• Journal of Microbiology and Biotechnology
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• v.17 no.6
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• pp.952-959
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• 2007

His-His-Leu (HHL), a tripeptide derived from a Korean soybean paste, is an angiotensin-I-converting enzyme (ACE) inhibitor. We report here a method of producing this tripeptide efficiently by expressing tandem multimers of the codons encoding the peptide in E. coli and purifying the HHL after hydrolysis of the peptide multimers. The HHL gene, tandemly multimerized to a 40-mer, was ligated with ubiquitin as a fusion gene (UH40). UH40 was inserted into vector pET29b; the UH40 fusion protein was then produced in E. coli BL21. The recombinant UH40 protein was purified by cation-exchange chromatography with a yield of 17.3mg/l and analyzed by matrixassisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry and protein N-terminal sequencing. Leucine aminopeptidase was used to cleave a 405-Da HHL monomer from the UH40 fusion protein and the peptide was purified using reverse-phase high-performance liquid chromatography (HPLC) on a C18 HPLC column, with a final yield of 6.2mg/l. The resulting peptide was confirmed to be HHL with the aid of MALDI-TOF mass spectrometry, glutamine-TOF mass spectrometry, N-terminal sequencing, and measurement of ACE inhibiting activity. These results suggest that our production method is useful for obtaining a large quantity of recombinant HHL for functional antihypertensive peptide studies.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.38 no.5
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• pp.286-290
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• 2005

Oxytocin (OT)-related peptide, isotocin was purified from the brain extract of conger eel (Conger myriaster) using reverse-phase, ion-exchange and size exclusion high performance liquid chromatography (HPLC). The sequence of the peptide, with a molecular weight of 967.30 Da, was determined as Cys-Tyr-Ile-Ser-Asn­Cys-Pro-Ile-Gly-$NH_2$, where the Cys between 1st and 6th residues made an intramolecular disulfide bridge by the automated amino acid sequence analysis and MALDI-TOF mass spectrometry. The sequence was confirmed by identical elution with the purified and synthetic peptide using the HPLC system. As a result of homology investigation, the primary structure of this peptide was the same as that of OT -superfamily member, isotocin. The synthetic peptide showed a contractile activity at a minimal effective concentration of $10^{-7}M$ on the intestinal smooth muscle of goldfish (Carassius auratus).

• Journal of Chitin and Chitosan
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• v.23 no.4
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• pp.220-227
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• 2018

Amyloid plaque is a product of aggregation of ${\beta}$-amyloid peptide ($A{\beta}$) and is an important factor in the pathogenesis of Alzheimer's Disease (AD). $A{\beta}$ is a major component of amyloid plaque and vascular deposits in the AD brain. The enzyme ${\beta}$-secretase is required for the production of $A{\beta}$; thus, prevention of the formation of $A{\beta}$ through the inhibition of ${\beta}$-secretase is a major focus in the study of the treatment of AD. In this study, we investigated ${\beta}$-secretase inhibitory activity of an Arctoscopus japonicus peptide. An Alcalase hydrolysate had the highest ${\beta}$-secretase inhibitory activity. A ${\beta}$-secretase inhibitory activity peptide was separated using ion exchange column chromatography (carboxy-methyl: CM, quaternary methyl ammonium: QMA) and reverse phase high performance liquid chromatography (RP-HPLC) on a C18 column. The $IC_{50}$ value of the purified peptide was $248.2{\pm}1.73{\mu}g/mL$. The ${\beta}$-secretase inhibitory peptide was identified as a six amino acid residue of Gly-Pro-Val-Gly-Ala-Pro (MW: 497.27 Da). In cell viability experiments, the final purified fraction, the carboxy-methyl ion exchange column fraction (CM-F1) showed no significant cytotoxic effect in SH-SY5Y cells at concentrations below $100{\mu}g/mL$ in 24 h. The results of this study suggest that peptides separated from Arctoscopus japonicus may be beneficial as ${\beta}$-secretase inhibitor compounds in functional foods.

• Journal of Plant Biology
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• v.27 no.1
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• pp.7-13
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• 1984

Activities of corn endosperm adenylate cyclase and phosphodiesterase were found right after germination, and phosphodiesterase activity was shown to increase steadily. Protease activity was also found. Corn peptide fraction purified by using Sephadex G-25 column was shown to enhance corn phosphodiesterase activity buy inhibit bovine phosphodiesterase activity. And the fraction inhibits corn adenylate cyclase activity. Trypsintreated peptide fraction was shown to enhance phosphodiesterase activity 80% compared to that of native peptide fraction. However, in case of DNase phosphodiesterase was shown to be innocuous. According to cumulative results, it is more likely that peptide fraction produced by protease inhibits adenylate cyclase activity and enhance phosphodiesterase, decreasing cAMP level.

• Preventive Nutrition and Food Science
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• v.15 no.4
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• pp.282-286
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• 2010

To isolate a calcium-binding peptide from chlorella protein hydrolysates, chlorella protein was extracted and hydrolyzed using Flavourzyme, a commercial protease. The degree of hydrolysis and calcium-binding capacity were determined using trinitrobenzenesulfonic acid and orthophenanthroline methods, respectively. The enzymatic hydrolysis of chlorella protein for 6 hr was sufficient for the preparation of chlorella protein hydrolysates. The hydrolysates of chlorella protein were then ultra-filtered under 5 kDa as molecular weight. The membrane-filtered solution was fractionated using ion exchange, reverse phase, normal phase chromatography, and fast protein liquid chromatography to identify a calcium-binding peptide. The purified calcium-binding peptide had a calcium binding activity of 0.166 mM and was determined to be 700.48 Da as molecular weight, and partially identified as a peptide containing Asn-Ser-Gly-Cys based on liquid chromatography/electrospray ionization tandem mass spectrum.

• The Journal of Natural Sciences
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• v.16 no.1
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• pp.1-13
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• 2005

The angiotensin I-converting enzyme (ACE) inhibitor has anti-hypertensive effects and has long been used as prevention or remedy of hypertension. This study were carried out to produce and purify a new ACE inhibitor from recombinant E. coli and further elucidate its structure-function relationship. Recombinant pGEX-4T-3 containing ACE inhibitory peptide gene of Saccharomyces cerevisiae was transformed into E. coli BL21(DE3). Glutathione-S transferase (GST) fusion protein from E. Coli BL21(DE3) harboring the recombination pGEX-4T-3 was obtained and the ACE inhibitory peptide was purified with Sephadex G-25 column chromatography. The purified ACE inhibitory peptide was a novel decapeptide with sequence Tyr-Asp-Gly-Gly-Val-Phe -Arg-Val-Tyr-Thr which shows very low similarity to the other ACE inhibitory peptide sequence. The purified ACE inhibitor competitively inhibited ACE.

• Journal of Microbiology and Biotechnology
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• v.30 no.9
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• pp.1387-1394
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• 2020

Clam worms (Marphysa sanguinea) are a rich source of bioactive components such as the antibacterial peptide, perinerin. In the present study, we explored the physiological activities of a novel NCWPFQGVPLGFQAPP peptide (NCW peptide), which was purified from clam worm extract through high-performance liquid chromatography. Tandem mass spectrometry (MS/MS) revealed that NCW was a new peptide with a molecular weight of 1757.86 kDa. Moreover, NCW peptide exhibited significant antioxidant effects, causing a 50% inhibition of DPPH radical at a concentration of 20 μM without showing any cytotoxicity. These were associated with a reduction in the activity of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) in LPS-stimulated RAW264. 7 cells. Furthermore, NCW peptide exhibited anti-inflammatory effects in LPS-stimulated RAW264.7 macrophages via inhibition of the abnormal production of pro-inflammatory cytokines including nitric oxide (NO), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). These anti-inflammatory effects of NCW peptide were associated with the inhibition of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). Our results therefore suggest that this novel NCW peptide with antioxidant and anti-inflammatory effects could be a good therapeutic agent against inflammation-related diseases.

• Applied Biological Chemistry
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• v.43 no.4
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• pp.247-252
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• 2000

As functionality investigation of Korean traditional soybean fermentation foods, an antihypertensive peptide was separated during Chunggugjang fermentation by Bacillus subtilis CH-1023 and investigated inhibitory effect against angiotensin converting enzyme. After incubation at $20^{\circ}C,\;30^{\circ}C,\;40^{\circ}C,\;50^{\circ}C,\;60^{\circ}C$ for the $0{\sim}72$ hrs, protein content, protease activity and angiotensin converting enzyme inhibitory rate were determined. The protein content and protease activity were increased and reached maximum at 60 hrs fermentation with $40^{\circ}C$ and decreased after the 60 hrs fermentation. The optimum condition for antihypertensive peptide from Chunggugjang was appeared for 60 hrs at $40^{\circ}C$. Crude extract of Chunggugjang was partially purified by Amicon YM-3 membrane filtration and Sephadex G-10, G-25 gel filtration. The purified peptide showed inhibitory rate of 94.3% with 0.5 mg peptide content. The most prominent amino acid composition of the peptide from Chunggugjang was alanine, followed by phenylalanine, histidine.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.54 no.6
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• pp.927-937
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• 2021

This study was performed to screen the antimicrobial activities of the extract from the Pacific oyster Crassostrea gigas against skin pathogens and to purify the relevant antibacterial peptide. The acidified extract showed potent antibacterial activities against gram-positive and gram-negative bacteria but showed no activity against Candida albicans and no significant cell toxicity. Among acne-causing pathogens, the acidified extract showed potent antibacterial activity only against Staphylococcus aureus, and its antibacterial activity was completely abolished by treatment with trypsin or chymotrypsin, and was inhibited by salt treatment. The acidified extract showed strong DNA-binding ability but did not show bacterial membrane permeabilizing ability. Based on antimicrobial activity screening and cytotoxic effects, a novel antibacterial peptide was purified from the acidified gill extract using solid-phase extraction, cation-exchange, and reversed-phase HPLC. The resulting peptide had a molecular weight of 4800.8 Da and showed partial sequence homology with the carbonic anhydrase 4 (CA4) protein in the hard-shelled mussel. Overall, we purified a novel antibacterial peptide, named cgCAFLP, which is related to carbonic anhydrase 4 (CA4) protein, against skin pathogens. Our results suggest that the Pacific oyster extract could be used as an additive to control some acne-related skin pathogens (S. aureus).

• Journal of the Korean Society of Food Science and Nutrition
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• v.32 no.5
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• pp.745-751
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• 2003

This study was carried out to investigate whether peptide produced from wheat protein by enzyme hydrolysis can be used as a natural antimicrobial agent. Antimicrobial peptide was obtained from wheat protein hydrolyzed by 7 of pretense. The produced antimicrobial peptide was purified through ultrafiltration, membrane filtration and HPLC and molecular weight and amino acid sequence of the purified antimicrobial peptide were determined. Among hydrolysate produced from wheat protein by 7 of protease, antimicrobial activity was observed for the peptide obtained from Asp. saito protease. The Asp. saito protease did produce antimicrobial hydrolysate showing the highest antimicrobial activity at reaction condition of 37$^{\circ}C$ and pH 6.0, but not at reaction condition above 5$0^{\circ}C$. Wheat protein hydrolysate was fractionated by membrane filtration and showed antimicrobial activity between molecular weight 1,000~3,000. The antimicrobial activity fraction obtained by membrane filtration was separated through HPLC and showed antimicrobial activity in the peak of retention time 31.1~31.8 min. We could convince this hydrolysate as heat-stable peptide since antimicrobial activity was maintained after treated with heat for 15 min at 121$^{\circ}C$. Molecular weight of antimicrobial peptide identified by MALDI-mass was 1,633. Amino acid sequence of antimicrobial peptide was cysteine, glycine, prolin, prolin, prolin, valine, valine, alanine, alanine and arginine.