Differentiation of Fungal Tyrosinases and Laccases Using Selective

Chapter 6

Differentiation of Fungal Tyrosinases and Laccases Using Selective Inhibitors and Substrates 1

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William H. Flurkey , Betty Ratcliff, Luis Lopez , Jill Kuglin , and Ruthellen M. Dawley Downloaded by UNIV OF ROCHESTER on August 15, 2013 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/bk-1995-0600.ch006

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Department of Chemistry, Indiana State University, Terre Haute, IN 47809 Department of Chemistry, University of Evansville, Evansville, IN 47722

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Fungal tyrosinases and laccases can utilize similar substrates and can be difficult to differentiate. In Agaricus bisporus, both enzymes can use dopa, p-cresol, and diaminobenzidine as substrates, albeit at different rates. Tolidine can be used as a selective substrate to differentiate Agaricus laccase from Agaricus tyrosinase. Oxidation of this substrate, however, did not appear to be inhibited by cetyltrimethylammonium bromide, a common laccase inhibitor. N-hydroxylglycine appeared to be a selective inhibitor of laccase since it did not inhibit tyrosinase activity. Tropolone, salicylhydroxamic acid, and 4-hexylresorcinol were more effective inhibitors of tyrosinase at low concentrations than cinnamic acid or 2,3-naphthalenediol. Similar patterns of substrate and inhibitor preferences were noted in other mushroom species such as Oyster, Enoki, and Shiitake mushrooms. Many varieties of cultivated mushrooms are available for commercial use and consumption. Some of these varieties include the common cultivated button mushroom, either as a white or brown (Crimini) strain, as well as Oyster, Shiitake, Padi Straw, Portabella and Enoki mushrooms. Browning of the common cultivated mushroom, Agaricus bisporus, can occur after mechanical damage, infection, bruising and desiccation to mushroom tissues. However, browning of the cream colored Oyster and Enoki mushrooms is not as apparent. The potential for browning is dependent on several factors, one of which is the presence of enzymes involved in browning reactions. These enzymes act upon endogeneous phenolic compounds to eventually generate discoloration within the tissue. Browning of food products, such as mushrooms, not only decreases consumer appeal but can also decrease the nutritive value of the product. Several enzymes capable of oxidizing phenolic compounds can be found in fungi. Three of these enzymes - laccase, peroxidase, and tyrosinase (polyphenol oxidase) - may be present in a given fungus and may display considerable overlap in their substrate specificity (1-7). For example, all three enzymes have been reported to be present in Agaricus bisporus (4,9-10). Differentiating between these three enzymes can be difficult if they are present at varying concentrations and if they can utilize similar substrates. If they do show a similar substrate preference, they can sometimes be differentiated with judicious use of selective 0097-6156/95/0600-0081$12.00/0 © 1995 American Chemical Society

In Enzymatic Browning and Its Prevention; Lee, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

Downloaded by UNIV OF ROCHESTER on August 15, 2013 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/bk-1995-0600.ch006

82

ENZYMATIC BROWNING AND ITS PREVENTION

inhibitors. Although there are selective inhibitors of tyrosinases, there are relatively few specific inhibitors of fungal laccases. Tyrosinase activity in fungi has been demonstrated using tyrosine, dopa, pcresol, and 4-methyl catechol as substrates (9). Some fungal laccases, however, can use either tyrosine, dopa or p-cresol as substrates (8). Substrates for assaying fungal laccases have included p-quinol, quaiacol, syringaldazine, toluquinol, pcresol and tolidine (8,10-16). In particular, quaiacol, tolidine, diaminobenzidine, and phenylenediamine derivatives have been used to locate and monitor intracellular and extracellular Agaricus laccase (9,10,13). Peroxidase can also oxidize many of the above substrates in the presence of hydrogen peroxide (4,17). Thus, it is sometimes difficult to distinguish between the three enzymes based soley on the use of a particular substrate. Selective inhibition of one or more of these enzymes has been achieved in the presence of multiple types of phenol oxidases. For example, inhibition of tyrosinase, but not laccase, has been reported using tropolone, salicylhydroxamic acid (SHAM), 4-hexylresorcinol (4HR), cinnamic acids, naphthalenediol, and phenylhydrazine (12,16-21). Three of these inhibitors - tropolone, SHAM, and 4hexylresorcinol - can be used at relatively low concentrations to differentiate tyrosinase from laccase. While selective inhibitors of tyrosinase have been identified, few specific inhibitors of laccase have been reported. For example, cetyltrimethylammonium bromide (CTAB) has been reported to be a selective inhibitor of laccase but not tyrosinase; however, this may vary according to the fungal source (12). N-hydroxylglycine has also been reported to be selective inhibitor of Coriolus versicolor laccase and incapable of inhibiting mushroom tyrosinase, plant polyphenol oxidase or Pénicillium sclerotium pyrogallol oxidase (22). The effects of hydroxylglycine on other fungal laccases are not known. Experimental Materials. Agaricus bisporus tyrosinase and Pyricularia oiyzae laccase were obtained from Sigma Chemical Co.(St. Louis, MO). An extracellular Agaricus bisporus laccase was a gift from R. Kerrigan (Sylvan Foods, Worthington, PA). Agaricus (common button and Crimini), Enoki, Oyster, and Shiitake mushrooms were obtained from local groceries. N-hydroxylglycine was synthesized by R. Kjonaas according to the method of Murao et al. (22). All other chemicals were of reagent grade. Enzyme extracts. Extracts from whole fresh mushrooms or mushrooms stored at - 80 °C were prepared as described previously (9,19). All samples were stored frozen in small aliquots and centrifuged before use. Protein content was determined as reported earlier (9,19). Tyrosinase assays. Tyrosinase activity was measured spectrophotometrically as described earlier (9,19) using either 1 mM tyrosine, 5 mM L-dopa, or 5 mM pcresol as substrates. Increases in absorbance were monitored at 280 nm for tyrosine, 475 nm for dopa, and 410 nm for p-cresol. Slopes were measured from the linear portion of the absorbance time curve and one unit of activity was defined as a change of 1.0 absorbance unit per min. When indicated, salicylhydroxamic acid (SHAM), tropolone, 4-hexylresorcinol (4HR), cinnamic acid, or 2,3-naphthalenediol were added to the assay as tyrosinase inhibitors. Laccase assays. Laccase activity was also measured spectrophotometrically as described (23) using either 5 mM p-cresol, 5 mM p-phenylenediamine, 1 mM 3,3diaminobenzidine, 20 uM syringaldazine, 2 mM toluquinol, or 2 mM tolidine as


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Differentiation of Fungal Tyrosinases and Laccases 83


substrates. Increases in absorbance were monitored at 410 nm for p-cresol, 3,3diaminobenzidine, and p-phenylenediamine, 525 nm for syringaldazine, 280 nm for toluquinol, and 630 nm for tolidine. Units of enzyme activity were defined as above. When indicated, cetyltrimethylammonium bromide (CTAB) or N hydroxylglycine were added to the assays as laccase inhibitors. Electrophoresis. Native polyacrylamide gel electrophoresis was carried out using preparative mini-gels (8%) as described previously (9,24). Crude extracts were mixed with bromophenol blue and glycerol and layered along the entire stacking gel surface. When electrophoresis was terminated, the gels were incubated for 10 min in 0.1 M sodium phosphate buffer (pH 6.0) for tyrosinase and 0.1 M sodium acetate buffer (pH 4.8) for laccase detection. At the end of 10 min, one cm wide strips were cut out of the gels and placed in the appropriate buffer, plus or minus the inhibitor, for 10 min. After this second incubation, the gel strips were placed in buffer containing the substrate, plus or minus the inhibitor, for color development and localization of enzyme activity. Photographs of zymograms were made using Kodak EDF duplicating film. Results and Discussion Crude extracts of Agaricus bisporus mushrooms, which contain tyrosinase and laccase activity, can oxidize a variety of phenolic compounds. Using dopa and pcresol as substrates for tyrosinase and p-cresol, p-phenylenediamine, and syringaldazine as substrates for laccase, specific inhibitors were able to distinguish between these oxidative activities (Table I). Table I.

Differential Inhibition of Phenol Oxidase Activities in Crude Extracts of Agaricus bisporus Inhibitor

Substrate

dopa p-cresol p-phenylenediamine syringaldazine

none cinnamic acid (500 uM)

naptha- SHAM lenediol (5 mM) (50 uM)

tropolone (100 uM)

CTAB

1.8 0.029 0.094

1.47 0.023 0.09

0.56 0.022 0.086

0 0.022 0.084

0 0.022 0.09

nd nd 0.057

0.045

nd

0.043

0.042

nd

0

(300 uM)

Adapted from reference (9). Numbers represent specific acitivity in units/mg. nd indicates not determined. At low concentrations SHAM and tropolone were very effective in inhibiting dopa oxidase or tyrosinase activity. This is in accord with reports by Kahn (18) and Walker (72). In contrast, cinnamic acid and 2,3-naphthalenediol required much higher concentrations for partial inhibition. All of these tyrosinase inhibitors showed partial inhibition when p-cresol was used as a substrate.



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Conversely, none of the tyrosinase inhibitors showed much inhibition when pphenylenediamine was used as a substrate, indicating that this substrate may be used by a laccase. CTAB, on the other hand, showed a significant amount of inhibition when p-phenylenediamine and syringaldazine were used as a substrates. These results indicated that crude Agaricus extracts contained at least two oxidative enzymes, tyrosinase and laccase, and that both can use similar and dissimilar substrates. This substantiates previous reports on the inhibition of SHAM to differentiate tyrosinase from laccase and the ability of SHAM to inhibit both mono and diphenolase activities of tyrosinase (9, 12,24). Interestingly, commercial Agaricus tyrosinase preparations can also oxidize a number of phenolic compounds. In some of these preparations tyrosinase activity could be detected usingtyrosine,dopa, and p-cresol as substrates while laccase activity was detected using p-phenylenediamine, 3,3-diaminobenzidine and toluquinol as substrates (Table II). Table II.

Inhibition and Differentiation of Tyrosinase and Laccase by SHAM and 4-Hexylresorcinol

Substrate

Tyrosinase tyrosine dopa p-cresol Laccase p-phenylenediamine 3,3 -diaminobenzidine toluquinol

Agaricus tyrosinase

Pyricularia laccase

none

4HR

none SHAM 4HR

SHAM

100 100 100

0 5 0

4 14 61

—

—

—

100

115

92

100 100 100

101 95 106

92 106 102

100 100 100

100 98 96

92 107 96

—

—

—

Adapted from reference (24). Numbers represent percent of control specific acitivity. (-) desiginates an activity to low to be measured accurately. SHAM and 4HR concentrations were 100 uM. SHAM effectively blocked tyrosinase activity but not laccase activity in these preparations. In addition, 4-HR was used as an inhibitor to determine if this compound could inhibit tyrosinase but not laccase. Although the inhibition by 4HR was not as great as SHAM, 4-HR is more soluble and can be used to differentiate tyrosinase from laccase using a variety of substrates. A commercial preparation of Pyricularia laccase was used to determine if SHAM and 4-HR could affect other types of laccase (Table Π). Pyricularia laccase apparently lacked a detectable tyrosinase activity but could still use dopa as a substrate. SHAM and 4-HR showed little, if any, inhibition of this laccase using toluquinol, diaminobenzidine, or phenylenediamine as substrates. Differential identification of tyrosinase and laccase was attempted in common button and Crimini (both Agaricus strains) mushrooms using a variety of substrates. In conjunction with these substrates, SHAM was used to inhibit tyrosinase and CTAB was used as a potential laccase inhibitor (16). Tyrosinase activity was severely inhibited with SHAM using p-cresol, dopa or tyrosine as substrates in both Agaricus strains (Table III, data not shown).


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FLURKEY ET AL.

Differentiation of Fungal Tyrosinases and Laccases


Table III. Differention of Tyrosinase and Laccase in Agaricus and Crimini Mushrooms using SHAM and CTAB

Tyrosinase dopa tyrosine p-cresol Laccase p-cresol diaminobenzidine phenylenediamine syringaldazine toluquinol

none

Agaricus SHAM

100 100 100

0 0 0

112 150 148

100 100 100

3 1 2

86 81 150

100 100

0 93

101 nd

100 100

7 80

232 nd

100

75

100

100

105

107

100

100

50

100

130

69

100

91

25

100

86

210

CTAB

none

Crimini SHAM

CTAB

Adapted from reference (23). Numbers represent percent of control specific activity, nd indicates not determined. SHAM and CTAB concentrations were 100 uM and 1 mM respectively. CTAB showed little inhibition and apparent activation was noted with some substrates. Laccase activity, when assessed using diaminobenzidine, phenylenediamine, syringaldazine or toluquinol as substrates, was relatively unaffected by SHAM. In contrast, utilization of p-cresol in both Agaricus and Crimini extracts was inhibited by SHAM. Except for syringaldazine, little inhibition of laccase activity was noted when CTAB was used as a laccase inhibitor. This suggests that CTAB may not be a preferential laccase inhibitor as reported earlier (16) or that CTAB inhibition may be dependent on the type of substrate employed. Oyster and Shiitake extracts showed low levels of activity using p-cresol, dopa or tyrosine as indicators of tyrosinase activity (Table IV, data not shown) . These results are in agreement with Oddson (25) and Bano and Rajarathnam (26) who found PPO activity to be much lower in Plerotus Florida than Agaricus. They also agree with those of Marr et al. (6) who classified Oyster mushroom into a high laccase low tyrosinase group. Laccase activity was detected in both extracts but was not inhibited by SHAM to a large extent. CTAB also had little effect on laccase in these two varieties except for when syringaldazine, p-cresol, and toluquinol were used as substrates for laccase in Oyster extracts. Tolidine has been used by Kerrigan et al. (73) and Choi et al. (27) to assay for laccase and locate laccase isoforms from mushroom cultures after electrophoretic separation. To determine if tolidine could be a useful and a selective substrate for laccase, but nonselective for tyrosinase, a spectrophotometric assay was developed which monitored the increase in absorbance due to product formation at 630 nm. A blue colored product, presumably a meriquinoid blue dye, was produced by the action of laccase on tolidine. A similar assay was reported by Hanke and Ebert (28) and Mohr et al. (77) for the oxidation of tolidine by peroxidase. As seen in Table V, oxidation of tolidine occured in extracts of five different varieties of mushrooms.


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Table IV. Differentiation of Tyrosinase and Laccase in Oyster and Shiitake Mushrooms using SHAM and CTAB


Oyster none SHAM Tyrosinase dopa tyrosine p-cresol Laccase p-cresol diminobenzidine phenylenediamine syringaldazine toluquinol

Shiitake none SHAM CTAB

CTAB

—

—

—

100 100

0 0

0 0

— — —

— — —

— — —

100 100

123 80

18 nd

100 100

100 90

200 nd

100

105

89

100

86

77

100

123

50

—

—

—

100

42

27

100

100

200

Adapted from reference (25). Numbers represent percent of control specific acitvity. (-) desiginates a value low to be determined accurately, nd means not determined. SHAM and CTAB concentrations were 100 uM and 1 mM respectively.

Table V. Differentiation of Tyrosinase and Laccase using Dopa and Tolidine as Substrates and SHAM N-hydroxylglycine as Inhibitors Source

Dopa -

Agaricus Crimini Enoki Oyster Shiitake

100 100 0 100 100

SHAM 1 1 0 48 10

Tolidine HG

-

93 90 0 44 89

100 100 100 100 100

SHAM

HG

81 94 100 91 66

88 87 1 19 28

Numbers represent percent of control specific activity. SHAM and HG concentrations were 200 uM each.



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Differentiation of Fungal Tyrosinases and Laccases 87

The highest level of tolidine oxidation was found in Shiitake (0.67 units/mg) followed by Oyster (0.4 units/mg) and Enoki (0.007 units/mg) extracts. The lowest level of tolidine oxidative activity was in Agaricus (0.0015 units/mg) and Crimini (.0015 units/mg) extracts. SHAM was relatively ineffective in inhibiting laccase activity using tolidine as a substrate, suggesting that tyrosinase cannot use tolidine as a substrate. In contrast, hydroxylglycine was very effective in blocking laccase activity in Shiitake, Oyster, and Enoki mushrooms but not as effective in Agaricus or Crimini mushrooms. The highest level of dopa oxidative activity was in Crimini (1.1 units/mg) extracts, followed by Agaricus (0.1 units/mg), Shiitake (0.04 units/mg) and Oyster (0.01 units/mg) (Table V). Very little tyrosinase activity was present in Enoki extracts. Tyrosinase activity was severely inhibited by SHAM in Agaricus, Crimini, Oyster and Shiitake extracts. Hydroxylglycine had little effect on tyrosinase activity except in Oyster extracts in which there was a significant percent inhibition. Differentiation of tyrosinase and laccase was also determined by electrophoretic separation of isozymes and selective inhibiton of isoforms by SHAM and HG. An extracellular filtrate of Agaricus laccase showed two major isozymes when separated by PAGE and stained with DAB (Fig. 1 a). These results are similar to those reported previously (4,13,24). SHAM, 4-HR, and tropolone showed no inhibition of staining or the number of observed bands. In fact, the presence of tropolone appeared to enhance staining intensity. CTAB also showed no inhibition of enzyme staining. No other oxidative enzyme, such as peroxidase, appeared to be present when hydrogen peroxide was added to the staining medium. Tolidine was compared to DAB for use as a selective substrate in the presence and absence of SHAM and HG (Fig. 1 b). Tolidine gave a stronger signal in a much shorter staining time compared to DAB (data not shown). SHAM had no effect on DAB oxidation but did show an apparent decrease in the coloration of tolidine staining. In fact, inclusion of SHAM caused the blue color to assume a blue-green hue instead of a bright blue color. Hydroxylglycine inhibited oxidation of both DAB and tolidine by laccase. We also examined the usefulness of tolidine, SHAM, and HG to differentiate tyrosinase from laccase in commercial tyrosinase preparations. As seen in Fig. 1 c, a single band of tyrosinase was observed and this band showed no staining with tolidine. SHAM blocked this dopa oxidase activity while HG appeared to decrease the intensity of dopa staining. Two apparent laccase isoforms were noted when stained with DAB and tolidine, and neither of these forms showed tyrosinase activity or staining with dopa. SHAM increased the staining intensity of DAB while HG inhibited the staining intensity. In contrast, SHAM decreased the intensity of tolidine staining while HG completely inhibited this reaction. These results suggest that some inhibitors may be reacting with the product or intermediates in the enzyme catalyzed reactions of tyrosinase and laccase. Conclusions Depending on the mushroom species, it may be relatively easy to differentiate tyrosinase from laccase using selective substrates. In other species, selective inhibitors, in conjunction with selective substrates, can be used to differentiate tyrosinase from laccase. Tolidine appears to be useful as a substrate for colorimetric determination of laccase activity and for differentiating laccase from tyrosinase and peroxidase. Hydroxylglycine appears to be a specific inhibitor of laccase when used with certain substrates and may be of general utility in distinguishing tyrosinase from laccase.




Figure 1. Localization of tyrosinase and laccase after native electrophoresis, (a) Extracellular Agaricus laccase was subjected to native electrophoresis as described in the Methods section and stained for activity using diaminobenzidine (DAB) in the presence and absence of 200 uM 4hexylresorcinol (4HR), 200 uM salicylhydroxamic acid (SHAM), 200 uM tropolone (TROP), 5 mM CTAB and 0.01% hydrogen peroxide, (b) Extracellular Agaricus laccase was subjected to native electrophoresis and stained for laccase activity using DAB and tolidine (TOL) in the presence and absence of 500 uM SHAM and 500 uM hydroxylglycine (HG). (c) Commercial tyrosinase was subjected to native electrophoresis and stained for tyrosinase activity using DOPA and for laccase activity using DAB and TOL. Staining was carried out in the presence and absence of SHAM and HG as above.


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Differentiation of Fungal Tyrosinases and Laccases

89

Acknowledgements This project was supported in part by an Indiana State University research grant to W.H.F. and a University of Evansville research grant to R.M.D. We gratefully acknowledge the preparation of hydroxylglycine by R.A. Kjonaas. We also appreciate the secretarial assistance of P. Archer and G. Till.


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Differentiation of Fungal Tyrosinases and Laccases Using Selective

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