1989 Fiscal Year Final Research Report Summary
Improvement of wood-rotting activity of basidiomycetes using cell fusion techniques
| Project/Area Number |
62560110
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
発酵・醸造
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| Research Institution | Kagawa University |
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Principal Investigator |
KUWAHARA Masaaki Kagawa University, Faculty of Agriculture Professor, 農学部, 教授 (40035978)
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| Project Period (FY) |
1987 – 1989
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| Keywords | Lignin / Basidiomycete / White rot fungus / Protoplast / Cell fusion |
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| Research Abstract |
The lignin-degrading basidiomycetes have potential application in a variety of schemes for the commercial processing of lignocellulose. Then, this research was intended to obtain the basic results for applying the cell fusion method to lignin-degrading basidiomycetes, especially Phanerochaete chrysosporium, for producing strains with superior capacities of lignin degradation.
Protoplasts of P. chrysosporium were obtained by treating mycelia in 0.6 M MgSO_4 or 0.8 M mannitol with the mixture of Novozyme 234 (1%) and Cellulose ONOZUKA RS (1%) at 37ﾟC for 3 h with gentle shaking. The protoplasts showed the spherical feature with a diameter of 3-10 mum. The frequency of protoplast regeneration was calculated as about 5.0%. Both of the usual polyethylene glycol (PEG) treatment method and electrofusion method were available for the protoplast fusion of P.chrysosporium. Treatment of the protoplasts from two of the auxotrophic mutants of P. chrysosporium with PEG or electrofuser led to fusion o
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f protoplasts as measured by complementation on a minimal medium plate. The fusants obtained maintained the complementarity (the ability to grow on a minimal medium plate) with relatively stability through subcultures of some passage numbers. The fusants between auxotrophic mutants and peroxidase less mutants were also obtained.
The protoplasts could be labeled with the fluorescent compounds, fluorescein isothiocyanate (FITC) and rhodamine B isothiocyanate (RITC). The labeled protoplasts with FITC and RITC were distinguishable to each other under the fluorescence microscope. The separately labeled protoplasts with FITC or RITC of P. chrysosporium and of Coriolus versicolor or Pleurotus ostreatus were observed to fuse by PEG treatment. The fusants between P. chrysosporium and Coprinus aff ellisii were also produced by electrofusion. The fusants produced phenol oxidases (or peroxidases), which are known to play important roles in lignin degradation, under the both culture conditions in which each parental strain did, indicating that these fusants might show the superior capacities of lignin dergadation. Less
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