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학술연구/단체지원/교육 등 연구자 활동을 지속하도록 DBpia가 지원하고 있어요.
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논문 기본 정보
- 자료유형
- 학술저널
- 저자정보
- 저널정보
- 한국미생물생명공학회 Journal of Microbiology and Biotechnology Journal of Microbiology and Biotechnology 제24권 제6호
- 발행연도
- 2014.1
- 수록면
- 748 - 755 (8page)
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In the genome annotation of Escherichia coli MG1655, the orf382 (1,149 bp) is designated as a gene encoding an alcohol dehydrogenase that may be Fe-dependent. In this study, the gene was amplified from the genome by PCR and overexpressed in Escherichia coli BL21(DE3). The recombinant 6×His-tag protein was then purified and characterized. In an enzymatic assay using different hydroxyl-containing substrates (n-butanol, L-threonine, ethanol, isopropanol, glucose, glycerol, L-serine, lactic acid, citric acid, methanol, or D-threonine), the enzyme showed the highest activity on L-threonine. Characterization of the mutant constructed using gene knockout of the orf382 also implied the function of the enzyme in the metabolism of L-threonine into glycine. Considering the presence of tested substrates in living E. coli cel ls and previous literature, we believed that the suitable nomenclature for the enzyme should be an L-threonine dehydrogenase (LTDH). When using L-threonine as the substrate, the enzyme exhibited the best catalytic performance at 39°C and pH 9.8 with NAD+ as the cofactor. The determination of the Km values towards L-threonine (Km = 11.29 μM), ethanol (222.5 μM), and n-butanol (8.02 μM) also confirmed the enzyme as an LTDH. Furthermore, the LTDH was shown to be an ion-containing protein based on inductively coupled plasma-atomic emission spectrometry with an isoelectronic point of pH 5.4. Moreover, a circular dichroism analysis revealed that the metal ion was structurally and enzymatically essential, as its deprivation remarkably changed the α-helix percentage (from 12.6% to 6.3%).
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참고문헌 (23)
참고문헌 신청
Ben-Shitrit T / 2012 / Systematic identification of gene annotation errors in the widely used yeast mutation collections / Nat. Methods 9 : 373 ~ 378
Cochrane G / 2011 / The international nucleotide sequence database collaboration / Nucleic Acids Res 39 : 15 ~ 18
Crow JP / 1995 / Sensitivity of the essential zinc-thiolate moiety of yeast alcohol dehydrogenase to hypochlorite and peroxynitrite / Biochemistry 34 : 3544 ~ 3552
Datsenko KA / 2000 / One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci / Proc. Natl. Acad. Sci. USA 97 : 6640 ~ 6645
Feng DD / 2012 / Molecular cloning, heterogenous expression and the induction profiles after organophosphate phoxim exposure of the carboxylesterase Bmae33 in the silkworm, B
Cochrane G / 2011 / The international nucleotide sequence database collaboration / Nucleic Acids Res 39 : 15 ~ 18
Crow JP / 1995 / Sensitivity of the essential zinc-thiolate moiety of yeast alcohol dehydrogenase to hypochlorite and peroxynitrite / Biochemistry 34 : 3544 ~ 3552
Datsenko KA / 2000 / One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci / Proc. Natl. Acad. Sci. USA 97 : 6640 ~ 6645
Feng DD / 2012 / Molecular cloning, heterogenous expression and the induction profiles after organophosphate phoxim exposure of the carboxylesterase Bmae33 in the silkworm, B
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