• Journal of Microbiology and Biotechnology
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• v.22 no.4
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• pp.501-509
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• 2012

A 990 bp full-length gene (xynAHJ2) encoding a 329-residue polypeptide (XynAHJ2) with a calculated mass of 38.4 kDa was cloned from Bacillus sp. HJ2 harbored in a saline soil. XynAHJ2 showed no signal peptide, distinct amino acid stretches of glycoside hydrolase (GH) family 10 intracellular endoxylanases, and the highest amino acid sequence identity of 65.3% with the identified GH 10 intracellular mesophilic endoxylanase iM-KRICT PX1-Ps from Paenibacillus sp. HPL-001 (ACJ06666). The recombinant enzyme (rXynAHJ2) was expressed in Escherichia coli and displayed the typical characteristics of low-temperature-active enzyme (exhibiting optimum activity at $35^{\circ}C$, 62% at $20^{\circ}C$, and 38% at $10^{\circ}C$; thermolability at ${\geq}45^{\circ}C$). Compared with the reported GH 10 low-temperature-active endoxylanases, which are all extracellular, rXynAHJ2 showed low amino acid sequence identities (<45%), low homology (different phylogenetic cluster), and difference of structure (decreased amount of total accessible surface area and exposed nonpolar accessible surface area). Compared with the reported GH 10 intracellular endoxylanases, which are all mesophilic and thermophilic, rXynAHJ2 has decreased numbers of arginine residues and salt bridges, and showed resistance to $Ni^{2+}$, $Ca^{2+}$, or EDTA at 10 mM final concentration. The above mechanism of structural adaptation for low-temperature activity of intracellular endoxylanase rXynAHJ2 is different from that of GH 10 extracellular low-temperature-active endoxylanases. This is the first report of the molecular and biochemical characterizations of a novel intracellular low-temperature-active xylanase.

• Journal of Microbiology and Biotechnology
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• v.20 no.3
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• pp.518-524
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• 2010

A mannanase gene (man26B) was obtained from a sea bacterium, Paenibacillus sp. BME-14, through the constructed genomic library and inverse PCR. The gene of man26B had an open reading frame of 1,428 bp that encoded a peptide of 475- amino acid residues with a calculated molecular mass of 53 kDa. Man26B possessed two domains, a carbohydrate binding module (CBM) belonging to family 6 and a family 26 catalytic domain (CD) of glycosyl hydrolases, which showed the highest homology to Cel44C of P. polymyxa (60% identity). The optimum pH and temperature for enzymatic activity of Man26B were 4.5 and $60^{\circ}C$, respectively. The activity of Man26B was not affected by $Mg^{2+}$ and $Co^{2+}$, but was inhibited by $Hg^{2+},\;Ca^{2+},\;Cu^{2+},\;Mn^{2+},\;K^+,\;Na^+$, and $\beta$-mercaptoethanol, and slightly enhanced by $Pb^{2+}$ and $Zn^{2+}$. EDTA did not affect the activity of Man26B, which indicates that it does not require divalent ions to function. Man26B showed a high specific activity for LBG and konjac glucomannan, with $K_m,\;V_{max}$, and $k_{cat}$ values of 3.80 mg/ml, 91.70 ${\mu}mol$/min/mg protein, and 77.08/s, respectively, being observed when LBG was the substrate. Furthermore, deletion of the CBM6 domain increased the enzyme stability while enabling it to retain 80% and 60% of its initial activity after treatment at $80^{\circ}C$ and $90^{\circ}C$ for 30 min, respectively. This finding will be useful in industrial applications of Man26B, because of the harsh circumstances associated with such processes.

• Applied Biological Chemistry
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• v.34 no.3
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• pp.225-230
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• 1991

In this study, several combined effect of KCl addition into bring solution, microwave heating of Chinese radish for 3 minutes and addition of two salt mixtures into half fermented Kakdugi were investigated to compare their improving effects of storage stability of Kakdugi, Kakdugi was prepared by salting in 15% NaCl solution for 2 hours and fermented at $25^{\circ}C$ and determined for the changed in pH, total acidity and reducing sugars. It was found from the result that the combined method of salts addition and heating treatment improved the storage stability by 6.4 folds based on pH change where salts mixture of phosphated, Ca EDTA, and Sod. citrate contributed the most effect. Changed in reducing sugar were greatly reduced by salt mixture addition while microwave heating and KCl addition affected little. Storage of Kakdugi at $4^{\circ}C$ after preparation with combined method could extend the storage life more than 8 folds of those Kakdugi stored at $25^{\circ}C$. However total acidity showed higher values for those prepared by combined method and stored at $4^{\circ}C\;or\;25^{\circ}C$.

• Journal of Microbiology and Biotechnology
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• v.24 no.4
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• pp.489-496
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• 2014

High levels of extracellular xylanase activity (211.79 IU/mg) produced by Paenibacillus sp. NF1 were detected when it was submerged-cultured. After three consecutive purification steps using Octyl-Sepharose, Sephadex G75, and Q-Sepharose columns, a thermostable xylanase (XynNF) was purified to homogeneity and showed a molecular mass of 37 kDa according to SDS-PAGE. The specific activity of the purified XynNF was up to 3,081.05 IU/mg with a 14.55-fold purification. The activity of XynNF was stimulated by $Ca^{2+}$, $Ba^{2+}$, DTT, and ${\beta}$-mercaptoethanol, but was inhibited by $Fe^{2+}$, $Zn^{2+}$, $Fe^{2+}$, $Cu^{2+}$, SDS, and EDTA. The purified XynNF displayed a greater affinity for oat spelt xylan with the maximal enzymatic activity at $60^{\circ}C$ and pH 6.0. XynNF, which was shown to be cellulose-free, with high stability at high temperature ($70^{\circ}C-80^{\circ}C$) and low pH range (pH 4.0-7.0), is potentially valuable for various industrial applications. The enzyme hydrolyzed oat spelt xylan to yield mainly xylooligosaccharides (95.8%) of 2-4 degree of polymerization (DP2-4). Moreover, the majority of the xylooligosacharides (DP2-4) products was xylobiose (61.5%). The thermostable xylanase (XynNF) thus seems potentially usefull in the production of xylooligosaccharides.

• Journal of Microbiology and Biotechnology
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• v.30 no.11
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• pp.1659-1669
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• 2020

1,3-α-3,6-anhydro-L-galactosidase (α-neoagarooligosaccharide hydrolase) catalyzes the last step of agar degradation by hydrolyzing neoagarobiose into monomers, D-galactose, and 3,6-anhydro-L-galactose, which is important for the bioindustrial application of algal biomass. Ahg943, from the agarolytic marine bacterium Gayadomonas joobiniege G7, is composed of 423 amino acids (47.96 kDa), including a 22-amino acid signal peptide. It was found to have 67% identity with the α-neoagarooligosaccharide hydrolase ZgAhgA, from Zobellia galactanivorans, but low identity (< 40%) with the other α-neoagarooligosaccharide hydrolases reported. The recombinant Ahg943 (rAhg943, 47.89 kDa), purified from Escherichia coli, was estimated to be a monomer upon gel filtration chromatography, making it quite distinct from other α-neoagarooligosaccharide hydrolases. The rAhg943 hydrolyzed neoagarobiose, neoagarotetraose, and neoagarohexaose into D-galactose, neoagarotriose, and neoagaropentaose, respectively, with a common product, 3,6-anhydro-L-galactose, indicating that it is an exo-acting α-neoagarooligosaccharide hydrolase that releases 3,6-anhydro-L-galactose by hydrolyzing α-1,3 glycosidic bonds from the nonreducing ends of neoagarooligosaccharides. The optimum pH and temperature of Ahg943 activity were 6.0 and 20℃, respectively. In particular, rAhg943 could maintain enzyme activity at 10℃ (71% of the maximum). Complete inhibition of rAhg943 activity by 0.5 mM EDTA was restored and even, remarkably, enhanced by Ca²⁺ ions. rAhg943 activity was at maximum at 0.5 M NaCl and maintained above 73% of the maximum at 3M NaCl. K_m and V_max of rAhg943 toward neoagarobiose were 9.7 mg/ml and 250 μM/min (3 U/mg), respectively. Therefore, Ahg943 is a unique α-neoagarooligosaccharide hydrolase that has cold- and high-salt-adapted features, and possibly exists as a monomer.

• Korean Journal of Soil Science and Fertilizer
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• v.48 no.1
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• pp.57-63
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• 2015

Bioavailability of cadmium (Cd) and arsenic (As) can be different depending on soil pH. For this reason, main purpose of this research was to compare bioavailability of Cd and As in agricultural field under varied soil pH and different extractants. Bioavailable fraction of Cd and As in soil was extracted with $CaCl_2$, $NaNO_3$, DTPA, EDTA, and low molecular weight organic acids (LMWOAs). Soil samples and cultivated crops were collected at the range of soil pH 4.5-8.5 and correlation analysis was conducted between bioavailable fraction of Cd and As in soil and total concentration of Cd and As in crops. Results showed that concentration of Cd and As in acidic soil was ranged $0.002-0.462mg\;kg^{-1}$ and $0.041-4.903mg\;kg^{-1}$ respectively. In alkaline condition, concentration of Cd and As were ranged $0.006-0.351mg\;kg^{-1}$ and $0.039-2.807mg\;kg^{-1}$ respectively. Comparing bioavailable fraction of Cd and As in acidic and alkaline soil condition, higher concentration was measured in acidic condition. Similarly, higher average concentration of Cd and Asin crops was observed in acidic condition (0.398 and $0.751mg\;kg^{-1}$ respectively) than alkaline condition (0.248 and $0.264mg\;kg^{-1}$). Among different extractants, LMWOAs method showed higher correlation ($r^2=0.545$) for Cd in acidic condition indicating that LMWOAs method could be applied for evaluating bioavailability of Cd in acidic soil. However, no high correlation was observed for As in both acidic and alkaline condition. Overall, bioavailable fraction of Cd and As can be higher in acidic condition of soil than alkaline condition resulting higher uptake of Cd and As from soil to crops. Therefore, efficient best management practice (BMPs) for Cd and As in acidic soil should be conducted for minimizing uptake of Cd and As into crops.

• Microbiology and Biotechnology Letters
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• v.46 no.4
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• pp.360-371
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• 2018

Extracts and fractions of Anemarrhena asphodeloides Bunge were prepared and their physiological activities and components were analyzed. Antimicrobial activities of the ethyl acetate and aglycone fractions were $78{\mu}g/ml$ and $31{\mu}g/ml$, respectively, for Staphylococcus aureus and $156{\mu}g/ml$ and $125{\mu}g/ml$, respectively, for Pseudomonas aeruginosa. 1,1-Diphenyl-2-picrylhydrazyl free radical scavenging activities ($FSC_{50}$) of 50% ethanol extract, ethyl acetate fraction, and aglycone fraction of A. asphodeloides extracts were $146.2{\mu}g/ml$, $23.19{\mu}g/ml$, and $71.06{\mu}g/ml$, respectively. The total antioxidant capacity ($OSC_{50}$) in an $Fe^{3+}$-EDTA/hydrogen peroxide ($H_2O_2$) system were $17.5{\mu}g/ml$, $1.5{\mu}g/ml$, and $1.4{\mu}g/ml$, respectively. The cytoprotective effect (${\tau}_{50}$) in $^1O_2$-induced erythrocyte hemolysis was 181 min with $4{\mu}g/ml$ of the aglycone fraction. The ${\tau}_{50}$ of the aglycone fraction was approximately 4-times higher than that of (+)-${\alpha}$-tocopherol (${\tau}_{50}$, 41 min). Analysis of $H_2O_2$-induced damage of HaCaT cells revealed that the maximum cell viabilities for the 50% ethanol extract, ethyl acetate fraction, and aglycone fraction were 86.23%, 86.59%, and 89.70%, respectively. The aglycone fraction increased cell viability up to 11.53% at $1{\mu}g/ml$ compared to the positive control treated with $H_2O_2$. Analysis of ultraviolet B radiation-induced HaCaT cell damage revealed up to 41.77% decreased intracellular reactive oxygen species in the $2{\mu}g/ml$ aglycone fraction compared with the positive control treated with ultraviolet B radiation. The findings suggest that the extracts and fractions of A. asphodeloides Bunge have potential applications in the field of cosmetics as natural preservatives and antioxidants.

• Journal of Periodontal and Implant Science
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• v.30 no.2
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• pp.213-231
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• 2000

이 연구의 목적은 다공성의 CPP 내부에 쥐의 장골의 골수에서 유래된 세포를 접종하고 3차 원적으로 배양하여 CPP가 골 형성을 위한 조직공학의 지지체로 적용가능한가를 연구하는 것과 Calcium PolyPhosphate(CPP)의 돌연변이 유발성을 검사하는 것이다. 무수 ($Ca(H_2PO_4)$)를 condensation하여 무결정의 ($Ca(PO_3)$)를 얻고 이를 용융하고 냉각시킨 후 분쇄하여 Calcium polyphosphate(CPP) powder를 얻었다. 다공성의 CPP는 5% $SiO_2$를 첨가하여 sponge 형태로 $450-550{\mu}m$ 소공의 크기를 가지는 것과(CPP-45ppi) $200-300{\mu}m$의 소공의 크기를 가지는 것(CCP-60ppi) 2가지로 제작하였다. 각각의 CPP matrices는 $5mm{\times}5mm{\times}1mm$의 블록 형태로 만들었다. 체중 100g 내외의 백서에서 장골(femur, tibia)을 채취하여 백서의 장골 골수 세포를 분리하여 배양한 후 24well에 CPP block을 넣고 CPP block 당 $10^5$개의 배양한 세포를 접종하였다. 배양 1, 7, 14, 및 21 일째에 각 well에서 trypsin EDTA를 이용하여 2회 반복하여 cell을 분리하였고, 원심분리한 후 hemacytometer로 측정하였다. 또, 45ppi와 60ppi, 그 리 고 Tissue Culture Polystyrene(control group)에 접종, 배양된 세포들의 염기성 인산분해효소활성도를 배양 7, 14, 및 21 일째에 각각 측정하였다. 각 기간별로 배양된 세포-CPP 혼합체내에서 세포의 부착 및 증식과 형성된 조직의 3차원적 형태를 관찰하기 위하여 주사전자현미경하에서의 관찰하였다. CPP의 돌연변이 유발성 검사 (mutagenicity test)를 위해 hypoxanthine-guanine phosphoribosyl transferase(HPRT) assay를 하였다. NIH3T3 cell line과 CHO-K1 cell line으로 각각 $1000{\mu}g/m{\ell}$, $100{\mu}g/m{\ell}$, $10{\mu}g/m{\ell}$ 그리고 $1{\mu}g/m{\ell}$의 CPP 농도에서 측정하였다. 통계적 분석을 위해서 모든 측정은 각군당 4개체 이상 시험하였고, 각 측정값은 평균값${\pm}$표준편차로 나타내었다. 각 군간의 통계적 유의성 검정을 위해서 Analysis of variance(ANOVA)를 이용하였고 Tukey의 방법으로 사후분석을 실시하였다. 제작된 CPP matrices 소공들이 서로간에 연결이 잘 되어있는 형태였다. 두 가지로 제조된 CPP(45ppi와 60ppi) 모두에서 세포의 부착이 잘 일어났고, 부착된 세포의 분열도 잘 일어났다. 2 가지의 CPP 모두에서 7, 14, 21일째의 세포 수는 1일째에 비해 유의성 있게 증가하였다(P<0.01). 3차원적 구조인 Calcium PolyPhosphate에서 배양한 세포는 24well dish(tissue culture polystyrene)에서 평면적으로 배양한 대조군의 세포에서 보다 염기성 인산분해효소 (Alkaline Phosphatase)를 유의성 있게 높게 나타냈다. 주사전자현미경에서 세포-CPP 혼합체를 관찰한 결과, CPP block에 세포들이 잘부착되어 있었고, 시간이 지남에 따라 세포가 여러 층을 형성하면서 뭉치는 현상을 보였다. 또, HPRT assay 결과 , Calcium PolyPhosphate는 돌연변이 유발성을 보이지 않았다. 이상의 결과로 볼 때 CPP에는 세포부착이 잘 일어나고, 지지체 상에서 세포의 분열도 활발하게 일어나므로 골조직을 위한 조직공학의 우수한 지지체가 될 수 있을 것으로 사료된다.

• Korean Journal of Food Science and Technology
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• v.39 no.2
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• pp.209-216
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• 2007

The present study investigated anti-oxidative and anti-inflammatory activity of glycoprotein isolated from Morus Indica Linne (MIL glycoprotein). We found that MIL glycoprotein has a molecular weight of 32 kD and consists of carbohydrate (40.03%) and protein (59.97%), and that it has a strong scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl radical $({\cdot}OH)$, and superoxide anion $(O_2{\cdot}\;^-)$ radicals. In addition, MIL glycoprotein had a stable character and an optimal DPPH radical scavenging activity in the alkaline and neutral pH solution, and up to at 105. However, the results indicated that it has a minimal scavenging activity in the metal ionic solution ($Ca^{2+}$, $Mn^{2+}$, and $Mg^{2+}$) in the presence of EDTA. In addition, we further investigated whether MIL glycoprotein scavenges oxygen radicals and blocks inflammation-related signals in the bisphenol A (BPA)-stimulated Raw 264.7 cells. The results in this study showed that it has a character to scavenge the productions of reactive oxygen species (ROS) and nitric oxide (NO) dose-dependently. Also it blocked the activities of inflammation-related signals such as nuclear factor-kappa B ($NF-{\kappa}B$) and inducible nitric oxide synthase (iNOS). For example, it had an inhibitory effect on the activation of $NF-{\kappa}B$ (p50) and iNOS proteins at 200 ${\mu}g/mL$ MIL glycoprotein. Here, we speculate that MIL glycoprotein is one of natural antioxidants and of modulators of the BPA-induced inflammation.

• Journal of the East Asian Society of Dietary Life
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• v.17 no.6
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• pp.808-815
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• 2007

Four different herbs, including rosemary, oregano, basil and sage, along with green tea were individually added to ground beef at different percentages: 0.5, 1.0, and 1.5, respectively. The sensory qualities of the five natural herbs and their antioxidant effects were evaluated using Beef-Yukwonjeon. For the sensory evaluation results, the 0.5% herb addition and the control group generally showed higher acceptability scores than the 1.0% or 1.5% herb addition groups. In particular, the Yukwonjeon made with 0.5% green tea showed higher preferences in the acceptance tests and ranking tests. However, the sensory differences among samples were reduced after 3 days of storage at $6^{\circ}C$. For the results of lipid oxidation without storage, the TBARS values of the herb addition groups were slightly lower than the value of the control group, but not significantly(p>0.05). However, the antioxidant effects of each herb showed higher values over a longer storage period as well as at the higher herb concentrations. The antioxidant effects of the herbs during 7 days' of storage at $6^{\circ}C$ were evaluated as $10.0{\sim}38.5%$ reductions in TBARS values as compared to the group without herbs. Furthermore, the antioxidant effects of the herbs during 30 days' of storage at $-20^{\circ}C$ were shown as $16.8{\sim}27.8%$ reductions in the TBARS value. The green tea showed the highest antioxidant activity against lipid oxidation, because it was acceptable at higher concentrations in the sensory evaluation. These results indicate the potential to produce better quality beef-Yukwonjeon, with herbs, especially green tea.