Culture-Based Strategies for Reduction of Protease Activity in Filtrates from Aspergillus niger NRRL-3

Abstract

While Aspergillus strains are also being considered as potential hosts for production of extracellular heterologous proteins, the proteases produced by the host are highly problematic in that they typically modify and degrade the recombinant proteins. Culture-based approaches for minimization of protease activity in culture supernatants of Aspergillus niger NRRL-3 included reduction or elimination of peptide nitrogen in the medium, preferential use of a defined salts medium rather than a non-peptide nitrogen medium containing yeast-nitrogen base, supplementation of the medium with carboxymethylcellulose and cultivation at pH 6.5 rather than 7.5. In general, increased proteolytic activity was observed after maximum biomass was observed and biomass was declining suggesting the majority of protease activity was released by cell lysis. Carboxymethylcellulose shifted mycelial morphology from pelleted to filamentous. Mycelium lysis in the centre of pellets, with resultant release of intracellular proteases, would explain why filamentous cultures exhibited much lower proteolytic activity than pelleted cultures.

Incorporation of pepstatin into culture media for reduction of protease activity in filtrates from Aspergillus niger NRRL-3

“…In a previous study in our laboratory we demonstrated that protease activity in culture filtrates could be substantially reduced by inclusion of CMC in the medium and yeast nitrogen base in place of peptide nitrogen [22].…”

“…Pepstatin has been tested as a therapeutic agent for control of gastric ulcer and hypertension and analogues have also been developed. In a previous paper we demonstrated how culture-based approaches could be exploited to reduce protease activity in culture supernatants of A. niger NRRL-3 including preferential use of a inorganic or non-peptide nitrogen rather than peptidecontaining nitrogen and maintenance of filamentous rather than pelleted morphology [22]. Dai et al [23] recently observed that in A. niger a gene encoding pepsin-like protease activity is differentially expressed during pelleted but not during filamentous growth.…”

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

“…By incorporation of pepstatin into the Aspergillus culture media designed to support Wlamentous growth protease activity was reduced by 80-90%, indicating a residual of 10-20% of activity was pepstatin resistant [1,2]. In this paper we have investigated the potential to completely eliminate extracellular protease activity by adding additional protease inhibitors to the pepstatin-supplemented culture medium.…”

Expressão heteróloga, análise bioquímica e avaliação da produção fermentativa de uma enzima exo-arabinanase da família GH93

“…Em um processo de produção heteróloga de proteínas onde há o conhecimento do tipo de proteases produzidas pelo fungo, manter o valor de pH em uma faixa que não seja adequada para atividade de proteases evita o efeito adverso destas sobre a proteína de interesse. Por outro lado, diminuir ao máximo o efeito do cisalhamento evitando a ruptura celular diminui a liberação de proteases intracelulares produzidas pelo organismo (AHAMED; SINGH; WARD, 2005;WANG et al, 2005b). Meios de cultura enriquecidos com peptídeos como fonte de nitrogênio também estimulam a produção de proteases, por outro lado, o esgotamento de nutrientes, como as fontes de carbono e de nitrogênio, podem induzir a superprodução de proteases para realizar a autólise dos micélios mais antigos para obtenção de energia.…”

“…Embora, a utilização de fungos recombinantes para produção industrial de enzimas se apresente vantajoso, devido a sua maior estabilidade genética (uma caraterística essencial nos bioprocessos) e não ser susceptíveis a bacteriófagos quando comparados com outros organismos geneticamente modificados, como bactérias, a produção simultânea de pigmentos e proteases nativas, ainda é um desafio que deve ser superado para aumentar a sua produtividade (AHAMED; SINGH; WARD, 2005;PARDO-PLANAS et al, 2017;WANG et al, 2005a;WARD, 2012).…”