Identification and discrimination between some contaminant enzyme activities in commercial preparations of mushroom tyrosinase

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Abstract

Tyrosinase is a copper-enzyme involved in important biological processes. Many studies investigating these topics have relied on commercial preparations of mushroom tyrosinase as a tool or use as a model system. In this study, several commercial preparations of tyrosinase have been examined with regard to their composition and purity. Enzyme activities different from tyrosinase were found. Laccase, β-glucosidase, β-xylosidase, and xylanase activities were found in almost all samples examined. In particular, laccase and β-glucosidase were investigated for their ability to affect tyrosinase activity under certain experimental conditions. Laccase activity was variable in different commercial preparations and its presence could lead to misinterpretation of results ascribed to tyrosinase activity. In fact, it could hide the inhibitory effect of tropolone and kojic acid in relation to tyrosinase activity. β-Glucosidase released the aglycon moiety from the glucoside esculin which in turn could become a tyrosinase inhibitor or substrate. SDS–PAGE of commercial mushroom tyrosinase showed a complex pattern of unidentified proteins with at least four major and many minor protein staining bands. Taken together, these findings confirm that investigators should use caution in interpreting data relying on commercial sources of mushroom tyrosinase.

Introduction

Tyrosinase, a copper-enzyme belonging to type-3 copper proteins, is involved in the initial step of melanin biosynthesis and it is widespread throughout microrganisms, plant and fungal species, invertebrates, and vertebrates [1], [2], [3], [4], [5]. This enzyme catalyses the o-hydroxylation of monophenols to the corresponding catechols (monophenolase or cresolase activity) and the oxidation of such catechols to the corresponding o-quinones (diphenolase or catecholase activity). Tyrosinase, also called polyphenol oxidase (PPO), is involved in other important biological processes, such as enzymic browning easily observed in vegetables [6], betalain biosynthesis [7], sclerotization of insect cuticle [8], [9], and defense responses in arthropods [10], plants [11], and also in fungi [12]. Also studies on enzyme immobilisation for biotechnological purposes make use of mushroom tyrosinase [13].

Most of investigations, which make use of tyrosinase, have relied on commercial sources of the enzyme from the champignon mushroom, Agaricus bisporus. Unfortunately, these commercial preparations do not contain purified enzyme. In this regard, we would like to clarify that suppliers of commercial tyrosinase do not claim the preparations be homogeneous. In the past, some investigators [14], [15] suggested the possibility that the presence of contaminant enzyme activity had to be taken in serious consideration in interpreting data using commercial sources of mushroom enzyme. These investigators, beyond the shadow of a doubt, demonstrated the presence of significant amount of laccase in such commercial preparations. Recently, the presence of contaminant laccase activity in tyrosinase commercial preparations was also confirmed [16].

Moreover, it is well-known that constitutive forms of laccase are present in white rot fungi and also in the Agaricus genus [17], [18], [19], [20], [21], [22]. Recently, purification and characterisation of both a tyrosinase and a laccase from the same bacterium species was achieved [23].

Laccases are copper-enzymes, containing four copper ions, that oxidize phenolic compounds (but also aromatic amines) with poor specificity by means of electron abstraction thus forming a radical [3]. Further studies reached the conclusion that white rot fungi invest part of their metabolic energy to produce laccase for the purpose of degrading lignin. To do this, they also use additional enzymes [24]. Among them, quinone reductase, a membrane bound flavo-enzyme that binds FAD as the prosthetic group and in turn reduces quinones produced by laccase back to phenolic compounds. This redox cycle leads to the release of reactive oxygen species that take part in lignin degradation. When performing in vitro investigations relying on the enzymic properties of tyrosinase, the presence of laccase should be accurately excluded, otherwise, it could lead to ambiguous results as laccases share several substrates in common with tyrosinase, i.e. o-diphenols and o-aminophenols [25], [26].

In addition, other contaminant enzymes could bring about misleading results. For example, β-glucosidase in the presence of a suitable β-glucoside could release either phenolic substrates or inhibitors utilized by tyrosinase. Formally, a glycoside is any molecule in which a sugar group is bonded through its anomeric carbon to another group via a glycosidic bond. Many authors often suggest that the sugar be bonded to a non-sugar for the molecule to qualify as a glycoside. The sugar group is referred to as the glycone and the non-sugar group (often containing a phenolic group) as the aglycone or genin part of the glycoside. In nature, a huge number of glycosides are known. Among these glycosides, many are β-glucosides and they behave as substrates for β-glucosidases. After hydrolysis of β-glucosides by β-glucosidases, the product may potentially interact interact with tyrosinase and laccase.

Based on these considerations, we have investigated the purity of several lots of commercial tyrosinase that were purchased in the course of the last several years for our research needs. The results of this investigation are reported and some considerations about the need to examine commercial tyrosinase preparations more carefully are given.

Section snippets

Preparation of commercial tyrosinase

Six commercial preparations (purchase in the last eight years) were under investigation for the purposes of this study. Two of them (lot 17H9557, lot 112H9580, in the following named as S1- and S2-lot, respectively) were purchased from Sigma–Aldrich (Milan, Italy); three (lot 421497/1, lot 1179697, lot unreadable, in the following named F1-, F2- and F3-lot) were purchased from Fluka (Milan, Italy); the sixth (lot 36E8802, named WMT-1) was from Worthington (Lakewood, NJ, USA). The contents of

Identification of enzyme activities

All commercial preparations of mushroom tyrosinase we examined appeared to be non-homogeneous in appearance. This non-homogeneity was also observed when different samples (10 mg dissolved in 10 mL) from the same bottle gave solutions with different tyrosinase activity (U mL−1). Six lots were tested and the amount of tyrosinase, laccase, peroxidase, quinone reductase, β-glucosidase, β-xylosidase, xylanase, pectinase and endoglucanase investigated. Positive results are reported in Table 1. It should

Discussion

The large use of commercial preparations of tyrosinase is connected with the versatility of physiological processes in which this enzyme takes part. Investigators, who make frequent use of commercial tyrosinase preparations, seldom hint that the presence of contaminating enzymes could lead to misinterpretations of their results. However, it has been already reported in recent years that commercial preparations of tyrosinase could contain other proteins, such as laccase [14], [15], [16].

Conclusion

Based on these observations, and along with the reports from several investigators in the last years, we confirmed the presence of laccase activity and identified new contaminant enzyme activities in commercial preparations of tyrosinase; among these, the effect of a contaminating β-glucosidase was remarkable. These contaminants could affect correct interpretation of tyrosinase activity. In some cases, a suitable method of tyrosinase purification would be desirable to eliminate these effects on

References (43)

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