• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.6
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• pp.510-515
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• 2006

The putative EPA synthesis gene cluster was mined from the entire genome sequence of Shewanella oneidensis MR-1. The gene cluster encodes a PKS-like pathway that consists of six open reading frames (ORFs): ORFSO1602 (multi-domain beta-ketoacyl synthase, KS-MAT-4ACPs-KR), ORFSO1600 (acyl transferase, AT), ORFSO1599 (multi-domain beta-ketoacyl synthase, KS-CLF-DH-DH), ORFSO1597 (enoyl reductase, ER), ORFSO1604 (phosphopentetheine transferase, PPT), and ORFSO1603 (transcriptional regulator). In order to prove involvement of the PKS-like machinery in EPA synthesis, a 20.195-kb DNA fragment containing the genes was amplified from S. oneidensis MR-1 by the long-PCR method. Its identity was confirmed by the methods of restriction enzyme site mapping and nested PCR of internal genes orfSO1597 and orfSO1604. The DNA fragment was cloned into Escherichia coli using cosmid vector SuperCos1 to form pCosEPA. Synthesis of EPA was observed in four E. coli clones harboring pCosEPA, of which the maximum yield was 0.689% of the total fatty acids in a clone designated 9704-23. The production yield of EPA in the E. coli clone was affected by cultivation temperature, showing maximum yield at $20^{\circ}C$ and no production at $30^{\circ}C$ or higher. In addition, production yield was inversely proportional to glucose concentration of the cultivation medium. From the above results, it was concluded that the PKS-like modules catalyze the synthesis of EPA. The synthetic process appears to be subject to regulatory mechanisms triggered by various environmental factors. This most likely occurs via the control of gene expression, protein stability, or enzyme activity.

• The KIPS Transactions:PartC
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• v.14C no.6
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• pp.471-474
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• 2007

In this paper, we propose a variant of Diffie-Hellman key exchange protocol to provide pre-computable session key and to give another version of Diffie-Hellman key exchange protocol that might be useful in designing more sophisticated cryptographic protocols. We prove the security of the key exchange protocol by reducing DH key exchange protocol to ours.

• Journal of Microbiology
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• v.43 no.6
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• pp.499-502
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• 2005

Methylophaga sp. strain SK1 is a new restricted facultative methanol-oxidizing bacterium that was isolated from seawater. The aim of this study was to characterize the electron carriers involved in the methanol oxidation process in Methylophaga sp. strain SK1. The gene encoding cytochrome $c_L$ (mxaG) was cloned and the recombinant gene was expressed in Escherichia coli $DH5\alpha$ under strict anaerobic conditions. The recombinant cytochrome $c_L$ had the same molecular weight and absorption spectra as the wild-type cytochrome $c_L$ both in the reduced and oxidized forms. The electron flow rate from methanol dehydrogenase (MDH) to the recombinant cytochrome $c_L$ was similar to that from MDH to the wild-type cytochrome $c_L$. These results suggest that recombinant cytochrome $c_L$ acts as a physiological primary electron acceptor for MDH.

• Microbiology and Biotechnology Letters
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• v.31 no.3
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• pp.216-223
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• 2003

A 1.3-kb of chitosanase gene (choA) encoding 45-kDa polypeptide was cloned, expressed, and characterized from a newly isolated Bacillus cereus H-1. The chitosanase protein (ChoA) of B. cereus H-l was purified to homogeneity by ammonium sulfate precipitation and CM-sephadex column chromatography. Optimum pH was around 7, and stable pH range in the incubation at 50 C was 4-11. Optimum temperature was around 50 C, and enzyme activity was relatively stable below 45 C. ChoA showed the activities toward carboxymethyl cellulose (CMC) in addition to soluble or glycol chitosan. Based on MALDI-TOF MS analysis of purified ChoA, the entire amino acid sequence of ChoA was interpreted by database searching of previously known Bacillus chitosanases. A 1.6 kb of PCR product of corresponding chitosanase gene was obtained and its DNA sequence was determined. The deduced amino acid of choA revealed that ChoA have a 98% homology with those of Bacillus sp. No.7-M strain and Bacillus sp. KCTC0377BP. The recombinant ChoA protein was expressed in E. coli DH5$\alpha$. Deduced amino acid comparison of choA with other chitosanases suggested that it belongs to family 8 microbial endo-chitosanase with chitosanase-cellulase activity.

• Journal of Microbiology and Biotechnology
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• v.29 no.3
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• pp.401-409
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• 2019

Heat-resistant microbial hosts are required for bioprocess development using high cell density cultivations at the industrial scale. We report that the thermotolerance of Escherichia coli can be enhanced by overexpressing ybeD, which was known to encode a hypothetical protein of unknown function. In the wild-type E. coli BL21(DE3), ybeD transcription level increased over five-fold when temperature was increased from $37^{\circ}C$ to either $42^{\circ}C$ or $46^{\circ}C$. To study the function of ybeD, a deletion strain and an overexpression strain were constructed. At $46^{\circ}C$, in comparison to the wild type, the ybeD-deletion reduced cell growth half-fold, and the ybeD-overexpression promoted cell growth over two-fold. The growth enhancement by ybeD-overexpression was much more pronounced at $46^{\circ}C$ than $37^{\circ}C$. The ybeD-overexpression was also effective in other E. coli strains of MG1655, W3110, DH10B, and BW25113. These findings reveal that ybeD gene plays an important role in enduring high-temperature stress, and that ybeD-overexpression can be a prospective strategy to develop thermotolerant microbial hosts.

• The Magazine of the Society of Air-Conditioning and Refrigerating Engineers of Korea
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• v.40 no.3
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• pp.28-40
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• 2011

본 논문에서는 열섬완화대책에 관한 수치목표를 설정했을 때, 각 지역에 필요 대기 열부하 삭감량과 구체기술의 필요투입량 파악을 목적으로, 대기 열부하 삭감 포텐셜과 목표설정의 관계에 대해 검토하였다. 먼저, mesoscale 기상모델에 의해 과거회귀로부터 구체적인 수치목표를 구한 결과, 열대야 DH의 필요저감량은 대판평야(大阪平野)부의 공간평균으로 $10.7^{\circ}C{\cdot}h$, 인구 중첩평균으로 $12.5^{\circ}C{\cdot}h$가 산출되었다. 또한, 필요열부하 삭감량은 대판평야부(大阪平野部) 전체에서 일률삭감시에는 공간평균으로 11.5 $W/m^2$, 인구 중첩평균으로 12.0 $W/m^2$인 한편, 열부하 삭감 포텐셜이 큰 도심부를 중심으로 삭감을 행한 지역배분조건에서는 개략적으로 같은 양의 열부하 삭감으로 목표를 달성함을 나타내었다. 대판부(大阪府)내의 어떤 시구를 대상으로, 논문에서 뽑은 각종 열섬완화대책을 도입률 100%로 실시한 경우, 목표를 달성한 시구가 존재함을 나타내었다.

• Journal of Dairy Science and Biotechnology
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• v.34 no.4
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• pp.231-237
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• 2016

Kefir is a probiotic food. Probiotics have shown to be beneficial to health, and are currently of great interest to the food industry. Hence, this study was carried out to evaluate the lactase activity of kefir-isolated lactic acid bacteria. Three strains, Lactobacillu kefiri DH5 isolated from the kefir grains and Bifidobacterium animalis subsp. Lactis and Bifidobacteria longum 720, commercial probiotic LAB, were fermented in 10% reconstituted nonfat dry milk suspensions and incubated at $37^{\circ}C$ for 48 h, and then analyzed for various saccarides by HPLC. The results showed that changes in the concentrations of lactose and galactose were significantly decreased and increased, respectively (p<0.05), but all 3 probiotic strains tested in this study showed no increase in glucose concentration during 48 h of incubation. Both DH5 and BL720 showed high lactase activities (p<0.05), whereas BLC exhibited the lowest activity. Additionally, all three lactic acid bacteria showed high tagatose, but did not show high xylose and sedoheptulose. Finally, DH5, a kefir-isolated LAB, may have similar characteristics and properties to typical Bifidobacterium spp. and showed higher lactase activity than commercial Bifidobacterium spp.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2002.10a
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• pp.18-25
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• 2002

The pikromycin biosynthetic system in Streptomyces venezuleae is unique for its ability to produce two groups of antibiotics that include the 12-membered ring macrolides methymycin and neomethymycin, and the 14-membered ring macrolides narbomycin and pikromycin. The metabolic pathway also contains two post polyketide-modification enzymes, a glycosyltransferase and P450 hydroxylase that have unusually broad substrate specificities. In order to explore further the substrate flexibility of these enzymes a series of hybrid polyketide synthases were constructed and their metabolic products characterized. The plasmid-based replacement of the multifunctional protein subunits of the pikromycin PKS in S. venezuelae by the corresponding subunits from heterologous modular PKSs resulted in recombinant strains that produce both 12- and 14-membered ring macrolactones with predicted structural alterations. In all cases, novel macrolactones were produced and further modified by the DesVII glycosyltransferase and PikC hydroxylase leading to biologically active macrolide structures. These results demonstrate that hybrid PKSs in S. venezuelae can produce a multiplicity of new macrolactones that are modified further by the highly flexible DesVII glycosyltransferase and PikC hydroxylase tailoring enzymes. This work demonstrates the unique capacity of the S. venezuelae pikromycin pathway to expand the toolbox of combinatorial biosynthesis and to accelerate the creation of novel biologically active natural products. The polyketide backbone of rifamycin B is assembled through successive condensation and ${\beta}$-carbonyl processing of the extender units by the modular rifamycin PKS. The eighth module, in the RifD protein, contains nonfunctional DH domain and functional KR domain, which specify the reduction of the ${\beta}$-carbonyl group resulting in the C-21 bydroxyl of rifamycin B. A four amino acid substitution and one amino acid deletion were introduced in the putative NADPH binding motif in the proposed KR domain encoded by rifD. This strategy of mutation was based on the amino acid sequences of the corresponding motif of the KR domain of module 3 in the RifA protein, which is believed dysfunctional, so as to introduce a minimum alteration and retain the reading frame intact, yet ensure loss of function. The resulting strain produces linear polyketides, from tetraketide to octaketide, which are also produced by a rifD disrupted mutant as a consequence of premature termination of polyketide assembly. Much of the structural diversity within the polyketide superfamily of natural products is due to the ability of PKSs to vary the reduction level of every other alternate carbon atom in the backbone. Thus, the ability to introduce heterologous reductive segments such as ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) into modules that naturally lack these activities would increase the power of the combinatorial biosynthetic toolbox. The dehydratase domain of module 7 of the rifamycin PKS, which is predicted to be nonfunctional in view of the sequence of the apparent active site, was replaced with its functional homolog from module 7 of rapamycin-producing polyketide synthase. The resulting mutant strain behaved like a rifC disrupted mutant, i.e., it accumulated the heptaketide intermediate and its precursors. This result points out a major difficulty we have encountered with all the Amycolatopsis mediterranei strain containing hybrid polyketide synthases: all the engineered strains prepared so far accumulate a plethora of products derived from the polyketide chain assembly intermediates as major products instead of just analogs of rifamycin B or its ansamycin precursors.

• Microbiology and Biotechnology Letters
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• v.33 no.4
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• pp.241-247
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• 2005

A new lactococcal shuttle/expression vector for lactococci, pWgal13T, was constructed using a $\beta$-galactosi-dase gene (lacZ) from Lacfococcus lactis ssp. lactis ATCC 7962 as a screening marker. The pWgal 13T was introduced into Escherichia coli DH5a and L. lactis MG1363, and was easily detected by the formation of blue colonies on a medium containing X-gal without any false transformants. Also, the quantitatively lacZ activity of pWgal13T was measured in L. lactis ssp. cremoris MG1363, and was found to be four times higher than that of L. lactis ssp. lactis ATCC7962 grown on a medium containing glucose, which shows that the lacZ gene of pWgal13T can be used for the efficient screening of L. lactis on general media. The pWgal13T was equipped with a lactococcal replicon of pWV01 from L. lactis Wg2, the new promoter P13C from L. lactis ssp. cremoris LM0230, multiple cloning sites, and a terminator for the expression of a relevant gene. The vee-tor pWgal13T was used for the expression of the EGFP gene in E. coli and L. lactis. These results show that the lactococcal expression/shuttle vector constructed in the present study can be used for the production of foreign proteins in E. coli and L. lactis.

• Bulletin of the Korean Chemical Society
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• v.34 no.6
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• pp.1800-1808
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• 2013

Dual-responsive amphiphilic block copolymers were synthesized by combining enzymatic ring-opening polymerization (eROP) of ${\varepsilon}$-caprolactone (CL) and ATRP of N,N-dimethylamino-2-ethyl methacrylate (DMAEMA). The obtained block copolymers were characterized by gel permeation chromatography (GPC), $^1H$ NMR and FTIR-IR. The critical micelle concentration (CMC) of copolymer was determined by fluorescence spectra, it can be found that with hydrophilic block (PDMAEMA) increasing, CMC value of the polymer sample increased accordingly, and the CMC value was 0.012 mg/mL, 0.025 mg/mL and 0.037 mg/mL for $PCL_{50}$-b-$PDMAEMA_{68}$, $PCL_{50}$-b-$PDMAEMA_{89}$, $PCL_{50}$-b-$PDMAEMA_{112}$, $PCL_{50}$-b-$PDMAEMA_{89}$ was chosen as drug carrier to study in vitro release profile of anti-cancer drug (taxol). The temperature and pH dependence of the values of hydrodynamic diameter (Dh) of micelles, and self-assembly of the resulting block copolymers in water were evaluated by dynamic light scattering (DLS). The result showed that with the temperature increasing and pH decreasing, the Dh decreased. Drug-loaded nanoparticles were fabricated using paclitaxel as model. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) had been explored to study the morphology of the hollow micelles and the nanoparticles, revealing well-dispersed spheres with the average diameters both around 80 nm. In vitro release kinetics of paclitaxel from the nanoparticles was also investigated in different conditions (pH and temperature, etc.), revealing that the drug release was triggered by temperature changes upon the lower critical solution temperature (LCST) at pH 7.4, and at $37^{\circ}C$ by an increase of pH.