Oriented in situ immobilization of a functional tyrosinase on microcrystalline cellulose effectively incorporates DOPA residues in bioengineered mussel adhesive protein

Abstract

Background: Catechol-containing polymers such as mussel adhesive proteins (MAPs) are attractive as biocompatible adhesive biomaterials, and the catecholic amino acid 3,4-dihydroxyphenyl-L-alanine (DOPA) is considered a key molecule in underwater mussel adhesion. Tyrosinases can specifically convert tyrosine to DOPA without any cofactors. However, their catalytic properties still need to be adjusted to minimize unwanted DOPA oxidation via their diphenolase activity and catechol instability at neutral and basic pH values in the reaction products. Methods and Results:In this work, we constructed a novel functional tyrosinase, mTyr-CNK_CBM, by fusion of mTyr-CNK with a cellulose-binding motif (CBM) for oriented in situ immobilization on microcrystalline cellulose via the C-terminal CBM without any additional purification steps. mTyr-CNK_CBM showed optimal catalytic activity at pH 4.5-6.5 and room temperature and had a high monophenolase/diphenolase activity ratio (V max mono/V max di = 2.08 at pH 6 and 25 • C). mTyr-CNK_CBM exhibited 2.17fold higher (as a unimmobilized free enzyme) and similarly high (upon immobilization) in vitro DOPA modification of a bioengineered MAP compared to a commercially available mushroom tyrosinase. Moreover, the immobilized mTyr-CNK_CBM showed longterm storability and improved reusability.Conclusions: These results clearly demonstrate a strong potential for practical use of immobilized mTyr-CNK_CBM as a monophenol monooxygenase in preparing biocompatible DOPA-tethered biomaterials and other catechol-containing polymers.

Semi-Biosynthetic Production of Surface-Binding Adhesive Antimicrobial Peptides Using Intein-Mediated Protein Ligation

“…An engineered orthogonal aa-tRNA–aaRS pair can incorporate DOPA, although specifically incorporating DOPA without Tyr may be challenging owing to their structural similarity [ 26 ]. DOPA also can be incorporated into proteins via the enzymatic conversion of Tyr; however, unwanted sequential DOPA oxidation to DOPA-quinone due to the diphenolase activity of tyrosinase can occur and is difficult to control [ 27 ]. In addition, there are two challenges to overcome to establish AMP expression in Escherichia coli : first, production host cells are susceptible to AMPs due to their highly positive charge, and second, AMPs are prone to proteolytic degradation due to their low molecular weight [ 28 , 29 ].…”