Recombinant Tyrosinase from Polyporus arcularius: Overproduction in Escherichia coli, Characterization, and Use in a Study of Aurones as Tyrosinase Effectors

Abstract

Tyrosinases act in the development of organoleptic properties of tea, raisins, etc., but also cause unwanted browning of fruits, vegetables, and mushrooms. The tyrosinase from Agaricus bisporus has been used as a model to study tyrosinase inhibitors, which are also indispensable in the treatment of skin pigmentation disorders. However, this model has disadvantages such as side enzyme activities and the presence of multiple isoenzymes. Therefore, we aimed to introduce a new tyrosinase model. The pro-tyrosinase from Polyporus arcularius was overproduced in Escherichia coli. Trypsin digestion led to a cleavage after R388 and hence enzyme activation. The tyrosinase was a homodimer and transformed L-DOPA and tert-butylcatechol preferentially. Various aurons were examined as effectors of this enzyme. 2'- and 3'-hydroxyaurones acted as its activators and 2',4'-dihydroxyaurone as an inhibitor, whereas 4'-hydroxyaurones were its substrates. The enzyme is a promising model for tyrosinase effector studies, being a single isoenzyme and void of side enzyme activities.

Heterologous expression and characterization of functional mushroom tyrosinase (AbPPO4)

“…Enzymatically functional protyrosinase from Pholiota microspora was expressed in Escherichia coli 48 but no value for the yield was reported. Recently, protyrosinase from Polyporus arcularius was produced in the same host with a yield of 54 mg latent tyrosinase per liter of culture medium 49 . Here, the latent form of Ab PPO4 is produced by E. coli at a yield of 110 to 140 mg per liter of culture.…”

“…For most of the characterized tyrosinases this range is considerably more narrow, e.g. for the heterologously expressed tyrosinase from Polyporus arcularius it is found between pH 5–6 49 and the homologously overexpressed T. reesei TYR2 was found to be almost fully active in the range of pH 6–9.5 45 .…”

“…For TYR1 from P. nameko the kinetic parameters on tyrosine could not be determined due to insufficient solubility of the substrate 52 . For L -DOPA a slightly higher number of values (enzyme name: K m in µM | k cat in s −1 ) are reported for the recombinant tyrosinases from A. bisporus ( Ab PPO2: 1.22 | 141) 58 , P. nameko (TYR1: 1930 | 478) 52 , P. arcularius (Photo-regulated tyrosinase: 1040 | 223) 49 and T. reesei (TYR2: 3000 | 22) 45 . In comparison to those enzymes activated Ab PPO4 is less specific for L -DOPA and much faster on this substrate.…”

Natural and Bioinspired Phenolic Compounds as Tyrosinase Inhibitors for the Treatment of Skin Hyperpigmentation: Recent Advances

“…Alternatives to mushroom tyrosinase have also been developed, such as recombinant tyrosinase from Polyporus arcularius overproduced in Escherichia coli [228].…”

Versatile Fungal Polyphenol Oxidase with Chlorophenol Bioremediation Potential: Characterization and Protein Engineering

“…Plant PPOs from the flower Coreopsis grandiflora ( 11 ) and the grape Vitis vinifera ( 12 ) were functionally expressed in E. coli , whereas PPOs from the tea tree Camellia sinensis were poorly expressed in both prokaryotic and eukaryotic hosts ( 13 ). When it comes to Basidiomycota, several PPOs from Agaricus bisporus have been expressed in E. coli ( 14 , 15 ) and Saccharomyces cerevisiae ( 16 ), while recently, a tyrosinase from Polyporus arcularius was expressed in E. coli cells ( 17 ). Bacterial tyrosinases from various genera ( Verrucomicrobium , Bacillus , Rhizobium , Ralstonia , and Marinomonas ) have been heterologously expressed and characterized ( 18 – 22 ).…”