Chymostatin can combine with pepstatin to eliminate extracellular protease activity in cultures of Aspergillus niger NRRL-3

Abstract

Aspergillus strains are being considered as potential hosts for recombinant heterologous protein production because of their excellent extracellular enzyme production characteristics. However, Aspergillus proteases are problematic in that they modify and degrade the heterologous proteins in the extracellular medium. In previous studies we observed that media adjustments and maintenance of a filamentous morphology greatly reduced protease activity and that a low concentration of the aspartic protease inhibitor pepstatin inhibited the latter protease activity to the extent of approximately 90%. In this paper we report that when the serine protease inhibitor chymostatin is used in combination with pepstatin 99-100% of total protease activity in Aspergillus cultures is inhibited. In protease assays a concentration of 30 microM chymostatin combined with 0.075 microM pepstatin was required for maximum inhibition. Inhibitor concentrations of chymostatin and pepstatin of 120 and 0.3 microM, respectively, when added to Aspergillus cultures, has no significant effect on biomass production, glucose utilization or culture pH pattern. The potential of using these protease inhibitors in cultures of recombinant Aspergillus strains producing heterologous proteins will now be investigated to determine if the previously observed recombinant protein denaturing effects of Aspergillus proteases can be negated.

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

“…In the aspergilli, selection of Aspergillus spp. with low levels of extracellular proteinases [6], construction of multiple proteinase gene deletion mutants [7 -8], inhibition of proteinase activities in the culture media [9] as well as optimization of bioprocess parameters [10 -11] represent suitable tools to control the deleterious proteinase effects under various fermentation conditions. Industrial fungi produce high proteinase activities in general, which is influenced by several fermentation parameters including the availability of nutrients, pH, temperature, oxygen supply, agitation intensity, inoculum size, fungal morphology including pellet formation and immobilization of mycelia [10 -11].…”