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Structural, functional and kinetic studies of the recombinant YjeA endonuclease of bacillus subtilis

Authors Song, Qi Xiu
Issue Date 2010
Summary The low transformation efficiency of chimeric plasmid DNA in Bacillus subtilis has been hindering the successful application of this organism as an alternative cloning and secretory system to E. coli. Our laboratory has previously cloned a novel DNase gene, designated yjeA, from B. subtilis. The recombinant YjeA was shown to be an endonuclease, which cleaved pBR322 and hampered the transformation of the plasmid into E. coli cells. The current study focuses on the characterization of the functional and structural domains of the YjeA protein. To identify the functionally important region(s) of the protein, deletion mutagenesis was employed to generate YjeA mutants, of which regions located in the N-terminus, the C-terminus, or the middle part of the protein had been deleted. The C-terminal region of YjeA appeared to be crucial for both of activity and stability. Moreover, mutants with deletions of 49a.a. (184-232) and 30a.a. (283-312) in the mid-region of YjeA were also shown to be DNase-negative, suggesting that these regions were also functionally important. Further study showed that over-expression of YjeA induces host cell stress responses. The harmful effects observed included slow growth rate, small colony morphology and reduced plasmid stability. Despite the susceptibility of YjeA to proteolysis in E. coli JM101, expression of it under the control of the lacUV5 promoter carried on a multi-copy plasmid, pWKW2, was 2-fold better than that expressed employing the pACYC184-pR promoter system. Kinetic studies have showed similarities between YjeA and DNase I. The hydrolytic activity of YjeA was found to be activated by Mg2+, and reinforced by the presence of Ca2+; moreover, divalent transition metal ions such as Co2+ and Mn2+ were also shown to be potent activators of YjeA. One unit of the recombinant YjeA-H enzyme was defined as the amount of enzyme that hydrolyzes 0.1μg/μl of pBR322 CCC DNA in a 30ul reaction per minute at 37℃. The Km and Vmax were 10.7μg/μl and 2.18U/min, respectively, and the specific activity of YjeA-H is 9.95U/μg protein.
Note Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2010
Language English
Format Thesis
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