Studies on the Production of Fungal Peroxidases in Aspergillus niger

Abstract

To get insight into the limiting factors existing for the efficient production of fungal peroxidase in filamentous fungi, the expression of the Phanerochaete chrysosporium lignin peroxidase H8 (lipA) and manganese peroxidase (MnP) H4 (mnp1) genes in Aspergillus niger has been studied. For this purpose, a protease-deficient A. niger strain and different expression cassettes have been used. Northern blotting experiments indicated high steady-state mRNA levels for the recombinant genes. Manganese peroxidase was secreted into the culture medium as an active protein. The recombinant protein showed specific activity and a spectrum profile similar to those of the native enzyme, was correctly processed at its N terminus, and had a slightly lower mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Recombinant MnP production could be increased up to 100 mg/liter upon hemoglobin supplementation of the culture medium. Lignin peroxidase was also secreted into the extracellular medium, although the protein was not active, presumably due to incorrect processing of the secreted enzyme. Expression of the lipA and mnp1 genes fused to the A. niger glucoamylase gene did not result in improved production yields.

Phenanthrene Removal from Soil by a Strain of Aspergillus niger Producing Manganese Peroxidase of Phanerochaete chrysosporium

“…The wild-type strain showed no coloration; however, the control strain of P. chrysosporium showed a greater purple halo. These results are similar to findings by other authors, who screened autochthonous or recombinant fungal strains for the formation of halos by o-anisidine oxidation on agar plates induced by manganese peroxidase activity [40,42,52]. The MnP +7 transformant strain showed higher Phe tolerance than wild-type strain when inoculated into Cove's medium in Petri dishes at different Phe concentrations.…”

“…Recombinant MnP production by MnP +7 strain was increased by adding hemoglobin to the culture medium. Similar results have been obtained by other research groups in their studies on mnp1 expression in Aspergillus [40,42,51] and are explained by how the recombinant protein is produced by Aspergillus, i.e., as an unstable apoprotein. This apoprotein requires the heme group to produce the active hemoprotein, which acquires a more stable conformation for proteolytic attack than the apoprotein.…”

“…These enzymes were obtained extracellularly and with catalytic activity but have only been studied as expression systems for the production of heterologous proteins and have not been applied to the bioremediation of contaminated soils, as presented in this research [40,42,50,51].…”

“…This apoprotein requires the heme group to produce the active hemoprotein, which acquires a more stable conformation for proteolytic attack than the apoprotein. Due to low availability of heme provided by the heme biosynthetic pathway, this fact is considered a limiting factor for the production of heme proteins in different expression systems [41][42][43]52]. If the amount of heme produced by a microorganism is low in relation to the amount of apoprotein produced, the apoprotein will accumulate in the culture medium and undergo proteolytic degradation lowering the yield of the active hemoprotein.…”

“…Gene expression of lipA in A. niger F38 has also been studied, however LiP H8 activity was still detected at low levels [41]. Other researchers have used the protease-deficient A. niger strain for the expression of mnp1 and lipA [42]. Several factors have been identified which hamper the overproduction of recombinant proteins in filamentous fungi.…”