Masaru Ohtsuru

The interaction of L-ascorbic acid with the active center of myrosinase.

Only L-ascorbic acid activated plant myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1), whereas ascorbic acid analogs did not. The enzyme protein was conformationally changed by the addition of L-ascorbic acid to the spectrophotometric analysis, approx. 1.5 amino residues appeared on the surface of the enzyme and about 2.3 tryptophan residues were buried in the molecule when 1 mM L-ascorbic acid was added. Optimum temperature for the myrosinase activity was approx. 55 degrees C without L-ascorbic acid, but with L-ascorbic acid it was about 35 degrees C; that for beta-glucosidase activity was the same (55 degrees C) with or without L-ascorbic acid. The effect of chemical modification of the functional groups of myrosinase on the interaction of L-ascorbic acid was investigated and the interaction of L-ascorbic acid with the active center of the enzyme is proposed.

Purification and general characteristics of bacterial myrosinase produced by Enterobacter cloacae.

Myrosinase in cell-free extract of Enterobacter cloacae, no. 506, was purified about 1,000 fold by precipitation with ammonium sulfate, chromatography on CM-Sephadex and gelfiltration on Sephadex G–200 and Sephadex G–100. The enzyme was shown to be homogeneous by chromatography and by ultracentrifuge. Molecular weight obtained by gelfiltration was 61,000 and the sedimentation coefficient was 4.5 S. Maximum activity occurred at pH 6.8, The enzyme was stable in a pH range of 5.0 to 7.0 at temperature below 40°C and for 24 hr. Copper(I) and (II), mercury (II) and ferrous(II) ions strongly inhibited the activity. Sulfhydryl reagents had little effect but EDTA was a strong inhibitor. In contrast to plant myrosinase, this enzyme was inhibited by l-ascorbic acid. Many glucosides and sugars inhibited the enzyme. The relation between bacterial myrosinase and β-gluco-sidase is discussed in comparison to plant and fungous myrosinases. Some comparative properties of bacterial, fungous and plant myrosinases are discussed.

Studies on Fungous Myrosinase: Part I. Production, Purification and Some CharacteristicsPart II. Effects of Various Reagents on Its Enzymatic ActivitiesPart III. On the β-Glucosidase Activity of Fungous Myrosinase and the Relationship of Fungous and Plant Myrosinases to β-Glucosidases

In order to obtain fungous myrosinase, Aspergillus sydowi IFO 4284 was cultured on a medium containing mustard seed extract for 2 weeks. Myrosinase in the broth was purified about 150 fold by precipitation with ammonium sulfate and chromatography on DEAE-cellulose and DEAE-Sephadex. Comparison of thioglucosidase and sulfatase activities of the myrosinase preparation using pH-activity, pH-stability and temperature-stability curves revealed no differences from each other. The chromatograms of the two activities on DEAE-Sephadex showed good agreement. Consequently, the myrosinase produced by Aspergillus sydowi was concluded to be a single β-thioglucosidase, not a mixture of thioglucosidase and sulfatase.The effects of various reagents on Aspergillus sydowi myrosinase were studied.The enzymatic activity was stimulated by cobalt (II), zinc (II) and magnesium ions and inhibited by mercury (II), iron (II) and copper (II) ions. However, metal-complexing agents, SH reagents and diisopropylfluorophosphate showed no...