• Journal of Life Science
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• v.15 no.3 s.70
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• pp.317-322
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• 2005

The cycloinulooligosaccharide fructanotransferase (CFTase) gene (cft) from Paenibacillus macerans was subcloned into an E. coli-yeast shuttle vector, pYES2.0, resulting in pYGECFTN. The plasmid pYGECFTN (8.6 kb) was introduced into Saccharomyces cerevisiae SEY2102 cells and then the transformants were selected on the synthetic defined media lacking uracil. The cft gene expression in yeast transformant was demonstrated by the analyses cyclofructan (CF) spots on thin-layer chromatogram. The recombinant CFTase was not secreted into the medium and localized in the periplasmic space. The production of CF was observed after 5 min of the enzymatic reaction with inulin. The optimun pH and temperature for CF production were found to be at pH 8.0 and $45^{\circ}C$, respectively. Enzyme activity was stably maintained up to $55^{\circ}C$. The CF was produced from all inulin sources and was most efficiently produced from dahlia tubers and Jerusalem artichokes.

• Journal of Microbiology and Biotechnology
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• v.10 no.6
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• pp.805-810
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• 2000

An antifungal protein antagonistic to the rice blast fungus, Pyricularia oryzae was purified from Paenibacillus macerans PM-1 by ammonium sulfate fractionation, Q Sepharose Fast Flow column chromatography, Phenyl Sepharose CL-4B column chromatography and Superose 12 gen filtration. An apparent molecular mass of the purified antifungal protein was determined as 8 kDa by SDS-PAGE and 9 kDa by analytical gel filtration, respectively, suggesting that the purified protein is a monomer. The antifungal protein was stable at pH range from 7-12 and up to $100^{\circ}C$. The protein was also stable at 0.1-1% Tween 20 and Triton X-100. The N-terminal amino acid sequence of the antifungal protein was Thr-Glu-Leu-Pro-Leu-Gly-Ile-Val-Met-Asp-Lys-Tyr-Thr-Asp-Ala-Phe-Lys-Phe-Asp-Met-Phe. Comparison of the determined sequence with other peptide and DNA sequences did not reveal homology at all. Therefore, the purified antifungal protein was speculated to be a novel protein. The condidial germination in vitro of P. oryzae KJ301:93-39 by the purified protein ($5.9{\mu} g/ml$) was limited to $9{\pm}3.2%$ only, compared with $69{\pm}2.4%$ of the control. Ungerminated conidia were swollen at basa and mid cell by the purified protein. In vivo bioassay for inhibition of conidial germination of P. oryzae KJ 301, one of the most predominating racesin Korea. the purified protein ($5.9{\mu} g/ml$)strongly inhibited the conidial germination. The conidia, even though germinated, could not develop any further to produce appressoria efficiently.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 1999.04a
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• pp.39.2-39
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• 1999

• Journal of Microbiology and Biotechnology
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• v.10 no.6
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• pp.811-816
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• 2000

An antagonistic bacterium PM-1 which strongly inhyibits the growth of Pyricularia oryzae was isolated and identified as paenibacillus macerans. The antifungal substances of the strain PM-1 showed the broad antifungal spectra against P.oryzae races. Relating to the localization test, it was found that the antifungal substances existed not only in the cytoplasm but also in the culture supernatant, and importantly the antifungal activity of the latter was stronger than that of the former. The extracellular antifungal substances were extremely heat-stable up to $121^{\circ}C$ for 15 min. The substances were optimally produced at $20^{\circ}C$ and pH 10.0 in a potato dextrose broth. The culture filtrate of the strain PM-1 caused a partial swelling of the mycelia of P.oryzae, and it prevents the normal growth of the fungus as well. This result suggested that the antifungal substances secreted by the strain PM-1 potentially inhibited the germination of P.oryzae.

• Journal of Microbiology and Biotechnology
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• v.17 no.4
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• pp.695-700
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• 2007

The cycloinulooligosaccharide fructanotransferase (CFTase) gene (cft) from Paenibacillus macerans (GenBank access code AF222787) was expressed on the cell surface of Saccharomyces cerevisiae by fusing with Aga2p linked to the membrane-anchored protein Aga1p. The surface display of CFTase was confirmed by immunofluorescence microscopy and enzymatic assay. The optimized reaction conditions of surface-displayed CFTase were as follows; pH, 8.0; temperature, $50^{\circ}C$; enzyme amount, 30 milliunit; substrate concentration, 5%; inulin source, Jerusalem artichoke. As a result of the reaction with inulin, cycloinulohexaose was produced as a major product along with cycloinuloheptaose and cycloinulooctaose as minor products.

• Journal of Life Science
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• v.17 no.2 s.82
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• pp.241-247
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• 2007

The cycloinulooligosaccharide fructanotransferase (CFTase) gene (cft) from Paenibacillus macerans was subcloned into the surface display vector, pCTcon (GAL1 promoter). The constructed plasmid, pCTECFTN (9.0 kb) was introduced to S. cerevisiae EBY100 cell and then east transformants were selected on the synthetic defined medium lacking uracil and on the inulin containing medium. The surface display of CFTase was confirmed by immunofluorescence microscopy and its enzymatic ability to form cycloinulooligosaccharides(cyclofructans, CFs) from inulin. The total activity of the CFTase was reached about 5.52 unit/1 by cultivation of yeast transformant on YPDG medium. The optimized conditions determined were as follows; pH, 8.0; temperature, $50^{\circ}C$ ; substrate concentration, 5%; inulin source, Jerusalem artichoke. By the reaction with inulin, CFs consisting of cycloinulohexaose (CF6), cycloinuloheptaose (CF7), and cycloinulooctaose (CF8) were produced and CF6 was the major product.

• The Korean Journal of Pesticide Science
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• v.14 no.4
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• pp.421-426
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• 2010

Many bacterial strains inhabit strong saline condition, such as tidal mudflat and salted seafoods, were identified and reported for the proposed protease activities and salt resistance; however antifungal activities against plant fungal pathogen have not well been studied until now. In this study, primary screening was performed for the isolation of promising strains against major plant pathogens like Sclerotinia sclerotiorum, Fusarium oxysporum, Phytophthora capsici, Botrytis cineria, Collectotrichum acutatum and Pythium ultimum. Totally 423 bacterial strain were isolated from laboratory media which was based on different morphological characteristics and all the strains were dual cultured against major fungal pathogens on PDA, finally 40 strains were selected as antifungal bacterial strain and identified by fatty acid phylogenic difference analysis from MIDI shorlock gas chromatography system. As a result, antifungal strains from tidal mudflat were 10 species of 6 genus. Paenibacillus macerans was dominant species; 5 strains among the 17 isolates from tidal mudflat. Antifungal strains from salted seafoods were 7 species of 3 genus and Bacillus atrophaeus was dominant species; 12 strains among the 23 isolates from salted fishes.