These results show that a simplified purification process is capable of producing comparable specific activity results for the screening of novel MnP enzymes. Culture supernatant concentration and anion exchange chromatography using a Mono Q column was enough to purify the enzyme to a high degree. Although the initial enzyme amounts were low and the losses during concentration were quite high, these results provide a basis for enzyme ranking. Based on these results there are significant differences in the specific activities of MnP enzymes from different white-rot fungi. Out of the white-rot fungi used in this study P. chrysosporium, P. rivulosus and Phlebia sp. Nf b19 MnPs were the most promising. For P. rivulosus and Phlebia sp. Nf b19 these results are also consistent with the MnP activity and protein absorbance (280 nm) ratio in anion exchange chromatography (Table 1). The MnP activity (U l-1) of Bjerkandera sp. had come down over 70% at the point of harvesting. If the lost activity is accounted as inactivated MnP in the total protein measurement, the specific activity could be at least three-fold higher. In addition to specific activity the stability of the purified MnP enzymes towards temperature, pH and inactivating compounds needs to be studied before candidates for recombinant production are selected. The reported specific activities are significantly lower than those previously reported for P. chrysosporium byUrek et al. (2004), Bjerkandera sp. byPalma et al. (2000) and Taboada-Puig et al. (2011) and for Phlebia sp. Nf b19 by Schneegaβ et al. (1997)
The production of MnP by native producers, as also observed in this study, is limited due to relatively low maximal enzyme activities, slow growth and the sensitivity of white-rot fungi towards shear forces, difficult induction strategies and low adaptivity to submerged fermentations. In this study MnP was in most cases induced with several supplements (Mn2+, VA, Tween, Sodium malonate). However, as reported byHakala et al. (2006), the regulation of different MnP isoforms can be largely dependent on the inducing compounds and nutrient (nitrogen and carbon) sources and amounts. This suggests that novel isoforms would be found by changing the culture conditions. Mn2+−ions are typically necessary inducers for MnP production although repressive effects of Mn2+−ions have also been reported (Martinez et al.
1996). Although high activity level was not the focus in this study, the culture conditions and inducing strategies (see the materials section) for this study were selected on previous literature to maximize enzyme production. Specified effects of individual inducing components cannot be separated for any strain, but clearly no MnP was produced before inducing components were added.
In recombinant MnP processes with Pichia pastoris (Jiang et al.
2008b), Aspergillus oryzae (Stewart et al.
1996), Aspergillus niger (Conesa et al.
2000) and Echerichia coli (Whitwam et al.
1995; Whitwam &Tien 1996) production of high amounts of active protein has been a problem. These processes suffer from incorrect protein folding, insufficient heme synthesis and heme incorporation. However, MnP yield might be significantly increased with the right combination of recombinant enzyme, production host, promoter system, protein secretion system and optimized process conditions.Jiang et al. (2008b) also suspected that P. pastoris lacks the proper heme escorts and reseptors to transport exogenous heme to the site of MnP synthesis. This problem might be relieved by inserting the genes for such heme transport from e.g. Shigella (S.) dysenteriae (Mills &Payne 1995). S. dysenteriae is a known pathogen causing shigellosis, which would make the GMO rating of the recombinant strain difficult. On the other hand P. pastoris synthesizes large amounts of a homologous heme-containing catalase protein under alchohol oxidase (AOX) promoted recombinant expression. Thus gene expression under AOX promoter might be beneficial for MnP production due to the added need for heme synthesis inside the cells. Coprinus cinereus produces a class II excreted fungal peroxidase (CiP) that readily oxidizes phenols, but is unable to oxidize veratryl alcohol or Mn2+−ions (Hofrichter et al.
2010). This enzyme is successfully produced in a recombinant process using P. pastoris (Kim et al.
2009a) with high productivities (peroxidase activity over 1200 U ml-1 and total protein over 1.6 g l-1) and is also sold as a commercial enzyme by Novozymes (Baylase®). The productivity of this heme-containing peroxidase was optimized by host and expression promoter selection (Kim et al.
2009b). Highest productivities were obtained by using the AOX promoter with a fast methanol utilization strain (Mut+) of P. pastoris. This supports the theory, that inducing the methanol utilization pathway in a production host can promote the recombinant production of heme-containing peroxidases. Commercially available MnP (from Phlebia sp. Nf b19) and VP (from Bjerkandera adusta) from Jena Bioscience GmbH are native enzymes and overly expensive for any kind of industrial use.
In this study several well known MnP enzymes were compared. MnP and laccase and their regulation in P. rivulosus has been well characterized byHakala et al. (2005& 2006). The Differential regulation of MnP isoforms in P. rivulosus was also noted in these articles. In this study only one MnP from P. rivulosus is characterized, but this may very well be a group of MnP isoenzymes with approximately the same isoelectric points and molecular sizes. In characterization studies by Hildén et al. (2008), the MnP2 enzyme from Phlebia sp. Nf b19 showed a 96% amino acid identity to the MnP2 enzyme of P. radiata in the primary structure. In this study the specific activities and sizes of the MnP enzymes produced by these related fungal strains were in the same range. Even in growth and MnP production these strains showed similarities (extremely slow growth and late onset of MnP production). Hildén et al. (2005) describe two MnP enzymes from P. radiata that are different in their primary structure, intron amount, length and crystal structure. The other isoenzyme being structurally related to LiP, but having an alanine residue instead of tryptophan while still having a conserved Mn2+−binding site. In this study three MnPs from P. radiata were separated, but they seemed to be highly similar in size and specific activity for Mn2+ oxidation.
Many technical applications for MnP have been reported with promising results. The utilization of the enzyme is still dependent on a cost-effective recombinant production process and possibly the discovery of more robust novel isoenzymes or modifications of the currently known ones. Furthermore, optimization of enzymatic treatment processes for various technical lignins (Vishtal &Kraslawski 2011), paper pulps (Maijala et al.
2008;Xu et al.
2010) and organopollutants (Sack et al.
1997) with proper process conditions and co-oxidants will probably increase the interest in MnP. Delignification of pinewood sawdust using a MnP treatment in this study was relatively inefficient. The use of several co-oxidants and other enzymes involved in biological ligninolysis may help to achieve more thorough enzymatic delignification demonstrated by many MnP producing and LiP-negative white-rot fungi (Hammel &Cullen 2008). In previous laboratory experiments byHofrichter et al. (1998) andKapich et al. (1999) Mineralization and solubilization of synthetic (14C-labeled) large molecular weight lignin by MnP has been reported. These and various other studies suggest that isolated MnP enzymes can be used to delignify biomasses. However, for now the utilization of class II peroxidases to degrade polluting substances in soils is technically more appealing.