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KMID : 0545120000100020251
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Journal of Microbiology and Biotechnology
2000 Volume.10 No. 2 p.251 ~ p.257
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Screening System for Chitin Synthase 2 Inhibitors from Natural Resources and its Inhibitor Prodigiosin
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Hwang, Eui Il
Kim, Young Kook/Lee, Hyang Bok/Kim, Hong Gi/Kim, Sung Uk
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Abstract
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Chitin synthases are identified as key enzymes of chitin biosynthesis in most of the fungi. Among them, chitin synthase Ⅱ has been reported to be an essential enzyme in chitin biosynthesis, and exists as a membrane-bound form. To search and screen new antifungal agents from natural resources to inhibit chitin synthase Ⅱ, the assay conditions were established using the enzyme isolated from Saccharomyces cerevisiae ECY38-38A(pAS6) that overproduces only chitin synthase Ⅱ. This enzyme was activated only by partial proteolysis with trypsin. Its activity reached the maximum at 80㎍/ml of trypsin and was strongly stimulated by 2.0mM Co^2+, 1.0mM UDP-[^14C]-G1cNAc, and 32mM free-GlcNAc. Under these assay conditions, the highest chitin synthase Ⅱ activity was observed by incubation at 30℃ for 90min. However, an extremely narrow range of organic solvents up to as much as 25% of DMSO and 25% of MeOH was useful for determining optimal assay conditions. After a search for potent inhibitors of chitin synthase Ⅱ from natural resources, prodigiosin was isolated from Serratia marcescens and purified by solvent extraction and silica gel column chromatographies. The structure of prodigiosin was determined by UV, IR, Mass spectral, and NMR spectral analyses. Its molecular weight and formula were found to be 323 and C_20,H_25,N_3,O, respectively. Prodigiosin inhibited chitin synthase II by 50% at the concentration of 115㎍/ml.
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