4 methylumbelliferyl α l iduronide 4mu 1

Turnip hairy root culture media coming from transformed hairy root cultures as well as the purified rIDUA_RLT protein were used to determine the activity of the recombinant protein of interest by using the fluorogenic substrate sodium 4‐methylumbelliferyl‐α‐L‐Iduronide (4MU‐I; Santa Cruz Biotechnology) as described in (Ou et al., 2014). The 4MUI substrate was diluted to a working solution of 400 μm 4MU‐I with the reaction buffer 0.4 m sodium formate, pH 3.5. Twenty‐five μL of sample were added to 25 μL of 400 μm 4MU‐I substrate. The mixture was incubated at 37 °C for 30 min and 200 μL glycine carbonate buffer (pH 9.8) was added to quench the reaction. 4‐Methylumbelliferone (4MU) (Sigma) was used to prepare the standard calibration curve. Fluorescence was measured using a plate reader (TECAN Infinite M1000, Männedorf, Switzerland) with excitation at 355 nm and emission at 460 nm. IDUA enzyme activity was expressed in units (μmol converted to product per minute) per sample volume (millilitres). The parameters K_M, k_cat and V_max were calculated by linear fit on a Lineweaver‐Burk plot (Ou et al., 2014).

Escherichia coli strain JM101 and R. rhizogenes strain ICPB TR7 were used for cloning and plant transformation, respectively, and B. rapa rapa cv ‘Navet des vertus marteau’ for hairy root production. Plant tissue culture media, vitamins and sucrose came from Duchefa Biochemie. 4‐methylumbelliferyl‐α‐L‐Iduronide (4MU‐I) came from Santa Cruz Biotechnology (Dallas, TX). The commercial recombinant IDUA protein used as positive control came from Antibodies‐online. The anti‐IDUA antibody used in the Western‐blot analyses came from Antibodies‐online. All reagents used to study the post‐translational modifications of the IDUA protein were of HPLC grade. Peptide N‐Glycosidase A was purchased from Roche Mannheim, Germany. All reagents used for SDS‐PAGE silver staining, ‐Methylumbelliferone (4MU) and Concanavalin A were purchased from Sigma (Saint‐Louis, MO). Antibodies directed against the anti‐xylose and anti‐fucose epitopes were from Agrisera Vännäs, Sweden.