• Journal of Microbiology and Biotechnology
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• v.25 no.2
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• pp.227-233
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• 2015

Two recombinant arabinosyl hydrolases, α-_L-arabinofuranosidase from Geobacillus sp. KCTC 3012 (GAFase) and endo-(1,5)-α-_L-arabinanase from Bacillus licheniformis DSM13 (BlABNase), were overexpressed in Escherichia coli, and their synergistic modes of action against sugar beet (branched) arabinan were investigated. Whereas GAFase hydrolyzed 35.9% of _L-arabinose residues from sugar beet (branched) arabinan, endo-action of BlABNase released only 0.5% of _L-arabinose owing to its extremely low accessibility towards branched arabinan. Interestingly, the simultaneous treatment of GAFase and BlABNase could liberate approximately 91.2% of _L-arabinose from arabinan, which was significantly higher than any single exo-enzyme treatment (35.9%) or even stepwise exo- after endo-enzyme treatment (75.5%). Based on their unique modes of action, both exo- and endo-arabinosyl hydrolases can work in concert to catalyze the hydrolysis of arabinan to _L-arabinose. At the early stage in arabinan degradation, exo-acting GAFase could remove the terminal arabinose branches to generate debranched arabinan, which could be successively hydrolyzed into arabinooligosaccharides via the endo-action of BlABNase. At the final stage, the simultaneous actions of exo- and endo-hydrolases could synergistically accelerate the _L-arabinose production with high conversion yield.

• Journal of Microbiology and Biotechnology
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• v.22 no.11
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• pp.1532-1539
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• 2012

The ${\alpha}$-galactosidase-coding gene agaAJB13 was cloned from Sphingomonas sp. JB13 showing 16S rDNA (1,343 bp) identities of ${\leq}97.2%$ with other identified Sphingomonas strains. agaAJB13 (2,217 bp; 64.9% GC content) encodes a 738-residue polypeptide (AgaAJB13) with a calculated mass of 82.3 kDa. AgaAJB13 showed the highest identity of 61.4% with the putative glycosyl hydrolase family 36 ${\alpha}$-galactosidase from Granulicella mallensis MP5ACTX8 (EFI56085). AgaAJB13 also showed <37% identities with reported protease-resistant or Sphingomonas ${\alpha}$-galactosidases. A sequence analysis revealed different catalytic motifs between reported Sphingomonas ${\alpha}$-galactosidases (KXD and RXXXD) and AgaAJB13 (KWD and SDXXDXXXR). Recombinant AgaAJB13 (rAgaAJB13) was expressed in Escherichia coli BL21 (DE3). The purified rAgaAJB13 was characterized using p-nitrophenyl-${\alpha}$-D-galactopyranoside as the substrate and showed an apparent optimum at pH 5.0 and $60^{\circ}C$ and strong resistance to trypsin and proteinase K digestion. Compared with reported proteaseresistant ${\alpha}$-galactosidases showing thermolability at $50^{\circ}C$ or $60^{\circ}C$ and specific activities of <71 U/mg with or without protease treatments, rAgaAJB13 exhibited a better thermal stability (half-life of >60 min at $60^{\circ}C$) and higher specific activities (225.0-256.5 U/mg). These sequence and enzymatic properties suggest AgaAJB13 is the first identified and characterized Sphingomonas ${\alpha}$-galactosidase, and shows novel protease resistance with a potential value for basic research and industrial applications.

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.36-36
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• 2017

Out of twenty common protein amino acids, there are many kinds of non protein amino acids (NPAAs) that exist as secondary metabolites and exert ecological functions in plants. Mimosine (Mim), one of those NPAAs derived from L. leucocephala acts as an iron chelator and reversely block mammalian cell cycle at G1/S phases. Cysteine (Cys) is decisive for protein and glutathione that acts as an indispensable sulfur grantor for methionine and many other sulfur-containing secondary products. Cys biosynthesis includes consecutive two steps using two enzymes-serine acetyl transferase (SAT) and O-acetylserine (thiol)lyase (OASTL) and appeared in plant cytosol, chloroplast, and mitochondria. In the first step, the acetylation of the ${\beta}$-hydroxyl of L-serine by acetyl-CoA in the existence of SAT and finally, OASTL triggers ${\alpha}$, ${\beta}$-elimination of acetate from OAS and bind $H_2S$ to catalyze the synthesis of Cys. Mimosine synthase, one of the isozymes of the OASTLs, is able to synthesize Mim with 3-hydroxy-4-pyridone (3H4P) instead of $H_2S$ for Cys in the last step. Thus, the aim of this study was to clone and characterize the cytosolic (Cy) OASTL gene from L. leucocephala, express the recombinant OASTL in Escherichia coli, purify it, do enzyme kinetic analysis, perform docking dynamics simulation analysis between the receptor and the ligands and compare its performance between Cys and Mim synthesis. Cy-OASTL was obtained through both directional degenerate primers corresponding to conserved amino acid region among plant Cys synthase family and the purified protein was 34.3KDa. After cleaving the GST-tag, Cy-OASTL was observed to form mimosine with 3H4P and OAS. The optimum Cys and Mim reaction pH and temperature were 7.5 and $40^{\circ}C$, and 8.0 and $35^{\circ}C$ respectively. Michaelis constant (Km) values of OAS from Cys were higher than the OAS from Mim. Inter fragment interaction energy (IFIE) of substrate OAS-Cy-OASTL complex model showed that Lys, Thr81, Thr77 and Gln150 demonstrated higher attraction force for Cys but 3H4P-mimosine synthase-OAS intermediate complex showed that Gly230, Tyr227, Ala231, Gly228 and Gly232 might provide higher attraction energy for the Mim. It may be concluded that Cy-OASTL demonstrates a dual role in biosynthesis both Cys and Mim and extending the knowledge on the biochemical regulatory mechanism of mimosine and cysteine.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.4
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• pp.607-615
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• 2018

Objective: This study was conducted to isolate the cellulolytic microorganism from the rumen of Holstein steers and characterize endoglucanase gene (Cel5A) from the isolated microorganism. Methods: To isolate anaerobic microbes having endoglucanase, rumen fluid was obtained from Holstein steers fed roughage diet. The isolated anaerobic bacteria had 98% similarity with Eubacterium cellulosolvens (E. cellulosolvens) Ce2 (Accession number: AB163733). The Cel5A from isolated E. cellulolsovens sp. was cloned using the published genome sequence and expressed through the Escherichia coli BL21. Results: The maximum activity of recombinant Cel5A (rCel5A) was observed at $50^{\circ}C$ and pH 4.0. The enzyme was constant at the temperature range of $20^{\circ}C$ to $40^{\circ}C$ but also, at the pH range of 3 to 9. The metal ions including $Ca^{2+}$, $K^+$, $Ni^{2+}$,$Mg^{2+}$, and $Fe^{2+}$ increased the endoglucanase activity but the addition of $Mn^{2+}$, $Cu^{2+}$, and $Zn^{2+}$ decreased. The Km and Vmax value of rCel5A were 14.05 mg/mL and $45.66{\mu}mol/min/mg$. Turnover number, Kcat and catalytic efficiency, Kcat/Km values of rCel5A was $96.69(s^{-1})$ and 6.88 (mL/mg/s), respectively. Conclusion: Our results indicated that rCel5A of E. cellulosolvens isolated from Holstein steers had a broad pH range with high stability under various conditions, which might be one of the beneficial characteristics of this enzyme for possible industrial application.

• 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.

• Parasites, Hosts and Diseases
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• v.52 no.1
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• pp.21-26
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• 2014

Dirofilaria immitis (heartworm) infections affect domestic dogs, cats, and various wild mammals with increasing incidence in temperate and tropical areas. More sensitive antibody detection methodologies are required to diagnose asymptomatic dirofilariasis with low worm burdens. Applying current transcriptomic technologies would be useful to discover potential diagnostic markers for D. immitis infection. A filarial homologue of the mammalian translationally controlled tumor protein (TCTP) was initially identified by screening the assembled transcriptome of D. immitis (DiTCTP). A BLAST analysis suggested that the DiTCTP gene shared the highest similarity with TCTP from Loa loa at protein level (97%). A histidine-tagged recombinant DiTCTP protein (rDiTCTP) of 40 kDa expressed in Escherichia coli BL21 (DE3) showed immunoreactivity with serum from a dog experimentally infected with heartworms. Localization studies illustrated the ubiquitous presence of rDiTCTP protein in the lateral hypodermal chords, dorsal hypodermal chord, muscle, intestine, and uterus in female adult worms. Further studies on D. immitis-derived TCTP are warranted to assess whether this filarial protein could be used for a diagnostic purpose.

• Horticultural Science & Technology
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• v.34 no.6
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• pp.924-939
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• 2016

The great economic losses caused by Cucumber mosaic virus (CMV) infection of peppers has led to the development of genetically modified (GM) CMV-resistant peppers. We developed virus-resistant pepper plants using Agrobacterium tumefaciens -mediated transformation. The expressed recombinant protein was purified using nickel-nitrilotriacetic acid resin and immunoaffinity chromatography, and purity was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Immunoblot analysis revealed the purified CMV coat protein (CMV-CP) had a molecular mass of 25 kDa. After in-gel digestion and desalting, the internal peptide fragments of CMV-CP were sequenced by matrix-assisted laser desorption/ionization-time of flight. Most GM pepper and Escherichia coli BL21 internal peptides had identical peptide sequences and contained 137 of 183 whole peptides in CMV-CP. A quantitative enzyme-linked immunosorbent assay was performed to detect CMV-resistant GM peppers. We also provide basic information about the expressed protein in GM peppers for further safety assessment. The contents of soluble protein and CMV-CP were measured in GM and control peppers cultivated in three different areas of Korea. Statistical significance in terms of cultivation areas, harvest times, generations, and plant tissue origin were determined based on a P value of 0.05. The highest amount of CMV-CP was detected at the seedling stage from plant grown in each region. T3 and T5 showed significantly different levels of CMV-CP from T4 in leaves in the whorl stage. No statistical differences were observed among GM peppers at different stages of maturity in any cultivation area. The results from this study contribute to the safety evaluation of newly designed CMV-resistant GM peppers and provide a standard against which to compare other virus-resistant GM peppers.

• 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 Microbiology
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• v.54 no.2
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• pp.126-135
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• 2018

The characteristics of enzyme and gene for mannanase B had been reported from Cellulosimicrobium sp. YB-43 producing some kind of mannanase. A gene coding for the enzyme, named mannanase C (ManC), was expected to be located downstream of the manB gene. The manC gene was cloned by polymerase chain reaction and sequenced completely. From this nucleotide sequence, ManC was identified to consist of 448 amino residues and contain a carbohydrate binding domain CBM2 besides a catalytic domain, which was homologous to mannanase belonging to the glycosyl hydrolase family 5. The catalytic domain of ManC showed the highest amino acid sequence similarity of 55% with the mannanases from Streptomyces sp. SirexAA-E (55.8%; 4FK9_A) and S. thermoluteus (57.6%; BAM62868). The His-tagged ManC (HtManC) lacking N-terminal signal peptide with hexahistidine at C-terminus was produced and purified from cell extract of recombinant Escherichia coli. The purified HtManC showed maximal activity at $65^{\circ}C$ and pH 7.5, with no significant change in its activity at pH range from 7.5 to 10. HtManC showed more active on konjac and locust bean gum (LBG) than guar gum and ivory nut mannan (ivory nut). Vmax and Km values of the HtManC for LBG were 68 U/mg and 0.45 mg/ml on the optimal condition, respectively. Mannobiose and mannotriose were observed on TLC as major products resulting from the HtManC hydrolysis of mannooligosacharides. In addition, mannobiose and mannose were commonly detected as the hydrolyzed products of LBG, konjac and ivory nut.

• Journal of Microbiology and Biotechnology
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• v.16 no.10
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• pp.1529-1536
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• 2006

We cloned a gene of ompH(D:4) from pigs infected with P. multocida D:4 in Korea [16]. The gene is composed of 1,026 nucleotides coding 342 amino acids (aa) with a signal peptide of 20 aa (GenBank accession number AY603962). In this study, we analyzed the ability of the ompH(D:4) to induce protective immunity against a wild-type challenge in mice. To determine appropriate epitope(s) of the gene, one full and three different types of truncated genes of the ompH(D:4) were constructed by PCR using pET32a or pRSET B as vectors. They were named ompH(D:4)-F (1,026 bp [1-1026] encoding 342 aa), ompH(D:4)-t1 (693 bp [55-747] encoding 231 aa), ompH(D:4)-t2 (561 bp [187-747] encoding 187 aa), and ompH(D:4)-t3 (540 bp [487-1026] encoding 180 aa), respectively. The genes were successfully expressed in Escherichia coli BL21(DE3). Their gene products, polypeptides, OmpH(D:4)-F, -t1, -t2, and -t3, were purified individually using nickel-nitrilotriacetic acid (Ni-NTA) affinity column chromatography. Their $M_rs$ were determined to be 54.6, 29, 24, and 23.2 kDa, respectively, using SDS-PAGE. Antisera against the four kinds of polypeptides were generated in mice for protective immunity analyses. Some $50{\mu}g$ of the four kinds of polypeptides were individually provided intraperitoneally with mice (n=20) as immunogens. The titer of post-immunized antiserum revealed that it grew remarkably compared with pre-antiserum. The lethal dose of the wild-type pathogen was determined at $10{\mu}l$ of live P. multocida D:4 through direct intraperitoneal (IP) injection, into post-immune mice (n=5, three times). Some thirty days later, the lethal dose ($10{\mu}l$) of live pathogen was challenged into the immunized mouse groups [OmpH(D:4)-F, -t1, -t2, and -t3; n=20 each, two times] as well as positive and negative control groups. As compared within samples, the OmpH(D:4)-F-immunized groups showed lower immune ability than the OmpH(D:4)-t1, -t2, and -t3. The results show that the truncated-OmpH(D:4)-t1, -t2, and -t3 can be used for an effective vaccine candidate against swine atrophic rhinitis caused by pathogenic P. multocida (D:4) isolated in Korea.