Characterisation of bacterial myrosinase

The degradation of glucosinolates (GLSs) are mediated by the enzyme commonly known as myrosinase (β-thioglucosidase glucohydrolase; EC 3.2.3.1). There has been limited work on bacterial myrosinases which is surprising since they must play a role in both soil and animal gut ecology. The only bacterial myrosinase that has been purified to homogeneity is that of Enterobacter cloacae (no 506) but no amino acid sequence is available and there is very limited information on the characteristics of these enzymes. In this study a bacterial myrosinase from a soil isolate Citrobacter spp was purified to homogeneity and characterised. A combination of ion exchange chromatography two steps, (Mono Q column followed by gel filtration Superdex 75) were used. The molecular mass of both denatured and native protein is approximately 66 kDa. The optimum temperature and pH of the crude extract is 25 oC and pH 6.0 respectively. The enzyme is slightly activated by ascorbate but inhibited by glucose. Metal ions Zn2+, Ca2+, Fe3+ and Mg2+ also inhibited β-O-glucosidase activity with Fe3+ causing the largest loss of (76%) of activity followed by Mg2+, Zn2+ and Ca2+ while the metal ion chelator EDTA reduced enzyme activity by 70%. In vivo cultures of Citrobacter with sinigrin as a carbon source produced an as yet an unknown metabolite, while cell free extracts incubated with sinigrin produce 2-propenyl isothiocyanate. Sinigrin is completely degraded within 12 h of incubation of the Citrobacter in M9 medium either alone or in the presence of glucotropaeolin or gluconasturtiin. The latter two glucosinolates were metabolised differently and approximately 39% of glucotropaeolin and 20% gluconasturtiin remained in culture medium after 25 h of incubation. None of the tested β configuration substrates induced myrosinase. The enzyme hydrolysed sinigrin and glucoerucin at a faster rate than glucotropaeolin and glucoraphanin. The enzyme has both β-O-glucosidase and β-S-glucosidase activity with a greater affinity to aryl-β-glucoside with a Km of 0.0183 mM and a Vmax of 0.6 nmole.L-1.min-1 while for sinigrin a Km of 0.54 mM and a Vmax of 3.1 nmole.L-1.min-1. The enzyme activity could not be recovered from SDS-PAGE gels but was detected and eluted from native PAGE. SDS-PAGE of the eluted protein showed fewer protein bands and was comparable to the ion exchange second run in terms of number of bands on SDS-PAGE gels. The N-terminal sequence of the pure Citrobacter myrosinase shows little similarity with β-glycosidases while internal peptide analysis revealed some similarity with other O-glucosidases. However, no homology with any known myrosinases was found. Peptide sequencing of bands of 71 and 72 kDa obtained by different purification techniques (likely the same proteins) show some similarity with β-glucosidase of Citrobacter 30_2, while the peptide sequence of the secreted myrosinase band of ≈57 kDa shows similarity with glycoside hydrolase, family 3 of Ktedonobacter racemifer DSM 44963. The genome sequence of Citrobacter is required in order to allow further study such as cloning.