Unusual Stability of a Recombinant Verrucomicrobium spinosum Tyrosinase to Denaturing Agents and Its Use for a Production of a Protein with Adhesive Properties

• Published in: Applied biochemistry and biotechnology 2018-07, Vol.185 (3), p.736-754
• Main Authors: Axambayeva, A. S., Zhaparova, L. R., Shagyrova, Zh. S., Ramankulov, E. M., Shustov, A. V.
• Format: Article
• Language: English
• Subjects: Adhesion; Adhesive strength; Adhesives; Amino Acid Sequence; Base Sequence; Biochemistry; Biocompatibility; Biosensors; Biotechnology; Catalysis; Cell adhesion & migration; Chemical agents; Chemistry; Coding; Dihydroxyphenylalanine; Dihydroxyphenylalanine - chemistry; E coli; Enzymatic activity; Enzyme Stability; Enzymes; Fruit bodies; Glues; Hydrogen-Ion Concentration; Industrial applications; Kinetics; Mimicry; Monophenol Monooxygenase - antagonists & inhibitors; Monophenol Monooxygenase - genetics; Monophenol Monooxygenase - metabolism; Mushrooms; Oxidation; Parameter estimation; Phenols; Polypeptides; Post-translation; Protein Denaturation; Protein Processing, Post-Translational; Proteins; Proteins - genetics; Proteins - metabolism; Quinones; Reaction kinetics; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Residues; Sarcosine - analogs & derivatives; Sarcosine - chemistry; Sodium; Tyrosinase; Tyrosine; Urea; Urea - chemistry; Verrucomicrobia - enzymology
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1007/s12010-017-2686-y

Tyrosinases catalyze oxidation of phenols with a formation of biphenols, quinones, and highly polymerized melanins. Tyrosinases have prospects for industrial use to remove phenols, also in biosensors, in bioorganic synthesis, and for a production of biocompatible adhesives (medical glues). Despite growing fields of potential applications, a selection of commercially available tyrosinases are currently limited to a single enzyme which is isolated from fruiting bodies of mushrooms. This article describes a preparation of recombinant tyrosinase from a bacterium Verrucomicrobium spinosum using a heterologous expression in Escherichia coli . Recombinant V. spinosum tyrosinase has high specific activity (13,200 U/mg). A resistance of the enzyme was investigated to chemical agents used to denature proteins and keep poorly solvable proteins in a solution. The enzyme preserves activity in the presence of urea and retains at least a fraction of its enzymatic activity at concentrations of urea up to 4.5 M. An addition of sodium lauroyl sarcosinate to 1 or 2% activates the tyrosinase. Novel means of quantitatively expressing tyrosinase activity is described in this article. The method uses a set of parameters obtained from non-linear estimation of the progress curves and is suitable for enzymatic reactions which do not comply with Michaelis–Menten kinetics. Tyrosinase may be used to introduce into proteins a post-translational modification which is a conversion of tyrosine residues (Tyr) into residues of 3,4-dioxyphenylalanine (DOPA). The presence of DOPA provides the polypeptides with a capability of strong molecular adhesion. Co-expression of tyrosinase and a recombinant protein mimicking marine mussel-encoded adhesive proteins resulted in obtaining of the protein in which at least a part of Tyr residues had been converted to DOPA. The DOPA-containing protein had high adhesion strength (2.5 MPa).