• The Plant Pathology Journal
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• v.38 no.6
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• pp.583-592
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• 2022

Root-knot nematode disease is a widespread and catastrophic disease of tobacco. However, little is known about the relationship between rhizosphere bacterial community and root-knot nematode disease. This study used 16S rRNA gene sequencing and PICRUSt to assess bacterial community structure and function changes in rhizosphere soil from Meloidogyne incognita-infected tobacco plants. We studied the rhizosphere bacterial community structure of M. incognita-infected and uninfected tobacco plants through a paired comparison design in two regions of tobacco planting area, Yuxi and Jiuxiang of Yunnan Province, southwest China. According to the findings, M. incognita infection can alter the bacterial population in the soil. Uninfested soil has more operational taxonomic unit numbers and richness than infested soil. Principal Coordinate Analysis revealed clear separations between bacterial communities from infested and uninfested soil, indicating that different infection conditions resulted in significantly different bacterial community structures in soils. Firmicutes was prevalent in infested soil, but Chloroflexi and Acidobacteria were prevalent in uninfested soil. Sphingomonas, Streptomyces, and Bradyrhizobium were the dominant bacteria genera, and their abundance were higher in infested soil. By PICRUSt analysis, some metabolism-related functions and signal transduction functions of the rhizosphere bacterial community in the M. incognita infection-tobacco plants had a higher relative abundance than those uninfected. As a result, rhizosphere soils from tobacco plants infected with M. incognita showed considerable bacterial community structure and function alterations.

• Journal of Microbiology and Biotechnology
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• v.33 no.7
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• pp.949-954
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• 2023

Type III polyketide synthase (PKS) found in bacteria is known as 1,3,6,8-tetrahydroxynaphthalene synthase (THNS). Microbial type III PKSs synthesize various compounds that possess crucial biological functions and significant pharmaceutical activities. Based on our sequence analysis, we have identified a putative type III polyketide synthase from Nocardia sp. CS682 was named as ThnA. The role of ThnA, in Nocardia sp. CS682 during the biosynthesis of 1,3,6,8 tetrahydroxynaphthalene(THN), which is the key intermediate of 1-(α-L-(2-O-methyl)-6-deoxymannopyranosyloxy)-3,6,8-trimethoxynaphthalene (IBR-3) was characterized. ThnA utilized five molecules of malonyl-CoA as a starter substrate to generate the polyketide 1,3,6,8-tetrahydroxynaphthalene, which could spontaneously be oxidized to the red flaviolin compound 2,5,7-trihydroxy-1,4-naphthoquinone. The amino acid sequence alignment of ThnA revealed similarities with a previously identified type III PKS and identified Cys¹³⁸, Phe¹⁸⁸, His²⁷⁰, and Asn³⁰³ as four highly conserved active site amino acid residues, as found in other known polyketide synthases. In this study, we report the heterologous expression of the type III polyketide synthase thnA in S. lividans TK24 and the identification of THN production in a mutant strain. We also compared the transcription level of thnA in S. lividans TK24 and S. lividans pIBR25-thnA and found that thnA was only transcribed in the mutant.

• Journal of Microbiology and Biotechnology
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• v.33 no.12
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• pp.1595-1605
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• 2023

Dehydroquinate dehydratase (DHQD) catalyzes the conversion of 3-dehydroquinic acid (DHQ) into 3-dehydroshikimic acid in the mid stage of the shikimate pathway, which is essential for the biosynthesis of aromatic amino acids and folates. Here, we report two the crystal structures of type II DHQD (CgDHQD) derived from Corynebacterium glutamicum, which is a widely used industrial platform organism. We determined the structures for CgDHQD^WT with the citrate at a resolution of 1.80Å and CgDHQD^R19A with DHQ complexed forms at a resolution of 2.00 Å, respectively. The enzyme forms a homododecamer consisting of four trimers with three interfacial active sites. We identified the DHQ-binding site of CgDHQD and observed an unusual binding mode of citrate inhibitor in the site with a half-opened lid loop. A structural comparison of CgDHQD with a homolog derived from Streptomyces coelicolor revealed differences in the terminal regions, lid loop, and active site. Particularly, CgDHQD, including some Corynebacterium species, possesses a distinctive residue P105, which is not conserved in other DHQDs at the position near the 5-hydroxyl group of DHQ. Replacements of P105 with isoleucine and valine, conserved in other DHQDs, caused an approximately 70% decrease in the activity, but replacement of S103 with threonine (CgDHQD^S103T) caused a 10% increase in the activity. Our biochemical studies revealed the importance of key residues and enzyme kinetics for wild type and CgDHQD^S103T, explaining the effect of the variation. This structural and biochemical study provides valuable information for understanding the reaction efficiency that varies due to structural differences caused by the unique sequences of CgDHQD.

• Applied Biological Chemistry
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• v.26 no.4
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• pp.222-230
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• 1983

This study was designed to develop a process for the immobilization of xylose isomerase(D-xylose ketol isomerase, EC 5.3.1.5) from Streptomyces griseolus previously isolated by the authors and its application on a pilot plant scale for the production of high fructose corn syrup. The biomass which has endo-excreted xylose isomerase was homogenized under a pressure of $500kg/cm^2$ and 90.8% of the enzyme recovery of the native activity was obtained as compared to 54.7% recovery by the lysozyme treatment. Ionic bonding method was adopted for the enzyme immobilization due to its many reported merits. It was found that the porous resins such as Diaion HP 20, Duolite A-7, Amberlite IRA 93 and 94 were effective in immobilizing the enzyme. In addition, it was disclosed that the regeneration form of $BO_4--$ is effective for Amberlite IRA 93 and $HCO_3-$ for Diaion HP 20. Optimal immobilization condition for Amberlite IRA 93 was pH 8.0 and $55^{\circ}C$ yielding 80.6% of immobilization. Activity decay test showed half life of the immobilized enzyme with Amberlite IRA 93 was more than 24 days at $65^{\circ}C$. The carrier was evaluated to be resuable and its result showed the relative immobilization yields were 98.2, 93.3, 90.7 and 87.5%, respectively at second, third, forth and fifth rebinding test of the enzyme on Amberlite IRA 93. Optimal temperature of the immobilized enzyme was slightly lowered and the range widened to $60\sim70^{\circ}C$, while optimal pH moved toward $8.0\sim8.3$ in its isomerization reaction. The trial production result of high fructose corn syrup in pilot scale immobilization showed that one liter of immobilized xylose isomerase (350 IXIU/ml-R) is capable producing about 293l high fructose corn syrup(75% dry substance) in 30 days.

• Korean Journal of Organic Agriculture
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• v.15 no.2
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• pp.195-205
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• 2007

This study was conducted to determine effects of probiotics on performance, nutrient digestibility and nitrogen retention in Korean black goats fed two diets differing in forage to concentrate (F:C) ratio (30:70 and 70:30), for the establishment of their more efficient feeding management system. The probiotics employed in this trial were mixtures of different microbial species, which consisted of Lactobacillus casei, Bacillus subtilis, Saccharomyces cerevisiae, Aspergillus oryzae and Streptomyces griseus. Additional levels of probiotics to each F:C ratio (70:30 or 30:70) were 0 and 0.2%, respectively. Thus, twelve Korean black male goats were allotted to treatments in four groups of three goats per treatment and then they were housed in individual metabolism cages with a randomized complete block design for 21 days. Dry matter (DM) intakes were not affected by dietary F:C ratio and probiotics. Digestible DM amounts were significantly (p<0.05) decreased with increasing levels of dietary forage, but they were not affected by probiotics addition. Dry matter intakes per metabolic body weight and their ratio per body weight had a similar trend to DM intakes with no significant difference by F:C ratio and probiotics addition. The nutrient digestibility was significantly (p<0.05) increased with decreasing levels of forage in the diet, but it was not affected by probiotics addition. Urinary nitrogen loss was significantly (p<0.05) decreased with decreasing levels of dietary forage, but there was no significant difference between probiotics-supplemented and unsupplemented groups within the same F:C ratio. On the contrary, nitrogen retention was decreased with increasing levels of dietary forage, and probiotics supplementation to two diets differing in F:C ratio showed slightly increasing trends in the nitrogen retention. From the above results, probiotics supplementation to two diets differing in F:C ratio did not have the significant influence on feed intakes, nutrient digestibility and nitrogen retention. Consequently, these parameters of Korean black goats were dominated rather by F:C ratio than by dietary probiotics.

• Research in Plant Disease
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• v.18 no.2
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• pp.117-122
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• 2012

In 2006, the effect of soil pH adjustment on control of common scab of potato caused by Streptomyces spp. was evaluated in the field. Soil with an original pH 5.7 was treated with sulfur of 42 kg/10a and calcium hydroxide of 81.6 kg/10a and 184.5 kg/10a to adjust pH 5.0, 6.0 and 6.5, respectively. Additionally, the effect of gypsum (522 kg/10a) on soil pH and disease severity was examined. The soil pH of sulfur treatment plot was continuously declined from 5.13 at tuber initiation time to 5.01 at harvest. The disease severity of sulfur treatment plot was relatively low (22.8%) and the marketable yield of that was high (90.5%). By contrast, the soil pHs treated with calcium hydroxide (81.6 kg/10a and 184.5 kg/10a) were increased (pH 6.06 and 6.49, respectively) at harvest. In addition, calcium hydroxide treatment plots showed higher disease severities (51.0% and 61.1%), and lower marketable yields (55.7% and 37.0%). Meanwhile, the soil pH of gypsum treatment plot was not changed until harvest, and the disease severity and the marketable yield were not significantly different from those of control. The effect of crop rotation was also evaluated with four crops such as welsh onion, soybean, corn and Chinese cabbage. These crops were planted in the infested field from 2005 to 2007, and potatoes were planted in the same field in 2008. The disease severities of potatoes produced after rotations with welsh onion, soybean, corn and Chinese cabbage for 3 years were 13.1%, 16.7%, 28.9% and 30.2%, respectively. However, the disease severity of 73.1% was shown in continuous cropping of potato for 3 years. In addition, similar effects were exhibited in the marketable yield. These results demonstrate that the adjustment of soil pH and crop rotation might be very useful tools to control common scab of potato.

• Food Science of Animal Resources
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• v.30 no.3
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• pp.504-511
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• 2010

This study was undertaken to investigate the effects of feeding complex probiotics (Lactobacillus casei, Bacillus subtilis, Saccharomyces cerevisiae, Aspergillus oryzae, Streptomyces griseus, $1.5{\times}10^{10}$ CFU/kg) and antibiotics (oxytetracycline (OTC), 110 ppm) on growth performance and meat quality characteristics of broiler chicks. In the experiment 1, 0.3% complex probiotics feeding level was chosen to be proper addition level due to better average daily gain (ADG), feed conversion (FC) and dressing percent (DP) results among 3 levels (0.1, 0.3 or 0.5%). In the experiment 2, 5 treatments (T1, no probiotics + no antibiotics; T2, probiotics 0.3% + no antibiotics; T3, probiotics 0.3% + antibiotics 50%; T4, probiotics 0.3% + antibiotics 100%; T5, no probiotics + antibiotics 100%) were investigated. In the growth performance of broilers, T5 (antibiotics 100% only) showed the highest (p<0.05) ADG and FC values while T1 (control) showed the worst growth performance. However, T3 (probiotics 0.3% + antibiotics 50%) showed higher ADG (p<0.05), FC (p<0.05) and DP (p>0.05) values compared to control. In the breast and leg meat quality, T3 showed similar pH, proximate composition, cooking loss and meat color values except shear force value compared to T5. Addition of 0.3% probiotics with 50% antibiotics (T3) tended to lower the blood cholesterol levels of broiler chicks and Escherichia coli or Salmonella counts in cecum microflora of broiler chicks compared to T5. In the residual antibiotics analysis, T3 contained 0.04 ppm of residual antibiotics in the breast meat while T4 or T5 contained 0.1 ppm of residual antibiotics and addition of 0.3% probiotics with 50% antibiotics in broiler diets could lower the residual antibiotics level to 40% in the meat. As a result, 0.3% probiotics addition with 50% antibiotics in the broiler diets could be recommended for the production of high quality broiler meat.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.31 no.4 s.54
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• pp.349-357
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• 2005

New antioxidative substances for cosmeceuticals were screened from natural resources such as microbial metabolites, mushrooms, and medicinal plants. Four antioxidants were isolated from the fungal metabolite of Eupenicillium shearii and their structures were determined to be new phenolic compounds. The compounds were designated as melanocins A, B, C, and D. Melanocins $A{\sim}D$ exhibited free radical scavenging activity on DPPH and superoxide with $EC_{50}$ values of $21{\sim}94\;and\;7{\sim}84{\mu}M$, respectively, which were stronger activity than those of ${\alpha}-tocopherol$ and BHA. Melanocin A showed anti-wrinkle effects on the UV-irrated hairless mouse skin. A novel hispidin antioxidative compound designated as inoscavin A was isolated from the fruiting body of the mushroom, Inonotus xeranticus. Inoscavin A scavenged superoxide radical with $EC_{50}$ values of $0.03{\mu}g/mL$, and inhibited rat liver microsomal lipid peroxidation with $EC_{50}$ values of $0.3{\mu}g/mL$. Benzastatins $A{\sim}G$, the novel antioxidants isolated from the culture of Streptomyces nitrosporeus showed potent lipid peroxidation inhibitory activity with $EC_{50}$ values of $3{\sim}30{\mu}M$. A cyclopentene compound with strong hypopigmentary effect was isolated from the fungal metabolite of Penicillium sp. and identifed as terrein. Terrein significantly reduced melanin levels in a melanomacyte cell line, Mel-Ab. It showed 10 times stronger activity than kojic acid, but exhibited no cytotoxic effect even in $100{\mu}M$. It was suggested that terrein reduced melanin synthesis by reducing tyrosinase production by MITF down-regulation.

• Journal of Life Science
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• v.28 no.12
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• pp.1416-1423
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• 2018

With strong biotin binding affinity ($K_D=10^{-14}M$), the tetrameric feature of streptavidin could be used to increase the antigen binding activity of a camel heavy chain (VHH) antibody through their fusion, here stained with biotinylated horseradish peroxidase and subsequent immunoassays ELISA and Western blot analysis. For this application, we cloned the streptavidin gene amplified from the Streptomyces avidinii chromosome by PCR, and this was fused to the gene of the 8B9 VHH antibody which is specific to green fluorescent protein (GFP) antigens. To express a soluble fusion protein in Escherichia coli, we used the pUC119 plasmid-based expression system which uses the lacZ promoter for induction by IPTG, the pelB leader sequence at the N-terminus for secretion into the periplasmic space, and six polyhistidine tags at the C-terminus for purification of the expressed proteins using an $Ni^+$-NTA-agarose column. Although streptavidin is toxic to E. coli because of its strong biotin binding property, this soluble fusion protein was expressed successfully. In SDS-PAGE, the size of the purified fusion protein was 122.4 kDa in its native condition and 30.6 kDa once denatured by boiling, suggesting the tetramerization of the monomeric subunit by non-covalent association through the streptavidin moiety fusing to the 8B9 VHH antibody. In addition, this fusion protein showed biotin binding activity similar to streptavidin as well as GFP antigen binding activity through both ELISA and Western blot analysis. In conclusion, the protein resulting from the fusion of an 8B9 VHH antibody with streptavidin was successfully expressed and purified as a soluble tetramer in E. coli; it showed both biotin and GFP antigen binding activity suggesting the possible production of a tetrameric and bifunctional VHH antibody.

• Korean Journal of Microbiology
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• v.54 no.4
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• pp.330-342
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• 2018

The Streptavidin and Biotin system has been studied most extensively as the high affinity non-covalent binding of Biotin to STR ($K_D=10^{-14}M$) and four Biotin binding sites in tetrameric Streptavidin makes this system useful for the production of multivalent antibody. For the application of this system, we cloned Streptavidin amplified from Streptomyces avidinii chromosome by PCR and fused to gene of hAY4 single-chain Fv antibody specific to death receptor 4 (DR4) which is a receptor for tumor necrosis factor ${\alpha}$ related apoptosis induced ligand. The hAY4 single-chain Fv antibody fused to Streptavidin expressed in Escherichia coli showed 43 kDa monomer in heated SDS-PAGE. However, this fusion protein shown in both non-heated SDS-PAGE and Size-exclusion chromatography exhibited 172 kDa as a tetramer suggesting that natural tetramerization of Streptavidin by non-covalent association induced hAY4 single-chain Fv tetramerization. This fusion protein retained a Biotin binding activity similar to natural Streptavidin as shown in Ouchterlony assay and ELISA. Death receptor 4 antigen binding activity of purified hAY4 single-chain Fv fused to Streptavidin was also confirmed by ELISA and Westernblot. In addition, surface plasmon resonance analysis showed 60-fold higher antigen binding affinity of the hAY4-STR than monomeric hAY4 ScFv due to tetramerization. In summary, hAY4 single-chain Fv fused to Streptavidin fusion protein was successfully expressed and purified as a soluble tetramer in E. coli and showed both Biotin and DR4 antigen binding activity suggesting possible production of bifunctional and tetrameric ScFv antibody.