Construction of a Lanthanum-Affinity Recombinant Strain Using a Cell Surface Display System and Its Applications

다국어 초록 (Multilingual Abstract)

The lanthanide group poses potential risks of environmental contamination, biological hazards, and wastewater release during mining and refining. As an approach to replace current methods or mitigate these risks, a recombinant E. coli strain with lanthanum-binding capability was developed. This was accomplished by employing cell surface display engineering to present the lanthanide-binding protein LanM, thereby creating a foundation for lanthanide adsorption through the binding of lanthanum, a representative lanthanide element. This study optimized expression and adsorption parameters to establish a stable system by strongly fusing the protein LanM to OmpC. Optimization reduced damage to the adsorbent across various inducer levels and expression temperatures in the strain carrying the recombinant plasmid, while stabilizing adsorption conditions through adjustments in metal concentration and adsorption temperature. The results showed an adsorption capacity of 4005.87 μmol/g DCW (556.43 mg/g DCW) at a metal concentration of 5 mM. Precursors were produced from lanthanum captured in the control and biosorption groups using precipitation methods that accounted for cell mineralization and desorption processes, followed by pyrolysis. Phosphate adsorption was carried out using the lanthanum-sintered product. Quantitative assessments were performed using ICP-OES, while qualitative characterization employed FT-IR and XRD. Morphological and elemental analyses were conducted via FE-SEM and EDS.
These findings demonstrate that proteins anchored through cell surface technology overcame limitations in intracellular accumulation and enabled synthesis.

국문 초록 (Abstract)

란타나이드 족은 채굴과 정제에 환경오염, 생체 위험성 그리고 폐수 배출에 대한 잠재적 위험을 가지고 있다. 기존 기술의 대체하거나 잠재적 위험에 대한 기술로써, 란타나이드와 결합하는 단백질 LanM을 세포 표면 디스플레이 공학 기술을 활용하여 란탄나이드의 대표적 금속인 란타넘 흡착을 통해 란타나이드 족 흡착의 기반을 만들기 위한 란타넘 흡착 능력을 가지는 대장균 재조합 균주를 구축하였다.
본 연구는 단백질인 LanM을 OmpC에 high-fusion하여 안정적인 시스템을 구축하기 위해 발현조건과 흡착조건의 최적화를 실시하였다. 최적화는 재조합된 플라스미드를 가지는 균주를 통해 여러 유도제 농도, 발현온도에서 흡착제에 손상을 최소화하였고, 금속농도와 흡착온도를 통해 흡착조건을 안정화시켰다. 연구 결과에 따르면 금속농도 5mM 조건에서 4005.87 μmol/g DCW(556.43mg/g DCW)의 흡착용량을 보였다. 대조군과 Biosorption으로 흡착된 란타넘을 셀광물화와 탈착 방법을 고려한 침전법으로 전구체를 만든 후 열분해를 수행했다. 란타넘 소성물을 이용하여 인산흡착을 수행하였다. 정량 분석은 ICP-OES를 이용하였으며 정성 분석은 FT-IR과 XRD를 수행했다. FE-SEM, EDS로 광학적 특성을 분석하였다.
연구결과 세포 표면 기술을 통해 앵커링 된 단백질이 세포 축적의 한계를 극복하였으며 합성에도 용이하였다.

목차 (Table of Contents)

• Acknowledgement I
• Abstract in Korean II
• Abstract in English III
• Contents IV
• Figure list VII
• Table list IX
• 1. Introduction 1
• 1.1. Rare-earth element 1
• 1.1.1. REE application and market 1
• 1.1.2. Risks of Lanthanide mining 4
• 1.1.3. Challenges in Lanthanide Separation 4
• 1.1.4. Biotoxicity of lanthanides 4
• 1.1.5. Lanthanum 5
• 1.1.5.1. Lanthanum Carbonate 5
• 1.1.5.2. Lanthanum Oxide 5
• 1.1.5.3. Lanthanum Phosphate 6
• 1.2. Methods for removal of REE metals in wastewater 7
• 1.2.1. Chemical methods 7
• 1.2.1.1. Chemical precipitation 7
• 1.2.1.2. Oxidation-reduction 8
• 1.2.2. Physical methods 8
• 1.2.2.1. Ion Exchange 8
• 1.2.2.2. Membrane separation 8
• 1.2.2.3. Magnetic separation 9
• 1.2.3. Biological method 9
• 1.2.3.1. Biosorption 9
• 1.2.3.2. Bioleaching 10
• 1.3. Mechanism of biosorption 11
• 1.3.1. Bioaccumulation 11
• 1.3.1.1. Protein 11
• 1.3.1.2. Enzyme 12
• 1.3.1.3. Peptide 12
• 1.3.2. Lanmodulin 12
• 1.3.3. Cell surface display 13
• 1.4. Factors affecting biosorption 16
• 1.4.1. pH 16
• 1.4.2. Temperature 16
• 1.4.3. Initial metal ion concentration 17
• 1.4.4. Biomass concentration 17
• 1.5. Phosphate 18
• 1.5.1. Toxicity of phosphate 18
• 1.5.2. Current Research Trends in Phosphate Adsorbent 19
• 1.5.2.1. Metal-based adsorbents 19
• 1.5.2.2. Carbon-based adsorbents 19
• 1.5.2.3. Lanthanum-based adsorbent 19
• 2. Experimental method 21
• 2.1. Strains and culture conditions 21
• 2.2. Plasmid construction 22
• 2.2.1. SDS-PAGE 22
• 2.3. Lanthanum adsorption, desorption and analysis 23
• 2.4. Calcined Lanthanum preparation 23
• 2.4.1. FE-SEM and EDS 24
• 2.5. Assessment of phosphate removal 24
• 3. Results and discussion 25
• 3.1. Expression condition of LanM 25
• 3.1.1. SDS-PAGE 26
• 3.1.2. Optimization of expression 26
• 3.1.3. Temperature of expression 26
• 3.2 Adsorption conditions of lanthanum 28
• 3.2.1. Temperature of adsorption 28
• 3.2.2 Initial metal concentration 28
• 3.3. Validation of E.coli Adsorption 30
• 3.4. The Lanmodulin mechanism studies 30
• 3.5. The assessment of detachment 32
• 3.6. Calcined lanthanum characterization 32
• 3.6.1. FE-SEM and EDS 32
• 3.6.2 FT-IR 35
• 3.6.3 XRD (X-ray Diffraction) 36
• 3.7 Removal of phosphate 38
• 4. Conclusion 41
• 5. Reference 42