Extensive screening for a robust producer of alpha-L-rhamnosidase activity from well-defined strains of filamentous fungi, including multifactorial optimization (inducers, cultivation conditions) was accomplished. Enzyme production of the optimal producer Aspergillus terreus (non-toxigenic) was scaled up to 50 L alpha-L-Rhamnosidase, which was fully characterized, proved to be thermo- and alkali-tolerant, thus enabling effective operation at 70 degrees C and pH 8.0. These conditions allow for a very high substrate (rutin) load up to 100-300 g/L, thus enabling very high volumetric productivity of the reaction product quercetin-3-beta-D-glucopyranoside (isoquercitrin). Here, a novel concept of "immobilised substrate" is used. Isoquercitrin is a highly effective and biocompatible antioxidant with strong anti-inflammatory activities. Rutin biotransformation was optimized and scaled up to ca 10 kg production and thus the robustness of the large-scale production was demonstrated. Isoquercitrin can be produced to a very high purity (98%) in multikilogram amounts, without any quercetin and directly applicable in nutraceuticals. (C) 2011 Elsevier Ltd. All rights reserved.