|Budget Amount *help
¥8,700,000 (Direct Cost : ¥8,700,000)
Fiscal Year 2000 : ¥2,700,000 (Direct Cost : ¥2,700,000)
Fiscal Year 1999 : ¥2,600,000 (Direct Cost : ¥2,600,000)
Fiscal Year 1998 : ¥3,400,000 (Direct Cost : ¥3,400,000)
In bacterial infections, typically found in the case of Pseudomonas aeruginosa infection, the infected bacterial cells synthesize and excrete highly viscous and spramolecular polysaccharides and multiply in the environments surrounded by the polymer. However, the biofilms has a property to inhibit transport of antimicrobes or function of phagocytes, thus makes it difficult to treat biofilm-dependent bacterial infection diseases. In order to overcome the difficulty and develop novel therapeutic means, we proposed the use of bacterial alginate lyase that liquefies and removes biofilms. However, before doing so, the antigenicity of the bacterial enzyme should be eliminated. Through biochemical and computer graphic analyses, we determined antigenic-epitope in the vicinity of the N-terminal region (27Ser-44Cys) of the enzyme. The region is on the enzyme surface and far from the active center. Among the amino acids in the region, 27Ser, 29Gln, 32Asp, and 40Lys were highly exposed to the surface of the enzyme, and they were converted to non-polar amino acid alanine by site-directed mutagenesis. The mutated enzymes (S27A, Q29A, D32A, and K40A) were expressed in Escherichia coli cells. The enzyme K40A formed firm inclusion body and was not renatured in active form. Other enzymes were purified and subjected to the antigenic study in mouse system. In addition to these studies, we determined the detailed active site structure through the analyses of enzyme/substrate complex.