The effects of bioprocess parameters on extracellular proteases in a recombinant Aspergillus niger B1-D

Abstract

Although host proteases are often considered to have a negative impact upon heterologous protein production by filamentous fungi, relatively little is known about the pattern of their appearance in recombinant fungal bioprocesses. In the present study, we investigated extracellular proteases from a filamentous fungus, Aspergillus niger B1-D, genetically modified to secrete hen egg white lysozyme (HEWL). Our findings indicate that extracellular protease activity is only detected after the carbon source is completely utilised in batch cultures. The proteases are predominantly acid proteases and have optimal temperature for activity at around 45 degrees C. Their activity could be partially inhibited by protease inhibitors, indicating the existence of at least four kinds of proteases in these culture fluids, aspartic-, serine-, cysteine-, and metallo-proteases. Oxygen enrichment does not have any noticeable effects on extracellular protease activity except that the onset of protease activity appears earlier in oxygen enrichment runs. Oxygen enrichment stimulates HEWL production substantially, and we propose that it is related to fungal morphology. Thermal stress imposed by raising process temperature (from 25 to 30 and 35 degrees C) in early exponential phase, led to appearance of protease activity in the medium following the heat shock. Continued cultivation at high temperatures significantly reduced HEWL production, which was associated with increased activity of the extracellular proteases in these cultures.

The effects of elevated process temperature on the protein carbonyls in the filamentous fungus, Aspergillus niger B1-D

“…Though A. niger could adapt to raised temperature quickly, we show stress occurs at cellular level as demonstrated by accumulation of proteins showing oxidative damage. This is of particular interest in fungal fermentation, when temperature gradients inevitably form in bioreactors due to the heterogeneity of the broth, especially at large scale [33]. Our results indicate variance of temperature results in protein damage which is detrimental to fungal cells.…”

Extracellular proteinase formation in carbon starving Aspergillus nidulans cultures – physiological function and regulation

“…the ambient pH [10,12]. Meanwhile considerable acidification of culture media is foreseeable in fermentation processes conducted by Aspergillus niger, which elicits the biosynthesis of acidic proteinases [6,11], neutralization (Aspergillus oryzae [7]), or even alkalinization (Penicillium chrysogenum [13]; Aspergillus nidulans, [14]) of the media have been reported for other fungal species with the predominant production of neutral and alkaline proteinases [7, 11, 15 -18].…”

The effect of environmental conditions on extracellular protease activity in controlled fermentations of Aspergillus niger

“…Complementary to strain improvement, manipulation of environmental conditions can help to reduce protease secretion and thus improve heterologous protein production, but this has not been investigated systematically (Braaksma & Punt, 2008). Examples of bioprocess parameters which have been investigated for their influence on extracellular protease activity include fungal morphology manipulation (Liu et al, 1998;Xu et al, 2000;, pH control (O'Donnell et al, 2001), oxygen enrichment and cultivation temperature (Li et al, 2008). Studies on the effect of medium components have mainly focused on the derepression of protease genes when transferring mycelia to medium lacking either carbon or nitrogen sources (Cohen, 1981;.…”