Expression and action pattern of Botryotinia fuckeliana (Botrytis cinerea) rhamnogalacturonan hydrolase in Pichia pastoris.

The cDNA sequence coding for the complete rhamnogalacturonan hydrolase (RGase) of Botryotinia fuckeliana (Botrytis cinerea) was introduced into Pichia pastoris and expressed under the control of the alcohol oxidase promoter. The RGase was secreted into the medium of the yeast driven by the alpha-factor secretion peptide and could be purified using the C-terminal His6-tag fusion. RGase activity was measured using a traditional reducing end assay with linseed rhamnogalacturonan (RG) as the substrate, or with an assay using a fluorescent RG oligomer as the substrate and detection and identification of hydrolysis products by capillary zone electrophoresis (CZE). Both methods showed the recombinant enzyme to have a specific activity of about ten units per milligram of protein. Since the CZE method allows identification of the hydrolysis products, it was used to show that the RGase lacks a multiple attack mechanism and needs at least five GalA-Rha repeating disaccharides to be active. This finding is contrary to the action pattern of the native RGase of Aspergillus aculeatus which has the same substrate length requirement, but exhibits multiple attack, leading to products containing only two and three Rha-GalA repeat units without the appearance of intermediate sized fragments. No plant cell wall degrading enzymes were detected in the culture medium of un-transformed P. pastoris, thus the recombinant enzyme, devoid of extraneous activities, can be applied for fine structural studies on cell walls.