A method for the laboratory-scale production and isolation of chitosan (polyglucosamine) by liquid and solidstate fermentation from Lentinus edodes was developed. The yields of isolated chitosan were 120 mg/L of fermentation medium under liquid fermentation conditions and 6.18 g/kg of fermentation medium under solid-state fermentation conditions. The latter method, which gives up to 50 times yields than other chitosan production methods from fungi, provides a new flexible and easily scaledup procedure for the production of low acetylation degree chitosan.
In previous studies, the white-rot basidiomycete Lentinus edodes, strain SC-495. was proved to be a "selective" lignin degrader and its extracellular crude preparations arising from solid-state cultures were successfully employed in biopulping experiments on annual plants. This fungus produced extracellular laccase as the predominant phenoloxidase when growing in solid-state fermentation on corn stalks. Laccase from this strain was purified and partially characterized. as an initial approach towards the study of its ligninolytic complex. Laccase was purified 69.6-fold by anion-exchange chromatography and two affinity-chromatography steps with an overall yield of 7.454. The native enzyme exhibited a molecular mass of 74 m a , an isoelectric point of 3.42 and a carbohydrate content of 7.5%. The absorption spectrum of laccase showed a maximum at 605 nm. typical of blue-copper oxidases. The optimum pH and temperature for the activity of laccase were 4.0-4.2 and 50 "C, respectively. Kinetic experiments, performed with a wide range of phenolic compounds, showed that the reaction rate and the substrate affinity greatly varied depending on the nature of substituents and their reciprocal positions on the aromatic ring. In particular, the enzyme showed high affinity to phenolic compounds bearing methoxyl or methyl groups, but no affinity to those bearing the nitro group directly attached to the benzene ring, nor to non-phenolic lignin-related compounds, such as trans-cinnmic acid or 3,4-dimethoxycinnamic acid. The huge differences in terms of reactivity of the enzyme towards phenolic compounds suggest that a preliminary systematic screening should be advisable when using laccase in effluent treatment applications.
Total and specific activities of extra-cellular laccases from Lentinus e&&s were enhanced by adding corn straw and chestnut juice to the liquid growth medium. The aqueous extracts were chemically characterized and revealed the presence of several phenolic and non-phenolic compounds. Extensive extraction of these components from the tested extracts completely annulled their stimulating properties on lactase production, suggesting that these compounds can act at micromole levels.
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