Bacillus subtilis ATCC 21332的改質與表面素生產相關蛋白質之研究

Surfactin is a cyclic lipopeptidic biosurfactant produced by Bacillus subtilis ATCC 21332 with nonribosomal peptide synthesis mechanisn.Surfactin is composed of seven amino acids of lipopeptide and a long-chain fatty acid. The synthesis of peptide moiety of surfactin is catalyzed by three surfactin synthetases, SrfA , SrfB, and SrfC. Although surfactin is the most powerful biosurfactant, it’s application and commerializating is restricted because of low productivity and high production cost. The possible reason which cause low productivity of surfactin is the promoter of surfactin synthetases genes only work under the particular condition. Only few surfactin synthetases express to catalyze the surfactin synthesis. In order to upgrade the surfactin productivity, we tried to use the P43 promoter to replace the promoter of surfactin synthetas operon by gene targeting technology. Because of the poor transformation efficiency, no plasmid was transferred into B. subtilis ATCC 21332 to proceed further studies. it is necessary to get surfactin-producing strains with the ability to receive to proceed experiments for genetic engineering easily. Some B. subtilis 168 derive clones derived with highest surfactin productivity and genetic stability were gotten. The transformation frequencies of the B. subtilis 168 derive clones were better than B. subtilis 168 and 21332. P43 promoter replacement for promoter of surfactin synthetase operon was also performed, surfactin productivity was not improved and only some unknown products were synthesized to generate lysis zones on Blood agar. The nonribosomal peptide synthesis has been studied for more than fifty years. In addition to peptide synthetase, it is not clear which proteins regulate or participate in the nonribosomal peptide synthesis. We have carried out the preliminary proteomic analysis of B. subtilis ATCC 21332 proteins related to surfactin synthesis. Using two-dimensional electrophoretic separations followed by MALDI-TOF mass spectrometry, expression of 20 proteins alter during the surfactin synthesis and 13 protein were identified. Futuremore, functions of the 13 identified proteins in the surfactin synthesis will be studied.