Abstract
Endoinulinases gene was expressed in recombinant Aspergillus niger for selective and high-level expression using an exponential fed-batch fermentation. The effects of the growth rate (µ), glucose feed concentration, nitrogen concentration and fungal morphology, on enzyme production were evaluated. A recombinant endoinulinases with a molecular weight of 66 KDa was secreted. Endoinulinases production was growth associated at µ> 0.04 h -1 , which is characteristic of the constitutive gpd promoter used for the enzyme production. The highest volumetric activity (670 U/ml) was achieved at a growth rate of 93% of µ max (0.07 h -1 ), while enzyme activity (506 U/ml) and biomass substrate yield (0.043 g biomassDW /g glucose ) significantly decreased at low µ (0.04 h -1 ). Increasing the feed concentration resulted in high biomass concentrations and viscosity, which necessitated high agitation for improved mixing and oxygen. However, the high agitation and low DO levels (ca. 8% of saturation) led to pellet disruption and growth in mycelial morphology. Enzyme production profiles, product (Y p/s ) and biomass (Y x/s ) yield coefficients were not affected by feed concentration and morphological change. The gradual increase in the concentration of nitrogen sources showed that, a nitrogen limited culture was not suitable for endoinulinases production in recombinant A. niger. Moreover, the increase in enzyme volumetric activity was still directly related to an increase in biomass concentration. An increase in nitrogen concentration, from 3.8 to 12 g/L, resulted in volumetric activity increase from 393 to 670 U/ml, but the Y p/s (10053 U/g glucose ) and Y x/s (0.049 g biomasDWs /g glucose ) did not significantly change. The data demonstrated the potential of recombinant A. niger and high cell density fermentation for the development of largescale endoinulinases production system.
Genome sequencing of the neotype strain CBS 554.65 reveals the MAT1–2 locus of Aspergillus niger
