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.

FIG. 1

lipA and mnp1 expression cassettes. For an explanation, see Materials and Methods and Table 1.

FIG. 2

Northern blotting analysis of total RNA isolated from one representative transformant per expression cassette after 48-h culturing on AMM-maltose. (a) pglaA probe. (b) gpd probe. (c) Ratio between each transformant corrected glaA signal and the N024(pAB6-10)B1 corrected glaA signal. The expected mRNA sizes are indicated.

FIG. 3

Western blotting analysis of MnP transformants. Culture samples at 48 h of one representative transformant per construct are shown. (a and b) medium samples; (c and d) mycelium extracts. Blots were probed with either a polyclonal anti-MnP antibody (a and c) or a monoclonal anti-GLA antibody (b and d).

FIG. 4

Western blotting analysis of LiP transformants. Culture samples at 48 h of one representative transformant per construct are shown. (a) Medium samples; (b) mycelium extracts. Blots were probed with the anti-LiP-Ma polyclonal antibody.

FIG. 5

Immunodetection of rLiP. A purification fraction containing the anti-LiP cross-reactive 42-kDa protein band was detected either with anti-LiP-Ma polyclonal antibody (a) or with anti-LiP-Fr polyclonal antibody (b). (c) P. chrysosporium LiPH8 control probed with anti-LiP-Fr polyclonal antibody.

FIG. 6

Western blotting analysis of the extracellular protein production of strain MGG029(pMnp1.I)#25 cultured in differently supplemented AMM-maltodextrin media. −, no supplementation; Fe, FeSO₄; APO, apohemoglobin; HEM, hemin; HMG, hemoglobin. (a) Production of MnP: blot was probed with an anti-MnP polyclonal antibody. (b) Production of amylase (AMY): blot was probed with a cross-reacting anti-GLA polyclonal antibody. (c) Band intensities of the MnP signals (R_MnP) and amylase signals (R_AMY), relative to the unsupplemented medium.

FIG. 7

Spectrum profile of rMnP. Numbers at left are absorbances expressed in arbitrary units.