Figure 1.
Colony color and growth of C. fumago wild type and cpo1 mutant on solid media.
C. fumago wild type strain and the cpo1 mutant were plated on fructose minimal medium plates with a piece of mycelium grown on a glucose potato agar plate and incubated at 24°C for 7 days.
Figure 2.
Comparison of CPO production characteristics of wild type and cpo1 strain.
A: Comparison of CPO activities in supernatant samples of C. fumago wild type and cpo1 strain during cultivation in fructose minimal medium. Strain cultivation and determination of enzyme activity via the MCD assay was performed as described in the materials and methods section. Data is expressed as mean of values from two shake flasks ± standard deviation. B: Analysis of CPO protein levels in supernatant samples of C. fumago wild type and cpo1 strain by SDS-PAGE. SDS-PAGE analysis was performed as described in the materials and methods section. M: molecular weight marker. The numbers on the left indicate the sizes of the molecular weight marker.
Figure 3.
UV-Vis spectra of C. fumago wild type and cpo1 CPO purified by aqueous biphasic systems.
Culture supernatants from fructose minimal medium cultures grown for 11 days on a rotary shaker at 24°C each were purified using aqueous biphasic systems and protein concentration was equalized by dilution of the samples with 0.1 M citric acid buffer solution (pH 4). CPO activity of the diluted samples was determined as 246.46 U mL−1 and 0.03 U mL−1 for wild type and cpo1, respectively.
Figure 4.
SDS-PAGE analysis of CPO protein sizes after deglycosylation.
Culture supernatants from fructose minimal medium cultures grown for 10 days on a rotary shaker at 24°C each were purified using aqueous biphasic systems and deglycosylation was performed as described in the materials and methods section. N+O: glycosidases were added to remove N- and O-linked oligosaccharides, O: glycosidases were added to remove O-linked oligosaccharides, N: glycosidases were added to remove N-linked oligosaccharides, ut: untreated sample, M: molecular weight marker. The numbers on the left indicate the sizes of the molecular weight marker.
Figure 5.
A: Analysis of CPO protein levels in supernatant samples of C. fumago wild type and cpo1 strain by SDS-PAGE. SDS-PAGE analysis was performed as described in the materials and methods section. The numbers on the left indicate the sizes of the molecular weight marker. M: molecular weight marker. CPO activity of fructose minimal medium cultures of wild type and cpo1 was determined as 60 U mL−1 and 38 U mL−1, respectively. B: UV spectra of C. fumago wild type and cpo1 CPO purified by aqueous biphasic systems. Culture supernatants from fructose minimal medium cultures grown for 11 days on a rotary shaker at 24°C each were purified using aqueous biphasic systems and protein concentration was equalized by dilution of the samples with 0.1 M citric acid buffer solution (pH 4).