Optimized conditions are needed to refold recombinant proteins from bacterial inclusion bodies into their biologically active conformations. In this study, we found two crucial requirements for efficient refolding of cationic tetrameric chicken avidin. The first step is to eliminate nucleic acid contaminants from the bacterial inclusion body. The electrostatic interactions between the remaining nucleic acids and proteins strongly enhanced protein aggregation during the refolding process. The cysteine specific reversible S-cationization procedure was successfully employed for largescale preparation of nucleic acid free denatured protein without purification tag system. The second step is the intramolecular disulfide formation prior to refolding in dialysis removing denaturant. Disulfide intact monomeric avidin showed efficient formation of biologically active tetrameric conformation during the refolding process. Using this optimized refolding procedure, highly cationic avidin derivative designed as an intracellular delivery carrier of biotinylated protein was successfully prepared.

Mutants of Gibberella fujihuroi were obtained by selection from a large number of conidia of a wild type field isolate of the fungus on agar plates containing lethal concentration of a sterol demethylation inhibitor, pefurazoate.The mutants isolated from the colonies grown on the plates had higher minimal growth-inhibitory concentrations of pefurazoate and the EC95 values were higher than that for the mother isolate but only a small increase was observed in the EC5a values. Though the slope of the dosage-response curve was changed in the mutants, the mechanism of action of the fungicide in a mutant proved to be its inhibition of sterol 14cc-demethylation as shown by an experiment on inhibition of ergosterol biosynthesis using 14C-acetate. Gibberellin production in the mutants in liquid culture was tested by bioassay of gibberellins and assay of gibberellin A3 using HPLC. Mutants were also tested for their virulence to rice plants.Decreased virulence due to decreased production of gibberellins was observed in the mutants.A mutational change in a site related to 14c -demethylation in ergosterol biosynthesis and to kaurene oxidation in gibberellin biosynthesis in common is discussed.

A new azole fungicide, pefurazoate (4-pentenyl (RS)-2-[(2-furylmethyl) (imidazol-lylcarbonyl)amino]butanoate, Healthied) has one Chiral carbon center in its structure. From optically active 2-aminobutanoic acid, we prepared two enantiomers of pefurazoate, (R)isomer and (S)-isomer, whose optical rotations in methanol were [aTh + 10. 7 and [a]D -9. 90, respectively, and studied their antifungal activity and ergosterol biosynthesis in Gibberella fujikuyoi.The antifungal activity of the (S)-isomer was about 30 times higher than that of the (R)-isomer. At 0.24ppm, the in vitro inhibition of ergosterol biosynthesis by the (S) isomer was four 'times higher than by the (R)-isomer. These results indicate that the (S)isomer of pefurazoate is an active form fungicidal to G. fujikuyoi.

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