Doreen Heerd

• The present study revealed the potential of Aspergillus sojae ATCC 20235 for pectinase production in solid-state fermentation (SSF). A microbial screening of various Aspergillus species for pectinase production in SSF identified A. sojae ATCC 20235 as potential production organism. Media design and optimization of solid-state process parameters traced the increase of polygalacturonase (PG) titers. Optimization was performed in several steps applying various statistical designs, which yielded with 909.5 ± 2.7 U/g PG activity in 10.9 times increased enzyme production after 8 days at 30 °C applying sugar beet pulp (30 %) in combination with wheat bran as medium, wetted at 160 % by 0.2 M HCl. Development and implementation of a classical mutation and selection strategy for the improved production of pectinases resulted in the generation of mutant M3 after three cycles of repeated treatment by UV irradiation. Mutant M3 yielded 1.7 times increased PG activity in SSF. Scale up of the optimized SSF process for PG enzyme production was successfully demonstrated without loss in the total amount of enzyme at a rotating drum type solid-state bioreactor with a scaling factor of 100. Additionally, focus was also set on the enzyme recovery and purification. Optimizing the enzyme leaching process resulted in efficient PG recovery. PG purification by combination of ion exchange chromatography, size exclusion chromatography and hydrophobic interaction chromatography yielded in the isolation of the enzyme by means of a single band on the SDS-polyacrylamide gel with a molecular weight of about 40 kDa. Commercial pectinase preparations are usually mixtures of various enzyme species. Mass spectrometric characterization of A. sojae proteins identified a broad spectrum of carbohydrate-active enzymes. The characterized enzyme extracts were tested in several application studies related to fruit juice production and winemaking, which indicated improved process performances by enzyme treatment.