| Summary: | 碩士 === 國立高雄海洋科技大學 === 海洋生物技術研究所 === 97 === Caldimonas taiwanensis, an amylase-producing bacterium isolated from hot-spring, was used in this study. The thermostable amylase gene has been cloned and analyzed. The gene possess a signal peptide, bacterial pullulanase associated domain, pullulanase domain N-terminus, and -amylase (catalytic domain). The Blast result show that the cloned amylase showed highest homology with isopullulanase. Here, we designed the cloned enzyme as isopullulanase. Three pairs of primers for constructing three truncated proteins are used to survey the function of each protein domain. The signal peptide contained in the overexpressed isopullulanase (Amy3063, full length isopullulanase) showed partial inhibition in enzyme activity. Once removed the signal peptide, the specificity of isopullulanase (Amy82, signal peptide removed) was enhanced 5.5 fold. Truncated isopullulanase lack of signal peptide and bacterial pullulanase associated domain show a high enzyme activity on digesting pullulan. Biochemical study analysis shows that the optimum reaction temperature and pH value of isopullulanase (Amy82) are 62.5 oC and 7.25, respectively. One valence metal ion such as potassium, sodium, lithium, and rubidium ions, shows a significant inhibition on the enzyme activity. However, divalence ion, such as calcium, enhances the enzyme activity. Furthermore, calcium ion enhances the thermostability of isopullulanase (Amy82). The amylopectin was also enhancing the thermostability of it. Ionic detergents, such as SDS or CTAB, inhibit the enzyme activity, but non-ionic detergents do not. The isopullulanase show the highest activity on amylopectin. Accordingly, we propose that the enzyme possess the ability of hydrolyzing -1,4 and -1,6 linkage simultaneously. Thin layer chromatograghy analysis on the digested product of pullulan further confirmed the -1,6 linkage digesting ability. Furthermore, analysis result on the digesting oligosaccharide shows that the enzyme was a type of endoamylase. Western blotting analysis on the cultured medium of wild-type bacterium, verify the cloned isopullulanase was derived from C. taiwanensis.
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