• The Korean Journal of Physiology and Pharmacology
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• v.13 no.1
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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.

• Microbiology and Biotechnology Letters
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• v.26 no.5
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• pp.379-386
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• 1998

Deterioration of food is in general caused by the presence of microorganisms and chemical compounds of food itself. There exists antimicrobial compound in the food, however, addition of food antiseptics, additives, or physico-chemical processing is a common practice. The safety of artificial chemical antiseptics became a serious public concern, therefore, new natural antiseptic compounds are in need to be developed. We have isolated a new natural antifungal protein (KBS-B2) from Astragal seed through ammonium sulfate precipitation and column chromatography using FPLC Mono-S and Superose 12HR. The purified protein inhibited growth of Candida albicans, and spore germination of food spoiling fungi such as Aspergillus ochraceus, Penicillium expensum, P. digitatum and Botrytis cineria. Antifungal effect of the KBS-B2 protein could be directly assayed by bioautography overlaying the fungal spores on the electrophoresed acrylamide gel. The comparison of N-terminal amino acid sequences of the KBS-B2 with known antifungal protein revealed that had 50% homology to thaumatin and zeamatin like proteins.

• The Korean Journal of Pesticide Science
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• v.10 no.1
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• pp.56-65
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• 2006

An antagonistic bacterial isolate, that inhibits the growth of plant pathogens, was selected and identified from 5,000 isolates screened from the rhizosphere of various crop plants. An isolate Bacillus sp. N32, tested against Colletotrichum gloeosporioides causing anthracnose disease in hot pepper, produced both a heat resistant antifungal protein and a heat sensitive antifungal protein. The heat resistant protein was partially purified by Ammonium sulfate fractionation and gel filtration chromatography. The bioautography showed that the proteins possessed high antifungal activity. The biosynthetic gene cluster responsible for the heat resistant antifungal protein was cloned from cosmid library using DNA probe obtained from PCR product with the primers targeting the conserved nucleotide sequence of the synthetic genes reported earlier, Most of the clones obtained showed higher homology to fengycin antibiotic synthetic gene family reported earlier. On the other hand, the heat sensitive protein was isolated from SDS-PAGE and electroblotting to determine the N-terminal amino acid sequences. The heat sensitive antifungal protein gene was cloned from the ${\lambda}-ZAP$ libraries using a DNA probe based on the N-terminal amino acid sequences of the heat sensitive protein. We are contemplating to clone and sequence the whole gene cluster encoding the heat sensitive protein for further analysis.

• Korean Journal of Environmental Biology
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• v.22 no.4
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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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• v.39 no.3
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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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• v.21 no.1
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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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• v.42 no.5
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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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• v.19 no.10
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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.

• Proceedings of the Korean Biophysical Society Conference
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• 1997.07a
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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)

• Korean Journal of Environmental Agriculture
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• v.22 no.3
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• pp.203-209
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• 2003

In the course of screening for antifungal agents, a bacterial strain, DM3 was isolated from a mud sample collected at Daechon in Chungnam province and identified as Bacillus licheniformis based on API 50CHB test. It has antifungal activity against 12 plant pathogenic fungi in paper disc assay. At the 95% lethal dose of gamma radiation ($^{60}Co$, 10 kGy, $D_{10}=2.32\;kGy$), the antifungal activity deficient mutant (mDM3) against Colletotrichum gloeosporioides was induced From 2-D electrophoresis analysis, serine hydroxymethyltransferase (45.0 kDa), hypothetical protein(40.7 kDa), NifU protein homolog(15.4 kDa), and resolvase(12.5 kDa) homologous proteins were detected only in B. licheniformis DM3. Lysozyme(18.1 kDa) and alkyl hydroperoxide reductase(15.6 kDa) homologous proteins were expressed uniquely in B. licheniformis mDM3. Further studies are needed to reveal that these proteins from B. licheniformis DM3 could be closely related to the antifungal activity against plant pathogenic fungi.