Chemical Modification Of Cold Protease Mcp-01 Structure And Function Of Mcp-01 Autolysis Mechanism

Major work of this thesis is concerned about a psychrophilic protease MCP-01 secreted by Pseudoalteromonas sp. SM9913, includes amino acid sequence comparison analysis, chemical modification's influence on protease's structure and function and autolysis mechanism of this protease.We compared the amino acid sequence of MCP-01 with homogenies and found that MCP-01 has highest identity (>40%) with two in the protease K subfamily. Besides, we found in MCP-01 the conserved sites of this subfamily. So we classified MCP-01 into protease K subfamily. In the study of amino composition, we found that both the catalytic domain and the entire enzyme have the characteristics of psychrophilic enzymes: higher percentage of acidic amino acid and Gly, lower percentage of basic amino acid and Pro, lower A-index. We also built the 3D models of the catalytic domain and PKD domain, taking 1DBI and 1R64 as temples respectively. The catalytic is mainly composed of a-helix and p-sheet It has a catalytic hole formed by a-helix and P-sheet, at the bottom of which are the catalytic triad residues Ser, Asp and His. The PKD domain is mainly composed of P-sheet with little a-helix or other secondary structures.In the researches on MCP-01 's autolysis mechanisms, we firstly explored the high-T valued SDS-PAGE and Tricine-SDS-PAGE's ability to separate autolysis segments. Then we computed the degradation rate of the entire MCP-01 by analyzing the autolysis at 35°C in 50mM pH 7.5 tris-HCl buffer, and found that the entire protease's degradation is a first-order plot, which means original MCP-01 has no interaction with the autolysis fragments. At last, we sequenced the N-terminal of autolysis fragments, and found that the N-terminal of MCP-01 and those of fragments M1-M4 are all the same, which means the autolysis is from the C-terminal. Then by sequencing the N-terminal of fragments M5-M8, we found 5 autolysis sites, which are all located in the catalytic domain: Thr38 is in the N-terminal of this domain, Ala63, Ala91, Ala203 are in the coils.We utilized a variety of methods, including reductive amination, NaIO₄ oxidation, glutaraldehyde cross-linking and EDC cross-linking, with some monosaccharide and oligosaccharides, to modify MCP-01. Then by examing the thermal stability of the products, we found that only the protease modified by 0,1% glutaraldehyde exhibit a slightly higher stability than the contrast. Therefore, we chose glutaraldehyde plus...