CN105255977A - Porphyra yezoensis antibacterial peptide purification method and amino acid sequence of porphyra yezoensis antibacterial peptide - Google Patents
Porphyra yezoensis antibacterial peptide purification method and amino acid sequence of porphyra yezoensis antibacterial peptide Download PDFInfo
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- CN105255977A CN105255977A CN201510448341.XA CN201510448341A CN105255977A CN 105255977 A CN105255977 A CN 105255977A CN 201510448341 A CN201510448341 A CN 201510448341A CN 105255977 A CN105255977 A CN 105255977A
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Abstract
The invention discloses a porphyra yezoensis antibacterial peptide purification method. The method comprises porphyra yezoensis water-soluble protein primary purification, water-soluble protein II enzymatic hydrolysis and molecular weight grading, antibacterial peptide purification and antibacterial peptide sequence analysis. A porphyra yezoensis water-soluble protein is subjected to salting-out grading under the action of ammonium sulfate with a saturation degree of 30-50% so that a water-soluble protein II is obtained, the water-soluble protein II is degraded by pepsin and is subjected to ultrafiltration grading so that an ingredient with molecular weight less than 5KD is obtained, the ingredient is treated by a Sephadex G-25 chromatography column and then is eluted by distilled water, and the elution curve peak 2 ingredient is purified by the Sephadex C-25 chromatography column and then is freeze-dried so that antibacterial peptide TPDSEAL is obtained and has strong inhibition effects on staphylococcus aureus. The purification method is simple and fast, utilizes porphyra yezoensis with an abundant resource in China as a raw material to prepare the antibacterial peptide and has an important meaning for porphyra yezoensis high-value application.
Description
Technical field:
The present invention relates to one and utilize yezoensis laver water soluble protein, through protein digestion, purifying and sequential analysis, obtain yezoensis laver antibacterial peptide and its aminoacid sequence clear and definite.
Background technology:
Laver (Porphyra) belongs to rhodophyta (Rhodophyta) Bangiales section (Bangiacea), is distributed widely in from frigid zone to semi-tropical intertidal waters.Yezoensis laver (Porphyrayezoensis) is one of important economical alga of China, and its protein content, up to 36.31%, occupies first of marine alga.Research shows, is clearly become the good source of biologically active peptides by certain enzyme degrade proteins.Therefore, yezoensis laver protein is expected the important sources becoming bioactive peptide, has potential application prospect.
Along with pathogenic bacterium resistance problems becomes increasingly conspicuous, the microbiotic finding brand new class is the effective way solving resistance problems.Due to the singularity of Living marine resources, people, to developing more antibacterial specific medicaments newly from marine organisms, have expressed great hope.
The present invention is conceived to take yezoensis laver as raw material, inhibiting polypeptide is had to streptococcus aureus through proteins extraction, purifying, enzymolysis repurity preparation one, and measure its aminoacid sequence, for the higher value application of yezoensis laver and porphyra haitanensis provides novel method.
Summary of the invention:
The object of the present invention is to provide a kind of take yezoensis laver as a kind of method that novel antimicrobial peptide prepared by raw material, and measures with regard to its aminoacid sequence.
To achieve these goals, main process of the present invention comprises the steps such as yezoensis laver water soluble protein preliminary purification, enzymolysis, classification, purifying and sequential analysis, wherein
The preliminary purification of yezoensis laver water soluble protein: first by yezoensis laver water soluble protein solution with 30% saturation ratio ammonium sulfate precipitation, centrifugal segregation precipitates, and supernatant liquor ammonium sulfate saturation ratio is adjusted to 50%, centrifugal, to precipitate through dialysis desalination, make water soluble protein II lyophilized powder.
The enzymolysis of water soluble protein II and molecular-weight gradation: by water soluble protein II with Pepsin degradation, stomach en-usage quantity was 1/1000 of substrate protein white matter quality, medium pH1.5,40 DEG C of enzymolysis 2 hours.By enzymolysis solution ultrafiltration, retain molecular weight < 5KD fraction and freeze-drying preservation.
Antibacterial peptide purifying: it is appropriate to get molecular weight < 5KD fraction, upper SephadexG-25 chromatography column, distilled water wash-out, coutroi velocity 0.7mL/min, within every 4 minutes, collect 1 pipe, after wash-out terminates, under 220nm and 280nm, colorimetric obtains elution curve (Fig. 1) respectively, after determination of activity, peak 2 freeze-drying is obtained peptide sample A.Get A and go up SephadexC-25 chromatography column in right amount, under 220nm and 280nm, colorimetric obtains elution curve (Fig. 2) respectively, and learn the strong bacteriostatic activity of peak 2 tool through determination of activity, it is for subsequent use that freeze-drying obtains peptide sample B;
Antibacterial peptide sequential analysis: carry out the sequencing data collection of mixing mass spectrum and data analysis to peptide sample B, determine that peptide section aminoacid sequence is: TPDSEAL, molecular weight: 732, this antibacterial peptide mass spectrum of iso-electric point (pI): 9.59. is shown in Fig. 3.
Purge process of the present invention is easy, and gained antibacterial peptide product purity is high.
Accompanying drawing explanation
Fig. 1 is SephadexG-25 chromatography elution curves
Fig. 2 is SephadexC-25 chromatography elution curves
Fig. 3 is antibacterial peptide mass spectrum
Embodiment:
Below by example, the present invention is further elaborated:
First by yezoensis laver water soluble protein solution (containing 100.0 grams, protein) with 30% saturation ratio ammonium sulfate precipitation, centrifugal segregation precipitates, and supernatant liquor ammonium sulfate saturation ratio is adjusted to 50%, centrifugal, to precipitate through dialysis desalination, make water soluble protein II lyophilized powder 21.1 grams; Get 10 grams with distilled water dissolve after with Pepsin degradation, stomach en-usage quantity is 1/1000 of substrate protein white matter quality, regulate medium pH1.5,40 DEG C of enzymolysis 2 hours, enzymolysis solution ultrafiltration classification is obtained molecular weight < 5KD fraction lyophilized powder 2.2 grams; Get molecular weight < 5KD fraction appropriate, upper SephadexG-25 chromatography column, distilled water wash-out, coutroi velocity 0.7mL/min, within every 4 minutes, collect 1 pipe, after wash-out terminates, under 220nm and 280nm, colorimetric obtains elution curve (Fig. 1) respectively, peak 2 freeze-drying is obtained peptide sample A; Get A and go up SephadexC-25 chromatography column in right amount, under 220nm and 280nm, colorimetric obtains elution curve (Fig. 2) respectively, peak 2 freeze-drying is obtained peptide sample B for subsequent use; The sequencing data collection of mixing mass spectrum and data analysis are carried out to peptide sample B, determines that peptide section aminoacid sequence is: TPDSEAL, molecular weight: 732, iso-electric point (pI): 9.59.
Claims (1)
1. the purification process of yezoensis laver antibacterial peptide and an aminoacid sequence thereof, comprise the steps:
The preliminary purification of yezoensis laver water soluble protein: first by yezoensis laver water soluble protein solution with 30% saturation ratio ammonium sulfate precipitation, centrifugal segregation precipitates, and supernatant liquor ammonium sulfate saturation ratio is adjusted to 50%, more centrifugal, to precipitate through dialysis desalination, make water soluble protein II lyophilized powder;
The enzymolysis of water soluble protein II and molecular-weight gradation: with Pepsin degradation after water soluble protein II is dissolved with appropriate distilled water, stomach en-usage quantity is 1/1000 of water soluble protein II quality, medium pH1.5,40 DEG C of enzymolysis 2 hours, by enzymolysis solution ultrafiltration, retain molecular weight < 5KD fraction and freeze-drying preservation;
Antibacterial peptide purifying: get molecular weight < 5KD fraction and dissolve rear upper SephadexG-25 chromatography column with distilled water in right amount, distilled water wash-out, coutroi velocity 0.7mL/min, within every 4 minutes, collect 1 pipe, after wash-out terminates, under 220nm and 280nm, colorimetric obtains elution curve respectively, peak 2 freeze-drying is obtained peptide sample A; Get A and go up SephadexC-25 chromatography column in right amount, under 220nm and 280nm, colorimetric obtains elution curve respectively, peak 2 freeze-drying is obtained peptide sample B;
Antibacterial peptide sequential analysis: carry out the sequencing data collection of mixing mass spectrum and data analysis to peptide sample B, determines that peptide section aminoacid sequence is: TPDSEAL.
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| CN201510448341.XA CN105255977A (en) | 2015-07-24 | 2015-07-24 | Porphyra yezoensis antibacterial peptide purification method and amino acid sequence of porphyra yezoensis antibacterial peptide |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154423A (en) * | 2011-01-12 | 2011-08-17 | 淮海工学院 | Method for preparing active peptide of laver |
| CN102495169A (en) * | 2011-11-16 | 2012-06-13 | 江南大学 | Purifying and analyzing identification method for anti-oxidative peptide after controlled-enzymatic hydrolysis of laver |
| CN103865973A (en) * | 2014-04-03 | 2014-06-18 | 江南大学 | Preparation method of porphyra protein antibacterial peptide |
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2015
- 2015-07-24 CN CN201510448341.XA patent/CN105255977A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154423A (en) * | 2011-01-12 | 2011-08-17 | 淮海工学院 | Method for preparing active peptide of laver |
| CN102495169A (en) * | 2011-11-16 | 2012-06-13 | 江南大学 | Purifying and analyzing identification method for anti-oxidative peptide after controlled-enzymatic hydrolysis of laver |
| CN103865973A (en) * | 2014-04-03 | 2014-06-18 | 江南大学 | Preparation method of porphyra protein antibacterial peptide |
Non-Patent Citations (4)
| Title |
|---|
| 刘冬冬等: "条斑紫菜蛋白酶解产物中抗菌肽的纯化及特征", 《天然产物研究与开发》 * |
| 宋惠平等: "条斑紫菜蛋白酶解多肽的抑菌活性", 《渔业科学进展》 * |
| 杜春梅著: "《芽孢杆菌在农业中的研究与应用》", 31 May 2013 * |
| 林元藻主编: "《生化制药学》", 30 June 1998 * |
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Application publication date: 20160120 |