title: 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.
authors.affiliation: Department of Biology, University of Waterloo, Waterloo, ON N2L 3G1, Canada.
authors.forename: Aftab
authors.initials: A
authors.lastname: Ahamed
authors.forename: Ajay
authors.initials: A
authors.lastname: Singh
authors.forename: Owen P
authors.initials: OP
authors.lastname: Ward
mesh_terms: D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D001234:Aspergillus niger / Q000201:enzymology* / Q000254:growth & development; D001709:Biotechnology / Q000379:methods; D003470:Culture Media; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism*; D010436:Pepstatins / Q000378:metabolism*; D011480:Protease Inhibitors / Q000378:metabolism*
chemical_list: D003470:Culture Media; D005656:Fungal Proteins; D009842:Oligopeptides; D010436:Pepstatins; D011480:Protease Inhibitors; C002101:chymostatin; D016282:Aspartic Acid Endopeptidases; C031375:pepstatin