• Journal of Microbiology and Biotechnology
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• 제28권9호
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• pp.1482-1492
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• 2018

Fusarium oxysporum trypsin (FOT) is a fungal serine protease similar to mammal trypsin. The FOT could be successfully expressed in Pichiapastoris by engineering the natural propeptide APQEIPN. In this study, we constructed two recombinant enzymes with engineered amino acid sequences added to the N-terminus of FOT and expressed in P. pastoris. The N-terminal residues had various effects on the structural and functional properties of trypsin. The FOT, and the recombinants TE (with peptide YVEF) and TS (with peptide YV) displayed the same optimum temperature ($40^{\circ}C$) and pH (8.0). However, the combinants TE and TS showed significantly increased thermal stability at $40^{\circ}C$ and $50^{\circ}C$. Moreover, the combinants TE and TS also showed enhanced tolerance of alkaline pH conditions. Compared with those of wild-type FOT, the intramolecular hydrogen bonds and the cation ${\pi}$-interactions of the recombinants TE and TS were significantly increased. The recombinants TE and TS also had significantly increased catalytic efficiencies (referring to the specificity constant, $k_{cat}/K_m$), 1.75-fold and 1.23-fold than wild-type FOT. In silico modeling analysis uncovered that the introduction of the peptides YVEF and YV resulted in shorter distances between the substrate binding pocket (D174, G198, and G208) and catalytic triad (His42, Asp102, and Ser180), which would improve the electron transfer rate and catalytic efficiency. In addition, N-terminal residues modification described here may be a useful approach for improving the catalytic efficiencies and characteristics of other target enzymes.

• 미생물학회지
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• 제32권4호
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• pp.264-270
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• 1994

세균의 RNA 중합효소에서 여러 ${\sigma}$ 인자들 간에 보존된 아미노산 서열중 2.3 부위와 4.2 부위의 아미노산 서열로부터 유 n하여 두가지의 PCR primer를 제작하였다. 이들을 이용하여 PCR을 수행하였을 때, E. coli와 Streptomyces coelicolor의 DNA로부터 예상되었던 480 bp 정도의 DNA가 증폭되는 것을 관찰하였다. E. coli DNA에서 증폭된 DNA를 클로닝하여 염기서열을 결정한 결과 E. coli의 rpoS 유전자로부터 유래하였음을 알았다. 이를 탐침으로 S. coelicolor에서 genomic DNA hybridization을 수행하였을 때, PvuII 절편 두가지 (3.5 kb, 2.0 kb) 와 SalI 절편 두가지(3.4kb, 1.5 kb)에 탐침이 결합하는 것을 관찰하였다. 3.5 kb의 pvuII 절편을 sublibrary로부터 클로닝하고, 탐침이 결합하는 1.0kb의 BamHI/HincII 절편의 염기서열을 분석하였다. 부분적으로 결정된 염기서열을 BLAST 프로그램을 이용하여 GenBank와 EMBL, PDB 등의 data library의 유전자들과 비교하여 본 결과Streptomyces속의 ${\sigma}$인자들을 비롯한 Synechococcus종, Anabaena종, Pseudomonas aeruginosa, Stigmatella aurantica 등의 주된 ${\sigma}$ 인자와 높은 유사성을 보였다. 현재까지 1.2 부위와 4 부위에 해당하는 부분의 염기서열을 결정하였는데, 이 부분은 S. coelicolor에서 알려진 다섯가지의 ${\sigma}$ 인자 유전자 중 hrdA와 가장 높은 유사성을 보이며, 아미노산의 유사성이 1.2부위에서는 88%, 4 부위에서는 75%인 것으로 나타났다.

• 한국물환경학회지
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• 제34권3호
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• pp.285-292
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• 2018

This study was performed in order to identify the red tide-causing organism and to understand the characteristics of the water quality during the winter of 2015 and 2016 in the Oncheon stream, a tidal river in Busan, where red tide often occurs in the wintertime. Two sites were selected on the stream and the surface water was sampled a total of 28 times during the experimental period. Twelve water quality characteristics, including water temperature, pH, DO, COD, total-N (T-N), total-P (T-P), and salinity were analyzed in order to test water quality. The cell numbers of cryptomonads were counted directly by microscopic observation. The nucleotide sequences of the partial 28S rRNA gene and psbA gene from metagenomic DNA, derived from each sampling site, were analyzed. According to the results, the alga most responsible for the bloom was identified as Teleaulax OC1 sp., which belongs to the cryptomonads. Three items of chl-a, pH, and DO were positively correlated with the cell numbers of the cryptomonads counted at the upper stream of the tidal area (St 1) while eight items of chl-a, TOC, BOD, total-N, COD, SS, pH, and DO were positively correlated with the cells located at the junction between the stream and Su-young river (St 2) in the order.

• Journal of Microbiology and Biotechnology
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• 제6권6호
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• pp.369-374
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• 1996

The ${\beta}$-lactamase inhibitory protein (BLIP) produced by Streptomyces exfoliatus SMF19 was purified(33 kDa) and the N-terminal amino acid sequence was determined as NH2-ATSVVAWGGNND. Genomic DNA library of S. exfoliatus SMF19 was constructed in pWE15 and recombinants harbouring the corresponding gene were selected by colony hybridization to the mixture of 36-mer oligonucleotide designed from the N-terminal amino acid sequence. The corresponding gene (bliX) was isolated on a 4-kb ApaI fragment of S. exfoliatus SMF19 chromosomal DNA and then sequenced. The bliX consisting of 1, 119bp encoded a mature protein with a deduced amino acid sequence of 342 residues and also encoded a 40-amino-acid signal sequence. No significant sequence similarity to bliX was found by pairwise comparison using various protein and nucleotide sequences.

• 한국미생물·생명공학회지
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• 제24권5호
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• pp.553-559
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• 1996

We have designed nucleotide sequences of hEGF structural gene to eliminate the N-terminal methionine residue incorporated during the translation initiation step, and constructed recombinant human epidermal growth factor (rhEGF) secretion plasmids pYHB101, and pYHB2 in which pelB signal sequence-hEGF gene was expressed under the control of the T7, and tac promoter, respectively. We also constructed pYHB1 vector which contains rhEGF gene controlled by T7 promoter. The transformant with pYHB101 showed relatively slow growth pattern compared to the transformant with pYHB1. However, we observed that the transformant with pYHB101 secreted rhEGF of 13 mg/l significantly after 5 hr induction with 1 mM IPTG and that the T7 promoter was more effective than tac promoter when connected to pelB signal sequence. The amount of rhEGF was 14 mg/l under the sub-optimized condition.

• Journal of Microbiology and Biotechnology
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• 제17권8호
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• pp.1262-1270
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• 2007

Some hot springs located in the west of Turkey were investigated with respect to the presence of thermophilic microorganisms. Based on phenotyping characteristics and 16S rRNA gene sequence analysis, 16 of the isolates belonged to the genus Geobacillus and grew optimally at about $60^{\circ}C$ on nutrient agar. 16S rRNA gene sequence analysis showed that these isolates resembled Geobacillus species by ${\ge}97%$, but SDS-PAGE profiles of these 16 isolates differ from some of the other species of the genus Geobacillus. However, it is also known that analysis of 16S rRNA gene sequences may be insufficient to distinguish between some species. It is proposed that recN sequence comparisons could accurately measure genome similarities for the Geobacillus genus. Based on recN sequence analysis, isolates 11, IT3, and 12 are strains of G stearothermophilus; isolate 14.3 is a strain of G thermodenitrificans; isolates 9.1, IT4.1, and 4.5 are uncertain and it is required to make further analysis. The presence of xylanase and arabinofuranosidase activities, and their optimum temperature and pH were also investigated. These results showed that 7 of the strains have both xylanase and arabinofuranosidase activities, 4 of them has only xylanase, and the remaning 5 strains have neither of these activities. The isolates 9.1, 7.1, and 3.3 have the highest temperature optima ($80^{\circ}C$), and 7.2, 9.1, AO4, 9.2, and AO17 have the highest pH optima (pH 8) of xylanase. Isolates 7.2, AO4, AC15, and 12 have optimum arabinofuranosidase activities at $75^{\circ}C$, and only isolate AC15 has the lowest pH of 5.5.

• Asian Pacific Journal of Cancer Prevention
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• 제15권5호
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• pp.2101-2107
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• 2014

Background: Single nucleotide polymorphisms (SNPs) affecting microRNA (miR) sequences may influence carcinogenesis. Our current study primarily aimed to confirm previously conducted association studies between rs2910164 found on miR-146a, and rs11614913 located on miR-196a2 polymorphisms and cancer phenotypes in the Japanese elderly population. rs2910164 (G/C) and rs11614913 (T/C) polymorphisms were determined by genotyping on the samples collected from 1,351 consecutive autopsy cases registered in the Japanese SNPs for geriatric research (JG-SNP) data base. Cancer samples were systematically reviewed, pathologically verified and assessed with respect to miR-146a and miR-196a2 genotypic variation. The current study covered 726 males and 625 females with a mean age of $80.3{\pm}8.9$ years. The study included 524 subjects without cancer and 827 subjects with at least one type of cancer, such as gastric (n=160), lung (n=148), colorectal (n=116) or others. Males with cancers (n=467) were more numerous than females (n=360). Both rs11614913 (CT: TT adjusted odds ratio (OR) 95% confidence interval (95%CI)=0.98 (0.75-1.28), p=0.873, CC: TT adjusted OR (95%CI)=1.06 (0.76-1.47), p=0.737, CT+CC: TT, adjusted OR (95%CI)=0.99 (0.77-1.29), p=0.990), and rs2910164 (CG: CC adjusted OR (95%CI)=1.12 (0.87-1.44), p=0.383, GG: CC adjusted OR (95%CI)=1.03 (0.71-1.48), p=0.887, CG+GG: CC adjusted OR (95%CI)=1.10 (0.87-1.39), p=0.446) polymorphisms did not show significant association with overall cancer in all subjects. However, "CC" genotype in rs11614913 polymorphism was significantly associated with increased gastric cancer (n=160) in all subjects (CC: CT+TT, adjusted OR (95%CI)=1.50 (1.02-2.22), p=0.040). We found that rs11614913 and rs2910164 do not pose general cancer risk, but rs11614913 may influence gastric cancer in Japanese elderly population. Confirmation of our study results requires further investigations with larger subject populations.

• 대한수학회논문집
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• 제14권4호
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• pp.789-795
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• 1999

Let $A_1$,A$_2$…, A\ulcorner and B$_1$,B$_2$…, B\ulcorner be two sequences of events on the same probability space. Let X= X\ulcorner(A) and Y-Y\ulcorner)(B), repectively, by the number of those A\ulcorner and B\ulcorner which oc-cur. We establish bivariate lower bounds on the distribution P(X$\geq$1, Y, $\geq$1)and P(X$\geq$i , $Y\geq$j)by linear combinations of the bino-mial moments S\ulcorner, \ulcorner, 1$\leq$i$\leq$j

• 대한수학회지
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• 제41권1호
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• pp.157-174
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• 2004

In this work we discuss "plank problems" for complex Banach spaces and in particular for the classical $L^{p}(\mu)$ spaces. In the case $1\;{\leq}\;p\;{\leq}\;2$ we obtain optimal results and for finite dimensional complex Banach spaces, in a special case, we have improved an early result by K. Ball [3]. By using these results, in some cases we are able to find best possible lower bounds for the norms of homogeneous polynomials which are products of linear forms. In particular, we give an estimate in the case of a real Hilbert space which seems to be a difficult problem. We have also obtained some results on the so-called n-th (linear) polarization constant of a Banach space which is an isometric property of the space. Finally, known polynomial inequalities have been derived as simple consequences of various results related to plank problems.

• Journal of Microbiology and Biotechnology
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• 제17권8호
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• pp.1291-1299
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• 2007

Two ${\beta}$-1,4-glucanases (DI and DIII fractions) were purified to homogeneity from the culture filtrate of a cellulolytic bacteria, Cellulomonas sp. CS 1-1, which was classified as a novel species belonging to Cellulomonas uda based on chemotaxanomic and phylogenetic analyses. The molecular mass was estimated as 50,000 Da and 52,000 Da for DI and DIII, respectively. Moreover, DIII was identified as a glycoprotein with a pI of 3.8, and DI was identified as a non-glycoprotein with a pI of 5.3. When comparing the ratio of the CMC-saccharifying activity and CMC-liquefying activity, DI exhibited a steep slope, characteristic of an endoglucanase, whereas DIII exhibited a low slope, characteristic of an exoglucanase. The substrate specificity of the purified enzymes revealed that DI efficiently hydrolyzed CMC as well as xylan, whereas DIII exhibited a high activity on microcrystalline celluloses, such as Sigmacells. A comparison of the hydrolysis patterns for pNP-glucosides (DP 2-5) using an HPLC analysis demonstrated that the halosidic bond 3 from the nonreducing end was the preferential cleavage site for DI, whereas bond 2, from which the cellobiose unit is split off, was the preferential cleavage site for DIII. The partial N-terminal amino acid sequences for the purified enzymes were $^1Ala-Gly-Ser-Thr-Leu-Gln-Ala-Ala-Ala-Ser-Glu-Ser-Gly-Arg-Tyr^{15}$-for DI and $^1Ala-Asp-Ser-Asp-Phe-Asn-Leu-Tyr-Val-Ala-Glu-Asn-Ala-Met-Lys^{15}$-for DIII. The apparent sequences exhibited high sequence similarities with other bacterial ${\beta}$-1,4-glucanases as well as ${\beta}$-1,4-xylanases.