Purification, Characterization and Partial Amino Acid Sequencing of β-thioglucosidase from Brassica napus L.

Abstract

Summary A new purification procedure for the isolation of myrosinases (β-thioglucosidases, EC 3.2.3.1) from rapeseed is presented. Myrosinases were extracted from seeds of Brassica napus and purified by a procedure including adsorbtion to concanavalin A linked to agarose, ion exchange chromatography on DEAE-cellulose and fast protein liquid chromatography (FPLC). Three different forms of the enzyme were separated to a high purity. Isoelectric focusing of the three myrosinases gave isoelectric points of 4.94, 4.96 and 5.00. After denaturation and reduction, the three forms migrated as one band in sodium dodecyl sulphate polyacrylamide gel electrophoresis. The results from sodium dodecyl sulphate and native polyacrylamide gel electrophoresis indicate that the M, of myrosinase is 154,000, consisting of two identical subunits each with a M r of 77,000. The purified proteins were subjected to amino acid and carbohydrate analyses. Analysis of the total amino acid composition revealed no marked differences between the three myrosinases, and the N-terminal amino acid sequences were found to be identical. All three myrosinases contained different amounts of fucose, mannose and N-acetyl-glucosamine. The carbohydrate content varied from 9.6% to 18.9% of the total molecular mass. pH optima for the enzyme forms were between 5.2 and 5.5, and temperature optima between 70 and 75 °C. Km for all three forms were approximately 70 μM, and increased 3—7 times in the presence of 0.4 mM ascorbic acid.