• Journal of Microbiology and Biotechnology
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• 제17권8호
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• pp.1308-1315
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• 2007

Lipase B from Candida antarctica (CalB) displayed on the cell surface of H. polymorpha has been functionally improved for catalytic activity by molecular evolution. CalB was displayed on the cell surface by fusing to a cell-wall anchor motif (CwpF). A library of CalB mutants was constructed by in vivo recombination in H. polymorpha. Several mutants with increased whole-cell CalB activity were acquired from screening seven thousand transformants. The two independent mutants CalB 10 and CalB 14 showed an approximately 5 times greater whole-cell activity than the wild-type. When these mutants were made as a soluble form, CalB 10 showed 6 times greater activity and CalB 14 showed an 11 times greater activity compared with the wild-type. Sequence analyses of mutant CALB genes revealed amino acid substitutions of $Leu^{278}Pro$ in CalB10 and $Leu^{278}Pro/Leu^{219}Gln$ in CalB14. The substituted $Pro^{278}$ in both mutants was located near the proline site of the ${\alpha}$10 helix. This mutation was assumed to induce a conformational change in the ${\alpha}$10 helix and increased the $k_{cat}$ value of mutant CalB approximately 6 times. Site-directed mutagenized CalB, LQ ($Leu^{219}Gln$) was secreted into the culture supernatant at an amount of approximately 3 times more without an increase in the CalB transcript level, compared with the wild-type.

• Molecules and Cells
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• 제28권3호
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• pp.167-174
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• 2009

Genistein has been reported to potentiate glucose-stimulated insulin secretion (GSIS). Inhibitory activity on tyrosine kinase or activation of protein kinase A (PKA) was shown to play a role in the genistein-induced potentiation effect on GSIS. The aim of the present study was to elucidate the mechanism of genistein-induced potentiation of insulin secretion. Genistein augmented insulin secretion in INS-1 cells stimulated by various energygenerating nutrients such as glucose, pyruvate, or leucine/glutamine (Leu/Gln), but not the secretion stimulated by depolarizing agents such as KCl and tolbutamide, or $Ca^{2+}$ channel opener Bay K8644. Genistein at a concentration of $50{\mu}M$ showed a maximum potentiation effect on Leu/Gln-stimulated insulin secretion, but this was not sufficient to inhibit the activity of tyrosine kinase. Inhibitor studies as well as immunoblotting analysis demonstrated that activation of PKA was little involved in genistein-induced potentiation of Leu/Gln-stimulated insulin secretion. On the other hand, all the inhibitors of $Ca^{2+}$/calmodulin kinase II tested, significantly diminished genistein-induced potentiation. Genistein also elevated the levels of $[Ca^{2+}]_i$ and phospho-CaMK II. Furthermore, genistein augmented Leu/Gln-stimulated insulin secretion in CaMK II-overexpressing INS-1 cells. These data suggest that the activation of CaMK II played a role in genistein-induced potentiation of insulin secretion.

• BMB Reports
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• 제32권3호
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• pp.239-246
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• 1999

A tripeptidase (PepT) was purified to homogeneity from Bacillus subtilis through four sequential chromatographies including DEAE-Sepharose ion exchange, hydroxylapatite, mono-Q FPLC ion exchange, and Superose-12 FPLC gel filtration. The apparent molecular mass of the enzyme was 49,200 Da and 51,400 Da as determined by sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography, respectively, and the enzyme exists in a monomeric form. The physicochemical properties of the enzyme were as follows: optimum pH at 7.5, optimum temperature at $60^{\circ}C$, and pI at 4.9. The $K_m$ and $V_{max}$ values of the enzyme were 4.3 mM and 2.5 mmol/min/mg, respectively, with MetAla-Ser as substrate. The B. subtilis PepT requires $Co^{2+}$ ion(s) for activation, while it is inactivated by EOTA and 1,10-phenanthroline, suggesting that it is a metalloprotein. The enzyme was not inhibited by any of serine protease, aspartic protease, or leucine aminopeptidase inhibitors. The enzyme showed comparable activities towards four different substrates including Met-Ala-Ser, Leu-Gly-Gly, Leu-Ser-Phe, and Leu-Leu-Tyr. The amino terminal sequence of PepT determined by Edman degradation was found to be MKEEIIERFTTYVXV and turned out to be identical to that of PepT deduced from a cloned B. subtilis pepT.

• 한국미생물·생명공학회지
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• 제31권2호
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• pp.191-196
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• 2003

본 연구는 김치로부터 분리한 젖산균의 다양성과 항균성을 조사하기 위하여 수행되었다. 희석한 김치 국물을 CaCO$_3$가 첨가된 MRS 고체 배지에 도말 한 후, $25^{\circ}C$에서 2일 동안 배양하여 여러 가지 김치로부터 27가지의 젖산균을 분리하였다. 이렇게 분리한 젖산균들을 Bergey 분류법과 BPB 배지를 통한 생화학적인 방법과 16S rDNA PCR을 이용한 분자 생물학적인 방법으로써 분석하였다. 그러나 이들 방법으로 분석한 결과가 일치하지 않은 경우가 많았다. 이들 분리 젖산균은 16S rDNA 부분 염기서열을 결정한 결과 Leuconostoc mesenteriodes(11균주), Leu. carnosum(3균주), Lactobacillus curvatus(8균주), Lac. pentosus(2균주), Weisselia kimchi(1균주), W. cibaria(1균주), Pediococcus pentosaceus(1균주) 등의 것과 각각 매우 유사한 것으로 분석되었다. 이들중 Leu. carnosum은 이전에 김치에서 분리되었다고 보고된 바가 없다. 그리고 이들 김치에서 분리 동정한 균들의 항균작용 활성을 검사 해 본 결과 균주에 따라서 활성의 정도는 다르게 나타났으나 Bacillus subtilis, Escherichia coli, Salmonella enteritidis, S. paratyphica, S. typhi, Staphylococcus aureus, Shigella boydii, S. sonnei 등에 대해 뚜렷한 항균효과를 나타내었다.

• Applied Biological Chemistry
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• 제24권2호
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• pp.101-104
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• 1981

콩나물질소화합물(窒素化合物)의 영양학적(營養學的) 평가(評價)를 위하여 제조과정별(製造過程別) 유리(遊離)아미노산의 조성변화(組成變化)로 자엽(子葉) 및 배유부(胚軸部)에서 조사(調査)하였다. 대부분(大部分)의 유리(遊離)아미노산은 콩나물 제조중(製造中)점차 증가(增加)되었고 특히 배유부(胚軸部)에서 더 큰 증가(增加)를 보이었다. Arginine, proline al methionine은 제조과정중(製造過程中) 배유부(胚軸部)에서는 나타나지 않았다. 총(總)아미노산에 대(對)한 배유부(胚軸部)아미노산의 함함비(含含比)는 대두(大部)에서 0.11% 이었으나 8일(日) 콩나물에서 8.8%로서 콩나물이 길어 짐에 따라 증가(增加)되었다. 콩나물제조중(製造中) 유리(遊離)아미노산의 변화(變化)는 Asp>His>Val>Ile>Thr>Ser>Lys>Tyr>Phe>Leu>Ala>Gly>Pro>Arg>Glu>Met 의 순(順)으로 증가(增加)되었다. 4일(日)콩나물에서 유리(遊離)아미노산의 함량(含量)은 Asp>Ser>Val>His>Ala>Ile>Lys>Thr>Arg>Leu>Glu>Phe>Tyr>Gly>Cys>Met>Pro의 순(順)이였다.

• 생명과학회지
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• 제22권1호
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• pp.7-15
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• 2012

이전 연구에서 Streptomyces sp. M3 균주로부터 polyguluronate에 기질특이성을 가지는 알긴산분해효소를 cloning하고 활성을 연구하였다. 이번 연구에서는 pColdI vector에 들어있는 M3 알긴산분해효소 유전자를 돌연변이시켜 알긴산분해효소의 활성을 증진시키고자 하였으며, 점-돌연변이 또는 무작위-돌연변이 방법을 사용하여 돌연변이를 실시하였다. Ser25Arg, Phe99Leu, Asp142Asn, Val163Ala, Lys191Glu 및 Gly194Cys 등 6 종류의 돌연변이 단백질을 얻을 수 있었다. Phe99Leu 및 Lys191Glu 돌연변이 단백질은 알긴산을 분해하는 능력을 완전히 잃었으나 Gly194Cys 돌연변이 단백질의 활성은 원래 단백질에 비하여 10배 증가하였다. 또한 돌연변이된 M3 알긴산분해효소 단백질의 3차 구조는 Swiss-Model 자동모델러를 이용하여 생성하였으며 다른 알긴산분해효소의 결정구조와 비교하였다. 194 번째 아미노산인 글리신은 알긴산의 C-말단 보존서열인 YFKAGXYXQ의 Gly193과 Tyr195 사이에 위치한다. 이 연구에서 돌연변이된 글리신과 페닐알라닌 잔기들은 활성자리로부터 많이 떨어져있음에도 불구하고 돌연변이에 의하여 알긴산 분해활성이 강하게 영향을 받는 것으로 나타났다.

• Nuclear Engineering and Technology
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• 제48권3호
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• pp.613-623
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• 2016

Molybdenum-99 ($^{99}Mo$) is the most important isotope because its daughter isotope, technetium-99m ($^{99m}Tc$), has been the most widely used medical radioisotope for more than 50 years, accounting for > 80% of total nuclear diagnostics worldwide. In this review, radiochemical routes for the production of $^{99}Mo$, and the aspects for selecting a suitable process strategy are discussed from the historical viewpoint of $^{99}Mo$ technology developments. Most of the industrial-scale $^{99}Mo$ processes have been based on the fission of $^{235}U$. Recently, important issues have been raised for the conversion of fission $^{99}Mo$ targets from highly enriched uranium to low enriched uranium (LEU). The development of new LEU targets with higher density was requested to compensate for the loss of $^{99}Mo$ yield, caused by a significant reduction of $^{235}U$ enrichment, from the conversion. As the dramatic increment of intermediate level liquid waste is also expected from the conversion, an effective strategy to reduce the waste generation from the fission $^{99}Mo$ production is required. The mitigation of radioxenon emission from medical radioisotope production facilities is discussed in relation with the monitoring of nuclear explosions and comprehensive nuclear test ban. Lastly, the $^{99}Mo$ production process paired with the Korea Atomic Energy Research Institute's own LEU target is proposed as one of the most suitable processes for the LEU target.

• Asian-Australasian Journal of Animal Sciences
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• 제29권4호
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• pp.523-529
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• 2016

An isotope dilution method using $[U-^{13}C]glucose$ and $[1-^{13}C]leucine$ (Leu) was conducted to evaluate the effects of rice straw supplemented with urea and molasses (RSUM-diet) on plasma glucose and Leu turnover rates in sheep. Nitrogen (N) balance, rumen fermentation characteristics and blood metabolite concentrations were also determined. Four sheep were fed either mixed hay (MH-diet), or a RSUM-diet with a crossover design for two 21 days period. Feed allowance was computed on the basis of metabolizable energy at maintenance level. The isotope dilution method was performed as the primed-continuous infusion on day 21 of each dietary period. Nitrogen intake was lower (p = 0.01) for the RSUM-diet and N digestibility did not differ (p = 0.57) between diets. Concentrations of rumen total volatile fatty acids tended to be higher (p = 0.09) for the RSUM-diet than the MH-diet. Acetate concentration in the rumen did not differ (p = 0.38) between diets, whereas propionate concentration was higher (p = 0.01) for the RSUM-diet compared to the MH-diet. Turnover rates as well as concentrations of plasma glucose and Leu did not differ between diets. It can be concluded that kinetics of plasma glucose and Leu metabolism were comparable between the RSUM-diet and the MH-diet, and rumen fermentation characteristics were improved in sheep fed the RSUM-diet compared to the MH-diet.

• BMB Reports
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• 제29권4호
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• pp.359-364
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• 1996

By both in vitro hydroxylamine mutagenesis of the wild type 3-phosphoglycerate kinase gene (PGK) promoter DNA and insertion of the leu2-d gene, we have created yeast expression vectors potentially useful for production of eukaryotic genes in yeast. The guanine (G) to adenine (A) change at the -3 position from the ATG start codon of the PGK promoter-based vector rendered a 6~7 times elevated expression of the adjacent eukaryotic gene, and insertion of the leu2-d gene in the vector containing the mutated PGK promoter further enhanced the expression of the gene. When expression of the AIDS virus HIV1-gagP17 gene in a lacZ fusion form was examined with this new vector, a 15 times higher level of expression than that from the original PGK promoter was observed. Northern and Southern analysis showed that this elevated expression is due to the production of a high copy number of mRNA by leu2-d gene functioning and by efficient translation of the produced mRNA. Thus, the vector that contained the A at the -3 position from the ATG start codon in the promoter region and the leu2-d gene shows increased expression capability and will be potentially useful for production of eukaryotic genes in yeast.

• 한국식품과학회지
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• 제16권4호
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• pp.443-450
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• 1984

온도와 식염농도를 달리하여 김치를 발효시키면서 생화학적 및 미생물의 변화를 검토하였다. 김치의 적숙시기는 발효온도와 식염농도에 따라 달랐다. 김치발효는 저온, 고식염농도에서 보다, 고온 저식염 농도에서 더 빨리 진행되었고 김치의 비휘발성 유기산에 대한 휘발성 유기산의 비는 김치 적숙시기에 최대로 되었다가 김치가 시어지면서 감소되었다. 식염농도와 발효온도를 달리 하여 숙성시킨 김치에서 Leu. mesenteroids, Lac. brevis, Lac. plantarum, Ped. cerevisiae, St. faecalis 및 산생성이 낮은 Lactobacilli가 분리되었다. 그러나 김치발효에 관여하는 주 미생물은 Leu. mesenteroides이며, Lac. plantarum은 김치의 숙성보다는 산패와 더 관련이 있는 것으로 추정되었다. 총 생균수는 발효초기에 급증하여 적숙기간에 최대로 되었다가, 그 이후 서서히 감소되었고, 호기성 세균과 사상균수는 발효가 진행되면서 계속 감소하였고, 효모는 $10^{\circ}C$ 전후의 온도와 고 식염농도에서 그 수가 증가하였다가 다시 감소되었다.