No abstract

No abstract

No abstract

A new method of screening for chitinase inhibitors in crude fermentation broths as a means of discovering new insecticidal leads has been developed. In this procedure soluble Remazol brilliant violet 5R dye-coupled chitin degradation products released from insoluble chitin azure substrate by hydrolysis with Streptomyces griseus chitinase are filtered in 0.45 [im Millititer HA96 well filtration plates and collected in 96 well microtiter plates. Inhibitors of this reaction are detected by a decrease in absorbance (570 nm) of the filtrate. A chitinase inhibitor, designated A82516, produced by culture A82516 was discovered using this screen. Purified A82516was found to have an IC50 of 3.7x 10~6 m for S. griseus chitinase. At a test concentration of 0.27 mg/ml, A82516 was 100 % effective in preventing development of house fly larvae to pupae. Allosamidin, a recently reported chitinase inhibitor in vitro, has spectral properties identical to A82516.Chitin, a polymer of unbranched chains of N-acetyl-D-glucosamine units in^-1 -^4 linkages, is one of the main constituents of insect exoskeleton. Insects grow by rebuilding the exoskeleton following molts between larval or juvenile instars. At the beginning of the molting process exuvial fluid containing chitinase is secreted between the partly formed new cuticle and the old skeleton. Chitinase hydrolyzes chitin into oligosaccharides, the smallest of which is the disaccharide chitobiose, allowing the old exoskeleton to be shed. Inhibition of chitinase should interrupt insect molting and thus prevent maturation to the adult reproductive stage. Wesought to develop a chitinase inhibition screen capable of accomodating large numbers of samples as a means of discovering novel fermentationderived compounds with insect life cycle disrupting capabilities, but low probability of mammalian toxicity.Older chitinase inhibition assays were cumbersomeand unsuitable for rapid fermentation broth screening. Since the end product of chitinase digestion of chitin substrate is chitobiose or diacetylchitobiose, an additional incubation step was required to degrade the disaccharide to a product that would react with^-dimethylaminobenzaldehyde for photometric detection15. More recent chitin degradation procedures utilizing radioactive chitin2»3), glycol chitin4), and colloidal chitin in agar5)were not adaptable to our large volumemicrobial screen due to the use of radioisotopes, the need for secondary colorimetric reactions, or our inability to measure zones of clearing in chitin agar. A recent procedure using^-Chitin red6>7) substrate overcame these difficulties but was not designed for r This report was presented in part at the 87th Annual Meeting of the American Soc. for Microb., Atlanta, Georgia, Mar. 1~6, 1987.

The quantity of some of the molecular components of Streptomyces orientalis was markedly altered during the biosynthesis of vancomycin. The addition of elevated levels of inorganic phosphate caused changes in the amount of total carbohydrates, nitrogen, phosphate, DNA, RNA, and alkaline phosphatase in the cell during the fermentation. An excess amount of this ion also caused a decrease in the quantity of vancomycin synthesized as well as a lag in the time when synthesis was initiated. The possibility of a correlation between the biosynthesis of alkaline phosphatase and vancomycin is discussed.