STUDIES ON THE SUPEROXIDE DISMUTASES(SOD)FROM Stenotrophomonas maltophilia 276

摘要

从不同种类的植物叶片中共分离并收集到27种优势细菌分离物.通过光化学方法对这些细菌中的SOD活性进行了检测,结果发现在所有细菌SOD的活性中,从水稻叶片中分离得到的276菌株中的SOD的活性最高.利用聚丙酰胺凝胶电泳方法检测SOD的活性,其结果也表明不同细菌菌株的SOD活性存在着差异,其中276菌株的SOD活性最大,这与利用光化学方法检测SOD活性的结果是一致的.同时,利用非变性聚丙烯酰胺凝胶(PAGE)电泳后的凝胶显色反应,发现一些样品出现了多条活性带,这可能是因为在这些细菌提取物样品中含有不同类型的SOD分子,或是同一类型的SOD含有多个亚基组成.

目录

文摘

DECLARATION

CERTIFICATION

ACKNOWLEDGEMENTS

外文摘要

Abstract

Chapter 1. A brief overview of reactive oxygen and antioxidant

1.1 Introduction

1.2 The nature of oxygen free radical

1.3 The antioxidant defense system

1.4 Regulation of antioxidant enzyme expression and activity

1.5 Antioxidant enzymes in health and disease

1.6 Antioxidant and foods

Chapter 2. Review of related literature on SOD

2.1Introduction

2.2 The properties and structures of SOD

2.2.1 Cytosolic Cu/Zn SOD

2.2.2 Periplasmic Cu/Zn SODs

2.2.3 Mn- SOD

2.2.4 Fe -SOD

2.2.5 Extracellular SOD

2.3 Assays for SOD activity

2.3.1 Spectrophotometric Detection

2.3.2 Detection SOD by PAGE

2.3.3 Chemiluminescence Method

2.3.4 Electron spin resonance spectroscopy (ESR) method

2. 4 The Isolation and Purification of SOD

2. 4. 1 Cytosolic Cu/Zn SOD

2.4. 2 EC SOD

2.4.3 Mn SOD

2.4.4 Fe SOD

2.5 Electrophoretic methods for the analysis of SODs

2. 5.1 Identification and quantitative analysis

2. 5. 2 Analysis of isozymes

2.6 The application of SOD

2.7 The Objectives of This Research

Chapter 3. Isolation of bacterial strains and detection of SOD

3.1 Material and methods

3.1.1 Material for bacterial isolation

3.1. 2 Chemicals

3.1. 3 Preparation of media

3. 1.4 Isolation of bacterial strains

3.1.5 Preparation of cell extract

3. 1.6 SOD activity assay by photochemical method

3.1.7 Detection of SOD activity in PAGE

3.2 Results

3.2.1 Isolation and collection of bacterial strains

3.2.2 Detection of SOD Activity by photochemical method

3.2.3 Detection SOD activities by PAGE

Chapter 4. Identification of bacterial strain 276

4.1 Material and methods

4.1.1 Bacterial staining

4.1.2 Motivity

4.1.3 The color of colony on blood agar plate

4.1.4 Biochemical Tests

4．2Results

4．2． 1Gram staining

4．2． 2 The color ofcolony on blood agarplate

4．2． 3 Biochemical tests

4．4Conclusion

Chapter 5． The Purification andthe property study ofSODproducedbySte nooOPhomonas maltOPhilla276

5． 1Materialandmethods

5． 1．1Theeroductionof SUDcultUredniQlllerentmeQl

5． 1．2Theg r owthot bacteria andt De pfoauctlon ors

5． 1．3 Preparation ofcelleex ract

5． 1．4 Precipitation of SOD by anunonium sulfate

5． 1．5 Ion- exchange chromatograPhy

5． 1．6 Gel filtration chromatography

5． L．7 Protein determinations by Bradford assay

5． l．8SODActivityassay byphotochemicalmethod

5． 1．9 Electrophoresis and Detection of SOD

5． L．10 The affect of chemicalinhibitors on SOD activit

5． 1 11The thermal stability of SOD

5． 1．12 The Stability of SOD in pH

5． 1．13 The Stability of SOD in Illumination

5． 2 RCSllltS

5． 2．1The production of SOD cultUrea in different media

5． 2．2The Growth of Bacteria and production of SOD

5． 2．4 Precipitation of soD by ammonium sulfates

5． 2．5 Purification of SOD

5.2.6 Effect of chemical inhibitors on Mn-SOD activity

4.2.6 Identification of Mn -SOD in the active gel

5.2.7 The stability of SOD in pH

5.2.8. The thermal stability of SOD

5.2.9 The Stability of SOD in Illumination

Chapter 6 Cloning and Analysis of SOD Gene in Stenotrophomonas maltophilia276

6.1 Materials and methods

6.1.1 The preparation of Genomic DNA from S. maltophilia 276

6.1.2 The Probe construction and PCR

6.1.3 The cloning of PCR products

6.1.4 The isolation and purification of plasmid DNA

6.1.5 DNAA sequencing

6.2 Results and Analysis

Chapter 7 Discussion

Conclusion

References

10 Equipment and chemicals used in this study

10.1 Apparatus and equipment

10.2 Chemicals

Appendix