• Bulletin of the Korean Chemical Society
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• v.28 no.12
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• pp.2303-2309
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• 2007

Expressed protein ligation (EPL) technique, joining recombinantly expressed proteins to polypeptides, has been widely adopted for addressing various biological questions and for drug discovery. However, joining two recombinant proteins together is sometimes difficult when proteins are expressed insoluble and unrefoldable, because ligation-active proteins via intein-fusion are obtainable when they are folded correctly. We overcame this limitation coexpressing target protein with additional methionine aminopeptidase (MAP) which enhances removal of the initiation methionine of recombinantly expressed protein. Our approach demonstrated that two domains of 46 kDa 5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase, a target of herbicide glyphosate, were successfully joined by native chemical ligation, although its C-terminal domain was expressed as an inclusion body. The intein-fused N-terminal fragment of EPSP synthase (EPSPSN, residues 1-237) was expressed and the ligation-active thioester tagged N-terminal fragment (EPSPSN-thioester) was purified using a chitin affinity chromatography and mercapto-ethanesulphonate (MESNA) as intein thiolysis reagent. Its Cterminal fragment (EPSPSC, residues Met237-238CYS-427), expressed as an inclusion body, was prepared from an additional MAP-expressing strain. Protein ligation was initiated by mixing ~1 mM of EPSPSN-thioester with ~2 mM of EPSPSCCYS (residues 238CYS-427). Also we found that addition of 2% thiophenol increased the ligation efficiency via thiol exchange. The ligation efficiency was ~85%. The ligated full-length EPSP synthase was dissolved in 6 M GdHCl and refolded. Circular dichroism (CD) and enzyme activity assay of the purified protein showed that the ligated enzyme has distinct secondary structure and ~115% specific activity compared to those of wild-type EPSP synthase. This work demonstrates rare example of EPL between two recombinantly expressed proteins and also provides hands-on protein engineering protocol for large proteins.

• Journal of Crop Science and Biotechnology
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• v.10 no.3
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• pp.167-174
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• 2007

Spongy Alphonso mangoes were found to be infected with Staphylococcus bacteria. A Gram positive Staphylococcus strain was isolated from spongy pulp and identified from CABI Bioscience, UK, by partial 16S rDNA sequence analysis and by morphological and biochemical characterization through IMTECH, Chandigarh, India. Although identification by both of these methods indicated the organism belonged to same genus, different species names were given. Changes in total phenolics, reducing, and non-reducing sugars, respiration rate, total carotenoids, peroxidase(POX), and catalase activities were monitored during ripening of these fruits. The climacteric rise in spongy fruits was marked by an increase in respiration rate and a decrease in sugar content. Total phenolics content increased in spongy fruits as compared to ripe non-spongy fruits. Development of corky white tissue in spongy fruits was associated with about a 2.5-fold reduction in total carotenoids and a concomitant increase in lipoxygenase-mediated, $\beta$-carotene co-oxidation. A marked decrease in soluble protein content and about a 1.5-fold increase in POX activity was observed. Maximum POX activity was confined to 50-70%$(NH_4)_2SO_4$ fraction. The intense dark bands visible after POX specific substrate staining of the Native gel indicated a high expression of isoenzymes of POX in spongy fruits. Similarly, changes in levels of catalase activity were also observed in spongy fruits. The results suggest that infection of Alphonso mangoes with Staphylococcus bacteria affects the normal ripening processes of the fruit interfering with the carbohydrate and carotenoid metabolism. Also, the studies indicate the expression of POX and catalase enzymes as a plant defense response to microbial invasion.

• Microbiology and Biotechnology Letters
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• v.22 no.5
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• pp.507-514
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• 1994

Acetyl xylan esterase was produced by E. coli HB101 harboring a recombinant plasmid pKMG6 which contained the estI gene of Bacillus stearothermophilus. The maximum production was observed when the E. coli strain was grown at 37$\circC for 12 hours in the medium containing 0.5% acetyl xylan, 1.0% tryptons, 1.0% sodium chloride, and 0.5% yeast extract. The esterase produced was purified to homogeneity using a combination of ammonium sulfate fractionation, DEAE Sepharose CL-6B ion exchange chromatography and Sephacryl S-200 gel filtration. The native enzyme had an apparent molecular mass of 60 kd and was composed of two identical subunits of 29 kd. The N-terminal amino acid sequence of the polypeptide was Ala-X-Leu-Gln- Ile-Gln-Phe-X-X-Gln. The acetyl esterase displayed a pH optimum of 6.5 and a temperature opti- mum of 45$\circC. The heavy metal ions such as Ag$^{++}$, Hg$^{++}$ and Cu$^{++}$ inhibited nearly completely the activity of the esterase, and no specific metal ion was found to be required for the enzyme activity. The enzyme readily cleaved MAS, $\beta$-D-glucose pentaacetate, $\alpha$-naphthyl acetate, $\rho$-nitrophenyl acetate as well as acetyl xylan, but had no activity on $\rho$-nitrophenyl propionate, $\beta$-nitrophenyl butyrate or $\beta$-nitrophenyl valerate. The Km and Vmax values for MAS were 2.87 mM and 11.55 $\mu$mole/min, respectively. Synergistic behavior was demonstrated with a combination of xylanase and esterase from B. stearothermophilus in hydrolyzing acetyl xylan.

• Korean Journal of Environmental Agriculture
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• v.20 no.1
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• pp.63-66
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• 2001

To investgate growth effect of tomato by Bacillus sp. WRD-1 isolated from soil, the Bacillus sp. WRD-1 cultures were treated into tomato cultivated soil with different dilutions (1:100, 1:300, and 1:500) and autoclaved Bacillus cultures as control. Growth and yeild of tomato enhanced in treatments of the Bacillus cultures compared to control. The populations of native bacteria and actinomyces were increased twice in field treated with Bacillus sp. WRD-1 cultures, but the number of mold was decreased. Since the Bacillus sp. WRD-1 promoted growth of tomato and affected population dynamics of microorganism in field, this strain is prominent candidate as a microbial biocide to improve soil potential.

• Proceedings of the Korean Vacuum Society Conference
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• 2011.02a
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• pp.428-429
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• 2011

Recently, we performed ab initio total energy calculation and tight-binding molecular dynamics (TBMD) simulation to study structures and the reconstruction of native defects in graphene. In the previous study, we predicted by TBMD simulation that a double vacancy in graphene is reconstructed into a 555-777 composed of triple pentagons and triple heptagons [1]. The structural change from pentagon-octagon-pentagon (5-8-5) to 555-777 has been confirmed by recent experiments [2,3] and the detail of the reconstruction process is carefully studied by ab initio calculation. Pentagon-heptagon (5-7) pairs are also found to play an important role in the reconstruction of vacancy in graphene and single wall carbon nanotube [4]. In the TBMD simulation of graphene nanoribbon (GNR), we found the evaporation of carbon atoms from both the zigzag and armchair edges is preceded by the formation of heptagon rings, which serve as a gateway for carbon atoms to escape. In the simulation for a GNR armchair-zigzag-armchair junction, carbon atoms are evaporated row-by-row from the outermost row of the zigzag edge [5], which is in excellent agreement with recent experiments [2, 6]. We also present the recent results on the formation and development of dislocation in graphene. It is found that the coalescence of 5-7 pairs with vacancy defects develops dislocation in graphene and induces the separation of two 5-7 pairs. Our TBMD simulations also show that adatoms are ejected and evaporated from graphene surface due to large strain around 5-7 pairs. It is observed that an adatom wanders on the graphene surface and helps non-hexagonal rings change into stable hexagonal rings before its evaporation.

• Korean Chemical Engineering Research
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• v.56 no.5
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• pp.711-717
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• 2018

Human epidermal growth factor (hEGF) can stimulate the division of various cell types and has potential clinical applications. Since the protein contains three intra-molecular disulfide bonds, the high expression of active hEGF in Escherichia coli has not been well researched, We fused the hEGF gene with a small ubiquitin-related modifier gene (SUMO) by synthesizing an artificial SUMO-hEGF fusion gene that was highly expressed in E. coli (DE3) strain. The optimal expression level of the soluble fusion protein, SUMO-hEGF with IPTG (Isopropyl-${\beta}$-D-Thiogalactopyranoside), was up to 38.9% of the total cellular protein. The fusion protein was purified by Ni-NTA affinity chromatography and cleaved by a SUMO-specific protease to obtain the native hEGF, which was further purified by Ni-NTA affinity chromatography. The result of the reverse-phase HPLC showed that the purity of the recombinant cleaved hEGF was greater than 98%.

• BMB Reports
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• v.35 no.2
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• pp.206-212
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• 2002

The NS2B-NS3(pro) polyprotein segment from the dengue virus serotype 2 strain 16681 was purified from overexpressing E. coli by metal chelate affinity chromatography and gel filtration. Enzymatic activity of the refolded NS2B-NS3(pro) protease complex was determined in vitro with dansyl-labeled peptide substrates, based upon native dengue virus type 2 cleavage sites. The 12mer substrate peptides and the cleavage products could be separated by reversed-phase HPLC, and were identified by UV and fluorescence detection. All of the peptide substrates (representing the DEN polyprotein junction sequences at the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 sites) were cleaved by the recombinant protease NS2B-NS3(pro). No cleavage was observed with an enzymatically inactive S135A mutant of the NS3 protein, or with a modified substrate peptide of the NS3/NS4A polyprotein site that contained a K2093A substitution. Enzymatic activity was dependent on the salt concentration. A 50% decrease of activity was observed in the presence of 0.1M sodium chloride. Our results show that the NS3 protease activity of the refolded NS2B-NS3(pro) protein can be assayed in vitro with high specificity by using cleavage-junction derived peptide substrates.

• ALGAE
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• v.33 no.4
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• pp.341-349
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• 2018

Sargassum horneri is edible brown seaweed abundant along the coasts of Jeju Island, South Korea. In addition to the native S. horneri population, a large amount of S. horneri has been found to invade Jeju Island from the east coast of China. Thus, S. horneri of both Korea (SK) and China (SC) strains now inhabits along with the shore of Jeju Island and have become a threat to the coastal biodiversity. However, they could be used in obtaining functional ingredients for industrial level applications provided an optimized cost effective strategy. In the present study, we compared SK and SC strains for the extraction efficiency, components, antioxidant, and anti-inflammatory properties of 80% methanolic extracts and their partially purified fractions. According to the results, two strains indicated similar bioactive properties such as DPPH and alkyl radical scavenging activity as well as anti-inflammatory activities on lipopolysaccharide-stimulated RAW 264.7 cells. The yield of 80% methanol extract from SC was higher than SK. However, the yields of the ethyl acetate and chloroform fractions from SK were higher than those of SC strain. The major peaks in the high-performance liquid chromatography chromatograms, which was identified as Apo-9 fucoxanthinone, indicated that both methanolic extracts of SK and SC contains major target peaks but with different amounts. This study might be useful for developing functional materials from SC and SK in future.

• Journal of the Korean Applied Science and Technology
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• v.35 no.4
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• pp.1276-1284
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• 2018

probiotics strains promoting the health are a collection of microorganisms that improve or restore microbial populations in the intestines. In this study, Leuconostoc probiotics was isolated from fermented gimchi and identified. Angelica dahurica, containing abundantly antioxidant activity, imperator, is a wildly grown species of angelica native. Before fermentation, total phenolics compound were $48.83{\pm}4.9GAE\;mg/g$ in the Angelica dahurica extract. After fermentation total phenolic compounds were $97.7{\pm}12.6GAE\;mg/g$. The total amount of phenol in the fermented product was 30.2% higher than that before fermentation. The total flavonoid content before fermentation was $9.86{\pm}4.3mg/g$ and the total flavonoid content was $37.17{\pm}7.4mg/g$ after fermentation, which was 82.3% higher than before fermentation. The DPPH radical scavenging activity, superoxide radical scavenging activity, hydroxy radical scavenging activity and $Fe^{{+}{+}}$ chelating antioxidative activity of the Angelica dahurica extract were $41.6{\pm}7.1%$, $65.7{\pm}8.4%$, $55.26{\pm}9.4%$ and $17.5{\pm}4.6%$, respectively. After fermentation, they were $60.3{\pm}12.6%$, $78.8{\pm}8.3%$, $56.9{\pm}4.9%$ and $36.6{\pm}8.9%$, respectively. Therefore, the present study suggests that the fermentation using the probiotics strain of the Angelica dahurica extract can be used as a functional health food and cosmetic material with increased antioxidant capacity.

• Korean Journal of Microbiology
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• v.54 no.4
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• pp.436-441
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• 2018

Ginseng are native traditional herbs, which exhibit excellent pharmacological activities. Probiotic Lactobacillus helveticus KII13 and Pediococcus pentosaceus strain KID7 were used for ginsenoside transformation by fermenting crude ginseng extract to enhance minor gisenoside content. Thin-layer chromatography (TLC) analysis of fermented ginseng extract showed that the minor ginsenosides Rg3, Rh1, and Rh2 were main products after 5 days of fermentation. HPLC analysis was performed to quantify the major and minor ginsenosides. The Rg3 peak appeared on the 3rd day while the appearance of Rh2 peak and Rh1 peak were observed on the 5th day. The co-culture of L. helveticus KII13 and P. pentosaceus KID7 converted major ginsenosides (Rb1 and Rg1) into minor ginsenosides (Rg3, Rh2, and Rh1).