Fine control of tyrosinase dependent monophenol oxidation: Production of catechol derivatives and development of functional hydrogels

Abstract

Tyrosinase, a type III copper containing polyphenol oxidase, is an essential enzyme that involves the synthesis of melanin, dark pigments found in most living organisms, for example, hairs of mammals and inks of cephalopods. The natural pigmentation is processed from small phenolic molecules, for instance, from one of the essential amino acids, L-tyrosine, via two different oxidative reactions of tyrosinase, which are consecutive and inseparable. Previous studies on this oxidase are mainly focused on searching inhibitors of the pigmentation reactions for the purpose of skin whitening and prevention of fruit browning. However, not much effort has been exerted to guide how to control this enzyme for developing it as a highly valuable catalyst. In this thesis, the two oxidative reactions of tyrosinase were studied intensively, and methodologies for shifting the three different states of tyrosinase (deoxy-tyrosinase, oxy-tyrosinase, and met-tyrosinase) and for controlling the inseparable two reactions (monophenolase and catecholase activity) were suggested. This thesis can be broadly divided into two themes in accordance with two applications depending on the types of the oxidation mechanisms. Simply put, one is inhibiting reactions related to the production of melanin by-product, and another is accelerating the reactions. The first theme, the selective inhibition, suggests universal instructions in the usage of tyrosinase in the regio-selective ortho-hydroxylation of monophenols to produce functional catechol derivatives, which are valuable in the markets of food additives, cosmetic ingredients, and even in the markets of fine drugs. And the second theme includes the fabricating methods of hydrogel from natural biomacromolecules and the development of it as sprayable/injectable sticky hydrogel for minimally invasive treatments. The first theme is present in the three chapters of this thesis, Chapter 2, 3 and 4. These chapters are composed of a long journey of designing new reaction paths for inhibiting the second oxidation of tyrosinase, searching or constructing novel tyrosinases, and improving the yield and productivity of the production of ortho-hydroxylated monophenol phytochemicals, from the enzyme engineering in lab scale to the mass production up to 400 L reaction. As a result, approximately 1.2 kg of ortho-hydroxylated isoflavones, 3-ODI and orobol, were successfully synthesized with 3.17 g·L-1·h-1 of productivity. The total yield (considering both conversion and recovery yield) for this reaction reached the theoretical values, over 99 %, and the products were extremely purely recovered with over 99% of purity. Furthermore, it was demonstrated that the methods could generally be applicable in hydroxylation of various phenolic phytochemicals such as phloretin, resveratrol, naringenin, apigenin, daidzin, polydatin, glycitin, genistin, etc. Contrary to the previous chapters (Chapter 2, 3, and 4) that are for inhibiting the second oxidative reaction, Chapter 5 and 6 are comprised of the studies for accelerating the tyrosinase activity for enzymatic crosslinking of macromolecules. Previously, tyrosinase from Agaricus bisporus was generally utilized for the enzymatic crosslinking for preparing hydrogels because of easy availability

the mushroom tyrosinase is the only tyrosinase which is commercially available. However, the applications of tyrosinase from A. bisporus in enzymatic crosslinking have been limited due to the low degree of crosslinking as a result of the steric hindrances between tyrosinase and macromolecules. Through the studies for previous chapters, it was found that the tyrosinase from Streptomyces avermitilis has a flat surface that is evolved for binding to a helper protein. Thus, the new tyrosinase from S. avermitilis was brought up for resolving the steric hindrance when crosslinking macromolecules. In these chapters, the method for preparing the tyrosinase-meditated hydrogel (composed of porcine gelatin, hyaluronic acid, and elastin-like polypeptide) is present, which showed significantly increased mechanical properties regarding storage modulus (strength) and adhesion work (stickiness). Especially, the adhesion work of this stick hydrogel made by gelatin and tyramine-conjugated hyaluronic acid was remarkable, which is 21.34 J·m-2, compared to previously studied catechol-conjugated polymers including mussel foot proteins, which in the range of 0.12 to 7 J·m-2.