티로시나아제를 이용한 모노페놀류 산화반응의 정밀제어: 카테콜형 구조 물질 생산과 기능성 하이드로겔의 개발

학위논문 (박사)-- 서울대학교 대학원 공과대학 협동과정 바이오엔지니어링전공, 2017. 8. 김병기.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 availabilitythe 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-2.1. Chapter 1. Introduction 1 1.1 Mechanism of tyrosinase 2 1.1.1 Copper metalloproteins 2 1.1.2 The active site of tyrosinase and the structural comparison of type III copper-containing proteins 5 1.1.3 Rate-determining step of tyrosinase reaction 12 1.1.4 Inhibitors of tyrosinase and the suicidal inactivation 17 1.2 Application of tyrosinases 21 1.2.1 Production of catechol derivatives 21 1.2.2 Tyrosinase as a crosslinking agent 28 1.2.3 Miscellaneous 31 1.2.3.1 Melanin synthesis and its usages 31 1.2.3.2 Waste water treatment 34 1.3 The scope of thesis 35 2. Chapter 2. Heterologous expression of tyrosinase (SAV1137) from Streptomyces avermitilis MA4680 in Echerichia coli and its application for ortho-hydroxylation of resveratrol to produce piceatannol 40 2.1 Abstract 41 2.2 Introduction 42 2.3 Materials and Methods 46 2.3.1 Materials 46 2.3.2 Construction of plasmids for the recombinant tyrosinase and a glucose dehydrogenase. 46 2.3.3 Expression and preparation of recombinant tyrosinase and glucose dehydrogenase (GDH) for reaction. 47 2.3.4 Constructing mutants of MelC1 and MelC2. 48 2.3.5 Oxidation reaction of resveratrol in E. coli. 48 2.3.6 Effects of NADH on the production of piceatannol through tyrosinase reaction. 49 2.3.7 Screening for mutants with high specificity in piceatannol production 51 2.3.8 Kinetics characterization of wild-type and I41Y on L-tyrosine, L-DOPA, resveratrol, and piceatannol. 55 2.3.9 Preparing samples for kinetic values of MelC2. 55 2.3.10 Computational modeling 56 2.3.11 Analytical methods 57 2.4 Results and Discussion 63 2.4.1 Expression of tyrosinase, MelC2, in E. coli 63 2.4.2 Effect of NADH in the reaction system. 65 2.4.3 Saturation mutagenesis of helper protein, MelC1 at Y91 68 2.4.5 Model prediction and characterization of mutants 77 2.5 Conclusion 84 3. Chapter 3. Using tyrosinase as a monophenol monooxygenase: a combined strategy for effective inhibition of melanin formation 87 3.1 Abstract 88 3.2 Introduction 90 3.3 Materials and Methods 95 3.3.1 Materials 95 3.3.2 Construction of plasmids for recombinant tyrosinases. 95 3.3.3 Conditions for cell cultures and reactions 97 3.3.4 Computational modeling 98 3.3.5 Identification of products 98 3.4 Results and Discussion 100 3.4.1 Comparison of Tys from three different organisms for the ortho-hydroxylation of daidzein 100 3.4.2 Protecting the diol moiety of 3-ODI by borate ester bonds 104 3.4.4 Whole cell biocatalysis of daidzein by BM_Ty to produce 3-ODI 116 3.4.5 ortho-Hydroxylation of monophenols by tyrosinase. 127 3.4.6 ortho-Hydroxylation of benzopyrone ring of isoflavones by tyrosinase6- and 8- hydroxylation. 141 3.4.7 Calculation of k1 and k2 of BM_Ty on daidzein 149 3.4.8 Calculation of oxygen transfer rate and coefficient of the 5 mL reaction in a 50 mL reactor without additional oxygen transfer. 154 3.4.9 Calculation of oxygen transfer rate and coefficient of the 400 mL reaction in a 1 L reactor with a continuous air flow rate of 12 Lmin-1 . 155 3.4.10 Mass production of 3-ODI and orobol 160 3.4.10.1 Enzyme expression for scale-up studies. 160 3.4.10.2 The optimization of the reaction conditions 164 3.4.10.3 The optimization of the purification of products. 170 3.4.10.4 Overall process of the mass production of catechol derivatives. 174 3.5 Conclusion 178 4. Chapter 4. A novel tyrosinase from Burkholderia thailandensis active active at acidic pH and its application for the ortho-hydroxylation of glyco-conjugated monophenolic phytochemicals 179 4.1 Abstract 180 4.2 Introduction 181 4.3 Materials and Methods 185 4.3.1 Materials 185 4.3.2 Construction of a cladogram of bacterial Tys. 185 4.3.3 Plasmid construction 186 4.3.4 Expression and purification of tyrosinases 189 4.3.5 Purification of tyrosinase from Agaricus bisporus, mushroom tyrosinase 189 4.3.6 Measurement of tyrosinase activities 190 4.3.7 ortho-Hydroxylation of monophenol glycosides 191 4.3.8 HPLC and LC/MS analysis 191 4.4 Result and Discussion 193 4.4.1 Reactivity depending on pH and measurements of kinetic parameters. 193 4.4.2 ortho-Hydroxylation of monophenol phyrochemicals by BT_Ty. 197 4.4.2.1 ortho-Hydroxylation of monophenol gycosides 197 4.4.2.2 ortho-Hydroxylation of monophenol aglycones 207 4.4.3 Structural analysis of BT_Ty 211 4.4.3.1 The morphology of BT_Ty based on the X-ray crystallography 211 4.4.3.2 Residues related to inter-interactions for maintaining the homo-tetrameric structure. 218 4.4.3.3 Residues related to intra-interactions between cap and body domains. 222 4.4.3.4 Identifying residues that enhance the Ty activity even at acidic pH. 227 4.5 Conclusion 233 5. Chapter 5. Tissue adhesive, rapid forming, and sprayable ECM hydrogel via recombinant tyrosinase crosslinking 234 5.1 Abstract 235 5.2 Introduction 236 5.3 Materials and Methods 239 5.3.1 Materials 239 5.3.2 Preparation of enzymes 239 5.3.2.1 Expression and purification of recombinant tyrosinases. 239 5.3.2.2 Purification of mushroom tyrosinase. 242 5.3.4 Measurement of the specific activity of tyrosinase. 243 5.3.5 Fabricating HG_gels. 243 5.3.5.1 Synthesis of modified HA. 243 5.3.5.2 Preparation of HG_gels. 244 5.3.6 Characterization of the tyrosinase-mediated hydrogels. 245 5.3.6.1 Measurement of the swelling ratio of hydrogels. 245 5.3.6.2 Rheological analysis. 245 5.3.6.3 Evaluation of gelation time of HG_gel. 246 5.3.6.4 Scanning electron microscopy (SEM) analysis. 246 5.3.7 Biocompatibility and tissue adhesiveness. 247 5.3.7.1 Live/Dead assay. 247 5.3.7.2 Synthesis of fluorescein isothiocyanate (FITC) conjugated gelatin. 247 5.3.7.3 Immunofluorescence assay. 247 5.3.7.4 Adhesion test of HG_gel to mouse skin tissue ex vivo. 248 5.3.7.5 Imaging HG_gels coated on Mouse Cardiac. 248 5.4 Results and Discussion 250 5.4.1 Structure of tyrosinase from Streptomyces avermitilis 250 5.4.2 The activities of tyrosinases from various organisms to macromolecules 253 5.4.3 Preparation of hydrogel 259 5.4.5 Swelling and in vitro degrading properties 264 5.4.6 The measurement of stickiness of HG_gel 270 5.4.7 Biocompatibility and the applications for organ coating 272 5.4.8 List of supplementary videos (Links to Google Drive) 275 5.5 Conclusion 276 6. Chapter 6. Elastin-like polypeptide for fabricating functional hydrogel 277 6.1 Abstract 278 6.2 Introduction 279 6.3 Materials and Methods 282 6.3.1 Nomenclature 282 6.3.2 Construction of plasmids for expressing ELPs 282 6.3.3 Expression of ELP in E. coli and purification by ITC 286 6.3.4 Preparation of the ECM hydrogel, HGE_gel (HG_gel + ELP) 288 6.3.5 Hydroxylation of Tyr residue of VY24V20 288 6.3.6 Preparation of mussel mimicking ELP hydrogel 289 6.3.7 Synthesis of reduced graphene oxide (rGO) 289 6.3.8 Preparation of rGO hydrogel and a thin rGO film. 290 6.4 Results and Discussion 291 6.4.1 Expression and purification of ELPs. 291 6.4.2 VY24V20 as a crosslinker of ECM hydrogel, HG_gel 294 6.4.3 Introducing DOPA moiety into gelatin and VY24V20 for mimicking the mussel foot protein 297 6.4.4 Synthesis of rGO, and fabrication of rGO-ELP hydrogel. 299 6.5 Conclusion 303 7. Chapter 7. Overall Conclusion 305 7.1 From the design of reaction paths to the mass production of functional catechol derivatives 306 7.2 Expanding the scope of target products and searching novel tyrosinase with different substrate specificity. 311 7.3 Enzymatic crosslinking for fabricating hydrogels and polymerization for producing dark pigments. 318 8. Reference 320 9. Abstract in Korean 339Docto