• The Korean Journal of Physiology and Pharmacology
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• 제13권1호
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• pp.49-54
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• 2009

The mushroom Cordyceps militaris has been used for a long time in eastern Asia as a nutraceutical and in traditional Chinese medicine as a treatment for cancer patients. In the present study, a cytotoxic antifungal protease was purified from the dried fruiting bodies of C. militaris using anion-exchange chromatography on a DEAE-Sepharose column. Electrophoretic analyses indicated that this protein, designated C. militaris protein(CMP), has a molecular mass of 12 kDa and a pI of 5.1. The optimum conditions for protease activity were a temperature of $37^{\circ}C$ and pH of $7.0{\sim}9.0$. The enzyme activity was specifically inhibited by the serine protease inhibitor phenylmethylsulfonyl fluoride. Amino acid composition of intact CMP and amino acid sequences of three major peptides from a tryptic digest of CMP were determined. CMP exerted strong antifungal effect against the growth of the fungus Fusarium oxysporum, and exhibited cytotoxicity against human breast and bladder cancer cells. These results indicate that C. militaris represents a source of a novel protein that might be applied in diverse biological and medicinal applications.

• 한국미생물·생명공학회지
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• 제26권5호
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• pp.379-386
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• 1998

본 연구에서는 천연 항균물질의 개발 이용을 위해 황기 종자로부터 인체에 무해한 천연 항균 단백질을 ion exchange chromatography 및 gel filteration을 이용하여, 순수 분리하고 특성을 조사하였다. 황기종자로부터 추출한 천연 항균 단백질은 Aspergillus ocraceus, Penicillium expensum, P. digitatum, Botrytis cineria의 포자 발아 및 효모인 Candida albicans의 생육을 현저하게 저해하였으며 ammonium sulfate 포화도가 0.4일 때 단백질의 침전량이 122.6 $\mu\textrm{g}$/$m\ell$로 가장 많았고 항균력도 15.2 mm로 가장 높게 나타났다. 강력한 cation exchange chromatography인 Mono-S를 이용하여 FPLC에서 단백질을 분획하였을때 첫번째 peak에서 분획된 단백질군이 항균력을 보였으며 Superose 12HR gel filteration column을 이용하여 2차 분획 하였을 때 분자량이 19 kDa되는 단일 단백질만을 순수분리 할 수 있었다. 전기 영동한 polyacrylamide gel위에 곰팡이 포자를 중층하는 bio autography로 19 kDa 단백질 band의 항균력을 직접 확인하였으며 분리된 항균 단백질의 아미노 말단의 아미 노산 22잔기를 sequencing하고 thaumatin 및 zeamatin 유사 단백질들과 상동성을 측정한 결과 50%내외의 homology를 나타내었다. 분리된 항균 단백질은 곰팡이 균사가 성장하는 선단부위에 가장 먼저 침투하여 channel을 형성함으로 osmolysis를 일으켜 곰팡이의 생육을 억제하는 것으로 추측할 수 있었다.

• 농약과학회지
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• 제10권1호
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• pp.56-65
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• 2006

작물 근권 토양으로부터 분리한 5,000여 길항 균주로부터 Erwinia 및 Pseudomonas등의 세균과 Trichoderma, Colletotrichum 등 곰팡이의 성장을 동시에 억제하는 Bacillus sp. N 32 균주를 선발 동정 하였다. 특히 Bacillus sp. N32 균주는 고추 탄저병균인 Colletotrichum gloeosporioides에 대하여 열에 저항성이 있는 단백질과 열에 민감한 단백질의 2종류의 항균 단백질을 동시에 생산함을 단백질 침전과 활성 검정을 통하여 확인하였다. 이 항균 단백질들을 FPLC를 이용한 gel filtration chromatography방법으로 분리한 후 SDS-PAGE와 bioautography로 항균력을 확인하였다. 또한 이 항균 단백질의 유전자들을 선발하기 위하여 기존의 알려진 그람양성 세균의 대표적인 열 저항성 항균 펩타이드 생합성 유전자 서열을 primer로 이용한 PCR 방법으로 fengycin의 생합성 유전자 단편을 분리하고 이 PCR 산물을 이용하여 Bacillus sp. N32 균주의 cosmid library로부터 fengycin의 생합성 유전자 cluster중 일부를 분리하여 염기서열을 분석하였다. 또한 열에 민감한 항균 단백질 생산 유전자는 이 항균 단백질을 SDS-PAGE 및 electroblotting으로 분리한 뒤 N-terminal 부위의 15개의 아미노산 서열을 분석하고 이를 DNA 염기배열로 치환한 다음 probe로 이용하여 ${\lambda}-ZAP$ library로부터 항균 단백질 생산 유전자가 포함된 다수의 clone을 선발하였다.

• 환경생물
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• 제22권4호
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• pp.487-493
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• 2004

To investigate the antifungal activity related protein in pesticidal bacteria, a bacterial strain LTD was isolated from soil collected at Gimje in Jeonbuk province, Korea, and identified as Bacillus subtilis LTD based on a API50 CHB kit and 168 rDNA sequencing. It has an antifungal activity against 9 plant pathogenic fungi in a paper disc assay. The antifungal activity- deficient mutant, B. subtilis mLTD was induced at a 5 kGy dose of $^{60}Co$ gamma radiation. Using the two-dimensional electrophoresis and the matrix assisted laser desorption ionization time-of-flight mass spectrometry, the comparison analysis of proteins between the wild and mutant were performed. A major intracellular serine proteinase IspA (MW: 32.5 kDa), a NAD (P) H dehydrogenase (MW: 20.0 kDa), and a stage II sporulation protein AA, SpoIIAA (MW: 14.3kDa) were detected only in the B. subtilis LTD. These results suggested that the functions of these proteins found only in the B. subtilis LTD could. be closely related to the antifungal activity against plant pathogenic fungi.

• BMB Reports
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• 제39권3호
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• pp.278-283
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• 2006

Defensins are small cysteine rich peptides with a molecular mass of 5-10 kDa and some of them exhibit potent antifungal activity. We have cloned the coding region of a cDNA of 225 bp cysteine rich defensin, named as Tfgd1, from the legume Trigonella foenum-graecum. The amino acid sequence deduced from the coding region comprised 74 amino acids, of which the N-terminal 27 amino acids constituted the signal peptide and the mature peptide comprised 47 amino acids. The protein is characterized by the presence of eight cysteine resisdues, conserved in the various plant defensins forming four disulphide bridges, which stabilize the mature peptide. The recombinant protein expressed in E coli exhibited antifungal activity against the broad host range fungus, Rhizoctonia solani and the peanut leaf spot fungus, Phaeoisariopsis personata.

• Preventive Nutrition and Food Science
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• 제21권1호
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• pp.52-56
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• 2016

This study was performed to investigate the antifungal substances and the antifungal activity against fungi of lactic acid bacteria (LAB) isolated from kimchi. LAB from kimchi in Imsil showed antifungal activity against Penicillium brevicompactum strain FI02. LAB LI031 was identified as Lactobacillus sakei subsp. Antifungal substances contained in L. sakei subsp. ALI033 culture media were unstable at high pH levels. Both, the control and proteinase K and protease treated samples showed clear zones, suggesting that the antifungal substances produced by ALI033 were non-protein substances unaffected by protesases. Both, the control and catalase showed clear zones, suggesting that the antifungal metabolite was not $H_2O_2$. The molecular weights of the antifungal substances were ${\leq}3,000Da$. The organic acid content of crude antifungal substances produced by L. sakei subsp. ALI033 showed high concentrations of lactic acid (502.47 mg/100 g). Therefore, these results suggest that antifungal substance produced by L. sakei subsp. ALI033 is most likely due to its ability in producing organic acid.

• BMB Reports
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• 제42권5호
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• pp.281-285
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• 2009

Peroxisomes play an important role in cellular defense systems and generate secondary messengers for cellular communication. Saccharomyces cerevisiae containing oleate-induced peroxisomes were subjected to buffer-soluble extraction and two chromatographic procedures, and a protein with antifungal activity was isolated. The results of MALDI-TOF analysis identified the isolated protein as peroxisomal 3-ketoacyl-CoA thiolase (ScFox3). Purified yeast ScFox3 exhibited thiolase activity that catalyzed the thiolytic cleavage of 3-ketoacyl-CoA to acetyl-CoA and acyl-CoA. ScFox3 protein inhibited various pathogenic fungal strains, with the exception of Aspergillus flavus. Using ScFox3-GFP and PTS2 signal-truncated ScFox3M-GFP, we showed that only ScFox3-GFP, with an intact PTS2 peroxisome signal sequence, was able to translocate into peroxisomes. Yeast ScFox3 is a natural antifungal agent found in peroxisomes.

• Journal of Microbiology and Biotechnology
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• 제19권10호
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• pp.1169-1175
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• 2009

An antifungal chitinase, ChiCW, produced by Bacillus cereus 28-9 is effective against conidial germination of Botrytis elliptica, the causal agent of lily leaf blight. ChiCW as a modular enzyme consists of a signal peptide, a catalytic domain, a fibronectin type-III-like domain, and a chitin-binding domain. When two C-terminal domains of ChiCW were truncated, $ChiCW{\Delta}FC$ (lacking the chitin-binding domain and fibronectin type III-like domain) lost its antifungal activity. Since $ChiCW{\Delta}C$ (lacking the chitin-binding domain) could not be expressed in Escherichia coli as $ChiCW{\Delta}FC$ did, a different strategy based on protein engineering technology was designed to investigate the involvement of the chitin-binding domain of ChiCW ($ChBD_{ChiCW}$) in antifungal activity in this study. Because ChiA1 of Bacillus circulans WL-12 is a modular enzyme with a higher hydrolytic activity than ChiCW but not inhibitory to conidial germination of Bo. elliptica and the similar domain composition of ChiA1 and ChiCW, the C-terminal truncated derivatives of ChiA1 were generated and used to construct chimeric chitinases with $ChBD_{ChiCW}$. When the chitin-binding domain of ChiA1 was replaced with $ChBD_{ChiCW}$, the chimeric chitinase named ChiAAAW exhibited both high enzyme activity and antifungal activity. The results indicate that $ChBD_{ChiCW}$ may play an important role in the antifungal activity of ChiCW.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 1997년도 학술발표회
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• pp.41-41
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• 1997

The conformation studies of tenecin 3, which has been purified from the hemolymph of the meal worms Tenebrio molitor, was carried out by CD and NMR. This highly Gly-rich protein consisting of 78 amino acid residues shows similar biochemical features such as heat stability, humoral existence, and high contents of Gly and His residues to other insect antifungal proteins. (omitted)

• 한국환경농학회지
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• 제22권3호
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• pp.203-209
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• 2003

항진균 활성균주를 선발하는 과정에서 DM3 균주를 대천 바닷가에서 수집된 진흙 시료로부터 분리하였으며 API 50CHB kit를 이용하여 동정한 결과 Bacillus licheniformis로 동정되었다. 이 균주는 12종의 식물병원성 진균에 대해 항균활성을 나타내었다. 감마선($^{60}Co$)을 조사하여 항진균 활성 결핍 돌연변이체(mDMB)를 유도한 후, 이차원전기영동으로 단백질을 분석한 결과 DM3와 mDM3에만 존재하는 각각 4종과 3종의 단 단백질을 확인할 수 있었다. 2-DE 결과 B. licheniformis DM3에서 spot 1은 serine hydroxymethyltransferase(45.0kDa), spot 2는 hypothetical protein(40.7 kDa), spot 3는 NifU protein homolog(15.4 kDa), 그리고 spot 4는 resolvase(12.5 kDa)와 상동성을 지닌 단백질로 동정되었고 B. licheniformis mDM3에서만 발현된 spot 5는 lysozyme(18.1 kDa)과 spot 6, 7은 alkyl hydroperoxide reductase(15.6 kDa)으로 동정되었다. B. licheniformis DM3에서 이들 단백질들의 항진균 활성 관련 여부를 규명하기 위해서 더 연구가 진행되어야 할 것으로 사료된다.