Highly efficient site-specific protein modification using tyrosinase from Streptomyces avermitilis: Structural insight.

Tyrosinase-mediated protein conjugation has recently drawn attention as a site-specific protein modification tool under mild conditions. However, the tyrosinases reported to date act only on extremely exposed tyrosine residues, which limits where the target tyrosine can be located. Herein, we report a tyrosinase from Streptomyces avermitilis (SaTYR), that exhibits a much higher activity against tyrosine residues on the protein surface than other tyrosinases. We determined the crystal structure of SaTYR and revealed that the enzyme has a relatively flat and shallow substrate-binding pocket to accommodate a protein substrate. We demonstrated SaTYR-mediated fluorescence dye tagging and PEGylation of a surface tyrosine residue that was unreacted by other tyrosinases with an approximately 95.2 % conjugation yield in 1 h. We also present a structural rationale that considers the steric hindrance from adjacent residues and surrounding structures along with the extent of solvent exposure of residues, as necessary when determining the optimal positions for introducing target tyrosine residues in SaTYR-mediated protein modification. The study demonstrated that the novel tyrosinase, SaTYR, extends the scope of tyrosinase-mediated protein modification, and we propose that site-specific tyrosine conjugation using SaTYR is a promising strategy for protein bioconjugation in various applications.

Development of a Stable Peptide-Major Histocompatibility Complex (MHC) via Sortase and Click Chemistry.

T cells play a crucial role in antitumor immune responses and the clearance of infected cells. They identify their targets through the binding of T-cell receptors (TCRs) to peptide-major histocompatibility complex (pMHC) molecules present in cancer cells, infected cells, and antigen-presenting cells. This interaction is often weak, requiring multimeric pMHC molecules to enhance the avidity for identifying antigen-specific T cells. Current exchangeable pMHC-I tetramerization methods may overlook TCRs recognizing less stable yet immunogenic peptides. In vivo applications targeting antigen-specific T cells demand the genetic synthesis of a pMHC fusion for each unique peptide antigen, which poses a significant challenge. To address these challenges, we developed a sortase and click chemistry-mediated approach for generating stable pMHC molecules. Leveraging sortase technology, we introduced an azide click-handle near the N-terminus of ß2m, proximal to the MHC-peptide-binding groove. Simultaneously, the peptide was engineered with a multi glycine linker and a C-terminal alkyne click-handle. Azide-alkyne click reactions efficiently immobilized the peptide onto the MHC molecule, providing a versatile and efficient method for pMHC generation. The resulting peptide-clicked-MHC specifically binds to its cognate TCR and remains stable for over 3 months at 4 °C in the absence of any additional free peptide. The stability of the pMHC and its affinity to cognate TCRs are influenced by the linker's nature and length. Multi glycine linkers outperform poly(ethylene glycol) (PEG) linkers in this regard. This technology expands the toolkit for identifying and targeting antigen-specific T cells, enhancing our understanding of cancer-specific immune responses, and has the potential to streamline the development of personalized immunotherapies.

Recent trends in the modification of polyphenolic compounds using hydroxylation and glycosylation.

Polyphenols are bioactive molecules that are used in therapeutics. Polyphenol hydroxylation and glycosylation have been shown to increase their bioavailability, solubility, bioactivity, and stability for use in various applications. Ortho-hydroxylation of polyphenols using tyrosinase allows high selectivity and yield without requiring a cofactor, while meta- and para-hydroxylation of polyphenols are mediated by site-specific hydroxylases and cytochrome P450s, although these processes are somewhat rare. O-glycosylation of polyphenols proceeds further after hydroxylation. The O-glycosylation reaction typically requires nucleotide diphosphate (NDP) sugar. However, amylosucrase (AS) has emerged as a promising enzyme for polyphenol glycosylation in large-scale production without requiring NDP-sugar. Overall, this review describes recent findings on the enzymatic mechanisms, enzyme engineering, and applications of enzymatic reactions.

Single-Walled Carbon Nanotube-Guided Topical Skin Delivery of Tyrosinase to Prevent Photoinduced Damage.

When the skin is exposed to ultraviolet radiation (UV), it leads to the degradation of the extracellular matrix (ECM) and results in inflammation. Subsequently, melanocytes are triggered to induce tyrosinase-mediated melanin synthesis, protecting the skin. Here, we introduce a proactive approach to protect the skin from photodamage via the topical delivery of Streptomyces avermitilis-derived tyrosinase (SaTy) using single-walled carbon nanotube (SWNT). Utilizing a reverse electrodialysis (RED) battery, we facilitated the delivery of SaTy-SWNT complexes up to depths of approximately 300 µm, as analyzed by using confocal Raman microscopy. When applied to ex vivo porcine skin and in vivo albino mouse skin, SaTy-SWNT synthesized melanin, resulting in 4-fold greater UV/vis absorption at 475 nm than in mice without SaTy-SWNT. The synthesized melanin efficiently absorbed UV light and alleviated skin inflammation. In addition, the densification of dermal collagen, achieved through SaTy-mediated cross-linking, reduced photoinduced wrinkles by 66.3% in the affected area. Our findings suggest that SWNT-mediated topical protein delivery holds promise in tissue engineering applications.

Synthesis of soluble melanin nanoparticles under acidic conditions using Burkholderia cepacia tyrosinase and their characterization.

Melanin nanoparticles (MNPs) used for biomedical applications are often synthesized via the chemical auto-oxidation of catecholic monomers such as dopamine and 3,4-dihydroxyphenylalanine (DOPA) under alkaline conditions. However, the synthetic method for the chemical synthesis of MNP (cMNP) is relatively straightforward and more robust to control their homogenous particle size and morphology than the corresponding enzymatic synthetic methods. In this study, we demonstrated that the simple enzymatic synthesis of MNPs (eMNPs) with homogenous and soluble (<20 nm diameter) properties is possible using dopamine and Burkholderia cepacia tyrosinase (BcTy) under acidic conditions (i.e., pH 3.0). BcTy was highly reactive under pH 5.0, where the natural and chemical oxidation of catechol is complex, and thus melanin was synthesized via the hydroxylation of phenolic substrates. The detailed chemical analysis and characterization of the physical properties of the eMNPs confirmed the higher preservation of the catechol and primary amine moieties in the monomer substrate such as dopamine under acidic conditions. The eMNPs showed enhanced antioxidant activity and conferred stickiness to the formed hydrogel compared to the chemical auto-oxidation method owing to the large number of hydroxyl groups remaining such as catechol and quinone moieties. Because of these advantages and characteristics, the synthesis of MNPs using BcTy under acidic conditions can open a new path for their biomedical applications.

Light-Triggered In Situ Biosynthesis of Artificial Melanin for Skin Protection.

Tyrosinase-mediated melanin synthesis is an essential biological process that can protect skin from UV radiation and radical species. This work reports on in situ biosynthesis of artificial melanin in native skin using photoactivatable tyrosinase (PaTy). The I41Y mutant of Streptomyces avermitilis tyrosinase (SaTy) shows enzymatic activity comparable to that of wild-type SaTy. This Y41 is replaced with photocleavable o-nitrobenzyl tyrosine (ONBY) using the introduction of amber codon and ONBY-tRNA synthetase/tRNA pairs. The ONBY efficiently blocks the active site and tyrosinase activity is rapidly recovered by the photo-cleavage of ONBY. The activated PaTy successfully oxidizes L-tyrosine and tyramine-conjugated hyaluronic acid (HA_T) to synthesize melanin particles and hydrogel, respectively. To produce artificial melanin in living tissues, PaTy is encapsulated into lipid nanoparticles as an artificial melanosome. Using liposomes containing PaTy (PaTy_Lip), PaTy is transdermally delivered into ex vivo porcine skin and in vivo mouse skin tissues, thus achieving the in situ biosynthesis of artificial melanin for skin tissue protection under UV irradiation. The results of this study demonstrate that this biomimetic system can recapitulate the biosynthetic analogs of naturally occurring melanin. It should therefore be considered to be a promising strategy for producing protective biological molecules within living systems for tissue protection.

Regioselective One-Pot Synthesis of Hydroxy-(S)-Equols Using Isoflavonoid Reductases and Monooxygenases and Evaluation of the Hydroxyequol Derivatives as Selective Estrogen Receptor Modulators and Antioxidants.

Several regiospecific enantiomers of hydroxy-(S)-equol (HE) were enzymatically synthesized from daidzein and genistein using consecutive reduction (four daidzein-to-equol-converting reductases) and oxidation (4-hydroxyphenylacetate 3-monooxygenase, HpaBC). Despite the natural occurrence of several HEs, most of them had not been studied owing to the lack of their preparation methods. Herein, the one-pot synthesis pathway of 6-hydroxyequol (6HE) was developed using HpaBC (EcHpaB) from Escherichia coli and (S)-equol-producing E. coli, previously developed by our group. Based on docking analysis of the substrate or products, a potential active site and several key residues for substrate binding were predicted to interpret the (S)-equol hydroxylation regioselectivity of EcHpaB. Through investigating mutations on the key residues, the T292A variant was verified to display specific mono-ortho-hydroxylation activity at C6 without further 3'-hydroxylation. In the consecutive oxidoreductive bioconversion using T292A, 0.95 mM 6HE could be synthesized from 1 mM daidzein, while 5HE and 3'HE were also prepared from genistein and 3'-hydroxydaidzein (3'HD or 3'-ODI), respectively. In the following efficacy tests, 3'HE and 6HE showed about 30∼200-fold higher EC50 than (S)-equol in both ERα and ERß, and they did not have significant SERM efficacy except 6HE showing 10% lower ß/α ratio response than that of 17ß-estradiol. In DPPH radical scavenging assay, 3'HE showed the highest antioxidative activity among the examined isoflavone derivatives: more than 40% higher than the well-known 3'HD. In conclusion, we demonstrated that HEs could be produced efficiently and regioselectively through the one-pot bioconversion platform and evaluated estrogenic and antioxidative activities of each HE regio-isomer for the first time.

Rationally Designed Eugenol-Based Chain Extender for Self-Healing Polyurethane Elastomers.

Bio-based polyurethane (PU) has recently drawn our attention due to the increasing interest in sustainability and the risks involved with petroleum depletion. Herein, bio-based self-healing PU with a novel polyol, i.e., eugenol glycol dimer (EGD), was synthesized and characterized for the first time. EGD was designed to have pairs of primary, secondary, and aromatic alcohols, which all are able to be involved in urethane bond formation and to show self-healing and antioxidant effects. EGD was incorporated into a mixture of the prepolymer of polyol (tetramethylene ether glycol) and 4,4'-methylene diphenyl diisocyanate to synthesize PU. EGD-PU showed excellent self-healing properties (99.84%), and it maintained its high self-healing property (84.71%) even after three repeated tests. This dramatic self-healing was induced through transcarbamoylation by the pendant hydroxyl groups of EGD-PU. The excellent antioxidant effect of EGD-PU was confirmed by 2,2-diphenyl-1-picrylhydrazyl analysis. Eugenol-based EGD is a promising polyol chain extender that is required in the production of bio-based, self-healing, and recyclable polyurethane; therefore, EGD-PU can be applied to bio-based self-healable films or coating materials as a substitute for petroleum-based PU.

Polyphenol-Hydroxylating Tyrosinase Activity under Acidic pH Enables Efficient Synthesis of Plant Catechols and Gallols.

Tyrosinase is generally known as a melanin-forming enzyme, facilitating monooxygenation of phenols, oxidation of catechols into quinones, and finally generating biological melanin. As a homologous form of tyrosinase in plants, plant polyphenol oxidases perform the same oxidation reactions specifically toward plant polyphenols. Recent studies reported synthetic strategies for large scale preparation of hydroxylated plant polyphenols, using bacterial tyrosinases rather than plant polyphenol oxidase or other monooxygenases, by leveraging its robust monophenolase activity and broad substrate specificity. Herein, we report a novel synthesis of functional plant polyphenols, especially quercetin and myricetin from kaempferol, using screened bacterial tyrosinases. The critical bottleneck of the biocatalysis was identified as instability of the catechol and gallol under neutral and basic conditions. To overcome such instability of the products, the tyrosinase reaction proceeded under acidic conditions. Under mild acidic conditions supplemented with reducing agents, a bacterial tyrosinase from Bacillus megaterium (BmTy) displayed efficient consecutive two-step monophenolase activities producing quercetin and myricetin from kaempferol. Furthermore, the broad substrate specificity of BmTy toward diverse polyphenols enabled us to achieve the first biosynthesis of tricetin and 3'-hydroxyeriodictyol from apigenin and naringenin, respectively. These results suggest that microbial tyrosinase is a useful biocatalyst to prepare plant polyphenolic catechols and gallols with high productivity, which were hardly achieved by using other monooxygenases such as cytochrome P450s.

Novel enzymatic cross-linking-based hydrogel nanofilm caging system on pancreatic ß cell spheroid for long-term blood glucose regulation.

Pancreatic ß cell therapy for type 1 diabetes is limited by low cell survival rate owing to physical stress and aggressive host immune response. In this study, we demonstrate a multilayer hydrogel nanofilm caging strategy capable of protecting cells from high shear stress and reducing immune response by interfering cell-cell interaction. Hydrogel nanofilm is fabricated by monophenol-modified glycol chitosan and hyaluronic acid that cross-link each other to form a nanothin hydrogel film on the cell surface via tyrosinase-mediated reactions. Furthermore, hydrogel nanofilm formation was conducted on mouse ß cell spheroids for the islet transplantation application. The cytoprotective effect against physical stress and the immune protective effect were evaluated. Last, caged mouse ß cell spheroids were transplanted into the type 1 diabetes mouse model and successfully regulated its blood glucose level. Overall, our enzymatic cross-linking-based hydrogel nanofilm caging method will provide a new platform for clinical applications of cell-based therapies.

Fabrication of polyphenol-incorporated anti-inflammatory hydrogel via high-affinity enzymatic crosslinking for wet tissue adhesion.

Epigallocatechin gallates (EGCGs), isolated from green tea, have intrinsic properties such as anti-oxidant, anti-inflammation, and radical scavenger effects. In this study, we report a tissue adhesive and anti-inflammatory hydrogel formed by high-affinity enzymatic crosslinking of polyphenolic EGCGs. A mixture of EGCG conjugated hyaluronic acids (HA_E) and tyramine conjugated hyaluronic acids (HA_T) was reacted with tyrosinase isolated from Streptomyces avermitillis (SA_Ty) to form that displayed fast enzyme kinetic to form a crosslinked adhesive hydrogel. A 1,2,3-trihydroxyphenyl group in EGCG displayed a high affinity to SA_Ty that allowed HA_E to be quickly oxidized and crosslinked with HA_T to form HA_T and HA_E mixed hydrogel (HA_TE). We then compared the HA_TE hydrogel with commercially available tissue adhesives, such as cyanoacrylate and fibrin glue. We report that the HA_TE exhibited the highest tissue adhesiveness both in wet and dry conditions. Furthermore, HA_TE successfully closed a skin wound and displayed insignificant host tissue responses. This demonstrates that polyphenol-incorporated anti-inflammatory hydrogel may provide a robust tissue adhesive platform for clinical applications.

The Electrochemical Performances of n-Type Extended Lattice Spaced Si Negative Electrodes for Lithium-Ion Batteries.

The electrochemical performances of lithium-ion batteries with different lattice-spacing Si negative electrodes were investigated. To achieve a homogeneous distribution of impurities in the Si anodes, single crystalline Si wafers with As-dopant were ball-milled to form irregular and agglomerated micro-flakes with an average size of ~10 µm. The structural analysis proved that the As-doped Si negative materials retain the increased lattice constant, thus, keep the existence of the residual tensile stress of around 1.7 GPa compared with undoped Si anode. Electrochemical characterization showed that the As-doped Si anodes have lower discharge capacity, but Coulombic efficiency and capacity retention were improved in contrast with those of the undoped one. This improvement of electrochemical characteristics was attributed to the increased potential barrier on the side of Si anodes, inherited from the electronic and mechanical nature of Si materials doped with As. We believe that this study will guide us the way to optimize the electrochemical performances of LIBs with Si-based anodes.

Recent advances in the microbial hydroxylation and reduction of soy isoflavones.

Soy isoflavones are naturally occurring phytochemicals, which are biotransformed into functional derivatives through oxidative and reductive metabolic pathways of diverse microorganisms. Such representative derivatives, ortho-dihydroxyisoflavones (ODIs) and equols, have attracted great attention for their versatile health benefits since they were found from soybean fermented foods and human intestinal fluids. Recently, scientists in food technology, nutrition and microbiology began to understand their correct biosynthetic pathways and nutraceutical values, and have attempted to produce the valuable bioactive compounds using microbial fermentation and whole-cell/enzyme-based biotransformation. Furthermore, artificial design of microbial catalysts and/or protein engineering of oxidoreductases were also conducted to enhance production efficiency and regioselectivity of products. This minireview summarizes and introduces the past year's studies and recent advances in notable production of ODIs and equols, and provides information on available microbial species and their catalytic performance with perspectives on industrial application.