• Journal of Plant Biotechnology
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• 제41권1호
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• pp.1-9
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• 2014

So far it has been generally considered that proteomic approaches are very useful for studying plant-microbes interaction. In this review, recent studies based on papers published from 2010 to 2013 have investigated proteomics analysis in various interaction during plant-fungal pathogen infection by means of gel-based proteomics coupled with mass spectrometry (MS)-based analysis. In rice, three papers focused on rice-Magnaporthe oryzae interaction were mainly reviewed in this study. Interestingly, another study showed proteomic changes in rice inoculated with Puccinia triticina, which is not only an fungal pathogen in wheat and but also results to the disease resistance with non-host defense manner in rice. Additionally, proteomics analysis has been widely subjected to understand defense mechanism during other crops (wheat, tomato, strawberry and mint) and their fungal pathogen interaction. Crops inoculated are analyzed to identify differentially regulated proteins at various tissues such as leaf and apoplast using 2-DE analysis coupled with various MS approaches such as MALDI-TOF MS, nESI-LC-MS/MS and MudPIT, respectively. Taken together, this review article shows that proteomics is applicable to various organisms to understand plant-fungal pathogen interaction and will contribute to provide important information for crop disease diagnosis and crop protection.

• 방사선산업학회지
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• 제6권2호
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• pp.129-138
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• 2012

Proteomic analysis was performed in order to identify proteomic changes by salt stress between the Japonica cv. Donganbyeo (WT) and two salt-tolerant (ST) mutant lines by using the SDS-PAGE and 2-DE. Two salt tolerant rice mutant lines, ST-87 and ST-301, were selected by in vitro mutagenesis with gamma-ray. Three-week-old seedlings were treated with 171 mM NaCl for 7 days. In the SDS-PAGE, three proteins with molecular weights of 27, 46 and 58 kDa were highly increased under salt treatment. Total proteins from shoots of both WT and ST-lines were separated by two-dimensional gel electrophoresis. In 2-DE, 201, 226, 217 and 213 protein spots were detected in the untreated-or treated-WT and untreated- or treated-ST-87, respectively. Of theses, 17 and 10 protein spots were up- and down-regulated under salt stress in the WT, respectively. While, 16 and 8 protein spots were up- and down-regulated under salt stress in the ST-87, respectively, compared with the untreated plants. High intensity or de novo synthesized proteins were analyzed by MALDI-TOF/MS analysis.

• 한국육종학회지
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• 제41권3호
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• pp.205-212
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• 2009

Wheat (Triticum aestivum L.) grain texture is an important determinant of milling properties and end product use. Two linked genes, puroindoline a (PINA) and puroindoline b (PINB), control most of the genetic variation in wheat grain texture. Wheat seed proteins were examined to identify PINA and PINB gene using two pre-harvest sprouting wheat cultivars; Jinpum (resistant) and Keumgang (susceptible).Wheat seed proteins were separated by two-dimensional electrophoresis with IEF gels over pH ranges: pH 3-10. A total of 73 spots were digested with trypsin resulting peptide fragmentation were analyzed by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/MS). Mass spectra were automatically processed and searched through NCBInr, SWISS-PORT and MSDB database with mono isotopic masses and complete gene sequence were found by UniProt database. Puroindoline a and puroindoline b that is responsible for grain texture related with baking performance and roughness. Two spots were found Pin b (16.7 kDa) and Pin a (16.3 kDa) in Jinpum compare to seven spots were identified Pin a (16.1 kDa, 16.3 kDa) and Pin b (16.7 kDa, 9.5 kDa and 14.4 kDa) in Keumgang. Some selected spots were identified puroindoline like grain softness protein (16.9 kDa, 17 kDa and 18.1 kDa) in Keumgang. Moreover, to gain a better inferring the identification of puroindoline related proteins using proteomics, we accomplished a complete gene sequence of PINA and PINB gene in pre-harvesting sprouting wheat seeds between resistant (Jinpum) and susceptible (Keumgang).

• 한국미생물·생명공학회지
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• 제47권2호
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• pp.259-268
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• 2019

In this study, the extracellular metalloprotease from Serratia marcescens PPB-26 was purified to homogeneity via ethanol fractionation and DEAE-cellulose column chromatography. Thus, a 3.8-fold purification was achieved with a 20% yield and specific activity of 76.2 U/mg. The purified protease was a 50-kDa monomer whose optimum pH and temperature for activity were 7.5 and $30^{\circ}C$ respectively; however, it was found to remain active in the 5-9 pH range and up to $40^{\circ}C$ for 6 h. The protease had a half-life of 15 days at $4^{\circ}C$, an optimum reaction time of 10 min, and an optimum substrate (casein) concentration of 0.25%. Furthermore, the Michaelis constant ($K_m$) and reaction velocity ($V_{max}$) of the protease were calculated to be 0.28% and $111.11{\mu}moles/(min{\cdot}mg)^{-1}$, respectively. The protease was stable when subjected to metal ions (2 mM), showing increased activity with most (especially $CoCl_2$ and $MgSO_4$ (30.54% increase)). It was also stable when exposed to oxidizing agents, bleaching agents, and detergents (5% v/v for 60 min). It retained 93% of its activity in non-ionic detergents (Tween-20, Tween-80, and Triton X-100). Moreover, wash performance analysis in commercial detergents (Ariel and Tide) showed that not only was the protease capable of protein stain removal, but also reduced cleaning time by 80% when added to detergents. Thus, the Serratia marcescens PPB-26 metalloprotease appears to be a promising new candidate as a laundry additive in the detergent industry.

• 한국해양바이오학회지
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• 제14권1호
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• pp.9-24
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• 2022

Low-molecular weight peptides derived from fish collagen exhibit several bioactivities, including antioxidant, antiwrinkle, antimicrobial, antidiabetic, and antihypertension effects. These peptides are also involved in triglyceride suppression and memory improvement. This study aimed to investigate the optimal processing condition for preparing low-molecular weight peptides from flounder skin, and the properties of the hydrolysate. The optimal processing conditions for peptic hydrolysis were as follows: a ratio of pepsin to dried skin powder of 2% (w/w), pH of 2.0, and a temperature of 50℃. Peptic hydrolysate contains several low-molecular weight peptides below 300 Da. Gly-Pro-Hyp(GPHyp) peptide, a process control index, was detected only in peptic hydrolysate on matrix-assisted laser desorption/ionization-time-of-flight(MALDI-TOF) spectrum. 2,2'-azinobis-(3-3-ethylbenzothiazolline-6- sulfonic acid(ABTS) radical scavenging activity of the peptic hydrolysate was comparable to that of 1 mM ascorbic acid, which was used as a positive control at pH 5.5, whereas collagenase inhibition was five times higher with the peptic hydrolysate than with 1 mM ascorbic acid at pH 7.5. However, the tyrosinase inhibition ability of the peptic hydrolysate was lower than that of arbutin, which was used as a positive control. The antibacterial effect of the peptic hydrolysate against Propionibacterium acne was not observed. These results suggest that the peptic hydrolysate derived from a flounder skin is a promising antiwrinkle agent that can be used in various food and cosmetic products to prevent wrinkles caused by ultraviolet radiations.

• Mycobiology
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• 제50권5호
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• pp.366-373
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• 2022

Regulation of proper gene expression is important for cellular and organismal survival, maintenance, and growth. Abnormal gene expression, even for a single critical gene, can thwart cellular integrity and normal physiology to cause diseases, aging, and death. Therefore, gene expression profiling serves as a powerful tool to understand the pathology of diseases and to cure them. In this study, the difference in gene expression in Flammulina velutipes was compared between the wild type (WT) mushroom and the mutant one with clogging phenomenon. Differentially expressed transcripts were screened to identify the candidate genes responsible for the mutant phenotype using the DNA microarray analysis. A total of 88 genes including 60 upregulated and 28 downregulated genes were validated using the real-time quantitative PCR analysis. In addition, proteomic differences between the WT and mutant mushroom were analyzed using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Interestingly, the genes identified by these genomic and proteomic analyses were involved in stress response, translation, and energy/sugar metabolism, including HSP70, elongation factor 2, and pyruvate kinase. Together, our data suggest that the aberrant expression of these genes attributes to the mutant clogging phenotype. We propose that these genes can be targeted to foster normal growth in F. velutipes.

• Journal of Microbiology and Biotechnology
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• 제33권6호
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• pp.753-759
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• 2023

The genus Paenibacillus contains a variety of biologically active compounds that have potential applications in a range of fields, including medicine, agriculture, and livestock, playing an important role in the health and economy of society. Our study focused on the bacterium SS4^T (KCTC 43402^T = GDMCC 1.3498^T), which was characterized using a polyphasic taxonomic approach. This strain was analyzed using antiSMASH, BAGEL4, and PRISM to predict the secondary metabolites. Lassopeptide clusters were found using all three analysis methods, with the possibility of secretion. Additionally, PRISM found three biosynthetic gene clusters (BGC) and predicted the structure of the product. Genome analysis indicated that glucoamylase is present in SS4^T. 16S rRNA sequence analysis showed that strain SS4^T most closely resembled Paenibacillus marchantiophytorum DSM 29850^T (98.22%), Paenibacillus nebraskensis JJ-59^T (98.19%), and Paenibacillus aceris KCTC 13870^T (98.08%). Analysis of the 16S rRNA gene sequences and Type Strain Genome Server (TYGS) analysis revealed that SS4^T belongs to the genus Paenibacillus based on the results of the phylogenetic analysis. As a result of the matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF/MS) results, SS4^T was determined to belong to the genus Paenibacillus. Comparing P. marchantiophytorum DSM 29850^T with average nucleotide identity (ANI 78.97%) and digital DNA-DNA hybridization (dDDH 23%) revealed values that were all less than the threshold for bacterial species differentiation. The results of this study suggest that strain SS4^T can be classified as a Paenibacillus andongensis species and is a novel member of the genus Paenibacillus.

• Mycobiology
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• 제51권6호
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• pp.452-462
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• 2023

Opportunistic infections due to Cryptococcus neoformans and C. gattii species complexes continue to rise unabated among HIV/AIDS patients, despite improved antifungal therapies. Here, we collected a total of 20 environmental and 25 presumptive clinical cryptococcal isolates from cerebrospinal fluid (CSF) samples of 175 patients enrolled in an ongoing clinical trial Ambition 1 Project (Botswana-Harvard Partnership). Identity confirmation of the isolates was done using MALDI-TOF MS and PCR. We describe the diversity of the isolates by PCR fingerprinting and sequencing (Oxford Nanopore Technology) of the intergenic spacer region. Mating types of the isolates were determined by amplification of the MAT locus. We report an unusual prevalence of 42.1% of C. neoformans × C. deneoformans hybrids Serotype AD (n = 16), followed by 39.5% of C. neoformans Serotype A (n = 15), 5.3% of C. deneoformans, Serotype D (n = 2), 7.9% of C. gattii (n = 3), and 5.3% of C. tetragattii (n = 2) in 38 representative isolates that have been characterized. Mating type-specific PCR performed on 38 representative environmental and clinical isolates revealed that 16 (42.1%) were MATa/MAT𝛼 hybrids, 17 (44.7%) were MAT𝛼, and five (13.2%) possessed MATa mating type. We used conventional and NGS platforms to demonstrate a potential link between environmental and clinical isolates and lay a foundation to further describe mating patterns/history in Botswana.

• 한국미생물·생명공학회지
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• 제52권1호
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• pp.44-54
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• 2024

One of the serious modern environmental problems is pollution caused by highly toxic pesticides. Only small amounts of applied pesticides reach their target, and the rest ends up in soil and water. Chlorpyrifos is a toxic, broad-spectrum organophosphate insecticide. In humans, chlorpyrifos inhibits acetylcholinesterase (AChE) in the peripheral and central nervous system, and particularly in children, small amounts of this pesticide cause neurotoxic damage. As the toxic effects of chlorpyrifos and its persistence in the environment require its removal from contaminated sites, it is essential to study the biological diversity of chlorpyrifos-degrading microorganisms. In this study, we sought to determine the chlorpyrifos-degrading ability of the bacterial strain Ochrobactrum intermedium PDB-3. This strain was isolated from soil contaminated with various pesticides and identified as PDB-3 based on morpho-cultural characteristics, MALDI-TOF MS, and 16S rRNA. Studies were conducted for 30 days in sterile soils containing initial concentrations of 50, 75, 100, and 125 mg/kg of chlorpyrifos. To determine the degradation of chlorpyrifos, a liquid culture of the strain was added to the soil at three optical densities: 0, and after 24 and 48 h (OD = 0.03, 0.2 and 0.32). Using GX-MS, we determined that chlorpyrifos was converted to 3,5,6-trichloro-2-pyridinol (TCP). We also found that with increasing optical density, rapid degradation of the initial concentration of chlorpyrifos occurred. Sterile soil without strain PDB-3 was used as a control sample.

• 생명과학회지
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• 제26권11호
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• pp.1259-1268
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• 2016

항생제 남용의 문제점 및 사용제한에 따른 천연물 유래 항생제 대체제 개발을 위하여 우리나라와 중국, 일본에서만 서식하는 고유종인 참담치의 족부 근육 추출물로부터 항균 펩타이드를 분리 및 정제하였다. $C_{18}$ 역상 컬럼을 사용하여 항균 펩타이드를 정제하였으며, MALDI-TOF/MS로 분석 결과 분자량은 6,701 Da에 해당하였다. edman degradation 방법으로 N-말단 아미노산 서열 분석을 통하여 20개의 아미노산 서열을 밝혔으며 이는 캘리포니아 홍합(Mytilus californianus)의 sperm-specific protein 또는 protamine-like PL-II/PL-IV precursor와 100% 상동성을 지님을 밝혀냈다. 이러한 정보를 바탕으로 ORF를 분석한 결과 60개의 아미노산을 코딩하는 183 bp로 캘리포니아 홍합의 protamine-like PL-II/PL-IV precursor와 아미노산 서열이 100% 일치하였고 유전자 염기서열은 97.2%의 상동성을 나타냈다. 합성된 항균 펩타이드는 그람 양성 균주 Bacillus cereus (MEC, $20.8{\mu}g/ml$), Bacillus subtilis (MEC, $0.2{\mu}g/ml$), Streptococcus mutans (MEC, $0.2{\mu}g/ml$), 그람 음성 균주 Pseudomonas aeruginosa (MEC, $5.7{\mu}g/ml$), Escherichia coli (MEC, $2.6{\mu}g/ml$) 그리고 진균류 Candida albicans (MEC, $56.3{\mu}g/ml$)의 항균활성을 나타냈다. 또한 높은 열안정성을 지녔으며, C. albicans를 제외하고 염안정성을 지닌 항균 펩타이드로 확인되었다. 이를 통해 참담치 유래의 항균 펩타이드는 항생제 대체제의 후보소재로 높은 가능성을 지녔다고 할 수 있다. 따라서 본 연구에서 분리한 참담치 족부 근육 유래 항균 펩타이드는 향후 추가적인 연구를 통하여 항생제를 대체할 수 있는 물질의 개발이 가능할 것으로 사료된다. 그리고 해양무척추동물의 선천성 면역 기작에 대한 정보를 제공할 것으로 기대한다.