Myrosinase activity in different plant samples; optimisation of measurement conditions for spectrophotometric and pH-stat methods

Abstract

Myrosinase (EC 3.2.3.1) found in Brassicaceae plants, is the enzyme responsible for hydrolysis of glucosinolates. As a result a variety of biologically active metabolites are liberated, whose importance in crop protection and especially in cancer chemoprevention is rapidly gaining recognition. The growing practical application of glucosinolate degradation products requires that sensitive and reliable methods of myrosinase activity determination in different types of plant samples are established. With the use of commercial myrosinase prep, we systematically optimised conditions of measurement of this enzyme activity by spectrophotometric and pH-stat methods. The parameters evaluated included: sample preparation, choice of substrate, its concentration, reaction temperature and detection wavelength.Two substrates with different spectral properties were chosen: sinigrin (SIN) and glucotropaeolin (GTL). For both substrates, the best reliability was achieved at reaction temperature of 37°C and substrate concentration of 0.2mM and 5mM for spectrophotometric and pH-stat methods, respectively. GTL exhibiting higher absorption at the recommended detection wavelength of 230nm ensured greater sensitivity of spectrophotometric determination of myrosinase activity in the case of transparent plant samples. GTL seemed to increase also the sensitivity of pH-stat method, however, in this case homogenisation of plant samples turned out to be most important. The optimised conditions were then verified for a range of plant samples. Based on these results, the optimised protocols of myrosinase activity determination for both methods are proposed.