This work is part of an ongoing investigation that studies the application of a solution rich in Zn and Mn, obtained from a recycling process, as a preservative for wood. The effects of Zn2+ and Mn2+ on the enzyme-expression level of manganese-dependent peroxidase (MnP) from Phanerochaete chrysosporium was studied by real-time PCR. The glyceraldehyde 3-phosphate dehydrogenase gene (gpd) was used as reference for the relative quantification of the expression of the three isoenzymes. The expression kinetics was studied for different media and culture conditions. The conditions and culture time leading to maximum gene expression and enzyme activity were determined for each isoenzyme. The effects of medium composition (replete, ligninolytic: carbon-limited, nitrogen-limited), temperature (28ºC and 37ºC), and shaking (static or agitated at 125 rpm) were studied. Incubation in a static carbon-limited medium at 37ºC showed the highest expression of the mnp genes. In these conditions, the concentration of Zn2+ and/or Mn2+ that stimulate or inhibit the expression of mnps was determined. The selection of suitable culture conditions and the sensitivity of real-time PCR enabled a differentiation in the expression pattern of the three studied MnP isoenzymes in the Zn2+ and/or Mn2+-containing media. Zn2+ concentrations lower than 0.5 mM led to variable expression levels for the different isoenzymes, but were found to have a clear inducing effect when Zn2+ was used in combination with 100 µM Mn2+. Results of the above analysis were compared with enzymatic activity data obtained spectrophotometrically.

Download document (390 kb)
free for the members of IRG. Available if purchased.

Purchase this document