Production of Aspergillus niger ß-mannosidase in Pichia pastoris.

ß-Mannosidase (EC 3.2.1.25) is an exoglycosidase specific for the hydrolysis of terminal ß-linked mannoside in various sugar chains. cDNA corresponding to the ß-mannosidase gene was cloned from Aspergillus niger, sequenced, and expressed in the yeast Pichia pastoris. The ß-mannosidase gene contains an open reading frame which encodes the protein with 933 amino acid residues. The wild type and recombinant proteins were purified to apparent homogeneity and biochemically characterized (K(M) 0.28 and 0.44 mmol/l for p-nitrophenyl ß-d-mannopyranoside, pI 4.2 and 4.0, and their pH optima were at pH 4.5 and 5.5 and 65°C, respectively).

Sequencing, cloning and high-yield expression of a fungal ß-N-acetylhexosaminidase in Pichia pastoris.

The ß-N-acetylhexosaminidase from Talaromyces flavus has a remarkable synthetic ability, processing even carbohydrates with various functionalities. Its broader use is partially hampered by low-yield production in the native fungus. Here, we present an optimized 3-day production of this enzyme in the eukaryotic host of Pichia pastoris, in ca 10-fold higher volume activity (10 U/ml) and close-to-perfect purity (one chromatographic step needed). Importantly, the recombinant enzyme features the same biochemical and catalytic properties, including the syntheses with derivatized carbohydrate substrates. This is the first example of the overexpression of a fungal ß-N-acetylhexosaminidase by a single-cell producer in liquid medium. It represents a promising solution for wider biotechnological applications of this outstanding enzyme.

Preparatory production of quercetin-3-ß-D-glucopyranoside using alkali-tolerant thermostable α-L-rhamnosidase from Aspergillus terreus.

Extensive screening for a robust producer of α-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 50L. α-L-Rhamnosidase, which was fully characterized, proved to be thermo- and alkali-tolerant, thus enabling effective operation at 70°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-ß-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.