Molecular cloning and Biochemical properties of GH-16 β-agarase from Gilvimarinus agarolyticus JEA5

Agar is complex polysaccharide founds in the cell walls of some red algae and up to 70 % of the algal cell wall can be agar polymers. Agar was formed by a mixture of two polysaccharides named agarose and agaropectin. Agarose can be hydrolyzed by α-agarase (E.C. 3.2.1.158) and by β-agarase (E.C. 3.2.1.81); the former cleaves the α-1, 3 linkage of agarose to generate agaro-oligosaccharides, and the latter cleaves the β-1,4 linkage to generate neoagaro-oligosaccharides. Agarases have been isolated from many sources, including seawater, marine sediments, marine algae, marine mollusks, fresh water and soil. Recently, Givimarinus chinensis, G. polysacchalyticus, G. agarilyticus were identified and their agarolytic activity also reported. However, there are no report published that molecular and functional characterization of agarase from Givimarinus genus. In this study, we first report molecular characterization and biochemical properties of agarase from Gilvimarinus genus. Please click Additional Files below to see the full abstract

Biochemical properties of a novel neoagarotriose-producing β-agarase from Gilvimarinus agarolyticus JEA5

An agar degrading bacterium was isolated from seawater, collected from the east coast of Jeju Island, republic of Korea and identified as Gilvimarinus agarolyticus JEA5. The β-agarase gene from Gilvimarinus agarolyticus JEA5 (rGaa16B) was identified from draft genome sequence by BLAST. Gaa16B has 1800 bp of open reading frame encoding 636 amino acids (aa), and include glycosyl hydrolase family 16 (GH16) β-agarase module and two carbohydrate binding module 6 (CBM6). The Gaa16b was cloned and overexpressed as a MBP-fusion recombinant β-agarase (without signal peptide and two CBM6) in E. coil. rGaa16B showed highest activity at 60°C and pH 7. After incubation at 45OC for 90 min, rGaa16B showed over than 95% of its initial activity. rGaa16B were enhanced in the presence of MnCl2, KCl2, MgCl2, FeSO4. rGaa16B showed 2112.1 unit/mg in the presence of 2.5 mM of MnCl2. rGaa16B produce mainly neoagartetraose (NA4) and neoagarobiose (NA2). Interestingly, we observed neoagartriose (NA3) from hydrolytic products of rGaa16B. LC/Mass analysis was performed to confirm the hydrolytic products containing neoagarotriose. We found three different hydrolytic products which showed 324.28, 468.41, 630.55 Da of molecular weight, respectively. Please click Additional Files below to see the full abstract

A newly identified glutaminase-free ʟ-asparaginase (ʟ-ASPG86) from the marine bacterium Mesoflavibacter zeaxanthinifaciens

ʟ-Asparaginase (EC 3.5.1.1) is an enzyme involved in asparagine hydrolysis and has the potential to effect leukemic cells and various other cancer cells. We identified the ʟ-asparaginase gene (ʟ-ASPG86) in the genus Mesoflavibacter, which consists of a 1035-bp open reading frame (ORF) encoding 344 amino acids (aa). Following phylogenetic analysis, the deduced amino acid sequence of ʟ-ASPG86 (ʟ-ASPG86) grouped as a type I asparaginase with respective homologs in Escherichia coli and Yersinia pseudotuberculosis. The ʟ-ASPG86 gene was cloned into the pET-16b vector to express the respective protein in E. coli BL21 (DE3) cells. Recombinant ʟ-asparaginase (r-ʟ-ASPG86) showed optimum conditions at 37-40oC, pH 9. Moreover, r-ʟ-ASPG86 did not exhibit glutaminase activity. In the metal ions test, its enzymatic activity was highly improved upon addition of 5 mM manganese (3.97-fold) and magnesium (3.35-fold) compared with the untreated control. The specific activity of r-ʟ-ASPG86 was 687.1 units/mg under optimum conditions (37oC, pH 9 and 5 mM MnSO4). Please click Additional Files below to see the full abstract

Synergistic effect of acetyl xylan esterase on xylanase reaction originated from Ochrovirga pacifica

Acetyl xylan esterase plays an important role in complete enzymatic hydrolysis of lignocellulosic materials into fermentable sugars. It hydrolyzes ester linkages of acetic acid in xylan polysaccharide and supports to enhance the activity of xylanase. This study was conducted to recognize and overexpress the acetyl xylan esterase gene found from Ochrovirga pacifica strain S85 which was isolated from Chuuk state, Micronesia. The genome sequence was analyzed with genome sequencer-FLX and acetyl xylan esterase gene (Axe) was detected. The gene had an open reading frame of 864 bp encoding a polypeptide of 287 amino acids. Theoretical molecular mass and isoelectric point (pI) were 32 kDa and 5.9, respectively. The deduced amino acid sequence of the Axe showed 35.1% similarity with both endo-1,4-β-xylanase B from Robiginitalea biformata HTCC2501. The mature protein displayed the catalytic residues classically found in enzymes belonged to GH16 family. Axe was cloned into pET11a vector and recombinant protein was expressed in E. coli BL21 (DE3), purified by nickel affinity chromatography and its purity was visualized on SDS-PAGE. Commercial xylanase activity was tested after treatment of recombinant acetyl xylan esterase (rAXE) to birchwood xylan substrate. The xylanase activity of rAXE treated sample was about 2 times higher than xylanase only treated sample. Please click Additional Files below to see the full abstract

Recombinant protein production in Escherichia coli by combining of signal peptide originated from Bacillus subtilis

We isolated chitosanase secreting B. subtilis CH2 and identified the chitosanase nucleotide sequence. Analyzed the sequence showed that it consisted of 813 bp, including 87 bp signal sequence. The signal sequence leads the target protein to the cell-membrane of the B. subtilis CH2 and then secret the chitosanase out of the cell. The signal peptide showed 6 amino acids deletion compared to other B. subtilis chitosanase signal peptides. The chitosanase sequence including signal peptide was cloned into pET11a vector without fusion and expressed in E. coli BL21(DE3). The expressed chitosanase in E. coli showed two distinct bands which represent the pro-chitosanase in cytoplasm and mature chitosanase in periplasm. Time frame induction and results showed that muture chitosanase was increased. Subsequently, we linked this chitosanase signal sequence in front of B. subtilis CH2 xylanase and human superoxide distimutase 1 (hSOD1) sequences, and expressed it in E. coli BL21(DE3). The recombinant xylanase and hSOD1 moved to periplasmic space with high efficiency. This signal sequence is useful for bio-medical protein production in E. coli. Please click Additional Files below to see the full abstract

20대 총선과 미디어 효과 그리고 유권자의 여론조사에 대한 인식

오늘날 선거에서 매스미디어는 유권자들의 선거에 대한 관심을 독려하고, 중요한 정치 정보를 제공하고, 나아가 정치 참여를 촉진하기도 하며, 한편으로 미디어가 선거나 정치과정을 여과 없이 유권자들에게 노출시킴에 따라 일반 시민들의 정치에 대한 냉소나 회의감을 증폭시키는 부정적 모습을 연출하기도 한다. 이에 본 연구는 20대 총선 과정과 관련된 다양한 미디어의 기능과 효과들에 대한 경험적 분석을 실시하였다. 20대 총선에 참여한 유권자 의식조사 결과 분석에 따르면, 이번 총선에서도 매스미디어는 주요 선거정보 제공자로서 큰 기능을 담당한 것으로 조사되었다. 또한 유권자들은 이번 총선에서 언론보도가 대체로 공정하게 진행되었다고 믿는 것으로 나타났다. 다만, 이러한 인식은 정당일체감, 이념성향 그리고 다양한 인구통계적 특성 등 유권자들의 다양한 정치사회적 배경에 따라 다소 차별화되는 것으로 나타났다. 뿐만 아니라, 유권자들이 언론매체에 노출되는 빈도가 높을수록 더 풍부한 정치지식과 더 높은 선거관심도를 갖는 것으로 조사되었는데, 이는 또한 유권자들의 전반적 정치참여에 대한 긍정적 효과로 이어지기도 하였다. 실제로 TV 토론회를 더 많이 시청한 사람들이나 TV 나 라디오를 더 자주 시청한 유권자들 그룹일수록 투표나 비인습적 정치참여를 할 확률이 올라가는 것으로 나타났다. 즉, 한국 선거과정에 있어서 매스미디어는 다양한 정치적 효과를 갖는다. 따라서 미디어의 다양한 정치적 효과를 감안해 향후에도 민주적 선거과정에서 미디어의 기능과 역할에 대한 지속적 관심과 후속연구가 필요할 것이다. Previous literature has shown that mass media tend to increase people’s interests in election and encourage more active participation of voters by providing useful political information. By utilizing the original survey of Korean voters during the 20th General election race of April 2016, this study examines if this positive media effect has occurred. In addition this paper also investigates how voters evaluate media coverage of election campaigns in terms of its fairness and accuracy. The findings of this study suggest that most voters believe media coverage of elections was fair, although this perception varies by respondents’ political and ideological backgrounds. Also this study finds that more exposure of individuals to media such as televised debates leads to active participation in political process as well as increased interests in elections.22Nkc

Dynamic modeling and numerical analysis of a cold rolling mill

The dynamic characteristics in a rolling process have been investigated with various methods, but a mathematical model to predict actual chatter vibration has not been established. In this paper, we propose a mathematical model of a cold rolling mill to predict chatter vibration. The mathematical model was verified by experiment and theoretical analysis. The chatter frequency predicted by the model was very similar to that determined by experiment and theoretical analysis. The model took into account the stiffness caused by roller bearings and contact between rolls. We considered not only the vertical, but also the horizontal rolling force, with the aim of investigating the added effect of the latter. The work and the intermediate rolls had the same periods of vibration because their masses were similar and the rolls were adjacent. Both the vertical and horizontal vibrations were investigated. The maximum displacement and amplitude of chatter frequency were much larger in the vertical than in the horizontal direction. To reduce chatter vibration, the rolling force had to be reduced within the range of efficient productivity. The chatter frequency that affected the performance of the rolling mill was found, and the dynamic characteristics were investigated.X111714sciescopuskc

Development of a Mathematical Model for the Prediction of Vibration in a Cold Rolling Mill Including the Driving System

To date, a variety of analytical and mathematical dynamic models of a rolling mill have been developed, but they were simplified models involving the vertical vibration of the rolls and were not enough to be compatible with actual chatter vibration. In this paper, a mathematical model of a cold rolling mill including the driving system is proposed. The model is reliable enough to be compatible with experimental and theoretical analysis. It took into account the frictional forces between the rolls and the stiffness caused by the roller bearings and the contact between rolls. The joint forces of the spindle were computed from the force equilibrium equations in which the contact stiffness between the gears was approximated in a Fourier series form. To solve the model efficiently, a novel combination of the direct integration method and quasistatic analysis was proposed. The principal frequencies, including the gear mesh and chatter frequencies, predicted by the model were very similar to that determined by experimental and theoretical analysis. Not only the vertical, but also the horizontal vibration was investigated to study the added effect of the horizontal rolling force, frictional forces, and joint forces. The horizontal chatter vibration had a strong effect on the dynamic characteristics, although the chatter frequency was generated both in the vertical and horizontal directions.ungraded118sciescopu

Optimising the DPPH Assay for Cell-Free Marine Microorganism Supernatants

Antioxidants prevent ageing and are usually quantified and screened using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. However, this assay cannot be used for salt-containing samples, such as the cell-free supernatants of marine microorganisms that are aggregated under these conditions. Herein, the DPPH solvent (methanol or ethanol) and its water content were optimized to enable the analysis of salt-containing samples, aggregation was observed for alcohol contents of >70%. The water content of methanol influenced the activities of standard antioxidants but did not significantly affect that of the samples. Based on solution stability considerations, 70% aqueous methanol was chosen as the optimal DPPH solvent. The developed method was successfully applied to the cell-free supernatants of marine bacteria (Pseudoalteromonas rubra and Pseudoalteromonas xiamenensis), revealing their high antioxidant activities. Furthermore, it was concluded that this method would be useful for the screening of marine microorganism–derived antioxidants, which also has numerous potential applications, such as salt-fermented foods