Abstract
Tyrosinases are an ubiquitous group of copper containing metalloenzymes that hydroxylate and oxidize phenolic molecules. In an application context the term ‘tyrosinase’ usually refers to ‘mushroom tyrosinase’ consisting of a mixture of isoenzymes and containing a number of enzymatic side-activities. We describe a protocol for the efficient heterologous production of tyrosinase 4 from Agaricus bisporus in Escherichia coli. Applying this procedure a pure preparation of a single isoform of latent tyrosinase can be achieved at a yield of 140 mg per liter of autoinducing culture medium. This recombinant protein possesses the same fold as the enzyme purified from the natural source as evidenced by single crystal X-ray diffraction. The latent enzyme can be activated by limited proteolysis with proteinase K which cleaves the polypeptide chain after K382, only one The latent enzyme can amino acid before the main in-vivo activation site. Latent tyrosinase can be used as obtained and enzymatic activity may be induced in the reaction mixture by the addition of an ionic detergent (e.g. 2 mM SDS). The proteolytically activated mushroom tyrosinase shows >50% of its maximal activity in the range of pH 5 to 10 and accepts a wide range of substrates including mono- and diphenols, flavonols and chalcones.
Highlights
Tyrosinases form an ubiquitous family of metalloenzymes and are found in all domains of life[1]
By cloning and sequencing both molecules were identified as the expected gene for AbPPO4
The loss of these bases occurred in such a manner that the reading frame was conserved and amino acids A436 to A580 were deleted from the translation product
Summary
Tyrosinases form an ubiquitous family of metalloenzymes and are found in all domains of life[1]. As tyrosinases can oxidize both small phenolic molecules and phenolic moieties of larger molecules (e.g. proteins) they have found a plethora of biotechnological applications in e.g. organic synthesis, determination of phenolic analytes, bioremediation as well as in medicine, food processing and engineering of (bio)materials[19, 20] Most of these application utilize tyrosinase isolated from fruiting bodies of the common white mushroom Agaricus bisporus (‘mushroom tyrosinase’)[20], supposedly mainly due to its ready commercial availability[21]. It should be noted that the purification protocols used to prepare the available commercial preparations do usually not yield homogenous tyrosinase and these are likely to contain one or more unspecified ‘extras’ like laccase, β-glucosidase, β-xylosidase, cellulase, chitinase and xylanase activities[22, 23] The purity of these preparations is further compromised by the fact that Agaricus bisporus possesses genes coding for six different tyrosinases (AbPPO1 - AbPPO6)[24, 25]. We describe such a protocol, yielding pure AbPPO4 in its latent form as well as providing access to the mature, active form of the enzyme
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