• Applied Biological Chemistry
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• v.37 no.2
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• pp.65-71
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• 1994

Chungkook-Jang was produced by fermenting Bacillus licheniformis CN-115. The changes of chemical composition, enzyme activity, and amino acids during the fermentation were investigated. The proximate composition was shown irregular fluctuation phenomenon during the fermentation, but only the moisture tended some reducing during the fermentation just after steaming. The content of amino nitrogen was increased radically after the 36 hours of fermentation and became the highest level at 18.072 mg/g at the 60 hours of it. In accordance with the fermentation of Chungkook-Jang, pH got to the 8.39 at 60 hours with increasing, protease activity was increased according to the fermentation and acid and neutral protease activity was reduced after being reached at the highest activity at 48 hours. The most suitable pH was 6.5 and temperature was $35^{\circ}C$ for dissolution-activated of protein in the process of fermentation of Chungkook-Jang. The content of water soluble protein and the content of salt soluble protein were increased at continuously according to the fermentation time of Chungkook-Jang the largest quantity. The molecular weight of water soluble protein of Chungkook-Jang fermented for 48 hours was about 19,000. The amino acids of water soluble protein just after steaming were totally 16 kinds and proline was amino acid and them was in series by glutamic acid and serine in that ordered. The amino acids salt soluble protein, just after steaming were totally 16 kinds and was the largest quantity phenylalanine, glutamic acid and aspartic acid and aspartic acid in that order.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.76.1-76
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• 2003

The coat protein (CP) of Turnip crinkle virus (TCV) is organized into 3 distinct domains, R domain (RNA-binding) connected by an arm, 5 domain and P domain. We have previously shown that the CP of TCV strongly suppresses RNA silencing, and have mapped N-terminal R domain of which is also the elicitor of resistance response in the Arabidopsis ecotype Di-17 carrying the HRT resistance gene. In order to map the region in the TCV CP that is responsible for silencing suppression, a series of CP mutants were constructed, transformed into Agrobacterium, coinfiltrated either with HC-Pro (the helper component proteinase of tobacco etch potyvirus) known as a suppressor of PTGS or GFP constructs into leaves of Nicotiana benthmiana expressing GFP transgenically. In the presence of HC-Pro, all CP mutants were well protected, accumulating mutant CP mRNAs and their proteins even 5 days post-infiltration (DPI). In the presence of GFP, some mutant constructs which showed the accumulation of CP mutants and GFP mRNAs at early stage but eventually degraded at 5 DPI. Only a mutant which carrying 4 amino acid deletion of R domain was tolerable to maintain suppressing activity, suggesting that the suppressing activity is not directly related with the eliciting activity. A transient assay also revealed that the mutants synthesized their proteins, suggesting that a full length of CP sequences and its intact structure are required to stabilize CP, which suppresses the RNA silencing.

• Development and Reproduction
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• v.17 no.3
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• pp.221-229
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• 2013

Cathepsins are members of the multigene family of lysosomal cysteine proteinases and have regulated function in several life processes. The potential role of cathepsin F cysteine gene was expected as protease in the yolk processing mechanism during early developmental stage, but expression analysis was unknown after fertilization. The alignment analysis showed that amino acid sequence of cathepsin F from olive flounder liver expressed sequence tag (EST) homologous to cathepsin F of other known cathepsin F sequences with 87-98% identity. In this study, we examined the gene expression analysis of cathepsin F in various tissues at variety age flounder. Tissue distribution of the cathepsin F mRNA has been shown to be ubiquitous and constitutive pattern regardless of age in each group, although derived from cDNA library using liver sample. The mRNA level of cathepsin F more increased as developmental proceed during embryogenesis and early developmental stage, especially increased in the blastula, hatching stage and 3 days post hatching (dph). As a result, it may suggest that the proteolysis of yolk proteins (YPs) has been implicated as a mechanism for nutrient supply during early larval stages in olive flounder.

• BMB Reports
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• v.34 no.2
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• pp.109-113
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• 2001

The Serratia marcescens metalloprotease inhibitor (SmaPI) is a proteinase inhibitor toward Serratia marcescens metalloprotease (SMP). The three-dimensional structure of SmaPI was calculated by computer modeling using the structure complex between SMP and the Erwinia chrysanthemi inhibitor as a template. Based on this model structure, the substitution of the amino acid residues, Ala4, Leu-5, Pro-6, and Thr-7, were located at the hinge region of the N-terminal segment by site-directed mutagenesis. Although the A4R and T7A mutant SmaPIs showed a nearly full inhibitory activity, the inhibitory activity of SmaPI decreased significantly when the Leu-5 was converted to Ala, Gly, Ile, or Val. Surprisingly, the L5I and L5V mutant SmaPIs showed less inhibitory activities than the L5A mutant. From these results, we suggested that the orientations and positions of respective aliphatic groups in the side-chain of position 5 mainly affected the inhibitory activity of SmaPI. The overall side-chain hydrophobicity was only slightly affected. The side-chain of the Leu-5 residue contributed approximately 0.79 kcal/mol out of 8.44 kcal/mol to the binding of SmaPI with SMP The inhibitory activities of P6A and F6G were also severely decreased. The Pro-6 may have a critical role in maintaining the strict conformation of the N-terminal portion that may be important in the inhibitory activity of SmaPI. In conclusion, Leu-5 and Pro-6 have crucial roles in the inhibitory function of SmaPI toward SMP.

• Journal of Microbiology and Biotechnology
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• v.25 no.9
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• pp.1493-1501
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• 2015

A Bacillus species, EMD4, with strong antibacterial activity was isolated from ganjang (soy sauce) and identified as B. subtilis. B. subtilis EMD4 strongly inhibited the growth of B. cereus ATCC14579 and B. thuringiensis ATCC33679. The antibacterial activity was stable at pH 3-9 but inactive at pH 10 and above. The activity was fully retained after 15 min at 80℃ but reduced by 50% after 15 min at 90℃. The activity was completely destroyed by proteinase K and protease treatment, indicating its proteinaceous nature. The bacteriocin (BacEMD4) was partially purified from culture supernatant by ammonium sulfate precipitation, and Q-Sepharose and Sephadex G-50 column chromatographies. The specific activity was increased from 769.2 AU/mg protein to 8,347.8 AU/mg protein and the final yield was 12.6%. The size of BacEMD4 was determined to be 3.5 kDa by Tricine SDS-PAGE. The N-terminal amino acid sequence was similar with that of Subtilosin A. Nucleotide sequencing of the cloned gene confirmed that BacEMD4 was Subtilosin A. BacEMD4 showed bactericidal activity against B. cereus ATCC14579.

• KSBB Journal
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• v.26 no.6
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• pp.513-516
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• 2011

The occurrence of dental caries is mainly associated with oral pathogens, especially cariogenic Streptococcus mutans. The aim of this study was to isolate and characterize lactic acid bacterium showing inhibitory activity against cariogenic Streptococcus mutans. As results, an isolate with strong inhibitory activity was obtained from Kimchi and it was identified as Lactobacillus sakei by API and 16S rRNA gene analyses. This strain secreted an inhibitory compound in cell growth medium and the activity of the compound was completely disappeared by proteinase K revealing the fact that the compound is proteinous substance, bacteriocin. Optimal culture condition for bacteriocin production by Lb. sakei K-7 was at pH 7.5 and $37^{\circ}C$ for 18 h. Oral administration of this isolate may give anticariogenic and probiotic effects on hosts.

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.131-131
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• 2017

Though the Platycodon grandiflorum, has a broad range of pharmacologic properties, but the mechanisms underlying these effects remain unclear. In order to profile proteins from the nodal segment, callus, root and shoot, high throughput proteome approach was executed in the present study. Two-dimensional gels stained with CBB, a total of 84 differential expressed proteins were confirmed out of 839 protein spots using image analysis by Progenesis SameSpot software. Out of total differential expressed spots, 58 differential expressed protein spots (${\geq}2-fold$) were analyzed using MASCOT search engine according to the similarity of sequences with previously characterized proteins along with the UniProt database. Out of 58 differential expressed protein, 32 protein spots were up-regulated such as ribulose-1,5-bisphosphate carboxylase, endoplasmic oxidoreductin-1, heat stress transcription factor A3, RNA pseudourine synthase 4, cysteine proteinase, GntR family transcriptional regulator, E3 xyloglucan 6-xylosyltransferase, while 26 differential protein spots were down-regulated such as L-ascorbate oxidase precursor, late embryogenesis abundant protein D-34, putative SCO1 protein, oxygen-evolving enhancer protein 3. However, the frequency distribution of identified proteins using iProClass databases, and assignment by function based on gene ontology revealed that the identified proteins from the explants were mainly associated with the nucleic acid binding (17%), transferase activity (14%) and ion binding (12%). Taken together, the protein profile may provide insight clues for better understanding the characteristics of proteins and its metabolic activities in various explants of this essential medicinal plant P. grandiflorum.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.108-108
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• 2002

포유동물의 암컷 생식기관에 존재하는 다양한 종류의 matrix metallo-proteinase (MMP)는 난소와 자궁의 구성성분의 주기적인 변화를 조절하며 이중 난소의 MMP는 난포의 성장과 배란 그리고 퇴화 동안 조직재구성에 매우 중요한 역할을 한다고 알려져 있다. 본 실험에서는 근래에 새로 발견된 사람의 난포액에 존재하는 분자량 약 110kDa인 MMP-2 isoform GA110을 동정하고 자 하였다. 난포액으로부터 GA110 단백질을 분리하기 위하여 난포액에 5mM ethylenediaminetetraacetic acid(EDTA)를 처리한 후 DEAE Sepharose Fast Flow를 이용한 chromatography를 시행하였다. 그 결과, 난포액 단백질들은 0.2M NaCl 의 분획에서 GA110 활성을 나타내었고 anti-human MMP-2 antibody에 대한 면역반응도 뚜렷이 나타났다. DEAE Sepharose Fast Flow에서 얻은 분획 중 GA110의 활성과 면역반응을 모두 나타내는 분획만을 모아 Gelatin Sepharose 4B affinity chromatography로 다시 분리하였다 분리한 결과 resin에 흡착된 단백질 (eluate) 분획들에서 매우 뚜렷한 GA110 gelatinase 활성을 나타내었으며 면역반응 또한 관찰되었다. 이 분획들의 단백질을 농축한 후 zymography를 시행하여 나타난 GA110 단백질 band를 잘라 내었으며 이로부터 단백질을 electroelution하여 농축한 후 reducing agent인 2-mercaptoethanol를 처리하였다. 이를 전기영동 후 MMP-2 (propeptide region) antibody를 사용하여 immunoblotting 한 결과 70-72kDa의 단백질만이 면역반응을 나타내었다. 마지막으로 위와 같이 준비된 70-72kDa 단백질의 아미노산 서열을 Edman degradation 방법으로 분석하였다. 그 결과 이 단백질의 N 말단의 10개의 아미노산 배열 순서가 알려진 사람의 proMMP-2의 전체 배열순서 중 propetide domain의 N 말단에서부터 다섯 번째에서 시작하여 10개의 아미노산의 서열과 정확하게 일치하였다. 위 결과들로 미루어 사람의 난포액에 존재하는 MMP-2의 새로운 isoform인 GA110은 70-72kDa의 ProMMP-2가 disulfide bond를 통해 homodimer 구조를 이루고 있는 것으로 여겨진다.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.60 no.1
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• pp.97-106
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• 2015

Platycodon grandiflorum, commonly known as Doraji in Korea, has a wide range of pharmacologic properties, such as reducing adiposity and hyperlipidemia, and antiatherosclerotic effects. However, the mechanisms underlying these effects remain unclear. In order to profile proteins from the nodal segment, callus, root and shoot, high throughput proteome approach was executed in the present study. Two dimensional gels stained with CBB, a total of 84 differential expressed proteins were confirmed out of 839 protein spots using image analysis by Progenesis SameSpot software. Out of total differential expressed spots, 58 differential expressed protein spots (${\geq}$ 2-fold) were analyzed using MASCOT search engine according to the similarity of sequences with previously characterized proteins along with the UniProt database. Out of 58 differential expressed protein, 32 protein spots were up-regulated such as ribulose-1,5-bisphosphate carboxylase, endoplasmic oxidoreductin-1, heat stress transcription factor A3, RNA pseudourine synthase 4, cysteine proteinase, GntR family transcriptional regulator, E3 xyloglucan 6-xylosyltransferase, while 26 differential protein spots were down-regulated such as L-ascorbate oxidase precursor, late embryogenesis abundant protein D-34, putative SCO1 protein, oxygen-evolving enhancer protein 3. However, frequency distribution of identified proteins using iProClass databases, and assignment by function based on gene ontology revealed that the identified proteins from the explants were mainly associated with the nucleic acid binding (17%), transferase activity (14%) and ion binding (12%). In that way, the exclusive protein profile may provide insight clues for better understanding the characteristics of proteins and metabolic activity in various explants of the economically important medicinal plant Platycodon grandiflorum.

• Mycobiology
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• v.34 no.4
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• pp.209-213
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• 2006

Agrocybe cylindracea, an edible mushroom belonging to Bolbitiaceae, Agaricales, is widely used as invaluable medicinal material in the oriental countries. This study was initiated to find the genes expressed during the fruiting body formation of A. cylindracea. The cDNAs expressed differentially during fruiting body morphogenesis of A. cylindracea were isolated through subtractive hybridization between vegetative mycelia and fruiting bodies. The cDNAs expressed in the fruiting body morphogenesis of A. cylindracea were cloned and twenty genes were identified. Eleven were homologous to genes of known functions, three were homologous to genes in other organism without any function known. Six were completely novel genes specific to A. cylindracea so far examined. Some genes with known functions were a pleurotolysin, a self-assembling poreforming cytolysins; Aa-Pril and Pir2p, specifically induced genes during fruiting initiation of other mushroom, Agrocybe aegerita; an amino acid permease; a cytochrome P450; a MADS-box gene; a peptidylprolyl isomerase; and a serine proteinase. For other clones, no clear function was annotated so far. We believe the first report of the differentially expressed genes in fruiting process of A. cylindracea will be great helps for further research.