• Journal of Environmental Health Sciences
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• v.46 no.4
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• pp.368-375
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• 2020

Objectives: Some animal studies have reported that methyl mercury causes developmental toxicities such as placental and fetal weight loss, but the mechanism is still unclear. This study aimed to investigate the developmental toxicities of methyl mercury, focusing on placental endocrine function and fetal growth retardation in rats. Methods: Positively same-time-mated female Sprague-Dawley rats were purchased on gestational day (GD) eight and treated with 0, 5, 10 and 20 ppm of methyl mercury (n=5) dissolved in tap water from GD eight through 19. During treatment, the drinking water (methyl mercury) intake and body weight of each pregnant rat was measured daily. On day 19, caesarean sections were performed and blood samples were collected. Developmental data such as placental and fetal weights, fetus numbers, and placental efficiency (fetal weight/placental weight) were also collected. Placental prolactin-growth hormone (PRL-GH) family, such as placental lactogen (PL) -Iv, II, and prolactin-like protein (PLP) -B, levels in serum were analyzed by ELISA. Also, placental tissues were assigned to histochemistry. Results: The mean cumulative methyl mercury exposure for the 5, 10, and 20 ppm groups were 2.37, 4.63, and 9.66 mg, respectively. The mean daily exposure of the 5, 10, and 20 ppm groups were 0.24, 0.47, and 0.97 mg, respectively. Maternal body weight increased in accordance with GD. There was no significant difference in weight gain among the experimental groups. Histopathologic changes were not observed in placental tissues among the experimental groups. However, mean placental and fetal weights were lower in the 10 and 20 ppm exposed groups compared to the control. Placental efficiency was also lower in the 10 and 20 ppm exposed groups compared to the control. Serum PL-Iv and II levels were lower in the 10 and 20 ppm exposed groups than the control, in accordance with the changing pattern of placental and fetal weights and placental efficiency. Conclusion: The inhibitory effects of methyl mercury on the serum levels of placental PRL-GH family such as PL-Iv and II may be secondary leads to the reduction of placental efficiency and fetal growth retardation in rats.

• Journal of Microbiology and Biotechnology
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• v.25 no.9
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• pp.1476-1484
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• 2015

Three endoxylanase-encoding genes from the moderately themophilic chemoorganotrophic bacterium Melioribacter roseus were cloned and expressed in Escherichia coli. Genes xyl2091 (Mros_2091) and xyl2495 (Mros_2495) encode GH10 family hydrolases, whereas xyl2090 (Mros_2090) represents the GH30 family. In addition to catalytic domains, Xyl2090 and Xyl2091 contain carbohydrate-binding modules that could facilitate their binding to xylans and Por sorting domains associated with the sorting of proteins from the periplasm to the outer membrane, where they are covalently attached. Recombinant endoxylanase Xyl2495 exhibited a high specific activity of 1,920 U/mg on birchwood xylan at 40℃. It is active at low temperatures, exhibiting more than 30% of the maximal activity even at 0℃. Endoxylanases Xyl2090 and Xyl2091 have lower specific activities but higher temperature optima at 80℃ and 65℃, respectively. Analysis of xylan hydrolysis products revealed that Xyl2090 generates xylo-oligosaccharides longer than xylopentaose. Xylose and xylobiose are the major products of xylan hydrolysis by the recombinant Xyl2091 and Xyl2495. No activity against cellulose was observed for all enzymes. The presence of three xylanases ensures efficient xylan hydrolysis by M. roseus. The highly processive "free" endoxylanase Xyl2495 could hydrolyze xylan under moderate temperatures. Xylan hydrolysis at elevated temperatures could be accomplished by concerted action of two cell-bound xylanases; Xyl2090 that probably degrades xylans to long xylo-oligosaccharides, and Xyl2091 hydrolyzing them to xylose and xylobiose. The new endoxylanases could be useful for saccharification of lignocellulosic biomass in biofuels production, bleaching of paper pulp, and obtaining low molecular weight xylooligosaccharides.

• Journal of Microbiology and Biotechnology
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• v.19 no.3
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• pp.257-264
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• 2009

A $\beta$-agarase gene, agaB34, was functionally cloned from the genomic DNA of a marine bacterium, Agarivorans albus YKW-34. The open reading frame of agaB34 consisted of 1,362 bp encoding 453 amino acids. The deduced amino acid sequence, consisting of a typical N-terminal signal peptide followed by a catalytic domain of glycoside hydrolase family 16 (GH-16) and a carbohydrate-binding module (CBM), showed 37-86% identity to those of agarases belonging to family GH-16. The recombinant enzyme (rAgaB34) with a molecular mass of 49 kDa was produced extracellularly using Escherichia coli $DH5{\alpha}$ as a host. The purified rAgaB34 was a $\beta$-agarase yielding neoagarotetraose (NA4) as the main product. It acted on neoagarohexaose to produce NA4 and neoagarobiose, but it could not further degrade NA4. The maximal activity of rAgaB34 was observed at $30^{\circ}C$ and pH 7.0. It was stable over pH 5.0-9.0 and at temperatures up to $50^{\circ}C$. Its specific activity and $k_{cat}/K_m$ value for agarose were 242 U/mg and $1.7{\times}10^6/sM$, respectively. The activity of rAgaB34 was not affected by metal ions commonly existing in seawater. It was resistant to chelating reagents (EDTA, EGTA), reducing reagents (DTT, $\beta$-mercaptoethanol), and denaturing reagents (SDS and urea). The E. coli cell harboring the pUC18-derived agarase expression vector was able to efficiently excrete agarase into the culture medium. Hence, this expression system might be used to express secretory proteins.

• Journal of Microbiology and Biotechnology
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• v.28 no.5
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• pp.776-783
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• 2018

The agarase gene gaa16a was identified from a draft genome sequence of Gilvimarinus agarilyticus JEA5, an agar-utilizing marine bacterium. Recently, three agarase-producing bacteria, G. chinensis, G. polysaccharolyticus, and G. agarilyticus, in the genus Gilvimarinus were reported. However, there have been no reports of the molecular characteristics and biochemical properties of these agarases. In this study, we analyzed the molecular characteristics and biochemical properties of agarases in Gilvimarinus. Gaa16A comprised a 1,323-bp open reading frame encoding 441 amino acids. The predicted molecular mass and isoelectric point were 49 kDa and 4.9, respectively. The amino acid sequence of Gaa16A showed features typical of glycosyl hydrolase family 16 (GH16) ${\beta}$-agarases, including a GH16 domain, carbohydrate-binding region (RICIN domain), and signal peptide. Recombinant Gaa16A (excluding the signal peptide and carbohydrate-binding region, rGaa16A) was expressed as a fused protein with maltose-binding protein at its N-terminus in Escherichia coli. rGaa16A had maximum activity at $55^{\circ}C$ and pH 7.0 and 103 U/mg of specific activity in the presence of 2.5 mM $CaCl_2$. The enzyme hydrolyzed agarose to yield neoagarotetraose as the main product. This enzyme may be useful for industrial production of functional neoagaro-oligosaccharides.

• Journal of Microbiology and Biotechnology
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• v.28 no.2
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• pp.284-292
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• 2018

A novel ${\beta}$-agarase, AgaJ5, was identified from an agar-degrading marine bacterium, Gayadomonas joobiniege G7. It belongs to the glycoside hydrolase family 86 and is composed of 805 amino acids with a 30-amino-acid signal peptide. Zymogram analysis showed that purified AgaJ5 has agarase activity. The optimum temperature and pH for AgaJ5 activity were determined to be $30^{\circ}C$ and 4.5, respectively. AgaJ5 was an acidic ${\beta}$-agarase that had strong activity at a narrow pH range of 4.5-5.5, and was a cold-adapted enzyme, retaining 40% of enzymatic activity at $10^{\circ}C$. AgaJ5 required monovalent ions such as $Na^+$ and $K^+$ for its maximum activity, but its activity was severely inhibited by several metal ions. The $K_m$ and $V_{max}$ of AgaJ5 for agarose were 8.9 mg/ml and 188.6 U/mg, respectively. Notably, thin-layer chromatography, mass spectrometry, and agarose-liquefication analyses revealed that AgaJ5 was an endo-type ${\beta}$-agarase producing neoagarohexaose as the final main product of agarose hydrolysis. Therefore, these results suggest that AgaJ5 from G. joobiniege G7 is a novel endo-type neoagarohexaose-producing ${\beta}$-agarase having specific biochemical features that may be useful for industrial applications.

• Journal of Microbiology and Biotechnology
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• v.23 no.7
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• pp.913-922
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• 2013

An agar-degrading bacterium was isolated from red seaweed (Gelidium amansii) on a natural seawater agar plate, and identified as Saccharophagus sp. AG21. The ${\beta}$-agarase gene from Saccharophagus sp. AG21 (agy1) was screened by long and accurate (LA)-PCR. The predicted sequence has a 1,908 bp open reading frame encoding 636 amino acids (aa), and includes a glycosyl hydrolase family 16 (GH16) ${\beta}$-agarase module and two carbohydrate binding modules of family 6 (CBM6). The deduced aa sequence showed 93.7% and 84.9% similarity to ${\beta}$-agarase of Saccharophagus degradans and Microbulbifer agarilyticus, respectively. The mature agy1 was cloned and overexpressed as a His-tagged recombinant ${\beta}$-agarase (rAgy1) in Escherichia coli, and had a predicted molecular mass of 69 kDa and an isoelectric point of 4.5. rAgy1 showed optimum activity at $55^{\circ}C$ and pH 7.6, and had a specific activity of 85 U/mg. The rAgy1 activity was enhanced by $FeSO_4$ (40%), KCl (34%), and NaCl (34%), compared with the control. The newly identified rAgy1 is a ${\beta}$-agarase, which acts to degrade agarose to neoagarotetraose (NA4) and neoagarohexaose (NA6) and may be useful for applications in the cosmetics, food, bioethanol, and reagent industries.

• Journal of the Korean Society of Hazard Mitigation
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• v.7 no.1 s.24
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• pp.1-9
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• 2007

This study does by purpose that propose shear resisting force equation of reinforced masonry wall that is reinforced by GFRP(glass fiber reinforced polymer) based on result that is noted through cyclic loading of masonry wall and a shaking table experiment of mock that reflect identifying marks of masonry building which is constructed in domestic. It was Rocking mode to dominate failure of masonry wall in the experiment results, and the equations of UBC show the most resemblant value with experiment results. Through this study, propose the shear force equation of GFRP strengthened masonry wall as following. $$V_n=0.02A_n{\sqrt{f'_m}}+0.022b_gh_g(1+2{\alpha})^3{\sqrt{f_g}}(N/mm^2)$$.

• Journal of Microbiology and Biotechnology
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• v.31 no.10
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• pp.1446-1454
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• 2021

Herein, we cloned and expressed an endo-β-1,4-glucanase gene (celA1805) from Bacillus subtilis B111 in Escherichia coli. The recombinant celA1805 contains a glycosyl hydrolase (GH) family 8 domain and shared 76.8% identity with endo-1,4-β-glucanase from Bacillus sp. KSM-330. Results showed that the optimal pH and temperature of celA1805 were 6.0 and 50℃, respectively, and it was stable at pH 3-9 and temperature ≤50℃. Metal ions slightly affected enzyme activity, but chemical agents generally inhibited enzyme activity. Moreover, celA1805 showed a wide substrate specificity to CMC, barley β-glucan, lichenin, chitosan, PASC and avicel. The K_m and V_max values of celA1805 were 1.78 mg/ml and 50.09 µmol/min/mg. When incubated with cellooligosaccharides ranging from cellotriose to cellopentose, celA1805 mainly hydrolyzed cellotetrose (G4) and cellopentose (G5) to cellose (G2) and cellotriose (G3), but hardly hydrolyzed cellotriose. The concentrations of reducing sugars saccharified by celA1805 from wheat straw, rape straw, rice straw, peanut straw, and corn straw were increased by 0.21, 0.51, 0.26, 0.36, and 0.66 mg/ml, respectively. The results obtained in this study suggest potential applications of celA1805 in biomass saccharification.

• Journal of Microbiology and Biotechnology
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• v.29 no.1
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• pp.37-43
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• 2019

The gene encoding an ${\alpha}-{\text\tiny{L}}-arabinofuranosidase$ (BvAF) GH51 from Bacillus velezensis FZB42 was cloned and expressed in Escherichia coli. The corresponding open reading frame consists of 1,491 nucleotides which encode 496 amino acids with the molecular mass of 56.9 kDa. BvAF showed the highest activity against sugar beet (branched) arabinan in 50 mM sodium acetate buffer (pH 6.0) at $45^{\circ}C$. However, it could hardly hydrolyze debranched arabinan and arabinoxylans. The time-course hydrolyses of branched arabinan and arabinooligosaccharides (AOS) revealed that BvAF is a unique exo-hydrolase producing exclusively ${\text\tiny{L}}-arabinose$. BvAF could cleave ${\alpha}-(1,2)-$ and/or ${\alpha}-(1,3)-{\text\tiny{L}}-arabinofuranosidic$ linkages of the branched substrates to produce the debranched forms of arabinan and AOS. Although the excessive amount of BvAF could liberate ${\text\tiny{L}}-arabinose$ from linear AOS, it was extremely lower than that on branched AOS. In conclusion, BvAF is the arabinan-specific exo-acting ${\alpha}-{\text\tiny{L}}-arabinofuranosidase$ possessing high debranching activity towards ${\alpha}-(1,2)-$ and/or ${\alpha}-(1,3)-linked$ branches of arabinan, which can facilitate the successive degradation of arabinan by $endo-{\alpha}-(1,5)-{\text\tiny{L}}-arabinanase$.

• Korean Journal of Animal Reproduction
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• v.20 no.1
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• pp.19-26
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• 1996

In the present study, we investigated devel-opmental ability and transgene expression of IVM/IVF derived porcine embryos following microinjection with SV40-LacZ. A total of 412 IVM/IVF derived embryos were used to examine developmental ability and transgene expression following DNA microinjection. After centrifugation, pronuclei were visible in 60.3% when examined between 18~21h after IVF. Development and transgene expression were assessed after 9 days in culture. The percentages of injected embryos reaching to the morula and blastocyst were significantly lower (P<0.05) than those of non-injected control embryos. However, the percentages of DNA microinjected embryos and non-injected embryos that developed to the blastocyst or hatched blastocyst stage in dual culture systems (NCSU23 and EMEM) were significantly higher (P<0.05) than those in NCSU23 medium alone. As the resuIt of X-gal staining, the proportion of positive embryos was 40~43% in morula and blastocyst stage embryos, however, mosaicism has been observed in the most putative transgenic morulae and blastocysts. In the PCR analysis, the percentages of embryos integrated gGH gene were 45.0 and 44.4% in morula and blastocyst stage, respectively. These results suggest that improved IVM /IVF system and culture condition increased the embryo viability and ex-pression of a microinjected transgene.