*3.6. E*ff*ect of Hydrogen Peroxide Concentration on the Metabolic Activity of the Parental and the Mutant Strain of C. pannorum A-1*

Identical amounts (2.65 g of dry weight per L) of two-day-old mycelia of the parental strain and mutant 1-6 were washed twice with sterile 0.1 M phosphate buffer and transferred to 100 mL Erlenmeyer flasks containing 40 mL dissolved BM with an addition of hydrogen peroxide at concentrations of 0.1, 0.5, 1.0, 1.5, 2.5, and 5% (*v*/*v*). After 60-min incubation at 20 ◦C with magnetic stirring (150 rpm), the residual content of H2O2 was estimated in 1 mL aliquots of the medium using potassium iodide and starch solution as an indicator. The pre-incubated biomasses were filtrated, washed twice with sterile 0.9% NaCl solution, and then fixed amounts of the mycelia were suspended uniformly in 0.05 M McIlvaine buffer (pH 6) with an addition of 1% glucose in order to measure the GMA of living mycelia, according to procedure described in the 'General metabolic activity assay'.

#### *3.7. Biotransformation Analysis*

After the specified biotransformation time, the biomass was harvested by filtration, and the liquid for product recovery was extracted twice with an equal volume of diethyl ether in a separation funnel for 10 min. Before extraction, 250 μL of a 0.1% internal standards (IS) solution in hexane was added to the filtrate. The ether fraction was collected and concentrated on rotary vacuum evaporators (BUCHI, Rotavapor R-200/205, Flawil, Schwitzerland) at a water bath temperature of 40 ◦C under mild pressure of 800 mbar. The residues obtained were dissolved in 6 mL of hexane. Gas chromatography with flame-ionization detector (GC-FID) (VARIAN, Palo Alto, California, USA) and mass specta coupled to gas chromatography (GC-MS) (Thermo Finnigan, Trace DSQ GC/MS Ultra Chromatograph, Austin, TX, USA) analyses of terpenes were conducted using the method reported previously [43]. Peaks were identified by fitting the mass spectra to those from standard library database systems (HP Wiley, NIST) and the MassFinder library and by comparing the GC retention indices to those of standard compounds. Biotransformations were performed in three replicate samples, and the analyses were carried out in duplicate. The error associated with the GC quantification of the samples was ±6% and was quoted for a confidence interval of 94%. The data are reported as mean values.
