In Brassica napus three different gene families with different temporal and tissue-specific expression and distribution patterns encode myrosinases (thioglucoside glucohydrolases, EC 188.8.131.52). Myrosinases encoded by the MA gene family are found as free and soluble dimers, while myrosinases encoded by the MB and MC gene families are mainly found in large insoluble complexes associated with myrosinase-binding proteins and myrosinase-associated proteins. These large complexes impede purification and characterization of MB and MC myrosinases from the plant. We used Pichia pastoris to express and secrete functional recombinant MYR1 myrosinase from B. napus to allow further characterization of myrosinase belonging to the MB gene family. The purified recombinant myrosinase hydrolyzes sinigrin with a Km of 1.0 mM; the specific activity and calculated kcat/Km were 175 U/mg and 1.9 × 105 s−1 M−1, respectively. A novel in-gel staining method for myrosinase activity is presented.