Characterization of Aspergillus niger Mutants Deficient of a Protease

Chung, Hea Jong; Park, Seung Moon; Kim, Dae Hyuk

Abstract

Aspergillus niger has been used as a host to express many heterologous proteins. It has been known that the presence of an abundant protease is a limiting factor to express a heterologous protein. The protease deficient mutant of A. niger was obtained using UV-irradiation. A total of 1x105 spores were irradiated with 10~20% survival dose of UV, 600 J/m2 at 280 nm, and the resulting spores were screened on the casein-gelatin plates. Ten putative protease deficient mutants showing the reduced halo area around colonies were further analyzed to differentiate the protease deficient mutant from other mutant types. Among ten putative mutants, seven mutants showed significant growth defect on nutrient rich medium and two mutants appeared to be the secretory mutants, which resulted in the impaired secretion of extracellular proteins including proteases. A mutant pro--20 showed reduced halo zone without any notable changes in growth rate. In addition, the starch-degrading and glucose oxidase activities in the culture filtrate of pro--20 mutant showed the similar range as that of the parental strain, which suggested that the pro--20 mutant ought to be the protease deficient mutant rather than a secretory mutant. The reduced proteolytic activity of the pro--20 was demonstrated using SDS-fibrin zymography gel. The reduced extracellular proteolysis was quantified by casein degradation assay and, comparing with the parental strain, less than 30% residual extracellular protease activity was detected in the culture filtrate of the pro--20 mutant. The bio-activity of an exogenously supplemented hGM-CSF (human Granulocyte-Macrophage Colony Stimulating Factor) in the culture filtrate of pro--20 mutant was detected until eight times more diluted preparations than that of the parental strain.