• Journal of Microbiology and Biotechnology
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• v.12 no.4
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• pp.603-609
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• 2002

DFA IV is di-D-fructose-2,6':6,2'-dianhydride, consisting of two fructose residues. It can be enzymatically synthesized from levan by levan fructotransferase, and can be used for mineral absorption. Understanding of the structure and composition of levan is important to obtain high-level production of DFA IV. A bacterial strain, Pseudomonas aurantiaca 5-4380, was identified to produce low-branched levan, and the levansucrase gene (lsch) from this bacterium was found to be composed of 1,275 Up coding for a protein of 424 amino acids, with an estimated molecular weight of 47 kDa. The bacterial levansucrase gene was expressed in Escherichia coli DH5${\alpha}$ by its own promoter and lac promoter. The recombinant levansucrase was produced in soluble form with 170U of levansucrase activity from 1-ml E. coii culture broth. The expressed enzyme from the clone showed similar biochemical properties, such as size of active levansucrase, degree of branching, and optimum temperature, with P.aurantiaca 5-4380 levansucrase.

• Journal of Microbiology and Biotechnology
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• v.22 no.3
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• pp.316-325
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• 2012

Xylanase has been used extensively in the industrial and agricultural fields. However, the low-yield production of xylanase from native species cannot meet the increasing demand of the market. Therefore, improving the heterologous expression of xylanase through basic gene optimization may help to overcome the shortage. In this study, we synthesized a high-GC-content native sequence of the thermostable xylanase gene xynB from Streptomyces olivaceoviridis A1 and, also designed a slightly AT-biased sequence with codons completely optimized to be favorable to Pichia pastoris. The comparison of the sequences' expression efficiencies in P. pastoris X33 was determined through the detection of single-copy-number integrants, which were quantified using qPCR. Surprisingly, the high GC content did not appear to be detrimental to the heterologous expression of xynB in yeast, whereas the optimized sequence, with its extremely skewed codon usage, exhibited more abundant accumulation of synthesized recombinant proteins in the yeast cell, but an approximately 30% reduction of the secretion level, deduced from the enzymatic activity assay. In this study, we developed a more accurate method for comparing the expression levels of individual yeast transformants. Moreover, our results provide a practical example for further investigation of what constitutes a rational design strategy for a heterologously expressed and secreted protein.

• Journal of Microbiology and Biotechnology
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• v.27 no.2
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• pp.271-276
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• 2017

A highly thermostable ${\beta}-(1-4)-glucanase$ (NA23_08975) gene (fig) from Fervidobacterium islandicum AW-1, a native-feather degrading thermophilic eubacterium, was cloned and expressed in Escherichia coli. The recombinant FiG (rFiG) protein showed strong activity toward ${\beta}-{\small{D}}-glucan$ from barley (367.0 IU/mg), galactomannan (174.0 IU/mg), and 4-nitrophenyl-cellobioside (66.1 IU/mg), but relatively weak activity was observed with hydroxyethyl cellulose (5.3 IU/mg), carboxymethyl cellulose (2.4 IU/mg), and xylan from oat spelt (1.4 IU/mg). rFiG exhibited optimal activity at $90^{\circ}C$ and pH 5.0. In addition, this enzyme was extremely thermostable, showing a half-life of 113 h at $85^{\circ}C$. These results indicate that rFiG could be used for hydrolysis of cellulosic and hemicellulosic biomass substrates for biofuel production.

• The Plant Pathology Journal
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• v.36 no.6
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• pp.591-599
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• 2020

Phytophthora root and stem rot reduce soybean yields worldwide. The use of R-gene type resistance is currently crucial for protecting soybean production. The present study aimed to identify the genomic location of a gene conferring resistance to Phytophthora sojae isolate 2457 in the recombinant inbred line population developed by a cross of Daepung × Daewon. Singlemarker analysis identified 20 single nucleotide polymorphisms associated with resistance to the P. sojae isolate 2457, which explained ~67% of phenotypic variance. Daewon contributed a resistance allele for the locus. This region is a well-known location for Rps1 and Rps7. The present study is the first, however, to identify an Rps gene locus from a major soybean variety cultivated in South Korea. Linkage analysis also identified a 573 kb region on chromosome 3 with high significance (logarithm of odds = 13.7). This genomic region was not further narrowed down due to lack of recombinants within the interval. Based on the latest soybean genome, ten leucine-rich repeat coding genes and four serine/ threonine protein kinase-coding genes are annotated in this region, which all are well-known types of genes for conferring disease resistance in crops. These genes would be candidates for molecular characterization of the resistance in further studies. The identified R-gene locus would be useful in developing P. sojae resistant varieties in the future. The results of the present study provide foundational knowledge for researchers who are interested in soybean-P. sojae interaction.

• Journal of Microbiology and Biotechnology
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• v.32 no.4
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• pp.484-492
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• 2022

Lichenase is an enzyme mainly implicated in the degradation of polysaccharides in the cell walls of grains. Emerging evidence shows that a highly efficient expression of a thermostable recombinant lichenase holds considerable promise for application in the beer-brewing and animal feed industries. Herein, we cloned a lichenase gene (CelA203) from Bacillus subtilis B110 and expressed it in E. coli. This gene contains an ORF of 729 bp, encoding a protein with 242 amino acids and a calculated molecular mass of 27.3 kDa. According to the zymogram results, purified CelA203 existed in two forms, a monomer, and a tetramer, but only the tetramer had potent enzymatic activity. CelA203 remained stable over a broad pH and temperature range and retained 40% activity at 70℃ for 1 h. The K_m and V_max of CelA203 towards barley β-glucan and lichenan were 3.98 mg/ml, 1017.17 U/mg, and 2.78 mg/ml, 198.24 U/mg, respectively. Furthermore, trisaccharide and tetrasaccharide were the main products obtained from CelA203-mediated hydrolysis of deactivated oat bran. These findings demonstrate a promising role for CelA203 in the production of oligosaccharides in animal feed and brewing industries.

• Journal of Veterinary Clinics
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• v.40 no.3
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• pp.175-181
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• 2023

Fucoidan extracted from brown seaweed has a variety of biological activities. Neutrophil extracellular traps (NETs) formation is an immune response for the invasion of pathogens. Neutrophils release granule protein and chromatin that form extracellular fibers that bind microbes. These NETs degrade virulence factors and kill bacteria. The aim of this study was to investigate the effect of fucoidan on NET formation of porcine peripheral blood polymorphonuclear cells (PMNs). The NET formation was determined by fluorescence emission of propidium iodide (PI) in PMNs by a fluorescence microplate reader. The production of tumor necrosis factor (TNF)-α from peripheral blood mononuclear cells (PBMCs) was measured by ELISA method. Fucoidan itself did not show any direct effect on NET formation. However, NET formation of PMNs was increased by the culture supernatant from PBMCs treated with fucoidan. The NET formation of PMNs were also enhanced by treatment with recombinant porcine (rp) TNF-α. The ability of culture supernatant from PBMCs treated with fucoidan to increase the NET formation of PMNs was inhibited by addition of goat anti-rp TNF-α polyclonal antibody (pAb) (IgG) prior to the culture. The increase of NET formation by rp TNF-α was also inhibited by goat anti-rp TNF-α pAb (IgG). The level of TNF-α in culture supernatant from PBMCs was increased by treatment with fucoidan. These results suggest that fucoidan increases porcine NET formation, which is mediated by TNF-α produced from PBMCs.

• Journal of Veterinary Clinics
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• v.29 no.3
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• pp.213-219
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• 2012

The recombinant bovine somatotropin (rbST) has been used for increasing milk production of dairy cows without adverse health effects. This study was conducted to compare effects of supplementation with $Boostin^{(R)}$-250 containing 250 mg of rbST on milk production with those of $Posilac^{(R)}$ and $Boostin^{(R)}$-S. And safety of rbST supplementation on target animals was also observed. Each twenty-five lactating dairy cows were assigned randomly to one of four groups. $Boostin^{(R)}$-250 and vehicle (control) were administered weekly. $Boostin^{(R)}$-S and $Posilac^{(R)}$ were administered two week intervals. Milk yield, milk components, milk somatic cell count, health status, and body condition score of cows were examined. Supplementation with $Posilac^{(R)}$, $Boostin^{(R)}$-S, and $Boostin^{(R)}$-250 induced more milk yield than control group by 2.9 kg/day (12.3%), 4.2 kg/day (17.9%), and 4.1 kg/day (17.4%), respectively. There was a significant difference in milk yield among three rbST treatment groups and control group (${\alpha}$ = 0.05). The rbST supplementation did not increase the incidence of clinical mastitis and milk somatic cell counts. Supplementation with rbST did not significantly affect milk components (milk fat, protein, and solid not fat). The rbST supplementation of the dairy cows after peak milk yield did not cause negative effect on BCS. However, some cows less than 100 days in milking had decreased BCSs after rbST supplementation. In conclusion, milk production in 250 mg of rbST administered cows every week was similar to that of 500 mg of rbST administered cows every 2 weeks. And supplementation of 250 mg of rbST every week could reduce metabolic stress in cows.

• Journal of Microbiology and Biotechnology
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• v.17 no.1
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• pp.123-129
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• 2007

The trehalose $({\alpha}-D-glucopyranosyl-[1,1]-{\alpha}-D-glucopyranose)$ biosynthesis genes MhMTS and MhMTH, encoding a maltooligosyltrehalose synthase (MhMTS) and a maltooligosyltrehalose trehalohydrolase (MhMTH), respectively, have been cloned from the hyperthermophilic archaebacterium Metallosphaera hakonesis. The ORF of MhMTS is 2,142 bp long, and encodes 713 amino acid residues constituting a 83.8 kDa protein. MhMTH is 1,677 bp long, and encodes 558 amino acid residues constituting a 63.7 kDa protein. The deduced amino acid sequences of MhMTS and MhMTH contain four regions highly conserved for MTSs and three for MTHs that are known to constitute substrate-binding sites of starch-hydrolyzing enzymes. Recombinant proteins obtained by expressing the MhMTS and MhMTH genes in E. coli catalyzed a sequential reaction converting maltooligosaccharides to produce trehalose. Optimum pH of the MhMTS/MhMTH enzyme reaction was around 5.0 and optimum temperature was around 70 C. Trehalose-producing activity of the MhMTS/ MhMTH was notably stable, retaining 80% of the activity after preincubation of the enzyme mixture at $70^{\circ}C$ for 48 h, but was gradually abolished by incubating at above $85^{\circ}C$. Addition of thermostable $4-{\alpha}-glucanotransferase$ increased the yield of trehalose production from maltopentaose by 10%. The substrate specificity of the MhMTS/MhMTH-catalyzed reaction was extended to soluble starch, the most abundant maltodextrin in nature.

• Reproductive and Developmental Biology
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• v.34 no.1
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• pp.47-53
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• 2010

Glycoprotein hormones have a common $\alpha$-subunit that is involved in the signaling pathway together with G protein, adenylcyclase and cAMP induction; however, it is an unclear how this common structure is related to hormonal action. To determine the biological functions of the COOH-terminal amino acids in the $\alpha$-subunit of these glycoprotein hormones, a tethered-molecule was constructed by fusing the $NH_2$-terminus of the $\alpha$-subunit to the COOH-terminus of the $\beta$-subunit of equine chorionic gonadotropin (eCG). The following deletion mutants were created by PCR; Ile was inserted at position 96 to form ${\Delta}96$, Lys was substituted at position 95 to form ${\Delta}95$, His was inserted at position 93 to form ${\Delta}93$ and Tyr was substituted at position 87 to form ${\Delta}87$. Each mutant was transfected into CHO-K1 cells. Tethered-wt eCG, and ${\Delta}96$, ${\Delta}95$, and ${\Delta}93$ mutants were efficiently secreted into the medium but the ${\Delta}87$ mutant was not secreted. Interestingly, the RT-PCR, real-time PCR, and northern blot analyses confirmed that the RNA was transcribed in the ${\Delta}87$ mutant. However, the ${\Delta}87$ mutant protein was not detected in the medium or the intracellular fraction of the cell lysates. The LH- and FSH-like activities of the recombinant proteins were assayed in terms of cAMP production using rat LH/CG and rat FSH receptors. The metabolic clearance rate (MCR) was determined by injecting rec-eCG (2 IU) into the tail vein. The ${\Delta}95$ and ${\Delta}93$ mutants were completely inactive in both the LH- and FSH-like activity assays. The ${\Delta}96$ mutant showed slight activity in the LH-like activity assay. In comparison to the wild type, the activity of the ${\Delta}96$ mutant in the FSH-like activity assay was the highest among all the mutants. The MCR assay in which rec-eCG was injected showed a peak at 10 min in all the treatment groups, which disappeared 4 h after injection. These results imply a direct interaction between the receptor and the COOH-terminal region of the a-subunit. The data also reveal a significant difference in the mechanism by which the eCG hormone interacts with the rLH and rFSH receptors. The COOH-terminal region of the $\alpha$-subunit is very important for the secretion and functioning of this hormone.

• KSBB Journal
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• v.31 no.4
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• pp.277-283
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• 2016

In this system, rice cells were genetically modified to express human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) using RAmy3D promoter induced by sugar depletion. Even though the target protein fused with signal sequence peptide, plant cell wall can be a barrier against secretion of recombinant proteins. Therefore, hCTLA4Ig can be trapped inside cell wall or remained in intracellular space. In this study, to enhance the secretion of hCTLA4Ig from cytoplasm and cell walls into the medium, permeabilizing agents, such as dimethyl sulfoxide (DMSO), Triton X-100 and Tween 20, were applied in transgenic rice cell cultures. When 0.5% (v/v) of DMSO was added in sugar-free medium, intracellullar hCTLA4Ig was increased, on the other hand, the secreted extracellular hCTLA4Ig was lower than that of control. DMSO did not give permeable effects on transgenic rice cell cultures. And Triton X-100 was toxic to rice cells and also did not give enhancing permeability of cells. When 0.05% (v/v) Tween 20 was added in rice cell cultures, however, intracellular hCTLA4Ig was lower than that of control cultures. And the maximum 44.76 mg/L hCTLA4Ig was produced for 10 days after induction, which was 1.4-fold increase compared to that of control cultures. Especially, Tween 20 at 0.05% (v/v) showed the positive effect on the secretion of hCTLA4Ig though the decrease of intracellular hCTLA4Ig. Also, Tween 20 as a non-toxic surfactant did not affect the cell growth, cell viability and protease activity. In conclusion, secretion of hCTLA4Ig could be increased by enhancing permeability of cells regardless of the cell growth, cell viability and protease activity.