• Journal of the Society of Cosmetic Scientists of Korea
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• v.49 no.3
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• pp.193-201
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• 2023

Hydrogen peroxide (H₂O₂) is a type of active oxygen species (ROS) that causes oxidative stress in cells and affects cell growth, proliferation, senescence, and death. The purpose of this study is to find active peptides that attenuate cytotoxicity of H₂O₂. A positional scanning synthetic tetrapeptide combinatorial library was screened to predict the sequence of potentially active peptides. As a result of comparing the effect of peptide pools on H₂O₂-induced death of human keratinocytes (HaCaT cells), various active peptide sequences were predicted. Especially, peptides containing cysteine (C) residue were predicted to be active. In follow-up experiments, the cytotoxicity and activity of cysteine-containing peptides of different lengths, such as C-NH₂, CC-NH₂, CCC-NH₂, and CCCC-NH₂ were examined. C-NH₂ and CC-NH₂ showed no significant cytotoxicity up to 1.0 mM, but CCC-NH₂, and CCCC-NH₂ showed relatively strong cytotoxicity. C-NH₂ and CC-NH₂ alleviated H₂O₂-induced cytotoxicity. CC-NH₂ was more cytoprotective compared to C-NH₂, C, N-acetyl cysteine (NAC), and glutathione (GSH). When intracellular ROS was measured by flow cytometry, H₂O₂ increased ROS production, and CC-NH₂ suppressed ROS production more effectively than C-NH₂, and it was as effective as C, NAC, and GSH. This study suggests that CC-NH₂ of the cysteine-containing peptides of different lengths has an antioxidant property that safely and effectively alleviates H₂O₂-induced cytotoxicity and ROS production.

• Journal of Microbiology
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• v.44 no.1
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• pp.54-63
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• 2006

Ferritin is a major eukaryotic protein and in humans is the protein of iron storage. A partial gene fragment of ferritin (255 bp) taken from the total RNA of Periserrula leucophryna, was amplified by RT-PCR using oligonucleotide primers designed from the conserved metal binding domain of eukaryotic ferritin and confirmed by DNA sequencing. Using the $^{32}P-labeled$ partial ferritin cDNA fragment, 28 different clones were obtained by the screening of the P. leucophryna cDNA library prepared in the Uni-ZAP XR vector, sequenced and characterized. The longest clone was named the PLF (Periserrula leucophryna ferritin) gene and the nucleotide and amino acid sequences of this novel gene were deposited in the GenBank databases with accession numbers DQ207752 and ABA55730, respectively. The entire cDNA of PLF clone was 1109 bp (CDS: 129-653), including a coding nucleotide sequence of 525 bp, a 5' -untranslated region of 128 bp, and a 3'-noncoding region of 456 bp. The 5'-UTR contains a putative iron responsive element (IRE) sequence. Ferritin has an open reading frame encoding a polypeptide of 174 amino acids including a hydrophobic signal peptide of 17 amino acids. The predicted molecular weights of the immature and mature ferritin were calculated to be 20.3 kDa and 18.2 kDa, respectively. The region encoding the mature ferritin was subcloned into the pT7-7 expression vector after PCR amplification using the designed primers and included the initiation and termination codons; the recombinant clones were expressed in E. coli BL21(DE3) or E. coli BL21(DE3)pLysE. SDS-PAGE and western blot analysis showed that a ferritin of approximately 18 kDa (mature form) was produced and that by iron staining in native PAGE, it is likely that the recombinant ferritin is correctly folded and assembled into a homopolymer composed of a single subunit.

• Journal of Microbiology and Biotechnology
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• v.17 no.1
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• pp.58-66
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• 2007

Isolation, expression, and characterization of a novel $endo-{\beta}-1,3(4)-D-glucanase$ with high specific activity and homology to Bacillus lichenases is described. One clone was screened from a genomic library of Paenibacillus sp. F-40, using lichenan-containing plates. The nucleotide sequence of the clone contains an ORF consisting of 717 nucleotides, encoding a ${\beta}-glucanase$ protein of 238 amino acids and 26 residues of a putative signal peptide at its N-terminus. The amino acid sequence showed the highest similarity of 87% to other ${\beta}-1,3-1,4-glucanases$ of Bacillus. The gene fragment Bg1 containing the mature glucanase protein was expressed in Pichia pastoris at high expression level in a 3-1 high-cell-density fermenter. The purified recombinant enzyme Bg1 showed activity against barley ${\beta}-glucan$, lichenan, and laminarin. The gene encodes an $endo-{\beta}-1,3(4)-D-glucanase$ (E. C. 3.2.1.6). When lichenan was used as substrate, the optimal pH was 6.5, and the optimal temperature was $60^{\circ}C$. The $K_m,\;V_{max},\;and\;k_{cat}$ values for lichenan are 2.96mg/ml, $6,951{\mu}mol/min{\cdot}mg,\;and\;3,131s^{-1}$, respectively. For barley ${\beta}-glucan$ the values are 3.73mg/ml, $8,939{\mu}mol/min{\cdot}mg,\;and\;4,026s^{-1}$, respectively. The recombinant Bg1 had resistance to pepsin and trypsin. Other features of recombinant Bg1 including temperature and pH stability, and sensitivity to some metal ions and chemical reagents were also characterized.

• Journal of Plant Biotechnology
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• v.1 no.1
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• pp.20-30
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• 1999

In order to modify lipid production of Perilla qualitatively as well as quantitatively by genetic engineering, genes involved in carbon metabolism were isolated and characterized. These include acyl-ACP thioesterases from Perilla frutescens and Iris sp., four different $\beta$-ketoacyl- ACP synthases from Perilla frutescens, and two $\Delta$15 a-cyl-ACP desaturases(Pffad7, pffad3). Δ15 acyl-ACP desa turase (Δ15-DES) is responsible for the conversion of linoleic acid (18:2) to $\alpha$-linolenic acid (ALA, 18:3). pffad 3 encodes Δ15 acyl-desaturase which is localized in ER membrane. On the other hand, Pffad7 encodes a 50 kD plastid protein (438 residues), which showed highest sequence similarity to Sesamum indicum fad7 protein. Northern blot analysis revealed that the Pffad7 is highly expressed in leaves but not in roots and seeds. And Pffad3 is expressed throughout the seed developmental stage except very early and fully mature stage. We constructed Pffad7 gene under 355 promoter and Pffad3 gene under seed specific vicillin promoter. Using Pffad7 construct, Perilla, an oil seed crop in Korea, was transformed by Agrobacterium leaf disc method. $\alpha$-linolenic acid contents increased in leaves but decreased in seeds of transgenic Perilla. Currently, we are transforming Perilla with Pffad3 construct to change Perilla seed oil composition. We isolated three ADP-glucose pyrophosphorylase (AGP) genes from Perilla immature seed specific cDNA library. Nucleotide sequence analysis showed that two of three AGP (Psagpl, Psagp2) genes encode AGP small subunit polypeptides and the remaining (Plagp) encodes an AGP large subunit. PSAGPs, AGP small subunit peptide, form active heterotetramers with potato AGP large subunit in E. coli expressing plant AGP genes.

• The Korean Journal of Food And Nutrition
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• v.15 no.2
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• pp.104-111
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• 2002

The peptidyl prolyl sis-trans isomerase (PPIase, EC 5.2.1.8) from bacillus stearothermophilus was extracted from the cells treated with by lysozyme. PPIase was purified from the cell extracts by heat treatment, ammonium sulfate precipitation, ion exchange chromatography and finally gel filtration, sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). The molecular weight of the purified PPIase was estimated as 18kDa by SDS-PAGE. The 39 amino acid residues from the N-terminus were determined by the protein sequencer. The enzyme showed the optimum pH at 8.0 and was stable at the range of pH 7.0∼8.0. The enzyme was considerably stable after heat treatment at 60$\^{C}$ for 30minutes, and the enzyme was quite stable up to 65$\^{C}$. The presence of the PPIase in the refolding solution accelerated the isomerization rate of the assay peptide. PPIase gene of Bacillus stearothermophilus was screened from a genomic library by plaque hybridization using the A-l primer as a probe. A PPIase positive plaque contained a 3.0kb insert of the chromosomal DNA. A 3.0kb fragment was subcloned into pUC18, resulting pPI-40. A DNA fragment encoding the N-terminal portion of the PPIase in pPI-40 was amplified by polymerase chain reaction(PCR) method using the A-1 and B-2 primers. The amplified fragment was cloned into the Sma I site of pUC18 and recombinant plasmid was designated as pSN-18. The nucleotide sequence of 167bp fragment was determined. The deduced amino acid sequence of PPIase was completely matched with the determined N-terminal amino acid sequence of PPIase B. stearothermophilus.

• Journal of Microbiology and Biotechnology
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• v.20 no.3
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• pp.518-524
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• 2010

A mannanase gene (man26B) was obtained from a sea bacterium, Paenibacillus sp. BME-14, through the constructed genomic library and inverse PCR. The gene of man26B had an open reading frame of 1,428 bp that encoded a peptide of 475- amino acid residues with a calculated molecular mass of 53 kDa. Man26B possessed two domains, a carbohydrate binding module (CBM) belonging to family 6 and a family 26 catalytic domain (CD) of glycosyl hydrolases, which showed the highest homology to Cel44C of P. polymyxa (60% identity). The optimum pH and temperature for enzymatic activity of Man26B were 4.5 and $60^{\circ}C$, respectively. The activity of Man26B was not affected by $Mg^{2+}$ and $Co^{2+}$, but was inhibited by $Hg^{2+},\;Ca^{2+},\;Cu^{2+},\;Mn^{2+},\;K^+,\;Na^+$, and $\beta$-mercaptoethanol, and slightly enhanced by $Pb^{2+}$ and $Zn^{2+}$. EDTA did not affect the activity of Man26B, which indicates that it does not require divalent ions to function. Man26B showed a high specific activity for LBG and konjac glucomannan, with $K_m,\;V_{max}$, and $k_{cat}$ values of 3.80 mg/ml, 91.70 ${\mu}mol$/min/mg protein, and 77.08/s, respectively, being observed when LBG was the substrate. Furthermore, deletion of the CBM6 domain increased the enzyme stability while enabling it to retain 80% and 60% of its initial activity after treatment at $80^{\circ}C$ and $90^{\circ}C$ for 30 min, respectively. This finding will be useful in industrial applications of Man26B, because of the harsh circumstances associated with such processes.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.36 no.3
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• pp.233-240
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• 2010

Derivatives of a novel natural compunds, melanostatin (PLG-$NH_2$) were prepared by solid phase synthesis[1,2] and assayed to evaluate their melanogensis inhibitory activity. Also, a small library (natural compound-XLG-$NH_2$, natural compound-X LG-OH) was prepared with same method for increasing synthetic yield and cost-reduction. PLG-$NH_2$ (Proline-Leucine-Glycine-$NH_2$) was well-known tripeptide as its $\alpha$-MSH release-inhibiting activity and tyrosinase inhibitory activity[3-5]. In order to choose best candidate for peptide derivatization, various natural compounds were screened by their tyrosinase inhibitory activity. As a result, caffeic acid and coumaric acid were selected. Most of these derivatives showed better activities than the parent natural compound, melanostatin.