| Summary: | 碩士 === 國立中興大學 === 植物病理學系 === 88 === (1)Studies on the Liquid Fermentation Production and the Purification of Antibiotics from Streptomyces saraceticus isolate 31 and the Mode of Action of its Antifungal Activity
Chin-Chih, Ko
The main objective of this investigation was to explore the cultural requirements for maximizing the antibiotic production and to illustrate the mode of action of the antifungal activity of Streptomyces saraceticus isolate 31. The antifungal activity of the antibiotic produced was determined by an bioassay in which Pythium aphanidermatum was used as the target. Using Czapek’s broth as a basal medium, starch, sucrose and NH4Cl were shown to be superior carbon and nitrogen sources for the antibiotic production. The addition of malt extract (ME) stimulated greatly the antibiotic production and substantially extends the antibiotic activity of the cultural broth. A total of 7 grain decoction broths were screened for the effectiveness in supporting the antibiotic production function. Among them, sorghum, corn and oat were in the order the best ones. The antibiotic production in sorghum and corn decoction broth culture was further enhanced by the addition of 1 % chitin. However, the addition of ME was not effective in this regard. For the extraction and purification of antibiotic, culture filtrates obtained from sorghum and corn decoction broth culture respectively were first clarified by C18 Sep-Pak cartridge (Waters) and then eluted by 30-100 % concentration series methanol (MetOH). The adjunct bioassay indicated the antibiotic was mainly recovered from 70 % MetOH elution. High performance liquid chromatography (HPLC) by C18 reverse phase column further demonstrated the recovery of a single antibiotic peak from the Sep-Pak clarified sample; the chromatography also indicated that the antibiotic extracted from sorghum decoction broth was identical to that from corn decoction broth. The antibiotic extracted was inhibitory to the growth of Pythium aphanidermatum, Alternaria brassicicola, and as well, Rhizoctonia solani AG4. The purified antibiotic preparation appeared to be better preserved at a low pH condition. Furthermore, the inhibitory effect of antibiotic was greatly enhanced with the supplementation of chitinase extracted from the test bacterium. Such synergistic effect by chitinase however worked specifically for A. brassicicola and R. solani AG4, whereas not for P. aphanidermatum, indicating that the antagonistic effect of the test bacterium to the later was due primarily the antibiotic function. For the commercialization of the tested bacterium as a biofungicide, the data presented provided a guide for the medium preparation for mass production, the formulation and also the improvement of the fungicidal efficacy.
(2)Production and molecular characterization of chitinase from Streptomyces saraceticus SS31 isolate
Chin-Chih, Ko
The production of chitinase and the characteristics of this enzyme from Streptomyces saraceticus SS-31 isolate were investigated. In a solid culture system, the enzyme production was found to have optimum temperature around 30 to 37 ℃ and having an optimum pH at 5.0. Using Czapek’s broth as a basal medium, it was demonstrated that sucrose was most favorable for chitinase production. The production of chitinase activity, on the contrary, was not affected by the kinds of inorganic nitrogen (ammonium or nitrate) provided. Among 6 grain decoction broths in which 1 % chitin was each amended, oat, corn and potato served best for supporting the enzyme production. In a 0.15 % chitin broth medium, production of chitinase by the tested bacterium was greatly enhanced by the addition of an appropriate amount of pectin, starch and cellulose respectively; pectin was among them worked best. From a 1 % chitin amended corn decoction medium chitinase produced by tested bacterium was extracted by 40-80 % ammonium sulphate precipitation. The enzyme appeared to have an optimum temperature at 37 ℃ and the activity was favored by a mild acidic condition. Enzyme purification by molecular sieving liquid chromatography with Sepharose CL-6B column and by isoelectric focusing in a Bio-Rad IEF Rotofor Cell indicated the existence of at least 7 isoenzymes with molecular weight at 31, 34.5, 38, 42, 47, 56 and 59 KDs respectively.
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