Improvement of fungal strain by repeated and sequential mutagenesis and optimization of solid state fermentation for the hyper-production of raw-starch-digesting enzyme.

A selected fungal strain, for production of the raw-starchdigesting enzyme by solid-state fermentation, was improved by two repeated sequential exposures to gamma-irradiation of Co60, ultraviolet, and four repeated treatments with Nmethyl- N'-nitrosoguanidine. The mutant strain Aspergillus sp. XN15 was chosen after a rigorous screening process, with its production of the raw-starch-digesting enzyme being twice that of usual wild varieties cultured under preoptimized conditions and in an unsupplemented medium. After 17 successive subculturings, the enzyme production of the mutant was stable. Optimal conditions for the production of the enzyme by solid-state fermentation, using wheat bran as the substrate, were accomplished for the mutant Aspergillus sp. XN15. With the optimal fermentation conditions, and a solid medium supplemented with nitrogen sources of 1% urea and 1% NH4NO3, 2.5 mM CoSO4, 0.05% (v/w) Tween 80, and 1% glucose, the mutant Aspergillus sp. XN15 produced the raw-starch-digesting enzyme in quantities 19.4 times greater than a typical wild variety. Finally, XN15, through simultaneous saccharification and fermentation of a raw rice corn starch slurry, produced a high level of ethanol with Yp/s of 0.47 g/g.