Agric. Biol. Chem., 43 (11) 22492255, 1979 2249

Studies on the Myrosinase from Wasabia japonica : Purification and Some Properties of Wasabi Myrosinase

Masaru OHTSURUand Harumi KAWATAN Departmentof Foodand Nutrition, Yamaguchi Women's University, Yamaguchi753, Japan ReceivedMarch 19, 1979

The myrosinasefrom Wasabi(Wasabia japonica) is effectivelyextracted by sonication. The enzymeis purifiedabout 100-foldby gel-filtrationon SephacrylS-200, Sepharose 6B and ion-exchangechromatography on DEAE-Sephadex.The enzymeis activeonly when L-ascorbicacid is addedto the reactionmixture. Generalreducing reagents (2-mercapto ethanoland dithiothreitol)do not activatethe enzyme. Sulfhydryland aminogroup dis criminatingreagents strongly inhibit the enzymeactivity. Theseresults suggest that amino groupsand sulfhydrylgroups constitute the activesites of the enzyme. Molecularweight as determinedby gel-filtrationis 580,000,and the enzymehas about 12 subunitsas assessedby SDS-polyacrylamideelectrophoresis. Km valuefor sinigrinis 4.7x10-4M. Comparativefindings on some propertiesof yellowmustard, microbial and Wasabi myrosinasesare reported.

Myrosinase [thioglucoside(glucosinolate)- myrosinase from yellow mustard (Brassica

glucohydrolase, EC 3.2.3.1] is the enzymes juncea). But there are few reports on the responsible for the hydrolysis of mustard oil myrosinase from Wasabi (Wasabia japonica).13) glucosides, which have been found in plants,1,2) In this study, we investigated the extraction,

fungi,3',4) bacteria5,6) and mammals.7) The purification and some properties of Wasabi action of myrosinase on the mustard oil com myrosinase and compared them with the re pounds is the hydrolysis of glucose from the sults from yellow mustard and microbial thioglucoside followed by Lossen rearrange myrosinases. ment of the aglycon to give isothiocyanate and sulfate.8,9) MATERIALS AND METHODS

Wasabi (Wasabia japonica) roots, Shimane No. 3,

were used as the enzyme source.

Chemicals. 2-Hydroxy-5-nitrobenzylbromide (HN- BB) was purchased from Sigma. 2-Methoxy-5-nitro

tropone (MNT) was purchased from Sankyo Co.

The myrosinase is responsible for the de (Japan). Bovine serum albumin, aldolase, catalase, velopment of the flavor and pungency of many ferritin, trypsin-inhibitor and RNA-polymerase as standard proteins on electrophoresis were purchased food products, such as mustard and horsera from Boehringer Mannheim GmbH. Trinitrobenzene dish, because of its hydrolysis of the thiogluco sulfonic acid (TNBS), p-mercuribenzoate (PCMB), sides. Thus, myrosinase is a kind of typical 5-5•L-dithio-bis(2-nitrobenzoic acid) (DTNB), diisopro flavor developing enzyme. Plant myrosinases pylfluorophosphate (DFP), diethylpyrocarbonate(DEP) are activated by L-ascorbic acid in general. ethylenediaminetetraacetate (EDTA) and other chemi cals were obtained from Nakarai Chemicals (Japan). In previous papers, we described the puri

fication and physicochemical properties,10) Enzyme assay. Sinigrin, obtained from NBC functional groups11) and active center12) of Chemicals (Ohio, U.S.A.), was used as the substrate 2250 M. OHTSURU and H. KAWATANI

for myrosinase. The assay mixture contained 2.5 Purification of myrosinase ƒÊ mol of substrate, 1 ƒÊmol of L-ascorbic acid, 0.1 mmol The enzyme solution was prepared from of phosphate buffer (pH 7.0) and enzyme solution in Wasabi roots. The juice (60 liters) from a total volume of 1 ml. Myrosinase activity was

measured by the liberation of glucose. Glucose was Wasabi roots (10 kg) with 0.2M sodium determined by Sumner's dinitrosalicylic acid method,14) phosphate buffer containing 1 mm 2-mercapto with a previously described modification.15) ethanol was devided into 30 units (2 liters

Assay of protein. Protein concentration was deter each), which were sonicated for 1 hr each in

mined by the method of Lowry et al.16) an ice bath. Then the juice was centrifugated, and its supernatant was precipitated by 0.8 Electrophoresis. Disc electrophoresis was accom saturation (satn.) of ammonium sulfate. After plished with 5.0% polyacrylamide gel in accor dance with the procedure of Davis,17) and sodium concentration by ammonium sulfate, gel dodecyl sulfate (SDS) polyacrylamide gel electrophore filtration on a Sephacryl S-200 column (5x sis was carried out by the methods of Weber and 90cm), equilibrated with 0.01M phosphate Osborn,1) and Kanda et al.19) buffer containing 0.1M NaCl, was carried out.

Sonication. The enzyme was prepared from 25g Then, each filtrate was precipitated by 0.8 satn.

of crushed Wasabi mixed with 25 ml of ice-cold 0 .2 M of ammonium sulfate and dialyzed against phosphate buffer, pH 7.0. The slurry was allowed to 0.01M phosphate buffer, pH 7.0. The dialy stand for about 10 min in an ice bath and then sonicated zate was applied to a DEAE-Sephadex column for 10 min at 250 mA with a 3.0 cm diameter probe (6x40cm), equilibrated with 0.01M phosphate (Kaijo Denki T-A-4201 ultrasonic oscillator). buffer, pH 7.0. The elution of the protein was made with 0.3M NaCl in the above buffer. RESULTS The active fractions were pooled. After con Extraction of myrosinase from Wasabi centration by 0.8 satn. of ammonium sulfate, Myrosinases from mustards and rapesseds gel-filtration on Sephacryl S-200 column (5x were easily extracted by crushing with a buffer, 90cm), equilibrated with 0.01M phosphate but it was too difficult to extract that from buffer containing 0.1M NaCl, pH 7.0, was Wasabi by the same method. However, the carried out. The active fractions were concen enzyme was able to be effectively extracted by trated by 0.8 satn. of ammonium sulfate and the sonication as shown in Table I. This dialyzed against the same buffer. The dialy zate was put on a DEAE-Sephadex column TABLE I. THE CONDITIONS OF EXTRACTION (4x25cm), equilibrated with 0.01M phosphate buffer, pH 7.0. The elution of protein was made with a linear gradient of 0 to 0.3M NaCl in the same buffer. Finally , the enzyme was purified by gel-filtration on a Sepharose 6B column (1.5x90cm) , equilibrated with 0.01M phosphate buffer containing 0.1M NaCl, pH 7.0. 1) Crushed and stirred for 1 hr at 5°C with 0 .2 M As shown in Fig. 1, a chromatographically phosphate buffer, pH 7. 2) Crushed with 0.2 M homogeneous pattern of the enzyme was ob phosphate buffer, pH 7.0, and sonicated for 10 min tained. The fractions indicated by the arrow in an ice bath. 3) Crushed and stirred for 1 hr were used throughout the following experi at 5°C with 0.2M phosphate buffer, pH 7 .0, con taining 1% Triton-X. ments as the purified enzyme preparation. 1 unit=1 ƒÊmol of glucose/min/ml of enzyme Summary of purification of the enzyme is shown in Table II. result indicates that Wasabi myrosinase is The enzyme was purified approximately a particle enzyme like the Crambe seed 100-fold compared to the first dialyzate . enzyme.20) Purification and Some Properties of Wasabi Myrosinase 2251

TABLE II. PURIFICATION OF WASABI MYROSINASE

FIG. 1. Gel-Filtration of Wasabi Myrosinase on a Column (1.5x90cm) of Sepharose 6B Fluted with 0.01 M Phosphate Buffer Containing 0.1M NaCl, pH 7.0.

General properties of Wasabi myrosinase Figure 2A through D show the pH-activity, pH-stability, temperature-stability and tem perature activity curves of Wasabi myrosinase, FIG. 2. Some Enzymatic Properties of Wasabi respectively. Optimum pH was about pH 6.5 N Myrosinase. 7.0. The enzyme was stable at about pH 7.0 A, pH-activity (pH 4- 5.5 Acetate buffer, pH 6- and a temperature of below 30°C. Optimum 7.5=Phosphate buffer, pH 8-10=Tris-HCl buffer); B, pH-stability (5°C, 41 hr); C, Temperature-activity temperature was 37°C. (pH 7.0, 20 min); D, Temperature-stability (pH 7.0, Figure 3 shows the Lineweaver-Burk plot 20 min). of the enzyme. The Km value for sinigrin was calculated to be 4.7x10-4M.

Effect of ascorbic acid on activation of myro sinase Myrosinase from mustard seeds21) has the enzymatic activity without the addition of L-ascorbic acid and is specifically activated by 1mM of ascorbic acid. However, myrosinase from Wasabi has the enzymatic activity only when the enzyme reaction mixture contains ascorbic acid. As shown in Fig. 4, the maxi mum activation was observed with around FIG. 3. Reciprocal Plot of Sinigrin Hydrolysis. 2252 M. OHTSURU and H. KAWATANI

2 mM ascorbic acid. Activation gradually de- stannous(II) ions stimulated the enzyme ac

creased at higher concentrations. The de- tivity at 10-3M, whereas mercury(II) and

crease of activation may also be explained by copper(II) ions strongly inhibited it (Table V). the result that ascorbic acid acts as a competi The activity was considerably inhibited by

tive inhibitor of mustard myrosinase.22) the chelating agents, EDTA and o-phenan

As shown in Table III, the enzyme activity throline. Glucono-ƒÂ-lactone, a specific inhi

was most activated by L-ascorbic acid in the bitor of ƒÀ-glucosidase, did not inhibit the

enzyme activity. TABLE III. EFFECTSOF THE ANALOGUEOF ASCOBRIC ACID ON ACTIVATIONOF MYROSINASE The functional group of Wasabi myrosinase was investigated with reagents for discrimi nating the states of amino acids in protein.

Effects of various reagents are summarized in

Table VI. TNBS and MNT, which should

TABLE V. EFFECTS OF INORGANIC SALTS ON ACTIVITY OF MYROSINASE

analogues tested. This enzyme, like yellow mustard enzyme,"' was not at all activated by dehydroascorbic acid. Nagashima and Uchi yama23) showed that mustard myrosinase was not activated by reducing agents (glutathione, cysteine etc.) except ascorbic acid. Wasabi myrosinase was scarcely activated by other reducing agents (Table IV). However, the

TABLE IV. EFFECTS OF REDUCING REAGENTS ON ACTIVATION OF MYROSINASE

The assay mixture contained 1 ƒÊmol of inorganic salt, 0.2 mmol of phosphate buffer (pH 7.0) or 0.1 mmol of *acetate buffer (pH 5.2), and the enzyme solution was pre-incubated for 10 min at 37°C. Then, 1 ƒÊmol of L-ascorbic acid and 2.5 ƒÊmol of sub strate were added. Enzymatic reaction was carried out at 37°C for 20 min.

generally react with amino groups, markedly inactivated myrosinase activity. PCMB and activation effect was maintained by ascorbic DTNB, which are well-known SH reagents, acid and 2-mercaptoethanol existing together. considerably inactivated the activity. Other This effect of 2-mercaptoethanol indicates that reagents, DFP (serine), HNBB (tryptophane) the reducing agent is protecting the oxidation and DEP (histidine), had almost no effect. of ascorbic acid. The above results suggest amino and sul fhydryl groups to be the functional groups of Effects of various reagents on activity of Wasabi myrosinase. myrosinase Manganese(II), calcium(II), cobalt(II) and Purification and Some Properties of Wasabi Myrosinase 2253

6B. Figure 5 shows the plots of Ve/Vo versus the logarithms of the known molecular weights of standard proteins. The molecular weight of the enzyme was estimated to be 580,000.

FIG. 4. Effect of Ascorbic Acid on Activity.

ઠO.D.=(O.D.-20 min)-(O.D.-0 min).

TABLE VI. EFFECTS OF VARIOUS REAGENTS ON

ACTIVTTY OF MYROSINASE FIG. 5. Determination of Molecular Weight of Wasabi Myrosinase by Gel-Filtration on Sepharose 6B.

A, Bovine serum albumin; B, Aldolase; C, Catalase D, Ferritin; E, Wasabi Myrosinase.

As Wasabi myrosinase was a large molecule, the molecular weight of the subunit of the

enzyme was estimated by SDS-polyacrylamide

gel electrophoresis. At first, standard electro phoresis with polyacrylamide gel was carried out, and then gels were actively dyed with sinigrin, ascorbic acid and BaCl2 solution.24)

After that, the active part was sliced and in

cubated with 0.1% SDS and 0.1% 2-mer captoethanol for 30 min at 45°C. Then it was

put on the top of a SDS-polyacrylamide gel column, and the electrophoresis was carried

out by the standard method.18,19,)

The active parts of Wasabi myrosinase are

indicated by arrows in Fig. 6. As can be seen

from Fig. 7; the relationship between protein mobility and log molecular weight of the pro The assay mixture contained 0.1•`10 ƒÊmol of tein used in the SDS-polyacrylamide gel system reagent, 0.1 mmol of phosphate buffer (pH 7.0) or

*0 .1 mmol of acetate buffer (pH 5.2), and the enzyme is linear within the range of 2x104•`1.6x105.

solution was pre-incubated for 10 or 20 min at 37°C. The molecular weight of Wasabi myrosinase

Then, 1 ƒÊmol of L-ascorbic acid and 2.5 ƒÊmol of sub was calculated to be about 45,000•`47,000. strate were added. Enzymatic reaction was carried This suggests that Wasabi myrosinase con out at 37°C for 20 min. sisted of about 12 subunits.

Estimation of the molecular weight DISCUSSION The determination of the molecular weight was performed by gel-filtration on Sepharose Wasabi myrosinase has at least three distinct 2254 M. OHTSURU and H. KAWATANI

and rapeseed myrosinases: a particle enzyme, a large molecule, and the development of the enzymatic activity only when ascorbic acid is added to the reaction mixture. As described in previous papers,10,25,26)plant myrosinases are, generally easily extracted by stirring with only a buffer. We have but one report that ultrasonic treatment releases thio glucosidase from insoluble particles of Crambe

FIG. 6. Polyacrylamide Disc Gel Electrophoresis of abyssinicaseed meal.20) As Wasabi myrosinase Wasabi Myrosinase. was effectively extracted by sonication, this

Myrosinase applied, 250 ƒÊg, 2 mA per column for enzyme is also considered to be a particle 3 hr (I, IA) or 4 hr (II, IIA). I and II were dyed with enzyme. Myrosinase is reported to exist in sinigrin, ascorbic acid (1 mM) and BaCl2 (5mg/ml) for myrosin cells of mustard plant.27) Therefore 5 hr at 37°C. IA and IIA were dyed with amido black we think that there are at least two types of 10B. cell walls in Cruciferae plants; one is hard to break and the other is easy to break. The molecular weight of Wasabi myrosinase is 580,000,which consists of 12 subunits. This molecular weight is the largest among plant and microbial myrosinases. This fairly large molecular weight may be related to the poor thermostability of the enzyme. The optimum temperature of this enzyme is the same, about 37°C, as that of yellow mustard myrosinase.12) The enzymatic activity of Wasabi myrosinase FIG. 7. Determination of Molecular Weight of can be determined only when ascorbic acid is Wasabi Myrosinase by SDS-Electrophoresis. added to the reaction mixture. This point is A, RNA-polymerase; B, Bovine serum albumin; different from mustard myrosinases. How C, RNA-polymerase a; D, Trypsin-Inhibitor; E, Wasabi Myrosinase. ever, like mustard enzymes, the enzyme is not activated by general reducing agents. It is characteristics in comparison with mustard impossible to conclude that ascorbic acid is

TABLE VII. COMPARISON OF SOME PROPERTIES OF MYROSINASES FROM VARIOUS ORIGINS

* The assay mixture contained L -ascorbic acid (1 mM). Purification and Some Properties of Wasabi Myrosinase 2255 either an activator or a co-factor of Wasabi 8) M. G. Ettlinger and A. J. Lundeen, J. Am. Chem. myrosinase; ascorbic acid is considered to be Soc., 78, 4172 (1956). 9) M. G. Ettlinger and A. J. Lundeen, ibid., 79,1764 an activator of mustard enzymes. On this (1957). point, further investigation will be required. 10) M. Ohtsuru and T. Hata, Agric. Biol. Chem., 36, Wasabi myrosinase is inhibited by TNBS, 2945 (1972). MNT, PCMB and DTNB. TNBS inhibited 11) M. Ohtsuru and T. Hata, ibid., 37, 269 (1973). the enzymatic activity very markedly. Yellow 12) M. Ohtsuru and T. Hata, Biochim. Biophys. Acta, 567, 384 (1979). mustard myrosinase is also inactivated by the 13) M. Kojima and K. Tamiya, Vitamine, 28, 380 above reagents, especially by -SH reagents.11) (1963). These facts suggest that the functional groups 14) J. B. Sumner, J. Biol. Chem., 65, 393 (1925). of Wasabi myrosinase are also amino groups 15) 1. Tsuruo, M. Yoshida and T. Hata, Agric. Biol. and sulfhydryl groups. Chem., 31, 18 (1967). 16) O. H. Lowry, N. J. Rosebrough, A. L. Farr and Some properties of various myrosinases are R. T. Randall, J. Biol. Chem., 193, 265 (1951). compared in Table VII. 17) B. J. Davis, Ann. N.Y. Acad. Sci., 121, 404 (1964). 18) K. Weber and M. Osborn, J. Biol. Chem., 244, 4406 (1969). REFERENCES 19) F. Kanda, Seikagaku, 50, 430 (1978). 1) A. Kjaer, J. Conti and I. Larsen, Acta Chem. 20) H. L. Tookey, Can. J. Biochem., 51, 1305 (1973). Scand., 7, 1276 (1953). 21) M. Ohtsuru and T. Hata, Agric. Biol. Chem., 37, 2) Z. Nagashima and M. Uchiyama, J. Agric. Chem. 1971 (1973). Soc. Japan, 33, 881 (1959). 22) 1. Tsuruo and T. Hata, ibid., 32, 1425 (1968). 3) E. T. Reese, R. C. Clapp and M. Mandels, Arch. 23) Z. Nagashima and M. Uchiyama, J. Agric. Chem. Biochem. Biophys., 75, 228 (1958). Soc. Japan, 33, 980 (1959). 4) M. Ohtsuru, I. Tsuruo and T. Hata, Agric. Biol. 24) D. B. MacGibbon and R. M. Allison, Phyto Chem., 33, 1309 (1969). chem, 9, 541 (1970). 5) E. L. Oginsky, A. E. Stein and M. A. Greer, 25) R. Bjorkman and B. Lonnerdal, Biochim. Bio Proc. Soc. Expt. Med., 119, 360 (1965). phys. Acta, 327, 121 (1973). 6) N. Tani, M. Ohtsuru and T. Hata, Agric. Biol. 26) B. Lonnerdal and J. C. Janson, ibid., 315, 421 Chem., 38, 1617 (1974). (1973). 7) I. Goodman, J.R. Fouts, E. Bresnick, R. Menegas 27) E. Werker, Planta, 116, 243 (1974). and G. H. Hitchings, Science, 1130, 450 (1959).