• BMB Reports
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• 제37권3호
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• pp.282-291
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• 2004

Phytases catalyze the release of phosphate from phytic acid. Phytase-producing microorganisms were selected by culturing the soil extracts on agar plates containing phytic acid. Two hundred colonies that exhibited potential phytase activity were selected for further study. The colony showing the highest phytase activity was identified as Aspergillus niger and designated strain 113. The phytase gene from A. niger 113 (phyI1) was isolated, cloned, and characterized. The nucleotide and deduced amino acid sequence identity between phyI1 and phyA from NRRL3135 were 90% and 98%, respectively. The identity between phyI1 and phyA from SK-57 was 89% and 96%. A synthetic phytase gene, phyI1s, was synthesized by successive PCR and transformed into the yeast expression vector carrying a signal peptide that was designed and synthesized using P. pastoris biased codon. For the phytase expression and secretion, the construct was integrated into the genome of P. pastoris by homologous recombination. Over-expressing strains were selected and fermented. It was discovered that ~4.2 g phytase could be purified from one liter of culture fluid. The activity of the resulting phytase was 9.5 U/mg. Due to the heavy glycosylation, the expressed phytase varied in size (120, 95, 85, and 64 kDa), but could be deglycosylated to a homogeneous 64 kDa species. An enzymatic kinetics analysis showed that the phytase had two pH optima (pH 2.0 and pH 5.0) and an optimum temperature of $60^{\circ}C$.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2001년도 추계학술대회
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• pp.46-53
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• 2001

A commensal thermophile, Symbiobacterium toebii, isolated from hay compost (toebii) in Korea commensally interacted with a thermophilic Geobacillus toebii sp. nov., which was a new species within the genus Geobacillus on the basis of the phenotypic traits and molecular systematic data. S. toebii required the crude extracts and/or culture supernatant of the Geobacillus toebii for axenic growth and could grow on the temperature between 45 and $70^{\circ}C$ (optimum: $60^{\circ}C$; 2.4 h doubling time) and pH 6.0 and 9.0 (optimum: pH 7.5). The G+C content of the genomic DNA was $65 mol\%$, and the major quinones were MK-6 and MK-7. A phylogenetic analysis of its 16S rDNA sequence indicated that Symbiobacterium toebii was closely related with solely reported Symbiobacterium thermophilum. The presence of the commensal thermophile 16S rDNA and accumulation of indole in all the enriched cultures indicate that Symbiobacterium toebii is widely distributed in the various soils. The genome of S. toebii constituted a circular chromosome of 3,280,275 base pairs and there was not an extra-chromosomal element (ECE). It contained about 4,107 predicted coding sequences. Of these protein coding genes, about $45.6\%$ was encoded well-known proteins and annotated the functional assignment of 1,874 open reading frames (ORFs), and the rest predicted to have unknown functions. The genes encoding thermostable tyrosine phenol-lyase and tryptophan indole-lyase were cloned from the genomic DNA of S. toebii and the enzymatic production of L-tyrosine and L-tryptophan was carried out with two thermostable enzymes overexpressed in recombinant E. coli.

• The Korean Journal of Physiology
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• 제17권2호
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• pp.125-133
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• 1983

Red cell glycolytic intermediates, metabolites and metabolic ratios were studied. Glycolytic intermediates were measured in neutralized perchloric acid extracts of red cell suspensions after 3 hr incubation at $37^{\circ}C$ in the presence and absence of saponin. Adenosine triphosphate(ATP), adenosine diphosphate(ADP), pyruvate and lactate were measured by enzymatic procedures involving stoichiometric oxidation or reduction of a pyridine nucleotide. Glucose was determined using glucose oxidase after zinc hydroxide extraction. The redox state was calculated from the lactate dehydrogenase equilibrium. Adenosine triphosphatase activity(ATPase) was measured by determining the amount of phosphate released from ATP by washed erythrocyte membranes(ghost) during 20 min. incubation. Both total hydrolysis and the amount of hydrolysis that occured in the presence of ouabain were measured. The second measurement yields Mg-ATPase and represents nonspecific ATPase activity of the membranes. The difference between total and Mg-ATPase activity can be attributed to Na-K-ATPase. For the measurement of sodium fluxes, human erythrocytes were preincubated in $^{22}Na$ for 3 hr at $37^{\circ}C$, washed and suspended in a tracer-free medium. The amount of $^{22}Na$ transported out of cells at any time was determined by analysis of supernatant samples taken at various time after addition of the labeled cells to isotope-free medium. The cells and medium were separated and the radioactivity appearing in the medium was measured. From the total radioactivity in the suspension and the radioactivity appearing in the medium at known time, the rate constant for sodium release was computed. The results are summarized as follows: 1) ATP and ATP/ADP were found to increase at every concentration of saponin tested whereas ADP declined at every cone. of saponin. The increase in pyruvate and lactate were observed at every cone, of saponin and thus $NAD^+/NADH$ computed from pyruvate/lactate also increased. Glucose utilization was stimulated by saponin. 2) $Na^+-K^+-ATPase$ activities showed a biphasic response to saponin, first increasing in lower concentration and then decreasing in higher concentration of saponin. 3) The efflux of sodium was significantly increased by saponin in the range of 5 to 10 mg%. The stimulatory effect of saponin on the rate constants for active(ouabain-sensitive) sodium efflux was inhibited by addition of ouabain.

• 한국수산과학회지
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• 제35권3호
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• pp.259-264
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• 2002

수산가공잔사의 효율적 활용, 새로운 수산가공용 중간소재의 개발이라는 관점에서 활용도가 거의 없는 붕장어 머리와 내장과 같은 가공잔사를 원료로 하여 수산가공용 유용 중간소재를 가공하기 위한 최적 추출조건을 구명하였고, 이의 관능적 특성에 대하여 실험하였다. 붕장어 잔사의 수분함량은 $73.1\%$, 조단백질은 $14.6\%$, 지방의 함량은 $4.6\%$였고, pH는 6.98, 휘발성염기질소 함량은 17.2mg/100g으로서 선도는 비교적 양호하였다. 붕장어 잔사의 주요 구성아미노산은 Asp, Glu, Leu, Gly, Ala, His 및 Arg 등이었으며, 총지방질의 구성지방산으로 16:0, 16:1n7, 18: 1n9 및 22:6n3 둥이 주요 성분이었다. 2단 효소분해소재의 제조를 위한 시판 alcalase의 1차 효소분해 시간은 3-4시간 정도가 가장 적합한 것으로 나타났으며, 시판 neutrase를 첨가해 2차 효소분해를 행한 결과 2차 효소 분해 시간은 4시간이 가장 적합하였다. 이 때의 최종수율은 효소별로 다소의 차이가 있었으나 대체로 80.9-$87.3\%$이었다. 각 추출 소재를 관능검사한 결과, 2단 효소분해소재는 열수나 가압추출 소재와는 달리 비린맛이나 붕장어 특유의 느끼한 누린내와 같은 이미의 생성이 적었으며, 감칠맛 및 종합평가 면에서 월등히 뛰어난 것으로 나타났다.

• 한국연초학회지
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• 제2권1호
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• pp.1-7
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• 1980

건조잎담배로부터 단백질로 간주되는 고분자 물질의 gel filtration column chromatography(Sephadex G-75), 투석 그리고 흡착 chromatography의 일종인 brushite column chromatography분리를 시도한 결과, 짙은 갈색의 고분자 물질을 얻었다. Sephadex column에 의한 분리 profile를 보면 분자량이 상이한 두종류의 갈색고분자 물질이 존재함을 확인하였고 brushite column의 분리 profile에 의하면 분자의 전자구조가 다른 두 종류의 고분자가 있음이 나타났다. Burley와 Hicks의 경우 단백질 분해효소에 의한 분해효과를 측정해본 결과, 가장 큰 분해효과를 보인 효소는 chymotrypsin으로 Burley에서 16-30%수준까지 분해현상을 나타내 주었으며 Hicks인 경우 38-57%까지 감소현상을 보여 주었다. Pepsin처리효과는 chymotrypsin처리구와 비슷한 수준을 보였으나 trypsin경우는 매우 낮은 분해현상을 나타냈다. 단백질 분해효소로 처리된 시료의 sephadex column분리 profile을 살펴보면, 분자량이 보다 큰 fraction의 경우 peak가 거의 완전하게 사라짐을 관찰할 수 있으나, 작은 분자량Peak는 약간의 감소현상만을 보여 주었다. 투석후의 갈색 고분자물질을 $300^{\circ}C$에서 연소시킨 경우, 연소전에 관찰할 수 없었던 강한 형광성 물질이 생성되었는데 TLC에 의한 분리후, 이 형광성 물질끈 흡수 스펙트라를 측정해 본 결과, 최대흡수 파장이 265. 275nm(benzene용매)로 나타났으며 스펙트럼 끈 모양이 polynuclear aromatic hydrocarbon(PAH) 계열 화합물의 것과 유사함을 보여 주었다.

• The Journal of Korean Physical Therapy
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• 제20권1호
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• pp.57-65
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• 2008

Purpose: Heat shock proteins (HSPs) are a group of proteins that are activated when cells are exposed to a variety of environmental stresses, such as infection, inflammation, exposure to toxins, starvation, hypoxia, brain injury, or water deprivation. The activation of HSPs by environmental stress plays a key role in signal transduction, including cytoprotection, molecular chaperone, anti-apoptotic effect, and anti-aging effects. However, the precise mechanism for the action of small HSPs, such as HSP27 and mitogen-activated protein kinases (MAPKs: extracellular-regulated protein kinase 1/2 (ERK1/2), p38MAPK, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), is not completely understood, particularly in application of cell stimulators including platelet-derived growth factor (PDGF), angiotensin II (AngII), tumor necrosis factor $\alpha$ (TNF$\alpha$), and $H_2O_2$. This study examined the relationship between stimulators-induced enzymatic activity of HSP27 and MAPKs from rat smooth and skeletal muscles. Methods: 2-dimensional electrophoresis (2DE) and matrix assisted laser desorption ionizationtime-of-flight/time-of-flight (MALDI-TOF/TOF) analysis were used to identify HSP27 from the intact vascular smooth and skeletal muscles. Three isoforms of HSP27 were detected on silver-stained gels of the whole protein extracts from the rat aortic smooth and skeletal muscle strips. Results: The expression of PDGF, AngII, TNF$\alpha$, and $H_2O_2$-induced activation of HSP27, p38MAPK, ERK1/2, and SAPK/JNK was higher in the smooth muscle cells than the control. SB203580 (30${\mu}$M), a p38MAPK inhibitor, increased the level of HSP27 phosphorylation induced by stimulators in smooth muscle cells. Furthermore, the age-related and starvation-induced activation of HSP27 was higher in skeletal muscle cells (L6 myoblast cell lines) and muscle strips than the control. Conclusion: These results suggest, in part, that the activity of HSP27 and MAPKs affect stressors, such as PDGF, AngII, TNF$\alpha$, $H_2O_2$, and starvation in rat smooth and skeletal muscles. However, more systemic research will be needed into physical therapy, including thermotherapy, electrotherapy, radiotherapy and others.

• 분석과학
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• 제12권3호
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• pp.190-195
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• 1999

Testosterone cypionate (750 mg) 주사제를 말에 투여한 후 testosterone의 대사과정을 살펴보았다. 채취한 뇨에서의 추출물은 효소와 산에 의해 가수분해한 후 oxime-t-butyldimethylsilyl(oxime-TBDMS)의 형태로 유도체화하여 gas chromatography/mass spectrometry(GC/MS)로 분석하였다. Testosterone의 체내 대사과정에서 생성되는 주요 대사체인 $5{\alpha}$-androstan-$3{\beta}$, $17{\alpha}$-diol, $5{\alpha}$-androstan-$3{\beta}$, $17{\beta}$-diol 그리고 $5{\alpha}$-androstan-$3{\beta}$-ol-17-one과 testosterone 및 DHEA의 구조는 표준물질과 질량스펙트럼을 비교하여 확인하였으며, 그들의 배설정도를 GC/MS의 selected ion monitoring (SIM) 방법으로 동시 분석하였다. 그 결과 본 실험방법에서 얻은 검정곡선은 정량 범위 내에서 직선성(r>0.984)을 나타내었고, 회수율은 86.3~94.7% 이었으며, 검출한계는 1~3 ppb 이었다. Testosterone의 배설정도는 주사제 투여 후 5일째 최고 농도에 이른 후 서서히 감소하였다. DHEA와 testosterone 및 그 대사체들의 비는 경주마 약물검사에서 testosterone의 투여 여부를 판단할 수 있는 방법으로 매우 유용함을 알 수 있었다.

• 생명과학회지
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• 제20권11호
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• pp.1569-1576
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• 2010

White-skinned sweet potato (WSSP, Ipomoea batatas L.)는 인도네시아 및 다른 나라 등에서 전통약제로 당뇨병 치료에 널리 사용되고 있다. 본 실험에서는 흰 쥐를 streptozotocin (45 mg/kg체중, i.p.)으로 당뇨병을 유발시킨 후 WSSP의 메탄올 추출물을 체중 1 kg당 Dose 100; 200 mg/kg을 경구로 투여하였다. 산화적 스트레스에 대한 보호효과를 평가하였고 그 효능을 인슐린 분비촉진제인 glimepiride (50 mg/kg 체중)와 비교해 보았다. 산화적 스트레스 평가는 WSP 메탄올 추출물과 glimepiride를 2주 투여 한 후 간장조직의 지질 과산화물(LPO)함량, 혈청 AST, ALT, total triglyceride (TG), total cholesterol (TC), 그리고 항산화효소들인 superoxide dismutase (SOD), 카탈라아제(CAT), 글루타치온 과산화물 분해효소(GPx), 글루타치온 S-전이효소(GST)활성도 등을 간장에서 측정하여 시행하였다. 당뇨 흰쥐에서 혈당, LPO 함량, AST, ALT, TG, TC 함량 등은 정상군에 비하여 그 값이 증가하였고, SOD, CAT, GPx, GST 활성도 값은 감소하였다. 당뇨 흰쥐에 WSSP 메탄올 추출물(200 mg/kg)을 2주일 동안 투여한 결과 의미있는 혈당 감소를 볼 수 있었고, LPO, TG, TC, AST, ALT 함량에서도 개선효과를 볼 수 있었다. 또한 SOD, GPx, 그리고 CAT등 항산화효소들의 활성도 증가도 나타났다. 따라서 WSSP 메탄올 추출물은 당뇨쥐의 혈당을 낮추어 산화적 스트레스를 약화시키고 당뇨로 유발된 손상을 보호해 주는 효과가 있다는 결과를 얻었다.

• 한국식품과학회지
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• 제30권1호
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• pp.199-205
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• 1998

우리의 식생활과 대장암이 밀접한 관계를 가지므로 일반적으로 이용되고 있는 식품과 한방에서 건강식품으로 사용되고 있는 재료들이 장내세균 효소인 ${\beta}-glucuronidase$ 및 tryptophanase 활성에 미치는 효과를 조사하였다. 한방식품 물추출물을 장내세균 배양배지에 첨가하였을때 pH 저하효과, ${\beta}-glucuronidase,{\;}tryptophanase$, 활성억제 효과를 측정한 결과, 대추, 감초는 pH 저하효과 뿐만 아니라 대장암 유발효소인 ${\beta}-glucuronidase$ 및 tryptophanase의 활성을 억제하는 효과도 우수하였다. 한방식품 물추출물의 ${\beta}-glucuronidase$ 및 tryptophanase의 효소를 직접저해하는 효과도 대추, 감초가 우수하였다. 감초, 대추, 수삼, 갈근의 유기용매 분획물의 ${\beta}-glucuronidase$ 및 tryptophanase 생산성 저해효과는 감초의 ether 분획과 대추의 ethylacetate 분획이 가장 우수하게 나타났다. ${\beta}-glucuronidase$ 효소저해활성이 가장 우수한 분획은 대추의 ether 및 ethylacetate 분획이였으며 그 다음이 인삼의 ether 분획과 감초의 ether 분획이였다. Tryptophanase 효소저해활성은 감초의 ether 분획과 대추의 ethylacetate 분획이 우수했다. ${\beta}-Glucuronidase$ 및 tryptophanase 저해효소가 가장 우수한 분획을 silica gel column chromtography로 성분 분획을 하여 6종(Rf=0.98, 0.65, 0.43, 0.34 0.22 0.16)을 분리 하였는데${\beta}-glucuronidase$ 저해효과는 Rf가 0.34인 화합물이 가장 우수 하였으며 tryptophanase 저해효과는 Rf가 0.43인 화합물이 가장 우수 하였다. 대추에는 장내세균 기인성 유해효소를 억제하는 효과가 있는 성분이 함유되어 대장암의 발암물질 생성이 낮아지고 이들 유해효소의 생산성을 억제하는 효과가 우수하게 나타났다 그러므로 대추는 우리 식생활에서 대장암과 같은 성인병 예방에 크게 기여하고 있는 것으로 생각된다.

• 한국수산과학회지
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• 제47권6호
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• pp.733-739
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• 2014

To develop an effective use for poor-quality individually quick-frozen (IQF) oysters Crassostrea gigas stored for a long period, the extract conditions, quality characteristics, and optimum reaction flavoring (RF) conditions of a complex extract from these IQF oysters were investigated. The moisture, pH, and volatile basic nitrogen contents of IQF oysters stored for 18 months (18M-IQFO) were 77.9%, 6.32, and 17.9 mg/100 g, respectively. Three different kinds of extract were prepared from 18M-IQFO: a hot-water extract (HE), scrap enzymatic hydrolysate (EH), and complex extract (CE). The respective extracts contained 5.5, 8.6, and 6.6% crude protein and 281.7, 366.0, and 343.0 mg/100 g amino nitrogen, and had 811, 359, and 1,170 mL/kg extraction yields. The CE was superior to the traditional HE in terms of the extraction yield, amino-nitrogen content, and organoleptic qualities, except for the odor. To improve flavor via the Maillard reaction, the reaction system used to produce a desirable flavor comprised CE (Brix $30^{\circ}$), 0.4 M glucose, 0.4 M glycine, and 0.4 M cysteine solution (4:2:1:1, v/v). The reaction time and pH were the independent variables, and the sensory scores for baked potato odor, masking shellfish odor, and boiled meat odor were the dependent variables. The surface response methodology (RSM) analysis of the multiple responses optimization gave a reaction time of 120.6 minutes and pH 7.33 at $120^{\circ}C$. The reaction improved the flavor of CE considerably, as compared to that of the unreacted extract.