Purification and comparative characterization of monophenolase and diphenolase activities from a wild edible mushroom (Macrolepiota gracilenta)

Process Biochemistry 47 (2012) 2449–2454

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Purification and comparative characterization of monophenolase and diphenolase activities from a wild edible mushroom (Macrolepiota gracilenta) Yakup Kolcuo˘glu ∗ Karadeniz Technical University, Science Faculty, Department of Chemistry, Trabzon, Turkey

a r t i c l e

i n f o

Article history: Received 31 August 2012 Received in revised form 2 October 2012 Accepted 19 October 2012 Available online 26 October 2012 Keywords: Polyphenol oxidase Monophenolase Diphenolase Macrolepiota gracilenta Mushroom Affinity chromatography

a b s t r a c t Polyphenol oxidases (PPO) are very important enzymes group in many industrial applications, especially in food, medicine and cosmetics. PPO from Macrolepiota gracilenta, a wild edible mushroom, was purified using a Sepharose 4B-l-tyrosine-p-amino benzoic acid affinity column and characterized in terms of mono- and diphenolase activity. The highest activities for pure enzyme were observed in the presence of PHPPA and DHPPA for monophenolase and diphenolase, respectively. The enzyme showed pH optimum values at 7.0 and 5.0, respectively, for monophenolase and diphenolase activities. Km values calculated as 0.8 mM for monophenolase and 1 mM for diphenolase activity at the presence of PHPPA and DHPPA as substrate, respectively. Vmax values were calculated as 2000 U/mg protein for both activity. Monophenolase and diphenolase activities were conserved approximately 40% and 60%, respectively, in their optimum pH at 4 ◦ C after 5 day incubation. The activities were inhibited most effectively by thiourea. The data obtained from this study showed that this enzyme could be useful for some industrial purposes. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction Most browning reactions are caused by enzymatic oxidation of natural phenolic compounds. Polyphenol oxidases (PPO), found in animals, plants, bacteria and fungi are a group of enzymes that include tyrosinases and catechol oxidase. They are responsible for the enzymatic browning during harvesting, handling, processing and storage of many plant materials [1]. All PPOs have a dinuclear copper center, and catalyze three different hydroxylation reactions (catechol oxidase or o-diphenol:oxygen oxidoreductase (EC 1.10.3.1), laccase or p-diphenol:oxygen oxidoreductase (EC 1.10.3.2) and cresolase or monophenol monooxygenase (EC 1.18.14.1)) and all of them use molecular oxygen in their reaction [2,3]. Reaction as a result of unstable o-quinones which are highly electrophilic molecules may auto-oxidize or cross-link to nucleophilic targets such as amino acids and nucleic acids. This lead to appearance of brown, red or black melanin-like pigments, a widespread and often undesirable and serious phenomenon especially for food, vegetable and beverage industry [1]. However, the physiological and biological functions of PPO are still unknown, not only in plants but also in fungi [4]. Recent studies suggested the participation of PPO in defense reactions against pathogens and

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insect pests, since the biosynthesis of floral pigments [5] protected membrane lipids against degradation in the forage during storage helping to improve fatty acid profile of ruminant products by reducing degradation of proteins [4]. Browning degrees of plants, vegetables and mushrooms depends upon the nature and amount of the phenolic compounds, reducing substances, pH, temperature, metal ions, presence of oxygen and the activity of the PPO [1]. PPO, especially monophenolase activity, is very important for use of the removal of phenols from wastewater [6]. In addition, monophenolase activity of PPO also attracts scientific interest for use in the synthesis of l-DOPA, used for the treatment of Parkinson’s disease [7]. Studies concerning the purification and characterization of PPOs from new sources are very important in explaining their biochemical properties and behavior. Thus, a particular enzyme may find its applications in the food or drug industries. Mushrooms are healthy foods. They have been used for centuries in various places for different purposes, particularly in traditional Chinese medicine. The nutritive and medicinal values of many wild species have long been known in the world. Some wild-growing mushrooms also possess pharmacological properties [8]. Macrolepiota gracilenta (Krombh.) Wasser (1978) (M. gracilenta) is a good wild and edible mushroom. It grows in the woods during summer and fall seasons. Cap 8–12 cm across, flattened convex, white to cream-ochre covered in minute pale ochraceous granular scales and gills white. Mushrooms are not only valuable foods but also

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good enzyme sources and PPO has many applications in industry. So, it is interesting to study PPO activity of different mushroom species. Agaricus bisporus (A. bisporus) has registered tremendous increase in interest over the recent years, due to its delicacy, flavor, nutritional value, and possible source of bioactive species. Among mushrooms, A. bisporus is the most studied species worldwide about PPO. The structure of tyrosinase from this mushroom is well explained and it is accepted as a model for other species [9,10]. The purpose of this study is to purify PPO of M. gracilenta by affinity chromatography and characterize it biochemically in detail and investigate the properties of the enzyme. Also, it is observed in literature that there is no knowledge about M. gracilenta as a source of enzyme. This study is the first containing polyphenol oxidase activity from this mushroom. So, the data extracted from this study are important and provide a new enzyme source for different industrial applications. 2. Materials and methods 2.1. Material and chemicals M. gracilenta (Krombh.) Wasser (1978) was harvested in September in the wooded areas of Karadeniz Technical University (KTU) in Turkey, and then carried into the laboratory in ice bath and stored there deep-frozen at −80 ◦ C. All the chemicals used were of reagent grade and purchased from Sigma (St. Louis, MO, USA). Affinity gel was synthesized at the laboratories in the department of chemistry according to the method reported previously [11]. 2.2. Enzyme extraction and purification Crude enzyme fraction was prepared as reported previously [2,3]. Firstly, the mushrooms were washed in distilled water three times. After that the mushrooms were placed in a dewar flask under liquid nitrogen for 10 min in order to disrupt cell membranes. The cold mushrooms were homogenized by using a porcelain mortar in 50 mM cold acetate buffer (pH 5.0) containing 2 mM EDTA, 1 mM MgCl2 , 1 mM phenylmethylsulfonyl fluoride (PMSF) and 3% (w/v) Triton X-114. The homogenate was filtered using three layers of cheesecloth. After filtration, the filtrate was centrifuged at 20,000 rpm for 30 min at 4 ◦ C. The crude extract was precipitated using an equal volume of cold acetone (chilled −30 ◦ C). This solution was stored at +4 ◦ C for 2 h. The acetone precipitate was collected and left for 24 h at 4 ◦ C to remove acetone. After that, the precipitate was resuspended in 50 mM acetate buffer (pH 5.0). After being stirred for 5 min, the suspension was centrifuged at 5000 rpm for 5 min. The enzyme solution was applied to an affinity column (1 cm × 15 cm) pre-equilibrated with 50 mM acetate buffer (pH 5.0). The affinity gel was washed with the same buffer. PPO was eluted with a solution of 50 mM phosphate buffer containing 1 M NaCl (pH 8.0). 2.3. Protein determination The soluble protein content of all steps was determined by the method of Lowry with some modifications [12] using bovine serum albumin as the standard. The absorbances obtained were evaluated by graphic interpolation on a calibration curve at 650 nm. 2.4. Enzyme assay PPO activity was assayed spectrophotometrically by measuring the initial rate of quinone formation from each of the five substrates specified below [13]. Activity was determined in the triplicates by measuring the increase in absorbance at 494 nm for 4-methylcatechol (4-MC) and 500 nm for all other substrates. Tyrosine and p-hydroxyphenyl propionic acid (PHPPA) were tested as monophenolic substrates and 4-MC, catechol, 3,4-dihydroxyhydrocinnamic acid (DHPPA) and l3,4-dihydroxyphenylalanine (l-DOPA) as diphenolic substrates. Enzyme activity was assayed using a 1 mL sample cuvette containing substrate (stock 100 mM), an equal volume of 3-methyl-2-benzothiazoline hydrazone (MBTH) (stock 10 mM) and dimethylformamide (DMF). The solution was diluted with buffer and subsequently added an appropriate volume of pure enzyme eluate. The reference cuvette included all the reactants except the enzyme [3]. One unit of PPO activity was defined as the amount of enzyme causing 0.001 increase of absorbance per minute in 1 mL of reaction mixture [3].

Laemli [14]. Coomassie brilliant blue R-250 was used for staining, and the molecular weight of the polypeptide was determined by using a calibration curve of the protein standards. Non-denaturing polyacrylamide gel electrophoresis was performed by using a 10% separating gel. After running, the gel was divided into two parts. The first part was stained by Comassie Brillant Blue R-250 and the second part by l-DOPA. For substrate staining, the gel was incubated in 24 mM l-DOPA for 1 h and then in 1 mM ascorbic acid until the appearance of protein band. 2.6. Determination of the enzyme properties 2.6.1. Substrate specificity and kinetics Catechol, 4-MC, l-DOPA, DHPPA, PHPPA and tyrosine, at the stock concentration of 100 mM, were used to monitor PPO activity. The rate of the reaction was measured in terms of the increase in absorbance at the absorption maxima of the corresponding quinone products for each substrate [13]. Monophenolase and diphenolase activity of M. gracilenta was monitored following concentration: 0.06–20 mM. The kinetic data were analyzed as 1/specific activity (1/V) versus 1/substrate concentration (1/[S]). The Michaelis–Menten constant (Km ) and maximum velocity (Vmax ) parameters were obtained from lineweaver–Burk plots [15]. 2.6.2. Influence of pH and temperature on monophenolase and diphenolase activity PPO activity of M. gracilenta, as a function of pH, was determined using DHPPA and PHPPA as the diphenolic and monophenolic substrate, respectively. The buffers were used to obtain the required overlapping pH ranges were: Mcilvaine (pH 3.0–7.0), phosphate (pH 7.0–8.0), Tris–HCl (pH 8.0–9.0) and glycine–NaOH (pH 10.0) [16]. The optimum temperature for monophenolase and diphenolase activity were determined by measuring enzyme activities at various temperatures over the range of 10–80 ◦ C with 10 ◦ C increments, in the presence of suitable substrates. The reaction medium containing substrate and buffer (at optimum pH) was incubated for 5 min at various temperatures as indicated above, prior to the addition of the enzyme solution. The results were expressed as the percentage of relative activity obtained at either the optimum pH or the optimum temperature [2]. 2.6.3. pH and thermal stability of the enzyme The effect of pH on the enzyme stability was determined by incubating the purified enzyme at 4 ◦ C and the own optimum temperatures for both monophenolase and diphenolase activities up to 5 days in different buffer solutions (Mcilvaine (pH 5.0 and pH 7.0) and Tris–HCl (pH 8.0)). At the end of the each storage periods, the monophenolase and diphenolase activities were assayed at incubation pHs. The percentage residual enzyme activities were calculated through comparison with non-incubated enzyme. In order to determine the thermal stability of the enzyme, the enzyme solutions in Eppendorf tubes were incubated at 4, 40 and 60 ◦ C for monophenolase activity and at 4, 30 and 60 ◦ C for diphenolase activity. Aliquots were withdrawn at times of 1, 2 and 5 days for 4, 30 and 40 ◦ C and 4 h at 60 ◦ C, rapidly cooled in an ice bath for 5 min, and then brought to 25 ◦ C. After reaching to room temperature, the enzyme activity was determined by standard assay conditions. Control with non-incubated enzyme was used under standard enzyme assay conditions for both activities to determine the 100% activity value [16]. 2.6.4. Effect of inhibitors and metal ions on PPO activity Inhibition effects of sodium azide, benzoic acid, sodium metabisulfite, ascorbic acid, thiourea, l-cysteine and phenylalanine on monophenolase and diphenolase activities were determined using PHPPA and DHPPA as substrate. Concentration of inhibitor causing 50% inhibition of PPO activity (IC50 ) was determined graphically by plotting the percentage of activities against the inhibitor concentration. The effect of metal ions on the enzyme activity was investigated by adding Na+ , Hg2+ , Mg2+ , Zn2+ , Ni2+ , Mn2+ , Co2+ , Ca2+ and Cu2+ directly to the standard reaction mixture in a final concentration of 1 and 10 mM. The remaining percentages of activities were identified through comparison with standard assay mixture with no metal ion added [2,16].

3. Results and discussion The results obtained in the present study indicate that PPO from M. gracilenta having monophenolase and diphenolase activities with good pH and thermal stabilities has been successfully purified and characterized biochemically.

2.5. SDS and natural polyacrylamide gel electrophoresis

3.1. Purification

SDS polyacrylamide gel electrophoresis was performed along with molecular size standard proteins in P8DS Electrophoresis Unit (Owl Scientific Inc., Woburn, USA). The gel, having 12% acrylamide concentration, was prepared according to

PPO was purified from M. gracilenta using a Sepharose 4Bl-tyrosine-p-aminobenzoic acid affinity column. Fractions were

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Table 1 Purification profile of PPO from M. gracilenta. Purification steps

Volume (mL)

Protein (mg/mL)

Activity (U/mL)

Specific activity (U/mg protein)

Purification (fold)

Crude extract Acetone precipitation Affinity chromatography

600 20 12

3.51 0.7 0.1

7.05 7.975 13.125

40.2 227.9 2625.0

1.0 5.7 65.3

120

A

100 Relative activity (%)

pooled as purified PPO with a high amount of protein (determined spectrophotometrically at 280 nm) and PPO activity. Table 1 summarizes the purification profile of PPO. All purification steps resulted in 65.3 fold purification. Up to now, PPO have purified using many different techniques such as ion exchange, hydrophobic interaction etc. The result is higher than the literature such as red Swiss chard 39 fold [17], Barbados cherry 60 fold [18], yali pear 10.8 fold [19]. PPO was also purified 74-fold from mulberry fruit using the same affinity column [11].

Diphenolase 80

Monophenolase

60 40 20 0

3.2. SDS and native polyacrilamid gel electrophpresis 120

3.0

4.0

5.0

6.0 pH

7.0

8.0

9.0

10.0

B

100 Relative activity (%)

PPO’s purity and molecular weight were determined by denaturating and non-denaturating polyacrilamid gel electrophoresis (Fig. 1). These electrophoregrams showed that the enzyme was efficiently purified and a single band on the SDS-PAGE corresponded approximately to 57.3 kDa molecular mass (Fig. 1A). These results suggest that PPO consists of subunits having same molecular weight. Similar results were shown by A. bisporus 67 kDa at single band after SDS denaturation [20], Chinese cabbage 65 kDa [21], Solanum melongena 112 kDa a homodimer [22] and potato PPO 57–60 kDa [23]. Non-denaturing polyacrylamide gel electrophoresis was performed at 4 ◦ C by using a 10% separating gel. The electrophoregrams showed that the enzyme was efficiently purified (Fig. 1B and C).

2.0

Monophenolase Diphenolase

80 60 40

20 0

0

10

20

30

40 50 Temparature ( C)

60

70

80

90

Fig. 2. pH-activity and optimum temperature profile on M. gracilenta PPO. Effect of pH on enzyme activity in the presence of PHPPA () and DHPPA () as a substrate were assayed at different pH values range from 3.0 to 10.0 using 50 mM Mcilvaine, Tris–HCl and glycine–NaOH buffers (A). The activity, as a result of temperature, was determined under standard assay conditions, within the range of 10–80 ◦ C (B).

3.3. Substrate specificity The M. gracilenta PPO was tested on six different phenolic substrates which were 4-MC, catechol, l-DOPA, DHPPA, tyrosine and PHPPA. The assays were performed pH 7.0 and at room temperature. The highest monophenolase and diphenolase activities of the purified enzyme were observed in the presence of PHPPA and DHPPA, respectively (Table 2). All the experiments were performed in the presence of both these substrates. In the literature, these substrates were studied less than other PPO substrates for mushroom PPOs.

Table 2 Substrate specificities of purified PPO. Substrate

Fig. 1. SDS and non-denaturing polyacrylamide gel electrophoresis. (A) Proteins were stained with Coomassie brilliant blue R-250. Lane M, molecular weight markers; lane 1: purified PPO; (B) Coomassie staining of purified enzyme; (C) activity staining of purified PPO with 24 mM l-DOPA.

Monophenols PHPPA l-Tyrosine Diphenols 4-Methylcatechol Catechol DHPPA l-DOPA

Wavelength (nm)

Relative activity (%)

500 500

85 6

494 500

90 12 100 1

500

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Y. Kolcuo˘glu / Process Biochemistry 47 (2012) 2449–2454

120

A

120

pH 5.0

80

pH7.0

Residual activity (%)

Residual activity (%)

100 pH8.0

60 40 20

C

100 80

pH 5.0 pH 7.0

60

pH 8.0

40 20 0

0 0

1

2

3

4

5

0

6

1

120

B

120

Residual activity (%)

Residual activity (%)

80 60

3

4

5

6

D

100

pH 5.0

2

Incubation time (Day)

Incubation time (Day)

pH7.0

pH8.0

40 20

100 80

pH 5.0

pH7.0

pH8.0

60 40 20 0

0 0

1

2

3

4

5

Incubation time (Day)

6

0

1

2

3

4

5

6

Incubation time (Day)

Fig. 3. pH stability profile of monophenolase and diphenolase activity. The enzyme incubated at different pH values for determining pH stability at 4 ◦ C for diphenolase (A) and monophenolase (B) activity; at 30 ◦ C for diphenolase (C) and at 40 ◦ C for monophenolase (D) up to 5 days.

3.4. Effect of pH and temperature on enzyme activities The previous studies have shown that PPO activity dependence on pH may be influence by several factors, such as source, extraction method, temperature and nature of phenolic substrate. pH alters either the ionization of amino acid side chains or the ionization of the substrates, hence it is the determining factor in the expression of enzymatic activity. The activities of pure PPO were analyzed different pH values between 3.0 and 10.0 by using 50 mM buffer systems described above in presence of PHPPA and DHPPA. The optimum pHs were found to be 7.0 and 5.0 for monophenolase and diphenolase activity, respectively (Fig. 2A). The acidic and neutral pH optima were reported for each activity. For example, Laurocerasus officinalis Roem. [2] at pH 5.0, Morus alba L. [11] between 5.0 and 7.0, and Stanley plum at pH 6.8 [24] for diphenolase activity; verdedoncella apple [13] at pH 4.6 and Haas avocado [25] at pH 5.0 for monophenolase activity. The temperature dependence of the both activities of purified PPO from M. gracilenta is presented in Fig. 2B. These activities were investigated over a temperature that ranges from 10–80 ◦ C at pH 5.0 and 7.0. The optimum temperatures were 40 and 30 ◦ C for monophenolase and diphenolase activity, respectively. It appears that the diphenolase activity is more sensitive to temperatures above 40 ◦ C. Monophenolase activity conserved approximately 85% at 50 ◦ C. Optimal temperatures for monophenolase and diphenolase activities were reported by other authors [2,17,18,26] and the values are between 18 and 50 ◦ C. 3.5. Enzyme kinetics Km and Vmax values for each activities of PPO from M. gracilenta were determined from the linear regression analysis of 1/V

versus1/[S]. Km value can change as depending on the structure of enzyme, pH, temperature and ion strength [27]. Using PHPPA and DHPPA as substrate, Km values were calculated 0.8 and 1 mM for monophenolase and diphenolase, respectively and Vmax values were calculated 2000 U/mg protein for each activity. It is well known fact that Km is measure of the affinity of an enzyme for particular substrate. The results obtained from this study, especially in terms of previously reported Km and Vmax values from mushroom [25,28,29] and other organism [2,3,13,25,30] make the study very interesting. It was reported that the Km values of most industrial enzymes varied within the range of 10−1 –10−5 M when acting on biotechnologically important substrates [31]. M. gracilenta having monophenolase and diphenolase activity could be utilized in various applications because of a low Km values.

3.6. pH and thermal stabilities The pH stabilities were examined by incubating the pure enzyme eluate at different pH values (5.0, 7.0 and 8.0) at 30 ◦ C for diphenolase activity, at 40 ◦ C for monopeholase activity and at 4 ◦ C for both activities up to 5 days (Fig. 3). The enzyme activity was assayed as mentioned above. Diphenolase activity maintained its original activity 100% and 70% at 4 and 30 ◦ C at pH 5.0 respectively, at the end of one-day incubation. After two-day incubation, the enzyme conserved its diphenolase activity by 63% in both temperatures at pH 5.0. At physiological pH, enzyme retained the diphenolase activity 80% at 4 ◦ C and 40% at 30 ◦ C. At the end of 5 days, at 4 ◦ C, enzyme conserve diphenolase activity 62% and 42% ratio at pH 5.0 and 7.0, respectively and the enzyme lost their activity at pH 8.0. The enzyme lost diphenolase activity all pH values at 30 ◦ C after 5 day incubation (Fig. 3A and C). For monophenolase activity, at the end of one-day incubation, the activity of M.

Y. Kolcuo˘glu / Process Biochemistry 47 (2012) 2449–2454

120

140

Residual activity (%)

100

Residual activity (%)

A Diphenolase 4 C

80

Monophenolase 4 C

60 40

2453

A

120 100

Mn2+ Ca2+ Hg 2+

Zn

2+

Ni

2+

80

Na+

Co2+

Cu2+

60 40 20

20

0

1mM 0 0

1

2

3

4

5

180

6

Incubation time (Day)

B

100

Diphenolase 30 C

80

Monophenolase 40 C

60

10 mM

B

Mn2+

160

Residual activity (%)

120

Residual activity (%)

Mg2+

140

Zn2+

120 100 80

Ca2+

Ni2+

Mg2+ Co2+

Cu2+

Hg2+

Na+

60 40 20

40

0

1mM

20

10 mM

Fig. 5. Effects of some metal ions on diphenolase (A) and monophenolase (B) activity.

0 0

1

2

3

4

5

6

Incubation time (Day) Fig. 4. The residual activity of the enzyme was measured after incubation of the enzyme solution at 4 ◦ C (A) (both activity), 30 ◦ C (diphenolase activity) and 40 ◦ C (monophenolase activity) (B) for 5 days.

gracilenta PPO retained only 20% at 4 ◦ C and 10% at 40 ◦ C of the activity at pH 5.0 and 7.0, respectively (Fig. 3B and D). The pH stability profile shows that diphenolase activity is more stable than monophenolase activity in examined pH values. Armillaria mellea and Hypholoma fasciculare lost their PPO activity at pH 5.0 25% and 60% ratio, respectively at 4 ◦ C for 24 h incubation. And at pH 7.0 and 8.0 these mushrooms lost their activity almost 20% ratio [29]. Macrolepiota mastoidea monophenolase conserved its activity 90% and dipenolase activity lost its original activity 65% ratio at pH 5.0. At pH 7.0 and 8.0 both activity conserved their original activities almost 90% ratio at 4 ◦ C after 24 h incubation [28]. Persimmon fruit [3] and cherry laurel [2] lost PPO activity and Barbados cherry [18] conserved PPO activity at their optimum pH in different values for 24 h incubation. Differently, Bramley’s seedling apple maintained more than 50% activity after 3 days incubation [32]. Given the literature, M. gracilenta PPO have high pH stability and this enzyme can be used easily in cases that require pH stability process. Profiles of the heat stability of monophenolase and diphenolase activities are shown in Fig. 4. The enzyme was stable at low temperature but unstable at high temperature. Diphenolase activity conserved approximately 40% and monophenolase activity lost 80% of its original activity after two days incubation period at its optimum temperature (Fig. 4B). At 4 ◦ C, PPO maintained 56% and 40% ratio after five days incubation for dipenolase and monophenolase activity, respectively (Fig. 4A). However, the enzyme, for both activities, was completely inactivated at 60 ◦ C after 4 h incubation (data not shown). These data show that monophenolase activity is more heat sensitive than diphenolase activity. It has been reported that A. bisporus was stable for 30 min 30 and 40 ◦ C and inactivated at 60 ◦ C [10]. Macrolepiota mastoidea mono-and diphenolase

activity were stable at 30 and 40 ◦ C and unstable at 60 ◦ C for 60 min [28]. Persimmon fruit PPO [3] was stable at 20–45 ◦ C for 30 min. Stanley plum PPO [24] was stable for 30 min at 70 ◦ C. The activities of the Oxygemmis PPO [2] dropped to 13% at the end of 30 min of incubation at 70 ◦ C. Zhou [19] reported that thermal stability of the PPO may be related to ripeness of plant and different molecular forms from the same source may also different thermostabilities. 3.7. Effect of various inhibitors and metal ions on PPO activity In order to examine the inhibitory effects of some chemicals on DHPPA and PHPPA oxidation, sodium azide, benzoic acid, sodium metabisulfite, ascorbic acid, thiourea, cysteine and phenylalanine were used. Each inhibitor was tested over wide range of concentrations sufficient to reduce PPO activity by 50% or more. Table 3 summarizes the IC50 values for each inhibitor when tested monophenolase and diphenolase activity. Results show that the most effective inhibitors on these activities were thiourea, ascorbic acid and cysteine with low IC50 values. In contrast, phenylalanine, sodium azide and benzoic acid were ineffective inhibitors at two activities of M. gracilenta PPO. These results were consistent with the earlier reports [26,28,29]. The mechanism of inhibition by ascorbate may involve reduction of quinonoid compounds Table 3 Inhibition of M. gracilenta monophenolase and diphenolase activities by some general PPO inhibitors. Inhibitors

IC50 (mM) Monophenolase activity

Sodium azide Benzoic acid Sodium metabisulfite Ascorbic acid Thiourea Cysteine Phenylalanine

0.5 0.9 0.06 0.03 0.008 0.3 16

± ± ± ± ± ± ±

1.8 2.1 0.6 0.5 0.6 1.4 2.2

Diphenolase activity 13.8 4.33 0.178 0.26 0.085 0.24 3.56

± ± ± ± ± ± ±

2.4 1.8 1.7 2.3 0.7 1.9 2.1

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produced by the diphenolases, and chelation or reduction of copper centers at the active site of the enzyme [33]. Ascorbic acid has also been reported to cause irreversible inhibition [34]. Inhibition by the thiol compounds is attributed to either the stable colorless products formed by addition reaction with o-quinones or binding to the active center of PPO [35]. Previous studies showed that various metal ions either inhibited or stimulated PPO activity. So, the effects of nine metal chlorides on the PPO activity were compared. The effects of various metal ions on the both activities of M. gracilenta PPO are shown in Fig. 5A and B. While Zn2+ , Ni2+ , Ca2+ and Mn2+ stimulated monophenolase activity by 27%, 20%, 23% and 53% respectively, at 1 mM final concentration, Hg2+ caused an inhibition in the range of 13% and 72% at 1 and 10 mM final concentration, respectively (Fig. 5B). M. gracilenta diphenolase activity was decreased by Hg2+ , Zn2+ and Ni2+ , 78%, 82% and 87% ratio respectively, at 10 mM concentration (Fig. 5A). Since metal ions may have different coordination numbers and geometries in their coordination compounds, and potentials as Lewis acids, they may behave differently toward proteins as ligands. These differences may also result in metal binding to different sites, and therefore, perturb the enzyme structure in different way [36]. 4. Conclusions The present study revealed the purification and characterization of PPO for the first time from M. gracilenta. The enzyme shares some catalytic characteristics such as optimum pH and temperature with other PPOs. In contrast, purified enzyme had greatest substrate specificity to PHPPA and DHPPA. PPOs have been used organic synthesis for producing fine chemicals and widespread industrial application. M. gracilenta PPO, having good pH and thermal stability, was very sensitive to some general PPO inhibitors. This indicates that the enzyme is highly suitable for this type of reaction conditions. One of the most important results is that this mushroom has monophenolase activity, which has been studied less than diphenolase activity. These results can make it a good candidate for some industrial or biotechnical applications. Acknowledgments This work was supported by KTU-BAP to Y.K. (2010.111.002). The author thanks to Prof. Dr. Ertu˘grul Sesli (Department of Science Education, Karadeniz Technical University, 61335 Trabzon, Turkey) for identifying the mushroom and Merve SARI (Department of English Language and Literature at Hacettepe University, Ankara, Turkey) for her kind assistance in improving the writing of this manuscript. References [1] Yoruk R, Marshall MR. Physicochemical properties and function of plant polyphenol oxidase: a review. J Food Biochem 2003;27:361–422. [2] Colak A, Özen A, Dincer B, Güner S, Ayaz FA. Diphenolases from two cultivars of cherry laurel (Laurocerasus officinalis Roem.) fruits at an early stage of maturation. Food Chem 2005;90:801–7. [3] Özen A, Colak A, Dincer B, Güner S. A diphenolase from persimmon fruits (Diospyros kaki L., Ebenaceae). Food Chem 2004;85:431–7. [4] Van Ranst G, Lee MRF, Fievez V. Red clover polyphenol oxidase and lipid metabolism. Animal 2010;5:512–21. [5] Nakayama T, Sato T, Fukui Y, Yonekura-Sakakibara K, Hayashi H, Tanaka Y, et al. Specificity analysis and mechanism of aurone synthesis catalyzed by aureusidin synthase, a polyphenol oxidase homolog responsible for flower coloration. FEBS Lett 2001;499:107–11.

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