• Journal of the Korean Magnetic Resonance Society
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• v.13 no.1
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• pp.27-34
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• 2009

HP (2-20), a 19-residue peptide derived from the N-terminus of Helicobacter pylori Ribosomal Protein L1, has antimicrobial activity but is not cytotoxic to human erythrocytes. Previously, we have synthesized several analogue peptides to investigate the effects of substitutions on the structure and antimicrobial activity. Substitution of $Gln^{16}$ and $Asp^{18}$ with Trp (Anal 3) caused a dramatic increase in bacterial and fungal lytic activities. In this study, analogue peptides were synthesized to investigate the effects of substitution of Gin and Asp with Phe (Anal 6) or Tyr (Anal 7) in HP (2-20) on its structure and antimicrobial activity. Substitution of Gin and Asp with hydrophobic aromatic residues at position 16 and 18 of HP (2-20) caused increase in antibiotic activity without hemolytic effect. Substitution of Gin and Asp with Trp and Try increased antibiotic activity of HP (220) twice more compared to substitution with Phe. The tertiary structures of Anal 6 and Anal 7 in SDS micelles has been investigated using NMR spectroscopy. The structures revealed that substitutions of the aromatic residues at C-terminus resulted in longer and well defined alpha-helix and improved their antibacterial activities

• Journal of Microbiology and Biotechnology
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• v.9 no.6
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• pp.832-838
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• 1999

Two different types of lipases (lipase I and lipase II) secreted into culture medium by Rhizopus sp. L-I were purified using a hydrophobic chromatography and were partially characterized. Both enzymes were monomeric as revealed by SDS-PAGE and gel filtration. The molecular masses of the enzymes were identified as 45 kDa (lipase I) and 69 kDa (lipase II). The isoelectric points were estimated to be 3.6 and 5.2 for lipase I and lipase II, respectively. pH and temperature activity optima for lipase I were as 7.5 and $50^{\circ}C$, respectively, whereas the corresponding parameters for lipase II were 6.0 and $45^{\circ}C$. The amino terminal sequences of lipase I and lipase II, determined by Edman degradation, were found to be Leu-Val-Met-Ile-Gln-Arg and Leu-Val-Met-Lys-Gln-Arg, respectively. By western blotting analysis, the two lipases were found to have a common antigenic determinant. Immuno-electron cytochemistry conducted with polyclonal anti-lipase I antibody indicated the enzyme located in both the periplasm and the adjacent vesicles of fungal hyphae. Fortunately, the sites on the cell envelope where lipase was exported into the culture medium was also identified.

• Tuberculosis and Respiratory Diseases
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• v.60 no.2
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• pp.171-179
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• 2006

Background : Despite the emerging danger of MDR-TB to human beings, there have only been a limited number of drugs developed to treat MDR-TB since 1970. This study investigated the cross-resistance rate between rifampicin (RFP) and rifabutin (RBU) in order to determine the efficacy of rifabutin in treating MDR-TB. In addition, the results of rifabutin were correlated with the rpoB mutations, which are believed to be markers for MDR-TB and RFP resistance. Methods : The MICs of RBU were tested against 126 clinical isolates of MDR-TB submitted to the clinical laboratory of National Masan TB Hospital in 2004. Five different concentrations ($10-160{\mu}g/ml$) were used for the MICs. The detection of the rpoB mutations was performed using a RFP resistance detection kit with a line probe assay(LiPA), which contains the oligonucleotide probes for 5 wide type and 3 specific mutations (513CCA, 516GTC, and 531TTG) The rpoB mutation was determined by direct sequencing. Results : The rate of cross-resistance between RFP and RBU was 70.5%(74/105) at $20{\mu}g/ml$ RBU(ed note: How much RFP?) Most mutations (86.3%) occurred in the 524~534 codons. The His526Gln, His526Leu, Leu533Pro, Gln513Glu, and Leu511Pro mutations(Ed note: Is this correct?) were associated with the susceptibilty to RBU. Conclusion : Based on the cross-resistance rate between RFP and RBU, RBU may be used effectively in some MDR-TB patients. Therefore, a conventional drug susceptibility test for RBU and a determination of the critical concentration are needed. However, rpoB gene mutation test may be have limited clinical applications in detecting RBU resistance.

• Journal of the East Asian Society of Dietary Life
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• v.7 no.3
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• pp.289-300
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• 1997

Newborn dog chymosin was extracted from the stomachs of dogs of 2 weeks of age, and was purified by ion exchange chromatography. Half of the sequence was determined by amino acid sequencing and the complete sequence was deduced from a cloned chymosin cDNA Results showed that the zymogen showed 79% sequence identity with calf prochymosin and 54% identity with porcine pepsinogen A The size of the propart and location of the residue which becomes the amino-terminus in the active enzyme was the same in the prochymosins. The maximum general proteolytic activity at pH 3.2 of newborn dog chymosin was 3-4% of that of porcine pepsin A at pH 2, whereas the milk clotting activity relative to the general proteolytic activity of newborn dog chymosin was much higher than that of calf chymosin. Agar gel electrophoresis at pH 5.2 of stomach extracts of individual dogs showed the existence of two predominant genetic variants of zymogen and enzyme. The two variants could not be distinguished by amino acid composition or amino-terminal sequencing, and no differences in the enzymatic properties of the genetic variants were observed. It was concluded that of the residues that participate in the substrate binding, calf and newborn dog chymosin differ in the following positions (porcine pepsin numbering, subsites in parentheses) : Ser 12 Thr(S$_4$), Leu 30 Val(S$_1$/S$_3$), His 74 Gln(S'$_2$), Val 111 Ile(S$_1$/S$_3$), Lys 220 Met(S$_4$). With regard to the low general proteolytic activity of newborn dog chymosin, the substitution Asp303 Val relative to calf chymosin may contribute to an explanation of this.

• Journal of Life Science
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• v.19 no.11
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• pp.1672-1678
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• 2009

The purified antifungal substances produced by Bacillus amyloliquefaciens IUB158-03 was positive to ninhydrin but negative to aniline, suggesting that the antifungal substance could be a peptide. FAB-MS, UV adsorption spectrum, and amino acid composition analysis revealed that the molecular weight of the antifungal substance was 1042 and that maximal adsorption was at 220 nm and 277 nm. The antifungal substance was composed of $Asn_3$, $Gln_2$, $Ser_1$, $Gly_1$, and $Tyr_1$. The composition and structural characteristics of antifungal substance were analysed by $^1H$-NMR spectrum, $^1H$-COSY, HMQC, which revealed that the compound belongs to the iturin A family. Temperature and pH had little effect on the stability of the antifungal substance in the ranges of $-70{\sim}121^{\circ}C$ and pH 6.0~10.0, respectively. It showed strong antibiotic activity against fungi. An in vitro cytotoxicity test using NIH3T3 cell showed that the antifungal substance does not have cytotoxicity. The number of circulating leukocytes and the hematobiological analysis of the mice administered with the antifungal substances was similar to those of the control group, indicating no cytotoxicity in vivo. Therefore, the antifungal substances extracted from culture broth of Bacillus amyloliquefaciens IUB158-03 have future potential as biocontrol agents against plant diseases caused by fungi.

• Korean Journal of Food Science and Technology
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• v.31 no.6
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• pp.1635-1641
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• 1999

Bacillus subtilis PCA20-3 was isolated from meju and was found to produce a protease. The strain produced the maximum amount of enzyme in the medium containing soytone (0.2%), soluble starch (2%), $(NH_4)_2SO_4\;(0.1%),\;CaCl_2(0.1%),\;yeast\;extract\;(0.01%),\;K_2HPO_4\;(0.1%),\;and\;KH_2PO_4\;(0.1%)$. Protease was first concentrated by ammonium sulfate (80% saturation, w/v) precipitation of culture supernatant. Then the enzyme was purified by column chromatography using CM Sephadex C-50. The collected proteins were rechromatographed using Sephadex G-100 gel filtration column. The fraction with protease active from Sephadex G-100 gel chromatography was found to be pure when examined by SDS-polyacrylamide gel electrophoresis and YMC-pak reverse phase chromatography. Specific activity, yield and purity were 76 U/mg. 2.7%, and 7.6 fold, respectively. The molecular weight of the enzyme was estimated to be 31.5 kDa by SDS-PAGE. The number of amino acids calculated from molecular weight was evaluated about 321 residues. N-terminal sequence of the enzyme was $Val^1-Pro^2-Tyr^3-Gly^4-Val^5-Ser^6-Gln^7-Gly^8-Lys^9-Ala^{10}$.

• Korean Journal of Environmental Agriculture
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• v.24 no.3
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• pp.245-252
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• 2005

The objective of the present work was to determine the corresponding uptake and assimilation of ${NO_3}^-$ in roots and shoots of leafy radish by applying of mixed amino acids (MAA). The amino acids used in this experiment were alanine (Ala), ${\beta}-alanine\;({\beta}-Ala)$, aspartic acid (Asp), asparagines (Asn), glutamic acid (Glu), glutamine (Gln), and glycine (Gly). Leafy radish was grown by conventional fertilization with macro- and micronutrients under controlled conditions. The 15-day-old seedlings were treated 0, 0.3 and 3.0 mM of MAA containing 5 mM ${NO_3}^-$ in growth medium. Nitrate uptake was determined by following ${NO_3}^-$ depletion from the uptake solution. The activity of the enzymes related to the process of ${NO_3}^-$ reduction (NR: nitrate reductase; NiR: nitrite reductase; GS: glutamine synthetase) and the content of ${NO_2}^-\;and\;{ND_3}^-$ were analyzed in shoots and roots. The results of this study showed that ${NO_3}^-$ uptake was inhibited 38% with treatment of 0.3 mM of MAA. However, there was more than three times increase of N03- uptake in 3.0 mM MAA. In addition, the enzymatic activities were positively affected by the high MAA rate. Finally, the ${NO_3}^-$ content was increased slightly both in shoots and roots of leafy radish by MAA treatments.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.34 no.2
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• pp.164-172
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• 2001

The angiotensin-I converting enzyme (ACE) inhibitors from laver hydrolysate was isolated. Among the 13 kinds of proteases, Maxazyme NNP was most effective for preparing the high ACE inhibitory compound. In extraction conditions of ACE inhibitory peptide from laver hydrolysate, ACE inhibitory activity of hydrolysate treated with diethylether for decolorization and that of $70\%$ ethanol soluble fraction among the different ethanol concentrations were higher than other preparations. Low molecular fraction less than 3,000 dalton of layer hydrolysate separated by ultrafiltration had the highest ACE inhibitory activity, for further separation of ACE inhibitory peptide from laver hydrolysate, gel filtration chromatography (Sephadex G-25), reverse-phase HPLC (ODS & Vydac C-18) and gel permeation chromatography (Superdex Peptide HR) were performed. The molecular mass of the ACE inhibitory peptide fractions of gel permeation chromatography determined by electrospray-mass spectrometer were 413.48 (S1O2V2V1P),346.86 (S1O2V2V2P) and 320.32 (S2O6V3V1P) dalton and their amino acid sequence were Val-Gln-Gly-Asn, Thr-Glu-Thr and Phe-Arg, respectively.

• Journal of the Korean Magnetic Resonance Society
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• v.12 no.1
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• pp.14-25
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• 2008

To investigate the 3-bond connectivity of human brain metabolites by scalar coupling interaction through 2D-correlation spectroscopy (COSY) techniques using high field NMR spectroscopy. All NMR experiments were performed at 298K on Unity Inova 500 or 600 (Varian Inc.) equipped with a triple resonance probe head with z-shield gradient. Human brain metabolites were prepared with 10% $D_2O$. Two dimensional 2D COSY spectra were acquired with 4096 complex data points in $t_2$ and 128 or 256 increments in $t_1$ dimension. The spectral width was 9615.4 Hz and solvent suppression was achieved using presaturation using low power irradiation of the water resonance during 2s of relaxation delay. NMR data were processed using VNMRJ (Varian Instrument) software and all the chemical shifts were referenced to the methyl resonance of N-acetyl aspartate (NAA) peak at 2.0 ppm. Total 10 metabolites such as N-acetyl aspartate (NAA), creatine (Cr), choline (Cho), glutamine (Gln), glutamate (Glu), myo-inositol (Ins), lactate (Lac), taurine (Tau), ${\gamma}$-aminobutyricacid (GABA), alanine (Ala) were included for major target metabolites. Symmetrical 2D-COSY spectra were successfully acquired. Total 14 COSY cross peaks were observed even though there were parallel/orthogonal noisy peaks induced by water suppression. Except for Cr, all of human brain metabolites produced COSY cross peaks. The spectra of NAA methyl proton at 2.02 ppm and Glu methylene proton ($CH_2(3)$) at 2.11 ppm and Gln methylene proton ($CH_2(3)$) at 2.14 ppm were overlapped in the similar resonance frequency between 2.00 ppm and 2.15 ppm. The present study demonstrated that in vitro 2D-COSY represented the 3-bond connectivity of human brain metabolites by scalar coupling interaction. This study could aid in better understanding the interactions between human brain metabolites in vivo 2D-COSY study. Also it would be helpful to determine the molecular stereochemistry in vivo by using two-dimensional MR spectroscopy.

• Biomedical Science Letters
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• v.12 no.3
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• pp.225-231
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• 2006

Fibrinolytic enzyme was purified from the fruiting bodies of Lepista nuda, using DEAE-Cellulose chromatography, Phenyl Sepharose chromatography, and Mono-S column chromatography. The substance has a molecular weight of 30006.62 Da as measured by MALD-TOF mass spectrometry. The N-terminal amino acid sequence of the enzyme was Tyr-Pro-Ser-Pro-Ser-His-Gln-Thr-Ala-Val-Asn-Ala-Ile-Ile-X. The activity of the enzyme was inhibited by PMSF, indicating that the enzyme is a serine protease. No inhibition was found with E-64, pepstatin, and EDTA. It has broad substrate specificity for synthetic peptides. The enzyme was stable up to $30^{\circ}C$. The enzyme hydrolyzes both Aa and y chains of human fibrinogen but did not show any reactivity for $B{\beta}$ chain of human fibrinogen.