Proteomic analysis of Saccharina japonica and biofunctional characterization of its glycoprotein SJGP
- Alternative Title
- Saccharina japonica의 proteome 분석 및 당단백질 SJGP의 생물학적 기능 연구
- Abstract
- Saccharina japonica which is a brown algae widely distributed in East Asia including Korea, China and Japan is useful in the industries such as food, cosmetics and medical products. Therefore, many researches are concentrated on photosynthesis, extraction, purification and application of S. japonica carbohydrate reported to have biochemical functions. Accordingly, this study has set up a database of S. japonica protein having most functionalities by participating in all of growth, metabolism, reproduction and motion, etc within the organism to verify the functionality of glycoprotein.
The situation is that the researches on protein of S. japonica is very insignificant by experiencing great deal of difficulty in the separation of protein due to various polysaccharides existing on the surface of S. japonica. In this study, an effective lysis of cell has been induced using various lysis buffers and detergents while clearly separating the total protein of S. japonica by attempting the elimination of polysaccharide. As a result of performing 2D-PAGE of the separated S. japonica protein, 200 or more protein spots have been gained and completed identification of protein on 60 spots through MALDI-TOF/MS/MS. Also, the proteins changing in various pH environment have been identified and the proteins increasing or decreasing expression in pH 7.5 and 9.5 have been verified. Such series of researches seem to be very useful in building up the protein database of S. japonica and seem to be possible to use as a biological marker detecting the change of sea environment by observing the change of sea organisms followed by various environmental changes.
Meanwhile, the protein was separated and purified from the S. japonica using only distilled water and ethanol while about 10kDa of protein was verified through SDS-PAGE and periodic acid-Schiff staining. In this study, glycoprotein separated from S. japonica was named ‘SJGP’. To verify the functionality of SJGP, an experiment on DNA damage prevention ability and increasing adhesiveness of lactic acid inside the intestines has been performed. To investigate the antioxidant activity of SJGP, SORS has been measured for the nonenzymatic antioxidant activity as well as the enzymatic antioxidant activities such as DPPH radical scavenging, ABTS radical scavenging, FRAP and SODA, etc while the antioxidant activity IC50 values of SJGP was shown as 0.12, 0.05, 0.3, 1.2, and 1.2 mg/mL, respectively. The xanthine oxidizing enzyme inhibition activity (max 85%) and NO scavenging activity (max 37%) of the separated SJGP also have been measured to prove on the antioxidant effect. Using Cu(II)-ascorbic acid, radical-inducing DNA was experimented while the protection of damage by SJGP has been verified. Such result enables development of antioxidant and DNA protector containing SJGP.
Finally, the improvement on adhesion ability of Lactobacillus plantarum which is lactic acid using SJGP has been verified. As a result of observing with light microscopy and scanning electron microscopy by treating L. plantarum and SJGP to the Caco-2 cell line, the improvement on the access of lactic acid toward the large intestine cells by SJGP has been verified. Also, the number of cells attached to the Caco-2 cell increasing by 1.13-1.86 times depending on the strain was shown. As a result of measuring the changing amount on cell surface hydrophobicity and cellular autoaggregation of L. plantarum, they have improved by maximum of 1.69 times and 2.7 times, respectively by the SJGP so that it seems to be increasing the adhesiveness toward cells. The SJGP was also verified as one of the factors improving adhesiveness by having lipase inhibitory activity. Meanwhile, as a result of verifying the genes participating in the adhesiveness of L. plantarum within the intestines, they have increased by 2.8 times, 3.4 times, 1.5 times and 1.9 times, respectively when 1mg/ml of SJGP has been processed. Through such series of results, the SJGP seems to be usable as a factor improving adhesiveness of lactic acid within the intestines.
- Issued Date
- 2013
- Awarded Date
- 2013. 2
- Type
- Dissertation
- Publisher
- 부경대학교
- Alternative Author(s)
- Kim, Eun Young
- Affiliation
- 부경대학교 대학원
- Department
- 대학원 생물공학과
- Advisor
- 공인수
- Table Of Contents
- LIST OF TABLES -------------------------------------------------------------------------ⅵ
LIST OF FIGURES ------------------------------------------------------------------------ⅶ
ABSTRACT ----------------------------------------------------------------------------------ⅸ
CHAPTER Ⅰ----------------------------------------------------------------------------------1
Literature review
1.1. Properties of seaweed......................................................................................2
1.2. Properties of Saccarina japonaca.....................................................................4
1.3. Proteomic analysis of seaweed.....................................................................6
1.4. Antioxidant activity..........................................................................................8
1.5. Lactic acid bacteria adhesion to intestinal cell......................................12
1.6. Propose of this study....................................................................................15
References..................................................................................................................16
CHAPTER Ⅱ--------------------------------------------------------------------------------20
An improved method of protein isolation and proteome analysis with Saccharina japonica incubated under different pH conditions
Abstract......................................................................................................................21
2.1. Introduction......................................................................................................23
2.2. Materials and methods..................................................................................27
2.2.1. Culture conditions...................................................................................27
2.2.2. Protein preparation..................................................................................27
2.2.3. SDS-PAGE and 2-DE..............................................................................28
2.2.4. MALDI-TOF/MS......................................................................................29
2.3. Results................................................................................................................31
2.3.1. Total protein isolation from S. japonica.............................................31
2.3.2. 2-DE and MALDI-TOF/MS analysis..................................................36
2.3.3. Effect of pH on protein expression....................................................41
2.4. Discussion..........................................................................................................44
References..................................................................................................................52
CHAPTER Ⅲ--------------------------------------------------------------------------------59
Antioxidant and DNA protection activities of a glycoprotein isolated from a seaweed, Saccharina japonica
Abstract......................................................................................................................60
3.1. Introduction......................................................................................................61
3.2. Materials and methods..................................................................................64
3.2.1. Isolation of glycoprotein from S. japonica.........................................64
3.2.2. Antioxidant activity of SJGP................................................................65
3.2.3. Nitric oxide scavenging assay of SJGP.............................................66
3.2.4. Xanthine oxidase inhibition activity of SJGP...................................66
3.2.5. DNA protective effect of SJGP ..........................................................67
3.2.6. Stability of SJGP in antioxidant activity...........................................67
3.3. Results and discussion..................................................................................69
3.3.1. Isolation of glycoprotein from S. japonica.........................................69
3.3.2. Antioxidant activity of SJGP................................................................72
3.3.3. DNA protective effect of SJGP............................................................85
3.3.4. Antioxidant stability of SJGP...............................................................87
References..................................................................................................................97
CHAPTER Ⅳ-------------------------------------------------------------------------------104
Effect of adhesion of Lactobacillus plantarum to intestinal cell by glycoprotein isolated from Saccharina japonica
Abstract....................................................................................................................105
4.1. Introduction....................................................................................................106
4.2. Materials and methods................................................................................110
4.2.1. Bacterial strains and culture conditions..........................................110
4.2.2. Isolation of SJGP ..................................................................................110
4.2.3. Culturing of Caco-2 cell......................................................................110
4.2.4. Direct counts of adhesion assay........................................................111
4.2.5. Microscopy of adhered cells...............................................................111
4.2.6. Cell surface hydrophobicity................................................................112
4.2.7. Autoaggregation assay..........................................................................113
4.2.8. Lipase inhibitory activity of SJGP....................................................114
4.2.9. RT-PCR assay for adhesive-related genes.......................................114
4.3. Results..............................................................................................................117
4.3.1. Adhesion of L. plantarum to Caco-2 cells.......................................117
4.3.1.1. Enumeration of adherent bacteria on selective media...........117
4.3.1.2. Morphological observation.............................................................119
4.3.2. Determination of adhesion properties in L. plantarum................121
4.3.2.1. Cell surface hydrophobicity...........................................................121
4.3.2.2. Cellular autoaggregation.................................................................121
4.3.2.3. Lipase inhibitory activity of SJGP...............................................121
4.3.2.4. Expression of adhesive-related genes..........................................124
4.4. Discussion........................................................................................................126
References................................................................................................................133
SUMMARY (in Korean)----------------------------------------------------------------142
ACKNOWLEDGEMENT----------------------------------------------------------------144
APPENDIX----------------------------------------------------------------------------------145
- Degree
- Doctor
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