Publications

Background: Methane (CH4) is an attractive carbon feedstock for sustainable biomanufacturing, but poor CH4 solubility and gas-liquid mass-transfer limitations continue to constrain methanotroph-based CH4 valorization. Here, we evaluated biocompatible bionanofluids as additive-based process intensification tools and examined whether two operationally distinct bionanofluid application strategies produce distinct time-dependent physiological responses beyond simple growth enhancement.

Results: Two bionanofluids, chitosan/oleamide (CS/OA) nanoparticles and tannic acid–Fe3+ complex-coated cellulose nanocrystals (TA-Fe3+CNCs), were evaluated in 5-L cultures of Methylosinus trichosporium OB3b using two different application approaches: CS/OA seed adaptation followed by cultivation in conventional medium, and TA-Fe3+CNCs supplementation in the main culture. In both approaches, time-resolved profiling of gas uptake, nitrogen species, extracellular metabolites, and poly(3-hydroxybutyrate) (PHB) showed higher CH4 and O2 utilization, with 1.5–1.6- and 2.0–2.1-fold increases in specific CH4 and O2 uptake rates, respectively, and faster growth, with 1.3–1.5-fold higher µmax, than in the conventional control. These changes were accompanied by consistently lower PHB content, faster nitrate depletion, higher ammonium accumulation, and distinct extracellular metabolite patterns. Notably, CS/OA seed adaptation was associated with transient succinate accumulation during exponential growth, whereas TA-Fe3+CNCs supplementation led to faster methanol accumulation without succinate release. These observations are consistent with a process-level response in which higher CH4/O2 uptake and faster growth coincided with lower PHB content under the tested conditions. As an initial proof-of-concept experiment, a consecutive strategy combining CS/OA seed adaptation with TA-Fe3+CNCs supplementation in Methylomonas sp. DH-1 was associated with greater transient succinate accumulation and a higher peak succinate titer than under the conventional condition.

Conclusions: Comparative time-resolved profiling of two different bionanofluid approaches indicated that bionanofluid application was associated with higher CH4/O2 uptake, faster growth, lower PHB accumulation, and distinct time-dependent metabolite patterns under the tested conditions. These findings support the use of biocompatible bionanofluids as practical media additives that can be applied without major bioreactor redesign and can motivate future component-controlled testing of sequential strategies for growth-linked CH4 bioconversion.

Microbial bioconversion of C1-gases, such as methane and carbon monoxide, is a promising approach for sustainable carbon utilization but faces challenges due to poor gas solubility and mass-transfer limitations. To address these issues, recent studies have explored enhancing mass transfer efficiency through modifications in bioreactor configuration and design, gas diffusion-enhancing additives, and biocompatible nanofluids. This review provides an overview of various bioreactor configurations and the role of surfactants and electrolytes in improving gas solubility and enhancing the mass transfer coefficient (kLa). Additionally, nanofluids with stably suspended nanoparticles can increase gas-liquid interfacial area, promote Brownian motion and convective diffusion, and enhance gas solubility. Biocompatible nanofluids composed of functional bio-nanomaterials may further mitigate mass transfer limitations through direct interactions with microorganisms, improving C1-gas bioconversion efficiency. Future developments will integrate these strategies with advanced bioreactor configurations, enabling scalable and eco-friendly C1-gas microbial bioconversion.

Biomass-derived aromatic compounds, including those obtained from lignin (which is the most abundant aromatic biopolymer on Earth), are valuable for sustainable chemical production. Various lignin-degrading approaches have been developed to cleave recalcitrant bonds. The incorporation of biocatalysts that operate under environmentally friendly and mild conditions with high substrate specificity is considered one of the emerging strategies for lignin valorization. In this study, an archaeal tyrosinase (Tyr-CNK), derived from the marine archaeon Candidatus nitrosopumilus koreensis, is characterized as a versatile biocatalyst for lignin biodegradation and valorization, based on kinetic studies, protein structure determination, and analysis. Notably, the extremely shallow active site pocket and the unique noncanonical caddy domain, which facilitate efficient copper incorporation without obstructing the active site, collectively empower Tyr-CNK with remarkable catalytic efficiency toward various lignin model compounds, such as p-coumaric acid, 4-phenoxyphenol, 4-(benzyloxy)phenol, and guaiacyl glycerol-β-guaiacyl ether. Together with molecular docking simulations, these catalytic and structural features indicate that Tyr-CNK serves as an efficient biocatalyst for the hydroxylation and oxidative degradation of lignin-derived phenolic compounds. Given its versatility, efficiency, and structural uniqueness, Tyr-CNK demonstrates great promise for expanding the catalytic repertoire for biomass conversions and offering new opportunities in sustainable biocatalysis, enzymatic and microbial biodegradation and biomass valorization.

Human bone morphogenetic protein-2 (hBMP-2) is a growth factor extensively used to promote bone regeneration. Nonetheless, challenges such as initial burst release, low solubility and aggregation under physiological conditions, and the requirement for high doses in clinical applications can limit its effectiveness and lead to adverse side effects. In this study, a simplified and efficient hBMP-2 formulation was developed by directly forming coacervates of hBMP-2 variants with hyaluronic acid (HA) without additional hBMP-2 encapsulation steps. Extracellular matrix (ECM) peptides such as RGD, PDSGR, and YIGSR were fused to hBMP-2 to create variants, in which the intrinsic disorder of ECM peptides facilitates the coacervation of hBMP-2 variants with HA. hBMP-2-YIGSR formed stable coacervates with HA and exhibited a controlled release profile on the coacervate-coated hydroxyapatite surfaces. Furthermore, the sustained release led to significantly enhanced alkaline phosphatase activity in preosteoblast MC3T3-E1 cells. This study highlights the stable coacervate formation of hBMP-2 fused with ECM peptides with HA and establishes the potential of this formulation to reduce the initial hBMP-2 burst release and provide a controlled release for extended hBMP-2 bioactivity in bone tissue engineering applications.

Carbon monoxide (CO) serves as a promising carbon source, but it adversely affects CO-metabolizing microorganisms. To address this issue, fermentation should occur under mass-transfer limitation conditions, where the rate of gaseous CO transfer is lower than its consumption rate. However, maintaining these conditions becomes challenging as microorganism physiology deteriorates during fermentation, requiring a novel strategy to sustain mass-transfer limitation conditions. In this study, cellulose-based nanocrystals coated with a tannic acid-Fe³⁺ complex (TA-Fe³⁺CNC) were developed. These nanocrystals exhibited reversible CO adsorption/desorption characteristics, enabling the transformation from kinetic to mass-transfer limitation conditions. This approach effectively mitigated substrate inhibition from CO with negligibly impacting cell physiology. Kinetic studies demonstrated the effectiveness of TA-Fe³⁺CNCs in mitigating CO inhibition under high CO partial pressures. Furthermore, computational modeling validated the successful shifting from kinetic to mass-transfer limitation conditions at the molecular level. Overall, this study introduces a novel platform for stabilizing CO metabolism by microorganisms.

Tyrosinases can directly catalyze the biosynthesis of catechol compounds without additional cofactors under mild conditions. However, the low stability of tyrosinases in organic media has limited their use. Herein, a tyrosinase (csTyr) was identified from Citreicella sp. SE45 that possesses a characteristic feature of halophilic proteins and can be functionally expressed in Escherichia coli. Remarkably, the enzymatic activity of csTyr was effectively induced by primary alcohols in cosolvent media, whereas csTyr did not have enzymatic activity in aqueous media, minimizing unwanted reactions during tyrosinase preparation. In particular, the catalytic efficiency and stability of csTyr in a 50 % (v/v) ethanol-water cosolvent were comparable to those of other tyrosinases in water. Using csTyr as a catalyst for ortho-hydroxylation reactions of acetaminophen and resveratrol, the high catalytic activity and stability in 50 % (v/v) ethanol efficiently enabled the production of 3-hydroxy-acetaminophen and piceatannol. Therefore, csTyr, an organophilic tyrosinase, can be greatly useful in the biosynthesis of catechol compounds by addressing the issue of poor enzymatic stability in organic media.

Numerous biological systems contain vesicle-like biomolecular compartments without membranes, which contribute to diverse functions including gene regulation, stress response, signaling, and skin barrier formation. Coacervation, as a form of liquid–liquid phase separation (LLPS), is recognized as a representative precursor to the formation and assembly of membrane-less vesicle-like structures, although their formation mechanism remains unclear. In this study, a coacervation-driven membrane-less vesicle-like structure is constructed using two proteins, GG1234 (an anionic intrinsically disordered protein) and bhBMP-2 (a bioengineered human bone morphogenetic protein 2). GG1234 formed both simple coacervates by itself and complex coacervates with the relatively cationic bhBMP-2 under acidic conditions. Upon addition of dissolved bhBMP-2 to the simple coacervates of GG1234, a phase transition from spherical simple coacervates to vesicular condensates occurred via the interactions between GG1234 and bhBMP-2 on the surface of the highly viscoelastic GG1234 simple coacervates. Furthermore, the shell structure in the outer region of the GG1234/bhBMP-2 vesicular condensates exhibited gel-like properties, leading to the formation of multiphasic vesicle-like compartments. A potential mechanism is proposed for the formation of the membrane-less GG1234/bhBMP-2 vesicle-like compartments. This study provides a dynamic process underlying the formation of biomolecular multiphasic condensates, thereby enhancing the understanding of these biomolecular structures.

Chitosan-based nanofibers have become attractive biomaterials for wound healing and dressing applications based on their intrinsic biocompatibility, biodegradability, and antibacterial properties. However, the unstable spinnability of chitosan-based nanofibers has impeded further applications. In this paper, a fatty acid amide oleamide was used as a blending material for nanofiber fabrication. The addition of oleamide into chitosan moderately decreased the viscosity of the electrospinning solution, resulting in enhanced spinnability when constructing chitosan/oleamide blended nanofibers. Remarkably, the 1 : 0.5 ratio of chitosan/oleamide nanofibers exhibited relatively high hydrophobicity, decreased tensile strength, and increased elongation at break compared to chitosan-only nanofiber. The nanofiber showed similar and slightly higher cell adhesion in the in vitro cell culture with mouse preosteoblast MC3T3-E1 and fibroblast NIH/3T3 cells, respectively; however, the cell proliferation levels were decreased on the blended nanofiber surfaces, presumably due to their increased hydrophobicity. These results suggest that chitosan/oleamide nanofibers with high spinnability can be applied to the preparation of wound dressing membranes or patches with intrinsic antibacterial properties and moderate hydrophobicity. We expect that oleamide, which has lubricant and antibacterial properties, can be utilized as a blending component of chitosan-based nanofibers for biomaterial and tissue engineering applications.

Microbial biotransformation of C1 gas feedstocks such as CH₄ and CO is a notable technique for sustainable, carbon–neutral chemical and fuel production. However, low C1-gas utilization efficiency in biological processes has hampered the efficient production of value-added materials, despite efficient, nonnative strains having been recently developed. Here, we constructed a nanofluid material mainly composed of chitosan and oleamide (CS/OA), which was stably suspended with a particle size of 120.7 ± 39.0 nm in aqueous culture media below pH 7.5 and attached to the cell surface, leading to a change in hydrodynamic properties mainly due to the decrease of surface tension and a decrease in mass-transfer resistance on the cell surface. The nanofluid was applied to three C1-gas-utilizing strains, Methylomonas sp. DH-1 (a type I methanotroph), Methylosinus trichosporium OB3b (a type II methanotroph), and Thermococcus onnurineus NA1 156 T (a hyperthermophilic CO-utilizing archaeon). Changes in culture media and cell membranes from the applied CS/OA nanoparticles increased specific cell growth rates (μ_(max)). Remarkably, the strains adapted from the seed culture using the CS/OA nanofluid media also had increased μmax in a subsequent subculture and the main culture using conventional culture media, resulting in higher C1 gas consumption, cell growth, and metabolite production, such as succinate production. These results showed that the CS/OA nanofluid could be an effective medium to increase gas utilization efficiency and cell growth, and it is expected that the nanofluid can be widely used for increasing the production of value-added metabolites in C1-gas microbial biotransformation.

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 2.17-fold 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 long-term 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.

Microbial biotransformation of CH₄ gas has been attractive for the production of energy and high-value chemicals. However, insufficient supply of CH₄ in a culture medium needs to be overcome for the efficient utilization of CH₄. Here, we utilized cellulose nanocrystals coated with a tannic acid-Fe³⁺ complex (TA-Fe³⁺CNCs) as a medium component to enhance the gas-liquid mass-transfer performance. TA-Fe³⁺CNCs were well suspended in water without agglomeration, stabilized gas bubbles without coalescence, and increased the gas solubility by 20% and the k_(L)a value at a rapid inlet gas flow rate. Remarkably, the cell growth rate of Methylomonas sp. DH-1 as model CH₄-utilizing bacteria improved with TA-Fe³⁺CNC concentration without any cytotoxic or antibacterial properties, resulting in higher metabolite production ability such as methanol, pyruvate, formate, and succinate. These results showed that TA-Fe³⁺CNCs could be utilized as a significant component in the culture medium applicable as a promising nanofluid for efficient CH4 microbial biotransformation.

Chitosan/gelatin and chitosan/gelatin/nanohydroxyapatite hydrogels were rapidly and stably prepared without any crosslinking materials by using an engineered tyrosinase (mTyr-CNK) with high catalytic activity for tyrosine/DOPA-tethered polymeric biomaterials throughout a broad pH range. A dual-barrel syringe with one part containing chitosan/mTyr-CNK solution and the other containing gelatin solution with/without nanohydroxyapatite was successfully used to form homogeneous hydrogels at room temperature followed by 37 oC to simulate an in situ injection approach. The obtained hydrogels exhibited an average pore size greater than 150 m and high swelling ratios with similar mechanical properties to other chemically crosslinked chitosan/gelatin hydrogels. The in vitro degradation properties and cellular viability suggested that the hydrogels could be used as biodegradable and biocompatible scaffolds for biomedical applications, such as space filling biomaterials and delivery vehicles for bioactive molecules and cells. These results demonstrated that mTyr-CNK-mediated hydrogels have remarkable promise as an injectable scaffold biomaterial.

The exquisite structure and attractive biological properties of biominerals have great potential and increased interest for use in a wide range of medical and industrial applications. Calcium carbonate biomineralization, mainly controlled by shell matrix proteins, has been used as a representative model to understand the biomineralization mechanism. In this study, in vitro calcium carbonate crystallization was carried out under room temperature and atmospheric pressure using recombinant shell matrix protein GRP_BA and artificial shell matrix protein GG1234. Both proteins inhibited the growth of typical rhombohedral calcite crystals in the calcium carbonate crystallization using CaCl2 solution and (NH4)2CO3 vapor, and spherulitic calcite crystals with rosette-like structures were synthesized in both the presence of GRP_BA and GG1234. These results might be caused by the properties of block-like domain structure and intrinsically disordered proteins. We expect that this study can contribute to enhance understanding of the calcium carbonate biomineralization controlled by shell matrix proteins.

An in situ injectable chitosan/gelatin hydrogel was formed under slightly acidic conditions (pH 4.0   4.5) using an acid-tolerant tyrosinase, tyrosinase-CNK. A homogeneous chitosan/tyrosinase-CNK solution was prepared in one part of a dual-barrel syringe, and highly soluble gelatin in distilled water was prepared in the other part of the syringe without any additional crosslinking materials. Chitosan/gelatin hydrogel was formed in situ by simple injection of the solutions at room temperature followed by curing at 37°C. However, conventional mushroom tyrosinase did not catalyze this permanent gel formation. Tyrosinase-CNK-catalyzed glycol chitosan/gelatin hydrogel was similarly formed by this in situ injection approach. The hydrogels exhibited a high swelling ratio of 20-fold their own weight, interconnected micropores with an average diameter of approximately 260 μm and in vitro biodegradability suitable for tissue engineering and drug delivery applications. These results showed that tyrosinase-CNKmediated chitosan/gelatin hydrogel formation has remarkable potential for the development of novel formulations for in situ injectable gel-forming systems.

With the help of recombinant DNA technology, many protein candidates have been investigated and engineered for biomaterial applications. Particularly, several repeat sequences with unique secondary structures have been selected as minimal building blocks for biosynthesis to improve the mechanical properties of biomaterials. However, most of these structural proteins have been limited to silk, elastin, collagen, and resilin for decades. In the present work, new repeat sequence found in sea anemone are characterized and biosynthesized into a recombinant protein (named anegen) for potential use as a load-bearing biomaterial. Because its repeat sequence unit has a unique polyproline II structure, which is prevalently found in the triple-helix of collagen, it is assumed to be a promising structural protein candidate that can provide conformational flexibility and elasticity. Because anegen has  10% tyrosine residues, inspiration is taken from di-tyrosine crosslinking in the hinge structures of insects, which can be initiated by light activation. It is found that the anegen hydrogel shows higher mechanical properties than a gelatin hydrogel and endures a compression series without deformation. Moreover, the mechanical properties of the anegen hydrogel are controllable through different crosslinking conditions in a wide range of material applications. Importantly, the anegen hydrogel exhibited suitable cell retainability and cell morphology as an implantable biomaterial. Thus, based on its mechanical properties and biocompatibility, the anegen hydrogel can be used as a potential load-bearing and cell-loading scaffolding biomaterial in the tissue and biomedical engineering fields.

Tyrosinases, as a representative type-3 copper enzyme (two copper ions in the active site), catalyze the orthohydroxylation of monophenols and the oxidation of diphenols. It is ubiquitously distributed in various organisms all around the nature world. The catalytic mechanism of tyrosinases was intensively studied for a long time. On the other hand, some structural data have been reported recently. This review summarizes the structural information of overall threedimensional structure and active site region. The overall structure can be divided into three domains and some characteristic conserved sequences are observed, although tyrosinases do not have a common structure in all species. The catalytic mechanism is based on the structural characteristics that are significantly important to understand the functional properties in tyrosinases, which can be efficiently applied into industrial and biomedical applications of the enzymes.

An acid-tolerant tyrosinase, tyrosinase-CNK, was used to form chitosan/gelatin hydrogels without any additional crosslinking materials at pH 4, where appropriate chitosan/gelatin blends were efficiently prepared based on the solubility of chitosan only under acidic conditions. The in vitro gelation degree and rheological studies revealed that the gelation of the chitosan/gelatin solution was successfully achieved within 12 min by the tyrosinase-CNK reaction at pH 4, and the glycol chitosan/gelatin blends were similarly crosslinked within 7 min at pH 6 (the optimum pH of tyrosinase-CNK). Remarkably, tyrosinase-CNK catalyzed the formation of permanent and stable chitosan/gelatin and glycol chitosan/gelatin gels, unlike the previous results of tyrosinase-mediated chitosan/gelatin gelation in which commercially available mushroom tyrosinase only catalyzed transient gel formation. In addition, the pore size distribution was greater than 100 m in the morphological structure, and the high swelling ratio and enhanced mechanical properties are expected to be suitable for tissue engineering applications. These results demonstrate that the acid-tolerant tyrosinase-CNK can efficiently mediate the in vitro formation of chitosan/gelatin conjugates, and the obtained hydrogels have the potential to be used as a promising scaffold.

Tyrosinase efficiently catalyzes the ortho-hydroxylation of monophenols and the oxidation of diphenols without any additional cofactors. Although it is of significant interest for the biosynthesis of catechol derivatives, the rapid catechol oxidase activity and inactivation of tyrosinase have hampered its practical utilization as a monophenol monooxygenase. Here, we prepared a functional tyrosinase that exhibited a distinguished monophenolase/diphenolase activity ratio (Vmax mono/ Vmax di = 3.83) and enhanced catalytic efficiency against L-tyrosine (kcat = 3.33 ± 0.18 s−1, Km = 2.12 ± 0.14 mM at 20 °C and pH 6.0). This enzyme was still highly active in ice water (>80%), and its activity was well conserved below 30 °C. In vitro DOPA modification, with a remarkably high yield as a monophenol monooxygenase, was achieved by the enzyme taking advantage of these biocatalytic properties. These results demonstrate the strong potential for this enzyme’s use as a monophenol monooxygenase in biomedical and industrial applications.

Molluscan nacre is a fascinating biomineral consisting of a highly organized calcium carbonate composite that provides unique fracture toughness and an iridescent color. Organisms elaborately control the biomineralization using organic macromolecules. Here, we propose the involvement of the matrix protein Pif80 from the pearl oyster Pinctada fucata in the development of the inorganic phase during nacre biomineralization, based on experiments employing the recombinant form of Pif80. Through interactions with calcium ions, Pif80 participates in the formation of polymer-induced liquid precursor-like amorphous calcium carbonate granules and stabilizes these granules by forming calcium ion-induced coacervates. At the calcification site, the disruption of Pif80 coacervates destabilizes the amorphous mineral precursors, resulting in the growth of a crystalline structure. The re-dissolved Pif80 controls the growth of aragonite on the polysaccharide substrate, which contributes to the formation of polygonal tablet structure of nacre. Our findings provide insight into the use of organic macromolecules by living organisms in biomineralization.

Coacervation is a liquid-liquid phase separation process of macromolecular polyelectrolytes. The formation of simple and complex coacervations of a synthetic acidic protein, GG1234, as a model shell matrix protein, was investigated with turbidity measurements and microscopic morphological observations. Simple coacervation of GG1234 was optimally induced at pH 3.75 and below 50 mM for all of the tested salts, and complex coacervates were prepared at pH 4–9 in sodium acetate solution at various ratios of GG1234 to lysozyme without inducing simple coacervation. The complex coacervates also had the ability to microencapsulate hydrophobic oil droplets in a similar manner to other complex coacervation systems. Remarkably, a thin film formed through in vitro CaCO₃ crystallization in the presence of complex coacervates, which was expected to be planar and poorly crystalline CaCO₃ guided at the interface of two immiscible liquid phases upon complex coacervation. Collectively, our results indicate that our synthetic acidic matrix protein could be used as the main protein for simple and complex coacervations, and the coacervates of this protein might be involved in thin film formation in CaCO₃ crystallization.

Primary fatty acid amides are commonly found in grasses, microalgae, and animal. Oleamide and erucamide are fatty acid amide derivatives of oleic and brassidic acids, respectively. They are the most frequently used slip agents in industrial applications. We evaluated their potential application as antifouling coatings on submerged surfaces. The deterrent properties of parafilm wax plates containing oleamide and erucamide against the surface recruitment of diatoms and biofilms and the attachment strength of abalones were assessed. Both amides were effective, but oleamide had 1.4-2.5-fold greater deterrent effects against all three groups of foulers. The amides produced slippery surfaces to which the biofoulers attached weakly. The benthic organisms were readily dislodged by water motion. Thus, oleamide has potential as an ingredient in antifoulant coating materials. We also developed a rapid abalone detachment assay that can be used to test the efficacy of antifouling agents against slow-growing members of the macrobenthos.

Tyrosinases catalyze the hydroxylation of a monophenol (monophenolase activity) and the conversion of an o-diphenol to o-quinone (diphenolase activity), which are mainly involved in the modification of tyrosine residues into 3,4-dihydroxyphenyl-alanine (DOPA) and DOPA/DOPAquinone-derived intermolecular cross-linking. Previously, we obtained a slightly acidic and cold-active tyrosinase, tyrosinase-CNK, by our recombinant protein approach. The enzyme showed optimal activity at pH 6.0 and 20 ℃ with an abnormally high monophenolase/diphenolase activity ratio and still had approximately 50% activity compared with the highest activity even in ice water. Here, we investigated reaction stability of the recombinant tyrosinase-CNK as a psychrophilic enzyme. The enzyme showed remarkable thermal stability at 0 ℃ and the activity was well conserved in repeated freeze-thaw cycles. Although water-miscible organic solvent as reaction media caused the activity decrease of tyrosinase-CNK as expected, the enzyme activity was not additionally decreased with increased concentration in organic solvents such as ethanol and acetonitrile. Also, the enzyme showed high salt tolerance in chaotropic salts. It was remarkably considered that 2+ metal ions might inhibit the incorporation of Cu2+ into the active site. We expect that these results could be used to design tyrosinase-mediated enzymatic reaction at low temperature for the production of catechols through minimizing unwanted self-oxidation and enzyme inactivation.

Recent adhesive technologies have focused on the development of high-quality and eco-friendly adhesives. Thus, there is a gradual shift from the currently used chemical-based adhesives toward harmless adhesives with improved quality and performance. Here, we evaluated the potential use of bacteria-produced recombinant mussel adhesive protein (MAP) as a harmless wood furniture adhesive. We formulated a MAP wood adhesive as an inclusion body type for economical preparation, and we confirmed its harmlessness through the non-detection of volatile organic compounds and heavy metals. The formulated MAP showed sufficiently strong bulk adhesive strength for the dried gluing of wood adherends. We also found that the formulated MAP wood adhesive exhibits robust adhesion in various environmental conditions, including open assembly times, incubation times, temperatures, and humidity levels. In summary, the developed recombinant MAP could be successfully used as a promising environmentally-friendly, harmless wood furniture adhesive.

In nature, marine mussels (Mytilus edulis) suffer less fouling colonization on the newly formed sides of their shells. Using settlement assays with algal spores of Porphyra suborbiculata, we determined that spore attachment and germination on the periostracum decreased to 36.8 and 3.3 %, respectively. Additionally, the spore settlement was considerably diminished by periostracum dichloromethane extracts containing 19 % oleamide, a major antifouling compound. A scanning electron micrograph of the surface revealed a regular ripple structure with approximately 1.4 μm between ripples. Based on these results, mussel periostraca or their associated biomimetic materials may become environmentally friendly, antifouling agents for preventing the settlement of soft foulants.

Objectives: The purpose of our study was to obtain an acidic and cold-active tyrosinase, which potentially minimizes unwanted self-oxidation of tyrosinase-catalyzed catechols including 3,4-dihydroxyphenyl-alanine (DOPA) at elevated pH and high temperature.

Results: A putative psychrophilic tyrosinase (named as tyrosinase-CNK) was identified from the genome information of the marine archaeon Candidatus Nitrosopumilus koreensis in the Arctic Circle. This protein contains key tyrosinase domains such as copper-binding domains and an oxygen-binding motif, and phylogenetic analysis revealed that it was distinct from other known bacterial tyrosinase types. Functional tyrosinase-CNK was successfully produced by applying a co-expression strategy together with chaperone proteins in E. coli with the final yield of approximately 30 mg L-1 and a purity greater than 95% in native conditions in a 50-mL flask culture. The purified enzyme showed optimal activity at pH 6.0 and 20 ºC and still had 50% activity compared with the highest activity in ice water. Surprisingly, the enzyme exhibited an abnormally high monophenolase/diphenolase activity ratio.

Conclusions: The acidic and cold-adapted tyrosinase-CNK, as a new type of tyrosinase, could expand potential applications of tyrosinases including the production of catechols through minimizing unwanted self-oxidation and the modification of existing materials at low temperature.

The biological structure of shells consists of highly organized calcium carbonate (CaCO3) crystals, which have remarkable mechanical and biological properties compared to the pure mineral form of CaCO3. It has been known that the organization of these biominerals is controlled by a relatively tiny amount of organic components such as shell matrix proteins. Here, we successfully produced a recombinant hypothetical acidic shell matrix protein in Escherichia coli, although the protein is composed of highly repetitive and biased amino acid sequences. About 15mg/L purified protein with greater than 95% purity was obtained in the 400 mL lab scale flask culture. The protein was able to efficiently form a complex with calcium ions, and spherulitic calcite crystals were synthesized in the presence of the recombinant protein. We expect that biomineralization using the recombinant protein could not only overcome the limited amount of protein available for biomineralization studies and biomineral preparation in practical aspects, but also provide opportunities to enable biomimetic synthesis of notable bio-composites based on organic-inorganic complexation.

Objectives: To overcome the limited production capability of shell matrix proteins and efficiently conduct in vitro CaCO3 biomineralization studies, a putative recombinant shell matrix protein was prepared and simply characterized.

Results: A glycine-rich protein (named GRP_BA) was found as a putative shell matrix protein (NCBI reference sequence; BAA20465), and the protein was genetically redesigned for the recombinant production in Escherichia coli. The recombinant protein was successfully obtained in the bacterial expression system with the final yield of approximately 30 mg L-1 and the purity of above 95% in a 400-mL lab scale flask culture. This protein was able to efficiently form a complex with calcium ions (Ca2+), and Ca2+-induced agglomeration was observed like other calcification-related proteins. Spherulitic calcite micro-particles of 20-30 µm diameter with rosette- and sphere-like structures were synthesized in the presence of the recombinant shell protein, which could be formed by stacking and/or aggregation of calcite nanograins and the bound protein.

Conclusions: Recombinant production of the shell matrix protein could overcome potential difficulties associated with limited amount of protein available for biomineralization studies and provide opportunities to fabricate notable biominerals in practical aspects.

Detection of inflammation in live cells is important because long-lasting inflammation is considered to be a primary cause of several diseases. However, few reports have been published on imaging analysis of inflammation in live cells. In this study, we developed an effective imaging system for detection of inflamed cells using a bradykinin ligand (BK) or a modified BK (mBK), which has specific affinity with the cellular B1R receptor. Synthetic BK or mBK labeled with FITC at the N-terminus was employed for discriminating between inflamed and normal cells; this method was found to be effective for detection of inflammation in live cells. In addition, using the mBK-based cell imaging system, we successfully per- formed flow-based analysis of live cell inflammation on a micro-chip channel, composed of a Starna flow cell and PDMS (Polydimethylsiloxane) walls. The BK-based cell imaging methods designed here would be a useful platform for development of a high-throughput live cell analysis system for investigating the factors underlying inflammation or for screening of anti-inflammation candidate drugs.

Carbonic anhydrase (CA) is a biocatalyst for CO2 sequestration because of its distinctive ability to accelerate CO2 hydration. High production and efficient immobilization of alkaline-active CAs are required, because one potential application of CA is its use in the alkaline solvent-based CO2 absorption/desorption process. Here, we designed and applied an a-type CA from Hahella chejuensis (HCA), which was reported as highly active in alkaline conditions, but was mostly expressed as insoluble forms. We found that the signal peptide-removed form of HCA [HCA(SP-)] was successfully expressed in the soluble form [70 mg of purified HCA(SP-) per L of culture]. HCA(SP-) also displayed high pH stability in alkaline conditions, with maximal activity at pH 10; at this pH, 90% activity was maintained for 2 h. Then, we prepared HCA(SP-)-encapsulated silica particles [HCA(SP-)@silica] via a spermine-mediated bio-inspired silicification method. HCA(SP-)@silica exhibited high-loading and highly stable CA activity. In addition, HCA(SP-)@silica retained more than 90% of the CA activity even after 10 cycles of use in mild conditions, and 80% in pH 10 conditions. These results will be useful for the development of practical CO2 sequestration processes employing CA.