참굴 패각의 생광물화 연구 : On Biomineralization in Crassostrea gigas : Mineral-organic matrix relationships and fine structure
- 발행기관 서강대학교 대학원
- 지도교수 최청송
- 발행년도 2006
- 학위수여년월 200608
- 학위명 박사
- 학과 및 전공 화학공학
- 식별자(기타) 000000103254
- 본문언어 한국어
초록/요약
생물기원 탄산칼슘으로 이루어진 참굴 (Crassostrea Gigas) 패각(shell)내 아라고나이트로 이루어진 미오스트라컴과 방해석으로 이루어진 folia의 형태·구조학적 특성 및 재료학적 특성을 결정하는 유기매트릭스의 역할과 탄산칼슘과의 상관관계를 연구하였다. 푸리에 셀프-디콘볼루션 (Fourier self-deconvolution, FSD)과 가우시안 커브피팅 (Gaussian curve fitting)을 이용하여 생광물화과정 (biomineralization)의 과정을 통해 형성된 참굴의 배아 및 유생의 생체복합재 (biocomposite)내에 존재하는 결정내부단백질 (intracrystalline proteins)이 탄산칼슘의 상전이 (phase transition) 과정에 관여 한다는 것을 확인 할 수 있었다. 탄산칼슘의 다형성 (polymorphism)이 공존하는 패각내 미오스트라컴과 folia 경계면으로부터 침상구조 (prismatic structure)인 미오스트라컴과 판상구조 (laminated structure)인 folia의 수용성 단백질을 각각 분리한 후 아미노산 조성분석을 통해 두층의 형태학적 특성 및 다형성을 결정하는 수용성 단백질의 차이를 확인할 수 있었다. 또한 미오스트라컴과 folia의 경계면을 이루는 유기매트릭스는 β 키틴으로 경계로하여 불용성 단백질과 β 구조가 풍부한 수용성 단백질이 적층구조로 존재하며 두 층 모두 (0 0 1) 면으로 성장한다는 것을 확인 할 수 있었다. 단백질 분리 및 정제를 통해 얻어진 패각내 수용성 단백질의 N-말단 단백질 서열분석을 바탕으로 RNA 정제 및 RT-PCR (Reverse Transcriptase - Polymerase Chain Reaction)을 이용하여 형태학적 특성 및 다형성을 결정하는 패각내 수용성 단백질의 아미노산 서열 (amino acid sequence)를 얻을 수 있었다. 또한, 리트벨트 refinement를 사용하여 생물기원 탄산칼슘과 무생물기원 탄산칼슘의 결정학적 특성을 비교한 결과 패각의 형태학적 특성 및 선택적 배향 (preferred orientation) 등은 패각내 유기매트릭스에 의해 설계되고 조절됨을 알 수 있었다. 분석된 아미노산 서열을 바탕으로 생물정보학 (bioinformatics) 관점에서 단백질의 구조 및 특성 등을 예측하고 이를 통해 패각내 유기매트릭스 (organic matrix)와 탄산칼슘의 상관관계를 논의하였다. 아라고나이트로 이루어진 미오스트라컴의 수용성 단백질 이차구조는 크게 N-말단 부분에 위치한 α-helix와 C-말단 부분에 위치한 β-구조로 구분할 수 있었으며 이들의 서열 및 구조 특성에 따른 패각생성 과정이 검증되었다. 본 연구를 통해 생광물화과정 (biomineralization)을 통한 미오스트라컴의 패각 형성과정은 미오스트라컴의 형태학적 특성 및 다형성을 결정하는 수용성단백질 이차구조내의 α 구조와 β 구조의 상호보완적이고 협동적인 상호 역할 분담에 의한 과정에 의해 설명될 수 있었다. 투과전자 현미경의 Kikuchi 패턴과 인덴테이션 (indentation) 분석을 통해 패각내 유기매트릭스의 재료학적 설계능력에 의해 합성된 생물기원 탄산칼슘의 선택적 배향 (preferred orientation)이 재료학적 특성에 미치는 영향과 역할이 논의 되었으며 이를 통해 방해석으로 이루어진 folia의 우수한 재료학적 특성을 확인할 수 있었다. 본 연구로부터 얻어진 결과와 정보들은 신물질 합성과 같은 생체모방공학 (biomimetics)의 응용을 위한 정보로 활용될 수 있으리라 판단된다.
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Biogenic calcium carbonates are associated with organic matrices such as proteins and polysaccharides, the principal constituents of bone and invertebrate shell organic material in nature. Oyster shells consist of mineralized composites of calcium carbonate and macromolecules in an exquisitely controlled manner, exhibiting exceptional nano-scale regularity and strength that can overcome the intrinsic brittleness of abiotic calcium carbonate. The higher-order architecture results from the presence of the organic matrix that creates defects in the perfect lattice which, in turn, enhance the fracture toughness by both absorbing stress and deviating the propagation of cracks along the cleavage planes of calcium carbonate, indicating the primary role in controlling the biominerals during nucleation and growth. The mineralization sequence with respect to development stages of Crassostrea gigas namely, from the embryonic and larval stages to the juvenile stage oyster, has been analyzed by X-ray Diffraction (XRD) and Fourier Transform Infrared Spectroscopy (FT-IR). At each stage, the oyster shell underwent a polymorphic change from embryos to juvenile, and there are significant alterations in their organic component and protein conformation. A linear increase in HCO3- with development is reflective of carbonic anhydrase activity. The β-sheet is the main component of the larvae in the prodissoconch II stage, which is related to the formation of aragonite. The composition of the organic matrix and the polymorphism of biominerals for the shell formation are almost completed at the dissoconch stage. Our results on phase transition of oyster shell suggest that the organic matrix, particularly the secondary structure of intracrystalline protein, changed the conformation of the protein at each stage, and could play a key role in determining the polymorphism of the Pacific oyster shell. The organic membrane at the interface between the myostracum (aragonite) and the folia (calcite) is obtained and identified as a chitin-like macromolecule after chemical treatment. The organic membrane is faced to the crystal (001) plane of the myostracum and folia. The secondary structures in soluble protein of the myostracum and the folia of oyster shell have been identified as a great quantity of β-structure. Soluble protein in the myostracum of oyster shell, Crassostrea gigas, has been characterized using biochemical and molecular biological techniques. From an analysis of secondary protein structure, The β-structure was predominant in myostracum. And via in vitro assays, the relation of myostracum to biomineral phase and morphology is studied. The amino acid sequence of 160 amino acids has been deduced for myostracum by characterization of the complementary DNA encoding the protein. The deduced protein was composed of a high proportion of Gly and Asp, typifying a calcium-binding protein for shell formation, and a relatively high proportion of Val, Ala and Ile, typifying an adhesive protein. In contrast to prevailing expectations, (ZXZ, Z: acidic amino acid; X: non-acidic amino acid)n-type sequence motifs exist in MPSP, demanding a revision of previous theories of protein–mineral interactions. Using by protein structure and characterization on the basis of analysed amino acid sequence and bioimformatics, the correlation between organic matrix and biogenic calcium carbonate is studied. Alpha-helix followed by a N-terminal region is comprised of -ZXZ- sequence clusters, where Z = Asp, Glu and X = nonanionic amino acids, which have high surface probability. Thus, the acidic amino acid residues in the clusters can act as ligands for calcium binding. Beta-structure followed by a C-terminal region possess hydrogen-bonding donar / acceptor amino acids (e.g., Asn, Gln, Thr, Ser, Arg) which may assist with displacement of waters of hydration or with the orientation of carbonate ions, which would change the activation barrier for aragonite formation. Thus, the shell formation of myostracum through biomineralization could be explained by cooperative processes involving alpha-helix and beta-structure in soluble protein to determine the morphology and polymorphism of myostracum. The thin sheets of calcite, termed folia, that make up much of the shell of an oyster are composed of foliated lath. Folia of the giant Pacific oyster have been examined using TEM (transmission electron microscopy) and tested using microindentation and nanoindentation techniques. Analysis of the Kikuchi patterns obtained from the folia showed that there are two types (type I and type II) of preferred orientation, with an angle of around 70° between them. Nanoindentation test shows that the folia exhibit a hardness of about 3 GPa and elastic modulus of about 73 GPa. Micro-cracks are generated using a microindenter in order to study the fracture mechanisms of the folia. Following on from these investigations, fracture mechanisms are discussed in conjunction with the correlation between preferred orientation and structural characteristics during cracking of the folia. Comparing the morphology and the polymorphism with nacre (also known as mother of pearl), the advantages of the relatively higher crystal growth and lower organic matrix in folia may have interesting implications for the development of sophisticated synthetic materials. The information obtained from this study is expected to help researchers devise new synthetic routes to improved materials synthesis.
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