• Journal of Microbiology and Biotechnology
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• 제24권6호
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• pp.748-755
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• 2014

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 $6{\times}$His-tag protein was then purified and characterized. In an enzymatic assay using different hydroxyl-containing substrates (n-butanol, $\small{L}$-threonine, ethanol, isopropanol, glucose, glycerol, $\small{L}$-serine, lactic acid, citric acid, methanol, or $\small{D}$-threonine), the enzyme showed the highest activity on $\small{L}$-threonine. Characterization of the mutant constructed using gene knockout of the orf382 also implied the function of the enzyme in the metabolism of $\small{L}$-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 $\small{L}$-threonine dehydrogenase (LTDH). When using $\small{L}$-threonine as the substrate, the enzyme exhibited the best catalytic performance at $39^{\circ}C$ and pH 9.8 with $NAD^+$ as the cofactor. The determination of the Km values towards $\small{L}$-threonine (Km = $11.29{\mu}M$), ethanol ($222.5{\mu}M$), and n-butanol ($8.02{\mu}M$) 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 ${\alpha}$-helix percentage (from 12.6% to 6.3%).

• 한국재료학회지
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• 제8권6호
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• pp.538-544
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• 1998

열차폐코팅층의 박리는 세라믹/금속접합층 계면에서 취성이 큰 스피텔의 생성, 금속과 세라믹의 열팽창계수의 차이, 세라믹층의 상변태, 코팅층의 잔류응력에 기인한다. 본 연구에서는 인코넬 713C에 전자빔 코팅 및 플라즈마 용사법으로 코팅된 안정화지르코니아/CoNiCrAIY 계면의 산화거동을 조사하기 위하여 $900^{\circ}C$에서 등온산화시험동안 생성되는 산화막층과 스피넬 생성 거동을 관찰, 분석하였다. 코팅 직후 코팅층에 고르게 분포하고 있는 Co,Ni,Cr,AI,Zr의 원소들이 산화시간에 따라 확산하여 산화반응을 하였다. 초기 20시간의 산화기시간에 안정화지르코니아/CoNiCrAIY 계면에 주요 성분이 $\alpha$-$AI_2O_3$인 산화막층이 생성되었고, CoNiCrAIY층에는 AI의 외부확산으로 인한 AI 결핍지역이 생성되었다. 산화시험동안 $\alpha$-AI2O층이 임계두께까지 성장한 후에 산화막층의 성장속도는 감소하였고, 안정화지르코니아/산화막층 계면에 스피넬, $Cr_2O_3$, $CO_2CrO_4$의 형성으로 인한 크랙이 관찰되었다.