• 한국버섯학회지
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• 제21권4호
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• pp.209-214
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• 2023

본 연구에서 Pleurotus ostreatus No.42는 이전에 보고된 새로운 유형의 회전식 통풍관 생물반응기(RTB)를 사용하여 포도당-펩톤-효모-밀기울(GPYW) 배지에서 배양하였다. 이 배지에서 7일 동안 펠렛형 균사체 배양 후, 리그닌 분해효소인 다목적 과산화 효소(VP)를 분리 및 정제하였다. 다목적 과산화 효소의 정제 과정은 한외여과, DEAE-Sepharose CL-6B 컬럼, Mono Q 컬럼을 순차적으로 적용하여 정제하였다. 그 결과, SDS-PAGE상에서 분자량(MW)은 36.4 KDa, 등전점 (IEF)은 3.65로 나타났으며, 아미노산 조성은 VTCATGQTT로 확인되었다. 정제된 다목적 과산화 효소는 Mn 이온을 산화시킬 뿐만 아니라 비페놀성 화합물인 베라트릴 알코올을 분해하는 특성을 갖는 것으로 나타났다.

• 분석과학
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• 제25권6호
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• pp.483-491
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• 2012

본 연구는 재조합 단백질 A를 생산하기 위한 정제공정 즉, 생산공정은 양이온교환칼럼(SP)과 음이온교환칼럼(Q)을 통하여 정제한 후 최종 재조합 단백질 A를 생산하게 되는데, 공정단계별로 생산된 시료를 바로 다음단계 공정으로 가지 못하는 경우 보관을 하는데 있어 안정성 확보 문제를 가지고 있다. 이에 본 연구에서 생산공정에서 생산된 시료를 보관 했을때 적정 보관조건과 시간을 알아보고자 하는데 목적이 있다. 즉, 공정시료의 저장방법 및 저장기간 설정과 경시변화에 따른 품질의 안정성의 평가를 목적으로 한다. 실험항목은 엔도톡신, SDS PAGE, HPLC 순도 그리고 농도이다. 실험결과 양이온교환칼럼 통과 후 냉장($4^{\circ}C$), 실온보관에서 SDS PAGE는 major band, 엔도톡신은 5.0 Eu/mg이하, 농도에서는 평균 8.21~8.24 mg/mL, RSD% 0.10~0.62%, 그리고 HPLC 순도 214 nm에서 평균 99.24~99.37%, RSD% 0.22~0.29%, 280 nm에서 평균 89.72~89.80%, RSD% 0.62~1.26%로서 다소 안정된 결과를 나타내었다. 음이온칼럼통과 후 냉장($4^{\circ}C$), 실온보관에서 SDS PAGE major band, 엔도톡신 0.5 Eu/mg이하, 농도는 평균 5.59 mg/mL, RSD% 0.03~0.10% 그리고 HPLC 순도 214 nm에서 평균 99.74%, RSD% 0.10~0.11%, 280 nm에서 평균 96.16~96.85%, RSD% 0.72~1.13%의 결과를 보였다. 상기의 실험결과로 재조합 단백질 A 제조공정시료의 실온과 $4^{\circ}C$에서 7~8 일 그리고 20~21 일 보관은 물질분해 특성이 없이 안정성이 확보 되었다는 결론을 얻을 수 있었다.

• Journal of Microbiology
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• 제45권6호
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• pp.510-514
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• 2007

Twenty-five bacterial strains that secrete mucous materials were isolated from sediment obtained from King George Island, Antarctica. Seven of these strains proved capable of producing cryoprotective exopolysaccharides. The strain KOPRI 21653 was selected for the further study of an anti-ice-nucleating polysaccharide (ANP), which originated from a polar region. KOPRI 21653 was identified as Pseudoalteromonas arctica as the result of 16S rRNA analysis. The exopolysaccharide, P-21653, was purified completely from the KOPRI 21653 cell culture via column chromatography and protease treatment. The principal sugar components of P-21653 were determined to be galactose and glucose, at a ratio of 1:1.5, via GC-MS analysis. The cryoprotective activity of P-21653 was characterized via an E. coli viability test. In the presence of 0.1% (w/v) P-21653, the survival ratio of E. coli cells was as high as 82.6% over three repeated freeze-thaw cycles. The survival ratio decreased drastically to 71.5 and 48.1 %, respectively, in five and seven repeated cycle conditions; however, the survival ratios were greater over three (96.6-92.1%) to seven (100.5-91.6%) freeze-thaw cycles in the presence of 0.5 and 1.0% (w/v) P-21653. In addition, at much lower concentrations (0.1-1.0%), P-21653 resulted in survival ratios (83.1-98.4%) similar to those of two commercially available cryoprotectants ($V_{EG}$ plus X-1000, 92.9% and $V_{M3}$, 95.3%), which were utilized at the recommended concentrations (90%). The biochemical characteristics of exopolysaccharide P-21653 reflect that this compound may be developed as a useful cryoprotectant for use in medical applications and in the food industry.

• Journal of Microbiology and Biotechnology
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• 제18권3호
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• pp.449-456
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• 2008

Phenotypic screening for bile salt hydrolase (BSH) activity was performed on Lactobacillus acidophilus PF01 isolated from piglet feces. A gene encoding BSH was identified and cloned from the genomic library of L. acidophilus PF01. The bsh gene and surrounding regions were characterized by nucleotide sequence analysis and were found to contain a single open reading frame (ORF) of 951 nucleotides encoding a 316 amino acid protein. The potential bsh promoter region was located upstream of the start codon. The protein deduced from the complete ORF had high similarity with other BSHs, and four amino acid motifs located around the active site, FGRNXD, AGLNF, VLTNXP, and GXGXGXXGXPGD, were highly conserved. The bsh gene was cloned into the pET21b expression vector and expressed in Escherichia coli BLR(DE3) by induction with 0.1mM of isopropylthiogalactopyranoside. The BSH enzyme was purified with apparent homogeneity using a $Ni^{2+}$-NTA agarose column and characterized. The overexpressed recombinant BSH enzyme of L. acidophilus PF01 exhibited hydrolase activity against tauroconjugated bile salts, but not glycoconjugated bile salts. It showed the highest activity against taurocholic acid. The maximum BSH activity occurred at approximately $40^{\circ}C$. The enzyme maintained approximately 70% of its maximum activity even at $60^{\circ}C$, whereas its activity rapidly decreased at below $37^{\circ}C$. The optimum pH was 6, and BSH activity was rapidly inactivated below pH 5 and above pH 7.

• Applied Biological Chemistry
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• 제36권5호
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• pp.370-375
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• 1993

벼잎의 산성 buffer(pH 2.8) 추출 조효소는 5개의 전기영동 band들이 PR protein으로 알려진 chitinase와 ${\beta}-1,3-glucanase$의 효소활성을 보유하고 있는 것으로 나타났다. 조효소는 DEAE-cellulose 및 chitin affinity chromatography로 염기성 및 산성 효소군으로 분획되었으며, 이들은 두 효소활성이외에도 lysozyme 활성을 보유하고 있었다. 분자량이 $14.3{\sim}66.0\;kd$ 범위인 이 두 효소군이 보유한 각 효소활성의 최적 pH와 온도를 조사해본 결과,${\beta}-1,3-glucanase$는 각각 pH 5와 $60^{\circ}C$로 동일하였으나, chitinase는 염기성 효소군에서 pH 4와 $50^{\circ}C$, 산성 효소군에서는 pH 3와 $60^{\circ}C$으로 다르게 나타나서, 전기영동 양상과 더불어 서로 상이한 효소분획인 것으로 추정되었다.

• Applied Biological Chemistry
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• 제36권2호
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• pp.86-92
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• 1993

무에서 DEAE Bio-Gel, Con-A 및 Superose-6 칼럼을 이용하여 myrosinase를 정제하고 그 효소학적 특성을 검토한 결과 myrosinase(II)는 2개의 subunits를 가졌으며, 이들의 분자량은 SDS-GAGE상에서 각각 53 및 39 KD였다. 정제된 무효소의 비활성도는 37,500 units/mg이었으며, 정제도는 44배였다. 최적 활성 pH는 phosphate 및 Tris-HCl 완충액에서 $6.5{\sim}7.0$이었으며, pH7.0에서 그 효소활성이 가장 안정하였다. 활성 최적온도는 $37{\sim}38^{\circ}C$였고, 열안정성은 $30^{\circ}C$ 이하였다. 무 myrosinase에 대한 무기염의 영향은 구리 및 수은 이온은 효소 활성을 매우 저해하며, ascorbic acid의 농도별 영향은 1 mM일 때 최대활성을 나타내었다. Ascorbic acid analogue에 대한 활성은 dehydroascorbic acid에 대해서는 거의 없었으며, 나머지 analogue들도 ascorbic acid보다 상당히 활성이 낮았다. 무 myrosinase에 대한 환원제의 영향은 2-mercaptoethanol과 dithiothreitol에 의해서는 활성을 나타내지 않았으나, 이들을 ascorbic acid 등 2-mercaptoethanol과 함께 첨가하면 활성을 나타내었다.

• 한국동물생명공학회지
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• 제35권3호
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• pp.245-257
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• 2020

Behavioral response to a diel photoperiodicity (500 lx for 16 h, 5 lx for 4 h and < 0.5 lx for 4 h) and phototactic characteristics in dark conditions were examined with Siberian sturgeon Acipenser baerii (Chondrostei, Actinopterygii) prelarvae. Siberian sturgeon prelarvae represented both qualitative and quantitative changes in their behavioral patterns according to different light intensities in a diel photoperiodicity. Under daylight conditions (500 lx), prelarvae displayed saltatory changes of behavioral features with ages (Day 0-Day 9) in a general order of swimming-up/drifting, swimming in the upper water column, benthic swimming with rheotaxis, schooling and post-schooling behavior. Compared to daylight conditions, prelarvae tended to show more benthic performances and quantitative reductions of schooling and post-schooling behaviors under dimlight conditions (5 lx). Under dark conditions (< 0.5 lx), prelarvae exhibited a fairly uniform behavioral pattern characterized by the benthic swimming across the bottom of the tank. From phototaxis tests under dark conditions, navigational responses of prelarvae to a spotlight illumination were quantitatively changed as their ages increased. The phototactic responses reached the peak on Day 2, continued until Day 4, and then gradually decreased until Day 8. A partial recovery of positive phototaxis was observed on Day 9. Data from this study suggest that the diel light cycle as well as the light intensity of each interval in the cycle should be considered as important components of a practical guide for evaluating fitness and developmental states of artificially propagated Siberian sturgeon prelarvae.

• Journal of Microbiology and Biotechnology
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• 제23권9호
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• pp.1327-1338
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• 2013

The humanized anti-hepatocyte growth factor (HGF) monoclonal antibody (mAb) YYB-101 is a promising therapeutic candidate for treating various cancers. In this study, we developed a bioprocess for large-scale production of YYB-101 and evaluated its therapeutic potential for tumor treatment using a xenograft mouse model. By screening diverse chemically defined basal media formulations and by assessing the effects of various feed supplements and feeding schedules on cell growth and antibody production, we established an optimal medium and feeding method to produce 757 mg/l of YYB-101 in flask cultures, representing a 7.5-fold increase in titer compared with that obtained under non-optimized conditions. The optimal dissolved oxygen concentration for antibody production was 70% $pO_2$. A pH shift from 7.2 to 7.0, rather than controlled pH of either 7.0 or 7.2, resulted in productivity improvement in 5 L and 200 L bioreactors, yielding 737 and 830 mg/ml of YYB-101, respectively. The YYB-101 mAb highly purified by affinity chromatography using a Protein A column and two-step ion exchange chromatography effectively neutralized HGF in a cell-based assay and showed potent tumor suppression activity in a mouse xenograft model established with human glioblastoma cells.

• 한국약용작물학회지
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• 제13권4호
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• pp.194-198
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• 2005

가시오갈피 유식물체의 함유성분을 밝히고, 분리된 화합물들을 대상으로 DPPH를 이용한 항산화활성을 측정하여 대체 약용자원으로서의 사용 가능성을 검토하기 위하여 가시오갈피 유식물체를 MeOH로 추출, 농축하고 Hexane, EtOAc 및 BuOH순으로 분획하여 얻은 각각의 분획을 대상으로 성분연구에 착수하였다. 각종 column chromatography를 이용하여 성분을 분리한 결과 EtOAc분획으로부터 1종의 화합물을 분리하였고, BuOH 분획으로부터 1종의 화합물을 분리하였다. 이들 화합물에 대하여 UV, IR, Mass, $^1H-NMR$ 및 $^{13}C-NMR$ spectrum과 문헌치를 대조하여 그 구조를 동정한 결과그 구조는 각각 Chlorogenic acld (1)과 1,4-di-O-caffeoyl-quinic acid (2)이였다. DPPH (1,1-diphenyl-2-picry-hydrazyl) free radical scavenging activity를 이용하여 측정한 항산화활성의 $RC_{50}$ value는 각각 1.2 및 $0.4\;{\mu}g/ml$였다. 이상의 결과로부터 가시오갈피 유식물체 (Eleutherococcus senticosus Max.)는 천연 항산화제로서의 개발가능성이 높다고 사료된다.

• Asian-Australasian Journal of Animal Sciences
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• 제16권3호
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• pp.342-347
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• 2003

The enzyme $20{\alpha}$-hydroxysteroid dehydrogenase ($20{\alpha}$-HSD) catabolizes progesterone to $20{\alpha}$-dihydroprogesterone ($20{\alpha}$-OHP), and is appeared in rat corpora luteal and placenta. A polled samples of 10-15 placental and ovarian tissues collected from each individual rat were subjected to measurement of $20{\alpha}$-HSD activity. A $20{\alpha}$-HSD activity in the cytosol fraction was based on the generation of NADPH. In this study, it is designed to study cytosolic $20{\alpha}$-HSD activity in rat ovarian and placenta during pregnancy, and its relationship to embryonic mortality. It was found that from days 5 to 18 of pregnancy the $20{\alpha}$-HSD activities steady by decreased but at parturition time rapidly increased in ovary. On the other hand, placental cytosolic $20{\alpha}$-HSD activities were high detected from days 8 to 10 of pregnancy, not detectable from days 11 to 20 of pregnancy, but again very high at the time of parturition. Analysis of DEAE column chromatography revealed that two different types of $20{\alpha}$-HSD (HSD-1 and HSD-2) were found with similar activity in the placental cytosol on day 10 of pregnancy. The number of fetuses on day 10 of pregnancy was 15.4 and decreased significantly to 12.9 on day 12. The results suggested that expression of $20{\alpha}$-HSD in the placental tissues seems to be related the number of fetal survived in the specific time (days 11 and 12) which spontaneous fetal loss occurs.