• Journal of Microbiology and Biotechnology
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• 제17권3호
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• pp.454-460
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• 2007

Enzymatic properties and substrate specificity of recombinant $\beta-glycosidases$ from a hyperthermophilic archaeon, Sulfolobus shibatae (rSSG), were analyzed. rSSG showed its optimum temperature and pH at $95^{\circ}C$ and pH 5.0, respectively. Thermal inactivation of rSSG showed that its half-life of enzymatic activity at $75^{\circ}C$ was 15 h whereas it drastically decreased to 3.9 min at $95^{\circ}C$. The addition of 10 mM of $MnCl_2$ enhanced the hydrolysis activity of rSSG up to 23% whereas most metal ions did not show any considerable effect. Dithiothreitol (DTT) and 2-mercaptoethanol exhibited significant influence on the increase of the hydrolysis activity of rSSG rSSG apparently preferred laminaribiose $(\beta1\rightarrow3Glc)$, followed by sophorose $(\beta1\rightarrow2Glc)$, gentiobiose $(\beta1\rightarrow6Glc)$, and cellobiose $(\beta1\rightarrow4Glc)$. Various. intermolecular transfer products were formed by rSSG in the lactose reaction, indicating that rSSG prefers lactose as a good acceptor as well as a donor. The strong intermolecular transglycosylation activity of rSSG can be applied in making functional oligosaccharides.

• Journal of Microbiology and Biotechnology
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• 제27권6호
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• pp.1120-1127
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• 2017

Marasmius scorodonius secretes an extracellular laccase in potato dextrose broth, and this enzyme was purified up to 206-fold using $(NH_4)_2SO_4$ precipitation and a Hi-trap Q Sepharose column. The molecular mass of the purified laccase was estimated to be ~67 kDa by SDS-PAGE. The UV/vis spectrum of the enzyme was nontypical for laccases, and metal content analysis revealed that the enzyme contains 1 mole of Fe and Zn and 2 moles of Cu per mole of protein. The optimal pH for the enzymatic activity was 3.4, 4.0, and 4.6 with 2,2'-azino-bis(3-ethylbenzothazoline-6-sulfonate) (ABTS), guaiacol, and 2,6-dimethoxy phenol as the substrate, respectively. The optimal temperature of the enzyme was $75^{\circ}C$ with ABTS as the substrate. The enzyme was stable in the presence of some metal ions such as $Ca^{2+}$, $Cu^{2+}$, $Ni^{2+}$, $Mg^{2+}$, $Mn^{2+}$, $Ba^{2+}$, $Co^{2+}$, and $Zn^{2+}$ at a low concentration (1 mM), whereas $Fe^{2+}$ completely inhibited the enzymatic activity. The enzymatic reaction was strongly inhibited by metal chelators and thiol compounds except for EDTA. This enzyme directly decolorized Congo red, Malachite green, Crystal violet, and Methylene green dyes at various decolorization rates of 63-90%. In the presence of 1-hydroxybenzotriazole as a redox mediator, the decolorization of Reactive orange 16 and Remazol brilliant blue R was also achieved.

• JSTS:Journal of Semiconductor Technology and Science
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• 제7권3호
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• pp.161-165
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• 2007

In this paper, mesoporous Pt on micro pillars Pt electrode is newly designed, fabricated, and characterized on silicon substrate for non-enzymatic electrochemical sensor micro-chip integrated with CMOS readout circuitry. The fabricated micro/nano Pt electrode has cylindrical hexangular arrayed nano Pt pores with a diameter of 3.2 nm which is formed on top of the micro pillars Pt electrode with approximately $6{\mu}m$ in diameter, $6{\mu}m$ in space, and $50{\mu}m$ in height. The measured current responses of the fabricated plane Pt, mesoporous Pt, and mesoporous Pt on the micro pillar Pt electrodes are approximately $9.9nA/mm^2,\;6.72{\mu}A/mm^2,\;and\;7.67{\mu}A/mm^2$ in 10mM glucose solution with 0.1M phosphate buffered saline (PBS) solution, respectively. In addition, the measured current responses of the fabricated plane Pt, mesoporous Pt, and mesoporous Pt on the micro pillar Pt electrodes are approximately $0.15{\mu}A/mm^2,\;0.56{\mu}A/mm^2,\;and\;0.74{\mu}A/mm^2$ in 0.1mM ascorbic acid (AA) solution with 0.1M phosphate buffered saline (PBS) solution, respectively. This experimental results show that the proposed micro/nano Pt electrode is highly sensitive and promising for CMOS integrated non-enzymatic electrochemical sensor applications. Since the micro-pillar Pt electrode can also be utilized with a micro-fluidic mixer in the sensor chip, the sensor chip can be much smaller, cheaper, and easier to be fabricated.

• Journal of Microbiology and Biotechnology
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• 제17권5호
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• pp.761-768
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• 2007

Serratia proteamaculans HY-3 isolated from the digestive tract of a spider produces an extracellular protease named arazyme, with an estimated molecular mass of 51.5 kDa. The purified enzyme was characterized as having high activities at wide pH and temperature ranges. We further characterized biochemical features of the enzymatic reactions under various reaction conditions. The protease efficiently hydrolyzed a broad range of protein substrates including albumin, keratin, and collagen. The dependence of enzymatic activities on the presence of metal ions such as calcium and zinc indicated that the enzyme is a metalloprotease, together with the previous observation that the proteolytic activity of the enzyme was not inhibited by aspartate, cysteine, or serine protease inhibitors, but strongly inhibited by 1,10-phenanthroline and EDTA. The araA gene encoding the exoprotease was isolated as a 5.6 kb BamHI fragment after PCR amplification using degenerate primers and subsequent Southern hybridization. The nucleotide sequence revealed that the deduced amino acid sequences shared extensive similarity with those of the serralysin family of metalloproteases from other enteric bacteria. A gene(inh) encoding a putative protease inhibitor was also identified immediately adjacent to the araA structural gene.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2010년도 정기총회 및 춘계학술발표회
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• pp.17-17
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• 2010

Ethylene, known as a stress hormone regulate wide developmental processes including germination, root hair initiation, root and shoot primordial formation and elongation, leaf and flower senescence and abscission, fruit ripening. The acceleration of ethylene biosynthesis in plant associated with environmental and biological stresses. 1-Aminocycloprophane-1-carboxlyate deaminase(ACCD) is an enzyme that cleaves ACC into and ammonia, a precursor of the plant hormone ethylene. Plant growth-promoting rhizobacteria (PGPR) having ACCD can decrease endogenous ACC level of tissue, resulting in reduced production of ethylene in plants. ACC deaminse was a key enzyme for protect stressed plants from injurious effects of ethylene. ACCD gene was encoded from Pseudomonas flourescens, PGPR and was cloned in Escherichia coli. We expressed the recombinant ACCD(rACCD) containing 357 amino acids with molecular weight 39 kDa that revealed by SDS-PAGE and western blot. The rACCD was purified by Ni-NTA purification system. The active form of rACCD having enzyme activity converted ACC to a-ketobutyrate. The optimal pH for ACC deaminase activity was pH 8.5, but no activity below pH 7.0 and a less severe tapering activity at base condition resulting in loss of activity at over pH 11. The optimal temperature of the enzyme was $30^{\circ}$ and a slightly less severe tapering activity at 15 - 30$^{\circ}$, but no activity over $35^{\circ}$. P. flourescens ACC deaminase has a highly conserved residue that plays in allowing substrate accessibility to the active sites. The enzymatic properties of this rACCD will provide an important reference for analysis of newly isolated ACCD and identification of newly isolated PGPR containing ACCD.

• 생명과학회지
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• 제25권5호
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• pp.507-514
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• 2015

D-Xylulose는 nonoxidative pentose phosphate 경로를 통해 glycolysis 과정으로 들어가기 전에 D-xylulose kinase에 의해서 D-xylulose-5-phosphate로 인산화 된다. K. gwangalliensis strain SJ2로부터 D-xylulose kinase (XK)를 암호화하는 유전자는 E. coli를 이용하여 서열분석 및 발현 하였으며, XK 유전자의 염기서열 1,419 bp로 구성되어 있으며 463개의 아미노산 잔기를 암호화하고 있다. 분석결과를 통해 XK 유전자가 진화과정 동안 잘 보존되었음을 보여 주었다. XK 유전자의 발현을 위해 pCold-II 발현 벡터에 클로닝 하였으며 클로닝 된 플라스미드는 E. coli strain BL21 (DE3)에 형질전환 하여 IPTG를 이용해 발현을 유도하였다. 재조합 된 XK 단백질의 크기는 약 48 kDa이었다. 이 발현된 단백질은 affinity chromatography를 이용하여 정제하였으며 D-xylulose kinase에 따른 enzymatic activity를 분석하였다. D-xylulose와 ATP로 실행한 XK enzyme kinetic 연구는 각각 250±20 μM과 1,300±50 μM의 Km value를 보였다. 본 연구를 통해 얻어진 결과는 분자적 수준에서 D-xylulose kinase의 특성연구의 보다 넓은 지식적 기초를 제공할 것으로 사료된다.

• 대한치과교정학회지
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• 제25권6호
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• pp.697-704
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• 1995

Chicken calvarial bone is known to contain various cell types, but their exact composition is unknown. By characterizing the chicken calvarial bone biochemically, it can be used to study biochemical, histochemical actions of bone cells in general. Calvaria of 18-day-old white leg horn embryo was aseptically dissected and bone cell populations were isolated by sequential enzymatic digestion. Histochemical study for osteoclast-like bone cell. population was performed with tartrate resistant acid phosphatase(TRAP) stain and for osteoblast-like bone cell population, alkaline phosphatase(ALP) stain was performed. Biochemical study for osteoblast-like bone cell population was performed using alkaline phosphatase(ALP) assay. Following conclusions were obtained from this study. 1. TRAP positive multi and mononuclear cells were mostly observed in group I and II, indicating that osteoclast-like bone cell population is mostly found in these groups. 2. All the cultured groups showed almost equal ALP activities and were positive for ALP stain, indicating that osteoblast-like bone cell population is evenly dispersed in all culture groups. 3. Experimental group treated with $1,25(OH)_{2}D_3$ showed increase in ALP activity in contrast to the control group, confirming previous studies that $1,25(OH)_{2}D_3$ increases ALP activities in in vitro bone cultures. 4. Results from von Kossa's stain indicated that in vitro bone formation had occured after 3 weeks of culture with beta-glycero phosphate.

• BMB Reports
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• 제31권4호
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• pp.345-349
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• 1998

Aspartase (EC 4.3.1.1) from Hafnia alvei was purified to homogeneity by a combination of DEAE-cellulose, Red A-agarose, and Sepharose 6B chromatography. The purified enzyme appeared homogeneous on denatured SDS-polyacrylamide gel electrophoresis. The purified enzyme was a tetrameric protein composed of identical subunits with a molecular weight of 55,000 daltons. The optimum pH for the enzymatic reaction was 8.5 and the optimum temperature for maximum activity was $45^{\circ}C$. The enzyme has an absolute requirement of divalent metal ions ($Mg^{2+}$, $Mn^{2+}$) at the alkaline pH. The enzyme, however, was inactivated in the presence of other divalent cations such as $Zn^{2+}$, $Ca^{2+}$. The helical content of the purified enzyme was estimated by CD spectropolarimetry to be 61%.

• Journal of Microbiology and Biotechnology
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• 제8권4호
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• pp.333-340
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• 1998

Streptomyces griseus trypsin (SGT) is an extracellular proteinase produced by S. griseus. The sprT gene, which encodes premature SGT protein, was cloned into the plasmid pWHM3, a Streptomyces-E. coli shuttle vector. When the recombinant plasmid was introduced into Streptomyces lividans TK24, two proteins with molecular weights of 28 kDa and 42 kDa were detected. The 28-kDa protein was a SGT protein while the larger 42-kDa protein is thought to have been a premature form of the SGT protein. The SGT protein was purified to homogeneity via ammonium sulfate fractionation and many column chromatographies, including CM -sepharose chromatography, Mono-S chromatography, and Superose-12 chromatography, from the culture broth of S. lividans TK24 harboring the sprT gene. The N-terminal amino acid sequence, isoelectric points, and stabilities at various conditions of the SGT proteins purified from the Pronase and transformant were almost identical. The amount of the expressed SGT in S. lividans TK 24 was determined to be 5 times more than that of S. griseus based on the enzymatic activity against artificial substrate.

• KSBB Journal
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• 제14권3호
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• pp.322-327
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• 1999

토양에서 분리된 Streptomyces sp으로부터 cholesterol oxidase의 정제 및 효소학적 특성을 조사하였다. 본 효소의 정제는 50~80% 포화의 황산암모늄 침전 및 cholesterol affinity column chromatography를 통하여 28.3%의 수율로 정제 되었다. 정제된 효소는 SDS-PAGE에서 단일한 밴드를 보였으며 분저량은 약 60,000 dalton으로 추정되었으며, HPLC분석 결과 단일의 peak로 검출되었다. 본 효소의 특성을 검토한 결과, 금속 이온으로 Hg와 Cu의 존재시 크게 저해를 받았고, dithiothreitol 과 mercaptoethanol과 같은 저해제에 의해서 상당히 실활되었다. 본 cholesterol oxidase의 Michaelis 상수는 cholesterol을 기질로하여 Lineweaver-Burk plot분석에서 1.38mM로 추산되었다. 정제효소의 아미노산 조정은 약 416개의 잔기로 추정되며 glycine, alanine, threonine의 함량이 높은 반면, methionine, isoleucine의 함량은 극히 낮았다.