Biochemical characterization, antioxidant and anticancer mechanism of medicinally effective protease from Bacillus siamensis with prominent therapeutic applications
- Issued Date
- 2024
- Abstract
- Bacillus siamensis가 생산하는 단백분해효소의 생화학적 특성 규명, 항산화 및 항암 메커니즘, 의학적 활용 연구 하산 타렉 지도교수:유진철 조선대학교 약학대학 약학과 단백분해효소는 다양한 필수 생물학적 기능에 중요한 역할을 하며 상업적으로도 큰 가능성을 가진 효소입니다. 이 연구에서는 한국의 전통발효식품 김치에서 분리된 Bacillus siamensis CSB55가 생산하는 세제 안정성을 갖춘 다기능 단백분해효소(SH21)의 정제 및 생화학적 특성을 보고합니다. SH21은 황산 암모늄 침전 (40-80%), Sepharose CL-6B, Sephadex G-75 컬럼을 통해 순수하게 정제되었습니다. SDS- PAGE 및 zymogram분석으로 그 분자 크기는 약 25 kDa로 확인되었습니다. 정제된 SH21의 N말단 서열은 QTGGSFFEPFNSYNSGLWQKANGYS입니다. 이 효소의 활성은 PMSF 및 DFP의 존재 하에서 거의 완전히 억제되었으며, 이는 SH21이 serine protease 계열에 속한다는 것을 나타냅니다. SH21은 다양한 pH와 온도 범위에서 뛰어난 활성을 보였으며, 최적 pH는 9.0, 최적 온도는 55 °C였습니다. 이 효소의 추정 Km 및 Vmax 값은 각각 0.197 mg/mL, 1.22×103 U/mg입니다. 또한, 다양한 유기 용매, 계면 활성제, 그리고 다른 시약들과 함께 좋은 활성을 유지했습니다. 이 효소는 다양한 병원균에 대한 MIC 측정을 통해 강력한 항균 활성을 나타냈습니다. MBIC 및 MBEC 실험을 통해 확인된 강력한 항생물막 활성도 보여주었으며, 공초점 현미경 연구를 통해 생물막을 분해하는 것이 확인되었습니다. SH21은 용량 의존적으로 강력한 항산화 및 ROS (reactive oxygen species) 생성 억제 활동을 보였습니다. SH21 처리된 샘플에서 SOD1(superoxide dismutase 1), CAT(catalase), GPx- 1(glutathione peroxidase 1)과 같은 항산화 효소의 단백질 및 mRNA 수준이 향상되었습니다. 또한, SH21은 Nrf2의 번역 및 전사 활동을 촉진하며, 이에 따라 해독 효소인 heme oxygenase-1 (HO-1)발달을 이끌었습니다. 더욱이, SH21은 LPS로 자극된 RAW 264.7 세포에서 NO 및 사이토카인(예: TNF-α, IL-6, IL-1β) 생성을 억제하는 방식으로 항염증 활동을 보였습니다. 60, 80, 100 µg/mL의 농도에서 SH21은 질산화효소(iNOS) 및 사이토카인 유전자 발현을 효과적으로 억제했습니다. 또한, SH21은 용량 의존적으로 암 세포 배양액에서 젖산 탈수소 효소(LDH)의 방출을 촉진하며 HL-60, Hela, A549 등의 테스트된 암 세피들에 대해 효과적인 항암 활성을 보였습니다. 이러한 연구 결과는 SH21이 항균, 항산화, 항염증 및 항암 효과를 갖추고 있으며, 염증 관련 질환에 대한 우수한 치료제가 될 수 있음을 시사합니다..|Biochemical characterization, antioxidant and anticancer mechanism of medicinally effective protease from Bacillus siamensis with prominent therapeutic applications Hasan Tarek Advisor: Prof. Jin Cheol Yoo Department of Pharmacy Graduate School of Chosun University Proteases are important enzymes that contribute to a range of essential biological functions and have a significant possibility for commercial applications. In this work, we reported the purification and biochemical description of a detergent-stable and multifunctional protease enzyme (SH21) produced by Bacillus siamensis CSB55 isolated from Korean fermented vegetable kimchi. SH21 was purified to obtain homogeneity via ammonium sulfate precipitation (40-80%), Sepharose CL-6B, and Sephadex G-75 column. By analyzing SDS-PAGE and zymogram, it was determined the molecular size was around 25 kDa. The NH2-terminal sequence of purified SH21 was QTGGSFFEPFNSYNSGLWQKANGYS. The enzymatic activity was almost entirely inhibited in the presence of PMSF and DFP, which specified that it is a member of serine protease family. SH21 showed excellent activity with a wide scale of pH and temperature, with its maximum pH of (9.0) and temperature of 55 °C. The enzyme had estimated Km and Vmax values of 0.197 mg/mL and 1.22×103 U/mg, separately. In addition, it preserved good activity with different organic solvents, surfactants, and other reagents. This enzyme also showed good antimicrobial activity that was evaluated by MIC against several pathogenic bacteria. It exhibited strong antibiofilm activity as determined by MBIC and MBEC assay and degraded the biofilms which were analyzed by confocal microscopic study. SH21 displayed very powerful antioxidant and ROS (reactive oxygen species) production inhibition activity in a dose-dependent approach. The mRNA expressions and protein levels of antioxidant enzymes including SOD1 (superoxide dismutase 1), CAT (catalase), and GPx-1 (glutathione peroxidase 1) were enhanced in the SH21-treated sample. SH21 also boosted the translational and transcriptional activities of NF-E2-related factor 2 (Nrf2) with the subsequent development of detoxifying enzyme heme oxygenase-1 (HO-1). In addition, SH21 showed potential anti-inflammatory activity via inhibition of nitric oxide (NO) and proinflammatory cytokines, such as TNF- α, IL-6, and IL-1β, generation in LPS stimulated RAW 264.7 cells. At concentrations of 60, 80, and 100 µg/mL, SH21 potentially suppressed nitric oxide synthase (iNOS) and cytokine gene expressions. Furthermore, SH21 significantly released lactate dehydrogenase (LDH) enzyme in cancer cell supernatant in a dose-dependent approach and exhibited effective anticancer activity against three tested cancer cell lines, including cells HL-60, A549, and Hela. Our results suggest that SH21 has effective antimicrobial, antioxidant, anti-inflammatory, and anticancer effects and could be an excellent therapeutic agent against inflammation-related diseases. Keywords: Protease SH21, SDS-PAGE, Antioxidant, Anticancer, Anti- biofilm, Mechanism of action, Confocal microscopy..
- Alternative Title
- Bacillus siamensis가 생산하는 단백분해효소의 생화학적 특성규명, 항산화 및 항암 메커니즘, 의학적 활용 연구
- Alternative Author(s)
- TAREK HASAN
- Affiliation
- 조선대학교 일반대학원
- Department
- 일반대학원 약학과
- Advisor
- Jin Cheol Yoo
- Awarded Date
- 2024-02
- Table Of Contents
- 1. CHAPTER ONE: GENERAL INTRODUCTION
1.1. Kimchi, a traditional Korean fermented food 1
1.2. Microbial enzymes 4
1.3. Protease enzymes 5
1.3.1. Sources of protease enzymes 5
1.3.2. Classifications of protease enzymes 6
1.3.3. Applications of protease enzymes 8
1.3.4. Physiological role of protease enzymes 12
1.3.5. Future prospects of protease enzymes 12
1.4. Objective of this study 13
2. CHAPTER TWO: SCREENING, ISOLATION, PURIFICATION AND BIOCHEMICAL CHARACTERIZATION OF PROTEASE SH21
2.1. INTRODUCTION 15
2.2. MATERIALS AND METHODS 17
2.2.1. Materials and bacterial strains 17
2.2.2. Screening, isolation, and phylogenic tree analysis of Protease enzyme 17
2.2.3. Media and Culture Conditions 18
2.2.4. Protease assay 18
2.2.5. Purifications of Protease SH21 19
2.2.6. Protein Measurements, SDS-PAGE, and Zymogram analysis 20
2.2.7. Determination of Optimum pH and Stability 20
2.2.8. Determination of Optimum Temperature and Thermal Stability 21
2.2.9. Effect of Inhibitors, Metals, Surfactants, and Bleaching agents on Protease Activity 21
2.2.10. Effect of Organic Solvents on Protease Activity 22
2.2.11. Substrate Specificity and Kinetics Parameters of Protease SH21 22
2.2.12. Stability and Compatibility of Protease with Commercial Laundry Detergent 22
2.3. RESULT AND DISCUSSIONS 23
2.3.1. Screening Identification and phylogenetic tree of protease-producing strain 23
2.3.2. Enzyme Production and Purification of Protease SH21 25
2.3.3. Molecular weight determination and N- terminal amino acid sequence 26
2.3.4. Effect of pH on the activity and stability of protease SH21 28
2.3.5. Effect of temperature on the activity and stability of protease SH21 29
2.3.6. Effect of inhibitors, metal ions, surfactants, and bleaches on protease stability 31
2.3.7. Effect of organic solvents on the activity and stability of protease SH21 34
2.3.8. Substrate specificity and enzyme kinetics of protease SH21 35
2.4. CONCLUSION 37
3. CHAPTER THREE: ANTIMICROBIAL AND ANTIBIOFILM ACTIVITY OF PROTEASE SH21
3.1. INTRODUCTION 39
3.2. MATERIALS AND METHODS 41
3.2.1. Determination of Minimum inhibitory concentration (MIC) of protease SH21 41
3.2.2. Inhibition of biofilm formation (MBIC assay) 41
3.2.3. Eradication of preformed biofilm (MBEC assay) 42
3.2.4. Confocal Laser Scanning Microscopy Analysis ..43
3.3. RESULT AND DISCUSSIONS 43
3.3.1. Antimicrobial activity of protease SH21 43
3.3.2. Antibiofilm activity of protease SH21 45
3.4. CONCLUSION 48
4. CHAPTER FOUR: ANTIOXIDANT, ANTIINFLAMMATORY AND ANTICANCER MECHANISM STUDY OF PROTEASE SH21
4.1. INTRODUCTION 50
4.2. MATERIALS AND METHODS 52
4.2.1. Materials 52
4.2.2. Cell Culture and Maintenance 53
4.2.3. DPPH Assay 53
4.2.4. ABTS Assay 53
4.2.5. Superoxide Radical Scavenging Activity Assay 54
4.2.6. Hydroxyl Radical Scavenging Activity Assay 54
4.2.7. FRAP Assay 55
4.2.8. CUPRAC Assay 55
4.2.9. Cell Viability and Intracellular ROS GenerationInhibition in Raw 264.7 cell 55
4.2.10. Cell Lysates Preparation and Western Blot Analysis 56
4.2.11. RT-PCR Analysis 57
4.2.12. Nitric Oxide (NO) Generation Inhibition and Cytokines Assay 58
4.2.13. MTT assay 59
4.2.14. The Lactate Dehydrogenase (LDH) Release Assay 60
4.2.15. Live/Dead Staining Assay 60
4.3. RESULT AND DISCUSSIONS 61
4.3.1. Antioxidant Activity of SH21 61
4.3.2. Cell Cytotoxicity Effect and ROS Generation Inhibition of SH21 63
4.3.3. Antioxidant Enzymes Expression by SH21 in Raw 264.7 Cells 64
4.3.4. SH21 Exerts Anti-Inflammatory Activity 68
4.3.5. Anticancer Activity of SH21 70
4.4. CONCLUSION 76
5. FUTURE DIRECTIONS 77
6. REFERENCES 78-93
7. LIST OF PUBLICATIONS 94
8. ACKNOWLEDGMENTS 95-96
- Degree
- Doctor
- Publisher
- 조선대학교 대학원
- Citation
- 타렉 하산. (2024). Biochemical characterization, antioxidant and anticancer mechanism of medicinally effective protease from Bacillus siamensis with prominent therapeutic applications.
- Type
- Dissertation
- URI
- https://oak.chosun.ac.kr/handle/2020.oak/17915
http://chosun.dcollection.net/common/orgView/200000742646
- Appears in Collections:
- General Graduate School > 4. Theses(Ph.D)
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