• 농업과학연구
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• 제29권2호
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• pp.78-90
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• 2002

밀 단백질에 효소가수분해 할 때 생산되는 peptide의 항균활성과 천연항균제로서의 이용가능성을 검토하기 위하여 실험을 행하였다. 밀 단백질에 7종의 단백질가수분해효소를 작용시켜 생성된 가수분해물의 항균활성을 측정하고 한외여과, membrane filtration, HPLC를 이용하여 항균성 peptide를 분리 정제한 후 분자량과 아미노산 결합순서를 측정한 결과는 다음과 같다. 밀 단백질에 7종의 단백질 분해효소를 적용시켜 제조한 가수분해물중 Asp. saito protease를 적용시켜 얻어진 peptide 만이 항균활성을 나타내었다. Asp. saito protease는 $37^{\circ}C$, pH 6.0에서 작용시킨 경우에 항균활성이 가장 높았으며, $50^{\circ}C$ 이상에서는 활성을 나타내지 않았다. 밀단백 효소가수분해물은 membrane filtration에 의하여 분자량 1,000~3,000 에서 항균활성이 나타났다. Membrane filtration으로 얻어진 항균활성분획을 HPLC로 분리한 결과 retention time 31.1~31.8 min에서 항균활성을 나타내었다. 밀단백 효소가수분해물은 $121^{\circ}C$에서 15분간 가열하여도 효소활성이 유지되는 매우 안정한 화합물이었다. 항균활성분획을 MALDI-mass로 질량을 분석한 결과 1,633이었다. 항균성 peptide의 아미노산 결합순서는 cysteine, glycine, prolin, prolin, prolin, valine, valine, alanine, alanine, arginine 의 순서였다.

• 한국축산식품학회지
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• 제40권1호
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• pp.87-95
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• 2020

In this study, we separated and purified lipase inhibitory peptide from fermented milk by Lactobacillus plantarum Q180 with the aim of developing a new functional anti-lipase activity yogurt product. L. plantarum 180 was inoculated into 10% reconstituted skimmed milk and incubated at 37℃ until the pH of the culture reached pH 4.4. The lipase activity was measured using porcine pancreatic lipase. The lipase inhibitory peptides were gradually isolated by ultrafiltration, reversed phase column chromatography (RPC), reversed phase high-performance liquid chromatography (RP-HPLC), and gel permeation high-performance liquid chromatography (GP-HPLC) from the fermented milk by L. plantarum Q180. An ODS-AQ column was used for the RPC, a Vydac C18 column for the RP-HPLC, and a Superdex Peptide HR column for the GP-HPLC. The peptide was composed of Asp, Thr, Ile, Ser, Ala, and Gln, and the anti-lipase activity (IC₅₀) was 2,817 ㎍/mL.

• 한국자기공명학회논문지
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• 제13권2호
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• pp.96-107
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• 2009

Lactophoricin (LPcin-I) is a cationic amphipathic peptide with 23-mer peptide, and corresponds to the carboxy terminal 113-135 region of Component-3 of proteose-peptone. LPcin-I is a good candidate as a peptide antibiotic, because it has an antibacterial activity, but no hemolytic activity. On the other hand, its shorter analog (LPcin-II), which corresponds to the 119-135 region of PP3, has no antibacterial activity. In order to understand the structure-activity relationship under the membrane environments, we succeed to produce large amounts of LPcin-I and LPcin-II peptides. Peptides were over expressed in the form of fusion protein in Escherichia coli, and purified with several chromatography techniques. In this paper, we introduce the optimizing processes of purification and NMR measurement.

• BMB Reports
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• 제37권4호
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• pp.460-465
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• 2004

A 16-residue polypeptide model with the sequence acetyl-YALSLAATLLKEAASL-OH was derived by rational de novo peptide design. The designed sequence consists of amino acid residues with high propensity to adopt an alpha helical conformation, and sequential order was arranged to produce an amphipathic surface. The designed sequence was chemically synthesized using a solid-phase method and the polypeptide was purified by reverse-phase liquid chromatography. Molecular mass analysis by electro-spray ionization mass spectroscopy confirmed the correct designed sequence. Structural characterization by circular dichroism spectroscopy demonstrated that the peptide adopts the expected alpha helical conformation in 50% acetonitrile solution. Liposome binding assay using Small Unilamellar Vesicle (SUV) showed a marked release of entrapped glucose by interaction between the lipid membrane and the tested peptide. The channel-forming activity of the peptide was revealed by a planar lipid bilayer experiment. An analysis of the conducting current at various applied potentials suggested that the peptide forms a cationic ion channel with an intrinsic conductance of 188 pS. These results demonstrate that a simple rational de novo design can be successfully employed to create short peptides with desired structures and functions.

• Journal of Microbiology and Biotechnology
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• 제16권12호
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• pp.1904-1911
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• 2006

The cell-free extracts of 250 yeasts were screened for their in vitro anti-angiogenic activity, to develop a new cancer metastasis inhibitor. Saccharomyces cerevisiae K-7 was selected as the producer of the anti-angiogenic agent, because it had the highest anti-angiogenic activity. The anti-angiogenic agent was produced maximally from hydrolysates of Saccharomyces cerevisiae K-7, when the yeast was cultured in yeast extract-peptone-dextrose medium at 30$^{\circ}C$ for 24 h, and cell-free extracts were than digested with pepsin for 4 h at 37$^{\circ}C$. The anti-angiogenic agent was further purified by ultrafiltration, Sephadex G-25 gel permeation chromatography and reverse-phase HPLC, and the anti-angiogenic activity of the final purified preparation was 72.7% at 10 $\mu$M/egg. The purified anti-angiogenic agent was found to originate from the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) molecule of Saccharomyces cerevisiae K-7, and its peptide sequence was Val-Ser-Trp-Tyr-Asp-Asn-Glu-Tyr-Gly-Tyr-Ser-Thr-Arg-Val-Val-Asp. In the MTT assay, the shape of the HT-l 080 cell was clearly changed to a circular type at 0.2 mM purified anti-angiogenic agent. This result indicated that the growth of the HT-I080 cell was significantly inhibited at 0.2 mM of the purified anti-angiogenic agent. The MMP activity of the treated HT-l080 cells was not affected, evidenced by the gelatin zymography, indicating that the anti-angiogenic mechanism of the purified anti-angiogenic agent is not mediated through MMP activity.

• KSBB Journal
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• 제29권5호
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• pp.380-384
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• 2014

Inhibition effects of peptide hydrolysates from Astragalus membranaceus Bunge. on the expression of the matrix metalloproteinases (MMPs) in human dermal fibroblasts were evaluated in vitro. Crude peptides were obtained by the hydrolysis of proteins extracted from A. membranaceus. Peptides were purified partially by the basis on the molecular weight using 40% polyacrylamide gel electrophoresis before treatment with human dermal fibroblasts. Basis on the doseeffect experiments, expressions of MMPs including MMP-1, MMP-3, MMP-8, MMP-13 in human dermal fibroblasts were evaluated. Expressions of MMP-1, MMP-3, MMP-8 and MMP-13 were reduced in 43%, 5%, 22% and 57% respectively. The mass spectrometric analysis of partially purified peptides from A. membranaceus, which strongly inhibit expressions of MMPs, indicated that the peptides were composed of molecules below 1500 Da.

• Journal of Microbiology and Biotechnology
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• 제14권6호
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• pp.1318-1323
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• 2004

This study describes the purification and characterization of a novel antihypertensive angiotensin 1­converting enzyme (ACE) inhibitory peptide from Saccharomyces cerevisiae. Maximal production of the ACE inhibitor from Saccharomyces cerevisiae was obtained from 24 h of cultivation at $30^{\circ}C$ and its ACE inhibitory activity was increased by about 1.5 times after treatment of the cell-free extract with pepsin. After the purification of ACE inhibitory peptides with ultrafiltration, Sephadex G-25 column chromatography, and reverse-phase HPLC, an active fraction with an $IC_{50}$ of 0.07 mg and $3.5\%$ yield was obtained. The purified peptide was a novel decapeptide, showing very low similarity to other ACE inhibitory peptide sequences, and its amino acid sequence was Tyr-Asp-Gly-Gly-Val-Phe-Arg-Val-Tyr-Thr. The purified inhibitor competitively inhibited ACE and also showed a clear antihypertensive effect in spontaneously hypertensive rats (SHR) at a dosage of 1 mg/kg body weight.

• Fisheries and Aquatic Sciences
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• 제19권2호
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• pp.11.1-11.7
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• 2016

In this study, purified peptides from shrimp waste hydrolysates (SWHs) were examined for their inhibitory effects against ${\beta}$-secretase. During consecutive purification using a Sephadex G-25 column chromatography and high performance liquid chromatography on a C18 column, a potent ${\beta}$-secretase inhibitory peptide Asp-Val-Leu-Phe-His (629 Da) was isolated and identified from SWH24 by Q-TOF MS/MS and the $IC_{50}$ value was determined to be $92.70{\mu}M$. The ${\beta}$-secretase inhibition patterns of the purified peptides were found to be competitive. Among synthesized ${\beta}$-secretase inhibitory peptides, Leu-Phe-His had higher ${\beta}$-secretase inhibitory activity than the others. The result of this study suggests that the ${\beta}$-secretase inhibitory peptide derived from SWH24 could be potential candidates to develop nutraceuticals and pharmaceuticals.

• Journal of Microbiology and Biotechnology
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• 제18권6호
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• pp.1076-1080
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• 2008

Glucagon, a peptide hormone produced by alpha-cells of Langerhans islets, is a physiological antagonist of insulin and stimulator of its secretion. In order to improve its bioactivity, we modified its structure at the C-terminus by amidation catalyzed by a recombinant amidase in bacterial cells. The human gene coding for glucagon-gly was PCR amplified using three overlapping primers and cloned together with a rat ${\alpha}$-amidase gene in plasmid pMGA. Both genes were expressed under control of the strong constitutive promoter of aph and secretion signal melC1 in Streptomyces lividans. With Phenyl-Sepharose 6 FF, Q-Sepharose FF, SP-Sepharose FF chromatographies and HPLC, the peptide was purified to about 93.4% purity. The molecular mass of the peptide is 3.494 kDa as analyzed by MALDI TOF, which agrees with the theoretical mass value of the C-terminal amidated glucagon. The N-terminal sequence of the peptide was also determined, confirming its identity with human glucagon at the N-terminal part. ELISA showed that the purified peptide amide is bioactive in reacting with glucagon antibodies.

• Molecules and Cells
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• 제42권3호
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• pp.262-269
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• 2019

The porcine myeloid antimicrobial peptide (PMAP), one of the cathelicidin family members, contains small cationic peptides with amphipathic properties. We used a putative lysozyme originated from the bacteriophage P22 (P22 lysozyme) as a fusion partner, which was connected to the N-terminus of the PMAP36 peptide, to markedly increase the expression levels of recombinant PMAP36. The PMAP36-P22 lysozyme fusion protein with high solubility was produced in Escherichia coli. The final purified yield was approximately 1.8 mg/L. The purified PMAP36-P22 lysozyme fusion protein exhibited antimicrobial activity against both Gram-negative and Grampositive bacteria (Staphylococcus aureus, Salmonella enterica serovar Typhimurium, Pseudomonas aeruginosa, and Bacillus subtilis). Furthermore, we estimated its hemolytic activity against pig erythrocytes as 6% at the high concentration ($128{\mu}M$) of the PMAP36-P22 lysozyme fusion protein. Compared with the PMAP36 peptide (12%), our fusion protein exhibited half of the hemolytic activity. Overall, our recombinant PMAP36-P22 lysozyme fusion protein sustained the antimicrobial activity with the lower hemolytic activity associated with the synthetic PMAP36 peptide. This study suggests that the PMAP36-P22 lysozyme fusion system could be a crucial addition to the plethora of novel antimicrobials.