aquatic science " studied, and finds that finally the increase of lower molecular weight peptidoglycan is conducive to the enhancing of immunological effect.Although peptidoglycan has good immuno-potentiation, research and the product development for it is very rare at present, traces it to its cause and is the low-solubility of itself, because solubility problem also makes peptidoglycan, only is confined to solid immunity enhancement adjuvant field.
It is a kind of covalent modification that phosphorylated molecules is modified, and is the process that the hydroxyl on side chain is replaced by phosphate radical, thereby helps immunological effect, the anti-tumor capacity of carbohydrate, the further enhancing of anticoagulation ability.But mainly concentrate on starch, chitosan and Mierocrystalline cellulose etc. about phosphorylation modification at present, the research of peptidoglycan phosphorylation modification aspect is so far there are no report.
Summary of the invention
Technical problem to be solved by this invention is to provide the preparation method of the phosphorylated peptide glycan that a kind of solvability significantly increases, immunological effect increases.
The present invention solves the problems of the technologies described above the technical scheme that adopts: a kind of preparation method of phosphorylated peptide glycan specifically comprises the following steps:
(1) preparation of peptidoglycan
A. microorganism collection: the Lactobacterium acidophilum that will activate the by volume inoculum size of per-cent 3-5% is inoculated in the MRS liquid nutrient medium, the 37-40 ℃ of standing cultivation of constant incubator 16-24h, the centrifugal 10-20min of 5000-8000r/min collects thalline, and with the distilled water repetitive scrubbing until thalline be creamy white;
B. dephosphorization Teichaic acid: the thalline that step a is obtained is suspended in the hydrofluoric acid solution that concentration is 40-50% by the solid-to-liquid ratio of 1g:9ml, 4 ℃ of placements are spent the night, the centrifugal 10-20min of 7000-9000 r/min collects the cell walls precipitation that does not contain teichoic acid, and with distilled water wash, is precipitated to neutrality;
C. enzymolysis broken wall: the precipitation that step b is obtained is dissolved in the Trypsin-chymotrypsin phosphoric acid buffer by the solid-to-liquid ratio of 2g:5ml, 37-40 ℃ of shaking table 110-130r/min vibration 15-17h, boiling water bath deactivation 4-6min, centrifugal 4-6min under the rotating speed of 1500-2500r/min, discard undissolved proteolytic enzyme precipitation, with supernatant liquor in the centrifugal 10-20min of 8000-12000r/min, the precipitation that obtains distilled water centrifuge washing;
D. grease removal: the precipitation that obtains after step c is washed is dissolved in ether by the solid-to-liquid ratio of 1g:1ml, and after stirring 3-5h, the centrifugal 10-20min collecting precipitation of 7000-9000r/min, be washed with distilled water to without the ether flavor and obtain peptidoglycan;
(2) reduce molecular weight:
The solid-to-liquid ratio of peptidoglycan by 1g:5ml is dissolved in the N,O-Diacetylmuramidase phosphoric acid buffer, 36-38 ℃ is stirred enzymolysis 6-10h, centrifugal 4-6min under the rotating speed of 1500-2500r/min, get supernatant liquor, again with supernatant liquor in the centrifugal 10-20min of 8000-12000r/min, get precipitation and use the distilled water centrifuge washing, obtain the small-molecular peptides glycan;
(3) mixed phosphate is modified
The solid-to-liquid ratio of small-molecular peptides glycan by 1g:10ml is dissolved in the compound phosphoric acid salts solution that concentration is 0.003-0.006g/ml, after 75-85 ℃ of reaction 4-6h, again with the 95% ethanol precipitation 20-28h of reaction solution with 8 times of reaction solution volumes, with the lyophilize of alcohol precipitation peptidoglycan, redissolve in 55-65 ℃ of water-bath again, it is 7000 dialysis tubing that solution after redissolution is placed in molecular weight cut-off, dialysis is after 2-3 days take distilled water as dialyzate, liquid concentration in dialysis tubing after 2-5ml, is carried out lyophilize and obtained the phosphorylated peptide glycan.
The compound method of described MRS liquid nutrient medium is as follows: with peptone 10g, extractum carnis 10g, yeast extract 5g, glucose 10g, triammonium citrate 2g, dipotassium hydrogen phosphate 2g, sodium-acetate 5g, sal epsom 0.58g, manganous sulfate 0.25, tween 80 1ml, adding distil water is settled to 1000ml, regulates pH to 7.2-7.4 and gets final product.
The compound method of described Trypsin-chymotrypsin phosphoric acid buffer is as follows: 150mg trypsinase and 25mg Chymetin are dissolved in the 0.1mol/L sodium phosphate buffer of 50mL and get final product, its pH is 8.0.
The compound method of described N,O-Diacetylmuramidase phosphoric acid buffer is as follows: 10mg N,O-Diacetylmuramidase and 200 μ g mutanolysin are dissolved in the 0.1mol/L sodium phosphate buffer of 50mL and get final product, its pH is 5.8.
Described composite phosphate is mixed by tripoly phosphate sodium STPP and the Trisodium trimetaphosphate mass ratio by 5:1.
Compared with prior art, the invention has the advantages that: the preparation method of a kind of phosphorylated peptide glycan of the present invention, at first the Lactobacterium acidophilum thalline is extracted former peptidoglycan as raw material, secondly make hydrofluoric acid first remove teichoic acid enzymolysis broken wall again under mild conditions, then adopt enzymolysis process to reduce its molecule aggregation degree, increase its solvability in solvent; Utilize finally the method for mixed phosphate to modify its structure, to promote its performance in immunological effect.Advantage is as follows:
(1) adopt hydrofluoric acid first to remove the teichoic acid method of enzymolysis again in the peptidoglycan leaching process, so not only increased the permeability of cell walls but also promoted the performance of Trypsin-chymotrypsin enzymolysis usefulness, Labor-saving high-efficiency on the basis of not damaging other materials;
(2) add Chymetin in the enzymolysis broken wall, can strengthen the performance of trypsinase effectiveness, the Chymetin vigor is high than trypsinase, at first the structural protein on cell walls surface are adhered to or sticked to an enzymolysis part, and then increase its permeability, the albumen to around the peptidoglycan skeleton that trypsinase can be single-minded is played effectiveness, improve enzymolysis efficiency;
(3) adopt mutanolysin to make N,O-Diacetylmuramidase damping fluid with the quality of 1:50 than compatibility with N,O-Diacetylmuramidase.At first be to cut scope because mutanolysin has the enzyme of high-efficiency broad spectrum, its glycosidic link that can not only make the N,O-Diacetylmuramidase effect that adds ruptures, and can also make some cardohydrata-peptide linkages and peptide bond rupture, cuts scope with regard to the enzyme that has improved the N,O-Diacetylmuramidase damping fluid like this; And mixed enzymolysis liquid is faster at same time internal ratio N,O-Diacetylmuramidase enzymolysis speed, better effects if.Secondly be best enzymolysis ratio by drawing after the optimization of response surface method with quality of 1:50 than compatibility, enzymolysis effectiveness will reach and fully play most with this understanding;
(4) mixing solutions that adopts tripoly phosphate sodium STPP and Trisodium trimetaphosphate is as the phosphorylation modification method, and is not only nontoxic but also can make phosphate radical obtain maximum replacement rate; Reaction conditions gentleness, reagent safety, immune effectiveness that phosphorylated molecules is modified are remarkable.
In sum, extraction process optimization of the present invention each step condition so that the reagent utilization maximizes; After adopting the method modified peptides glycan of phosphorylation, not only solved the poor solubility problem of peptidoglycan, and because of the introducing of phosphate radical, also give peptidoglycan better immunological effect, this has opened up a new thinking for peptidoglycan in the research aspect medicines and health protection, immunological adjuvant.Preparation method of the present invention has low cost, high production, and is practical, operates the advantages such as reasonable, is fit to very much plant size production.
Description of drawings
Fig. 1 is the Infrared spectrum scanning spectrogram of the present invention without the peptidoglycan of phosphorylation;
Fig. 2 is the Infrared spectrum scanning spectrogram of phosphorylated peptide glycan of the present invention;
Fig. 3 is physiological saline group mice spleen slice map;
Fig. 4 is peptidoglycan group (not phosphorylation) mice spleen slice map;
Fig. 5 is phosphorylated peptide glycan group mice spleen slice map;
Fig. 6 is physiological saline group mouse thymus slice map;
Fig. 7 is peptidoglycan group mouse thymus slice map;
Fig. 8 is phosphorylated peptide glycan group mouse thymus slice map.
Embodiment
Embodiment is described in further detail the present invention below in conjunction with accompanying drawing.
One, specific embodiment
Embodiment 1
The present invention is used common commercially available Lactobacterium acidophilum bacterium powder and is carried out the extraction of peptidoglycan as raw material.At first 2mg bacterium powder is inoculated in 200mL MRS liquid nutrient medium, 37 ℃ of standing cultivation 24h are with activated spawn, and the preparation method of a kind of phosphorylated peptide glycan of the present invention specifically comprises the following steps:
(1) preparation of peptidoglycan
A. microorganism collection: the Lactobacterium acidophilum that will activate the by volume inoculum size of per-cent 4% is inoculated in the MRS liquid nutrient medium, 38.5 the standing cultivation of ℃ constant incubator 20h, the centrifugal 15min of 6500r/min collects thalline, and with the distilled water repetitive scrubbing until thalline be creamy white; Wherein the compound method of MRS liquid nutrient medium is as follows: with peptone 10g, extractum carnis 10g, yeast extract 5g, glucose 10g, triammonium citrate 2g, dipotassium hydrogen phosphate 2g, sodium-acetate 5g, sal epsom 0.58g, manganous sulfate 0.25, tween 80 1ml, adding distil water is settled to 1000ml, regulates pH to 7.2-7.4 and gets final product;
B. dephosphorization Teichaic acid: it is that in 45% hydrofluoric acid solution, 4 ℃ of placements are spent the night that the thalline that step a is obtained is suspended in concentration by the solid-to-liquid ratio of 1g:9ml, and the centrifugal 15min of 8000r/min collects the cell walls precipitation that does not contain teichoic acid, and with distilled water wash, is precipitated to neutrality;
C. enzymolysis broken wall: the precipitation that step b is obtained is dissolved in the Trypsin-chymotrypsin phosphoric acid buffer by the solid-to-liquid ratio of 2g:5ml, 38.5 ℃ shaking table 120r/min vibration 16h, boiling water bath deactivation 5min, centrifugal 5min under the rotating speed of 2000r/min, discard undissolved proteolytic enzyme precipitation, with supernatant liquor in the centrifugal 15min of 10000r/min, the precipitation that obtains distilled water centrifuge washing; Wherein the formula of Trypsin-chymotrypsin phosphoric acid buffer is as follows: 150mg trypsinase and 25mg Chymetin are dissolved in the 0.1mol/L sodium phosphate buffer of 50mL and get final product, its pH is 8.0;
D. grease removal: the precipitation that obtains after step c is washed is dissolved in ether by the solid-to-liquid ratio of 1g:1ml, and after stirring 4h, the centrifugal 15min collecting precipitation of 8000r/min, be washed with distilled water to without the ether flavor and obtain peptidoglycan;
(2) reduce molecular weight:
The solid-to-liquid ratio of peptidoglycan by 1g:5ml is dissolved in the N,O-Diacetylmuramidase phosphoric acid buffer, 37 ℃ are stirred enzymolysis 8h, and centrifugal 5min, get supernatant liquor under the rotating speed of 2000r/min, again with supernatant liquor in the centrifugal 15min of 10000r/min, get the precipitation obtain the small-molecular peptides glycan with the distilled water centrifuge washing; Wherein the formula of N,O-Diacetylmuramidase phosphoric acid buffer is as follows: 10mg N,O-Diacetylmuramidase and 200 μ g mutanolysin are dissolved in the 0.1mol/L sodium phosphate buffer of 50mL and get final product, its pH is 5.8;
(3) mixed phosphate is modified
be dissolved in the small-molecular peptides glycan in the compound phosphoric acid salts solution (composite phosphate is mixed by tripoly phosphate sodium STPP and the Trisodium trimetaphosphate mass ratio by 5:1) of 0.0045g/ml by the solid-to-liquid ratio of 1g:10ml, after 80 ℃ of reaction 5h, again with the 95% ethanol precipitation 24h of reaction solution with 8 times of reaction solution volumes, with the lyophilize of alcohol precipitation peptidoglycan, redissolve in 60 ℃ of water-baths again, it is 7000 dialysis tubing that solution after redissolution is placed in molecular weight cut-off, dialysis is after 2.5 days take distilled water as dialyzate, with liquid concentration in dialysis tubing after 3.5mL, carry out lyophilize and obtain the phosphorylated peptide glycan.
Embodiment 2
With embodiment 1, its difference is:
(1) preparation of peptidoglycan
With the Lactobacterium acidophilum of activation by volume the inoculum size of per-cent 3% be inoculated in the MRS liquid nutrient medium, 37 ℃ of standing cultivations of constant incubator 16h, the centrifugal 20min of 5000r/min collection thalline, and with the distilled water repetitive scrubbing until thalline be creamy white; It is that in 40% hydrofluoric acid solution, 4 ℃ of placements are spent the night that thalline is suspended in concentration by the solid-to-liquid ratio of 1g:9ml, and the 7000 centrifugal 20min of r/min collect the cell walls precipitation that does not contain teichoic acid, and with distilled water wash, are precipitated to neutrality; The solid-to-liquid ratio of precipitation by 2g:5ml is dissolved in the Trypsin-chymotrypsin phosphoric acid buffer, 37 ℃ of shaking table 110r/min vibration 17h, boiling water bath deactivation 4min, centrifugal 6min under the rotating speed of 1500r/min, discard undissolved proteolytic enzyme precipitation, with supernatant liquor in the centrifugal 20min of 8000r/min, the precipitation that obtains distilled water centrifuge washing; The precipitation that obtains after step c is washed is dissolved in ether by the solid-to-liquid ratio of 1g:1ml, and after stirring 3h, the centrifugal 20min collecting precipitation of 7000r/min, be washed with distilled water to without the ether flavor and obtain peptidoglycan;
(2) reduce molecular weight
The solid-to-liquid ratio of peptidoglycan by 1g:5ml is dissolved in the N,O-Diacetylmuramidase phosphoric acid buffer, and 36 ℃ are stirred enzymolysis 10h, and centrifugal 6min, get supernatant liquor under the rotating speed of 1500r/min, then with supernatant liquor in the centrifugal 20min of 8000r/min;
(3) mixed phosphate is modified
The solid-to-liquid ratio of small-molecular peptides glycan by 1g:10ml is dissolved in the compound phosphoric acid salts solution that concentration is 0.006g/ml, after 75 ℃ of reaction 6h, 95% ethanol precipitation 20h with 8 times of volumes, with the lyophilize of alcohol precipitation peptidoglycan, redissolve in 55 ℃ of water-baths again, dialysis is 2 days in dialysis tubing, and liquid concentration in dialysis tubing after 2mL, is carried out lyophilize and obtained the phosphorylated peptide glycan.
Embodiment 3
With embodiment 1, its difference is:
(1) preparation of peptidoglycan
With the Lactobacterium acidophilum of activation by volume the inoculum size of per-cent 5% be inoculated in the MRS liquid nutrient medium, 40 ℃ of standing cultivations of constant incubator 24h, the centrifugal 10min of 8000r/min collection thalline, and with the distilled water repetitive scrubbing until thalline be creamy white; It is that in 50% hydrofluoric acid solution, 4 ℃ of placements are spent the night that thalline is suspended in concentration by the solid-to-liquid ratio of 1g:9ml, the 9000 centrifugal 10min collecting cell of r/min walls precipitations, and with distilled water wash, be precipitated to neutrality; The solid-to-liquid ratio of precipitation by 2g:5ml is dissolved in the Trypsin-chymotrypsin phosphoric acid buffer, 40 ℃ of shaking table 130r/min vibration 15h, boiling water bath deactivation 6min, centrifugal 4min under the rotating speed of 2500r/min, with supernatant liquor in the centrifugal 10min of 12000r/min, the precipitation that obtains distilled water centrifuge washing; The solid-to-liquid ratio of precipitation by 1g:1ml is dissolved in ether, and after stirring 5h, the centrifugal 10min collecting precipitation of 9000r/min, be washed with distilled water to without the ether flavor and obtain peptidoglycan;
(2) reduce molecular weight:
The solid-to-liquid ratio of peptidoglycan by 1g:5ml is dissolved in the N,O-Diacetylmuramidase phosphoric acid buffer, and 38 ℃ are stirred enzymolysis 6h, and centrifugal 4min, get supernatant liquor under the rotating speed of 2500r/min, then with supernatant liquor in the centrifugal 10min of 12000r/min;
(3) mixed phosphate is modified
The solid-to-liquid ratio of small-molecular peptides glycan by 1g:10ml is dissolved in the compound phosphoric acid salts solution that concentration is 0.003g/ml, after 85 ℃ of reaction 4h, with the 95% ethanol precipitation 28h of reaction solution with 8 times of reaction solution volumes, with the lyophilize of alcohol precipitation peptidoglycan, redissolve in 65 ℃ of water-baths again, be placed in the dialysis tubing dialysis after 3 days, liquid concentration in dialysis tubing after 5mL, is carried out lyophilize and obtained the phosphorylated peptide glycan.
Two, measuring
1, experimental determining method
1.1 infrared spectra detects: former peptidoglycan and phosphorylated peptide glycan are adopted KBr compressing tablet, 400-4000cm
-1Between scanning, measure infrared spectra.
1.2 immunological effect
1.2.1 Immune Organs Index: choose 24 of normal male kunming mices, body weight is all at 30 ± 2g.Mouse is divided into these 3 groups of control group (physiological saline), peptidoglycan group and phosphorylated peptide glycan groups, 8 every group at random.And adopt the primary sterilization syringe, with the dosage of 20mg/kgd, mouse is carried out the gavage processing, inject a week continuously, stop injecting and next day all mouse are weighed and put to death, thymus gland, the spleen weight of mouse respectively organized in record, statistics mouse immune organ (thymus gland, spleen) Gain weight, formula is as follows: Immune Organs Index=immune organ fresh weight (mg)/body weight (g), and with SPSS software, statistic data is carried out variance analysis.
1.2.2 tissue slice: in the fresh immune organ that takes out every group select one coated with tinfoil, throw quick-frozen 5min in liquid nitrogen, then put into immediately the fixing section of slicing machine, use the dyeing of HE method after section, observation of cell changing conditions under opticmicroscope.
2, experimental result
2.1 Infrared spectrum scanning spectrogram
By Fig. 1 and Fig. 2 as can be known, former peptidoglycan and phosphorylated peptide glycan all have 3415.1cm
-1, 3395cm
-1-the 0H stretching vibration, 2960.9cm
-1, 2927cm
-1The charateristic avsorption band of the glucides such as C-H stretching vibration, and at 905cm
-1-
876cm
-1There is the characteristic peak of β-D-glucopyanosyl at place.Can find to exist 1290cm in the structure iron of phosphorylated peptide glycan
-1The P=O key, prove that phosphate radical is grafted on peptidoglycan.
2.2 immunological effect
2.2.1 Immune Organs Index
By following table 1 as can be known, with control group relatively, peptidoglycan group and phosphorylated peptide glycan group are to thymus index, the index and spleen index of mouse all be significantly increased (P＜0.05); Phosphorylated peptide glycan group also has certain increase (P＜0.05) to the thymus index of mouse than the peptidoglycan group.
The impact of table 1 peptidoglycan on the mouse immune organ
| Group | Index and spleen index (mg/g) | Thymus index (mg/g) |
| Control group | 4.0436±0.2015 a | 2.1450±0.2299 c |
| The peptidoglycan group | 4.8978±0.5238 b | 2.5268±0.0892 d |
| Phosphorylated peptide glycan group | 5.0506±0.5464 b | 2.8093±0.1747 e |
Annotate: in same mensuration project, significant difference between different letters (P＜0. 05); Difference not significantly (P〉0.05) between same letter.
2.2.2 immune slice analysis
The tangent plane situation of mouse thymus after the tangent plane situation of mouse spleen and three group gavages of Fig. 6-Fig. 8 after three group gavages of observation Fig. 3-Fig. 5, compared to the physiological saline control group, two groups of test group spleen white pulps, red pulps are demarcated, and obviously (white pulp: stain dense arrangement and color are darker; Red pulp: stain arrangement evacuation and color are more shallow), the white pulp area increases, and lymph trifle and acini lienalis (being positioned at white pulp, visible germinal center) increase; Thymus gland shows as cortex/medullary substance (cortex: the dark zone of stain dense arrangement, color; Medullary substance: stain distribution evacuation, the more shallow zone of color) ratio significantly improves.Compared to phosphorylation modification peptidoglycan group not, the spleen of phosphorylation modification group, thymus gland changing conditions are more obvious.In addition, each organizes the appearance of mouse immune slices of organs no abnormality seen changes in microstructure.As seen the phosphorylated peptide glycan plays certain enhancement to immunizing power.
Certainly, above-mentioned explanation is not limitation of the present invention, and the present invention also is not limited to above-mentioned giving an example.Those skilled in the art are in essential scope of the present invention, and the variation of making, remodeling, interpolation or replacement, also should belong to protection scope of the present invention.
Claims (5)
1. the preparation method of a phosphorylated peptide glycan, its feature specifically comprises the following steps:
(1) preparation of peptidoglycan
A. microorganism collection: the Lactobacterium acidophilum that will activate the by volume inoculum size of per-cent 3-5% is inoculated in the MRS liquid nutrient medium, the 37-40 ℃ of standing cultivation of constant incubator 16-24h, the centrifugal 10-20min of 5000-8000r/min collects thalline, and with the distilled water repetitive scrubbing until thalline be creamy white;
B. dephosphorization Teichaic acid: the thalline that step a is obtained is suspended in the hydrofluoric acid solution that concentration is 40-50% by the solid-to-liquid ratio of 1g:9ml, 4 ℃ of placements are spent the night, the centrifugal 10-20min of 7000-9000 r/min collects the cell walls precipitation that does not contain teichoic acid, and with distilled water wash, is precipitated to neutrality;
C. enzymolysis broken wall: the precipitation that step b is obtained is dissolved in the Trypsin-chymotrypsin phosphoric acid buffer by the solid-to-liquid ratio of 2g:5ml, 37-40 ℃ of shaking table 110-130r/min vibration 15-17h, boiling water bath deactivation 4-6min, centrifugal 4-6min under the rotating speed of 1500-2500r/min, discard undissolved proteolytic enzyme precipitation, with supernatant liquor in the centrifugal 10-20min of 8000-12000r/min, the precipitation that obtains distilled water centrifuge washing;
D. grease removal: the precipitation that obtains after step c is washed is dissolved in ether by the solid-to-liquid ratio of 1g:1ml, and after stirring 3-5h, the centrifugal 10-20min collecting precipitation of 7000-9000r/min, be washed with distilled water to without the ether flavor and obtain peptidoglycan;
(2) reduce molecular weight:
The solid-to-liquid ratio of peptidoglycan by 1g:5ml is dissolved in the N,O-Diacetylmuramidase phosphoric acid buffer, 36-38 ℃ is stirred enzymolysis 6-10h, centrifugal 4-6min under the rotating speed of 1500-2500r/min, get supernatant liquor, again with supernatant liquor in the centrifugal 10-20min of 8000-12000r/min, get the precipitation obtain the small-molecular peptides glycan with the distilled water centrifuge washing;
(3) mixed phosphate is modified
The solid-to-liquid ratio of small-molecular peptides glycan by 1g:10ml is dissolved in the compound phosphoric acid salts solution that concentration is 0.003-0.006g/ml, after 75-85 ℃ of reaction 4-6h, again with the 95% ethanol precipitation 20-28h of reaction solution with 8 times of reaction solution volumes, with the lyophilize of alcohol precipitation peptidoglycan, redissolve in 55-65 ℃ of water-bath again, it is 7000 dialysis tubing that solution after redissolution is placed in molecular weight cut-off, dialysis is after 2-3 days take distilled water as dialyzate, liquid concentration in dialysis tubing after 2-5ml, is carried out lyophilize and obtained the phosphorylated peptide glycan.
2. the preparation method of a kind of phosphorylated peptide glycan according to claim 1 is characterized in that the compound method of described MRS liquid nutrient medium is as follows: with peptone 10g, and extractum carnis 10g, yeast extract 5g, glucose 10g, triammonium citrate 2g, dipotassium hydrogen phosphate 2g, sodium-acetate 5g, sal epsom 0.58g, manganous sulfate 0.25, tween 80 1ml, adding distil water is settled to 1000ml, regulates pH to 7.2-7.4 and gets final product.
3. the preparation method of a kind of phosphorylated peptide glycan according to claim 1, the compound method that it is characterized in that described Trypsin-chymotrypsin phosphoric acid buffer is as follows: 150mg trypsinase and 25mg Chymetin are dissolved in the 0.1mol/L sodium phosphate buffer of 50mL and get final product, its pH is 8.0.
4. the preparation method of a kind of phosphorylated peptide glycan according to claim 1, the compound method that it is characterized in that described N,O-Diacetylmuramidase phosphoric acid buffer is as follows: 10mg N,O-Diacetylmuramidase and 200 μ g mutanolysin are dissolved in the 0.1mol/L sodium phosphate buffer of 50mL and get final product, its pH is 5.8.
5. the preparation method of a kind of phosphorylated peptide glycan according to claim 1, it is characterized in that: described compound phosphoric acid salts solution is mixed by the mass ratio by 5:1 by sodium tripolyphosphate solution and Trisodium trimetaphosphate solution.
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| CN108003254A (en) * | 2017-12-14 | 2018-05-08 | 浙江海洋大学 | A kind of preparation method and application of phosphorylation carrageenan oligosaccharide |
| CN108396042A (en) * | 2018-03-05 | 2018-08-14 | 郑州安图生物工程股份有限公司 | A kind of preparation method of small soluble molecules peptide glycan |
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