| Summary: | 碩士 === 國立清華大學 === 生物資訊與結構生物研究所 === 94 === Chitin, a β-(1,4)-linked polymer of N-acetyl D-glucosamine (GlcNAc), is widely distributed in nature, particularly as a structural polysaccharide in fungal cell walls in the exoskeleton of arthropods, the outer shell of crustaceans, nematodes, etc. Chitinases which hydrolyze chitin as carbon and nitrogen nutrient, occur in a wide range of organisms include in viruses, bacteria, fungi, insects, higher plants, and animals. A gene of family 18 chitinase from Bacillus cereus NCTU2 encodes a signal peptide (27 amino acids) and a mature protein (333 amino acids), The gene of family 18 chitinase from Bacillus cereus NCTU2 was constructed in pET-22b(+) and over-expressed by E. coli BL21 (DE3) strain.. Amino acid multi-alignment reveals that E145 and Y227 are the potential residues mediating the catalytic function of ChiNCTU2. ChiNCTU2 and mutant E145Q of MW 36 kDa have been crystallized using the hanging-drop vapor diffusion method with solution consisted of polyethene glycerol 8000、sodium cacodylate and zinc acetate dihydrate. According to diffraction of ChiNCTU2 crystals at resolution 1.20 Å, the unit cell belongs to space group P21 and has parameters a = 50.789 Å, b = 48.788 Å and c = 66.867 Å. And E145Q crystal at resolution 1.49, the unit cell belongs to space group P1 and has parameters a = 61.306 50.820 Å, b =72.888 Å and c = 76.343 Å. The protein structure of ChiNCTU2 is monomer by using multiwavelength anomalous dispersion method and the crystal packing of E145Q is tetramer by using molecular replacement method. The structure of ChiNCTU2 comprises 12 α-helices and 10 β-sheets . Five residues Asp143、Glu145、Glu190、Gln225 and Tyr227 bind with zinc atoms in the catalytic domain of ChiNCTU2 protein structure. We proved that zinc atoms will cause declined activity of ChiNCTU2 by detecting the amount of chitobioside using DNS (3,5-Dinitrosalicylic acid) .We find an angular difference between residue E145 and Q145 when doing superimposition with dimer form ChiNCTU2 and E145Q protein structures reveals the declined activity of mutant E145Q. We also find that S.marcescens ChiA has greater catalytic velocity than Bacillus cereus chitinase when interact with colloidal chitin after doing DNS (3,5-Dinitrosalicylic acid) experiment .After comparing sequences and structures with other 18 family chitinase, we discover that ChiNCTU2 is the smallest protein among microbe chitinases.
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