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.

Polyphenol oxidase from apricot (Prunus armeniaca) (PaPPO) was purified in its latent form (L-PaPPO), and the molecular weight was determined to be 63 kDa by SDS-PAGE. L-PaPPO was activated in the presence of substrate at low pH. The activity was enhanced by CuSO₄ and low concentrations (≤ 2 mM) of SDS. PaPPO has its pH and temperature optimum at pH 4.5 and 45 °C for catechol as substrate. It showed diphenolase activity and highest affinity toward 4-methylcatechol (K_M = 2.0 mM) and chlorogenic acid (K_M = 2.7 mM). L-PaPPO was found to be spontaneously activated during storage at 4 °C, creating a new band at 38 kDa representing the activated form (A-PaPPO). The mass of A-PaPPO was determined by mass spectrometry as 37 455.6 Da (Asp102 → Leu429). Both L-PaPPO and A-PaPPO were identified as polyphenol oxidase corresponding to the known PaPPO sequence (UniProt O81103) by means of peptide mass fingerprinting.

Tyrosinases and catechol oxidases belong to the polyphenol oxidase (PPO) enzyme family, which is mainly responsible for the browning of fruits. Three cDNAs encoding PPO pro-enzymes have been cloned from leaves of Malus domestica (apple, MdPPO). The three pro-enzymes MdPPO1-3 were heterologously expressed in E. coli yielding substantial amounts of protein and have been characterized with regard to their optimum of activity resulting from SDS, acidic and proteolytic activation. Significant differences were found in the kinetic characterization of MdPPO1-3 when applying different mono- and diphenolic substrates. All three enzymes have been classified as tyrosinases, where MdPPO1 exhibits the highest activity with tyramine (k_cat = 9.5 s⁻¹) while MdPPO2 and MdPPO3 are also clearly active on this monophenolic substrate (k_cat = 0.92 s⁻¹ and k_cat = 1.0 s⁻¹, respectively). Based on the activity, sequence data and homology modelling it is proposed that the monophenolase and diphenolase activity of PPOs can be manipulated by the appropriate combination of two amino acids, which are located within the active site cleft and were therefore named “activity controllers”.

In this study, we investigate the effect of enzymatic browning on the phenolic composition of apricot in vivo and in vitro. The in vitro browning was caused by the recombinant latent apricot polyphenol oxidase (L-PaPPO). Successful heterologous expression of PaPPO in Escherichia coli yielded substantial amounts of enzyme containing both copper ions in the catalytic active site. The expressed L-PaPPO was characterized with regard to its molecular mass (56531.3 Da), pH optimum (7.0), activation by SDS, and enzyme kinetics. LC-MS/MS was used to compare the phenolic profiles of brown and non-brown apricots. The browning reactions did significantly decrease total phenolics and antioxidant capacity (measured with DPPH and CUPRAC assays). Catechin, epicatechin, and B-type procyanidins were the individual phenolics most affected by browning, followed by chlorogenic and neochlorogenic acid. These phenolics are most likely the main endogenous substrates of L-PaPPO, as they were oxidized much faster than the other identified phenolics.