Spontaneous Transition of Spherical Coacervate to Vesicle-Like Compartment.

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.

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

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 (Vmax mono/Vmax 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.

Cellulose nanocrystals coated with a tannic acid-Fe3+ complex as a significant medium for efficient CH4 microbial biotransformation.

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 kLa 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.

Bioinspired Load-Bearing Hydrogel Based on Engineered Sea Anemone Skin-Derived Collagen-Like Protein.

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.

A tyrosinase, mTyr-CNK, that is functionally available as a monophenol monooxygenase.

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 (V max mono/ V max di = 3.83) and enhanced catalytic efficiency against L-tyrosine (k cat = 3.33 ± 0.18 s-1, K m = 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.

Control of nacre biomineralization by Pif80 in pearl oyster.

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 biomineralization using organic macromolecules. 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 using 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 redissolved 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.

Correction: Comparison of Therapeutic Effect of Extracorporeal Shock Wave in Calcific Versus Noncalcific Lateral Epicondylopathy.

[This corrects the article on p. 294 in vol. 40, PMID: 27152280.].

Comparison of Therapeutic Effect of Extracorporeal Shock Wave in Calcific Versus Noncalcific Lateral Epicondylopathy.

OBJECTIVE: To assess the therapeutic effect of extracorporeal shock wave therapy (ESWT) in lateral epicondylopathy with calcification, and compare it to the effect of ESWT in lateral epicondylopathy without calcification. METHODS: A retrospective study was conducted. Forty-three patients (19 with calcific and 24 with noncalcific lateral epicondylopathy in ultrasound imaging) were included. Clinical evaluations included the 100-point score, Nirschl Pain Phase scale before and after ESWT, and Roles and Maudsley (R&M) scores after ESWT. ESWT (2,000 impulses and 0.06-0.12 mJ/mm(2)) was performed once a week for 4 weeks. RESULTS: The 100-point score and Nirschl Pain Phase scale changed significantly over time (p<0.001), but there was no significant difference between groups (p=0.555). The R&M scores at 3 and 6 months after ESWT were not significantly different between groups. In the presence of a tendon tear, those in the calcific lateral epicondylopathy group showed poor improvement of 100-point scores compared to the noncalcific group (p=0.004). CONCLUSION: This study demonstrated that the therapeutic effect of ESWT in calcific lateral epicondylopathy was not significantly different from that in noncalcific lateral epicondylopathy. When a tendon tear is present, patients with calcific lateral epicondylopathy might show poor prognosis after ESWT relative to patients with noncalcific lateral epicondylopathy.

A cold-adapted tyrosinase with an abnormally high monophenolase/diphenolase activity ratio originating from the marine archaeon Candidatus Nitrosopumilus koreensis.

OBJECTIVES: To obtain an acidic and cold-active tyrosinase, which potentially minimizes unwanted self-oxidation of tyrosinase-catalyzed catechols, including 3,4-dihydroxyphenylalanine 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. This protein contains key tyrosinase domains, such as copper-binding domains and an O2-binding motif, and phylogenetic analysis revealed that it was distinct from other known bacterial tyrosinases. Functional tyrosinase-CNK was produced by applying a co-expression strategy together with chaperone proteins in Escherichia coli with a yield of approx. 30 mg l(-1) and a purity >95 %. The purified enzyme showed optimal activity at pH 6 and 20 °C and still had 50 % activity at 0 °C. 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.

Recombinant production of a shell matrix protein in Escherichia coli and its application to the biomimetic synthesis of spherulitic calcite crystals.

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 characterized. RESULTS: A glycine-rich protein (GRP_BA) was found in Pinctada fucata as a putative shell matrix protein (NCBI reference sequence; BAA20465). It was genetically redesigned for the production in Escherichia coli. The recombinant protein was obtained in a 400 ml shake-flask culture at approx. 30 mg l(-1) with a purity of >95 %. It efficiently formed a complex with Ca(2+). Ca(2+)-induced agglomeration was like other calcification-related proteins. Spherulitic calcite micro-particles, 20-30 µm diam. 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 a shell matrix protein could overcome potential difficulties associated with the limited amount of protein available for biomineralization studies and provide opportunities to fabricate biominerals in practical aspects.

High expression and biosilica encapsulation of alkaline-active carbonic anhydrase for CO2 sequestration system development.

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 α-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 [∼70mg 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 2h. 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.

Highly effective detection of inflamed cells using a modified bradykinin ligand labeled with FITC fluorescence.

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 performed 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.

Biocatalytic conversion of methane to methanol as a key step for development of methane-based biorefineries.

Methane is considered as a next-generation carbon feedstock owing to the vast reserves of natural and shale gas. Methane can be converted to methanol by various methods, which in turn can be used as a starting chemical for the production of value-added chemicals using existing chemical conversion processes. Methane monooxygenase is the key enzyme that catalyzes the addition of oxygen to methane. Methanotrophic bacteria can transform methane to methanol by inhibiting methanol dehydrogenase. In this paper, we review the recent progress made on the biocatalytic conversion of methane to methanol as a key step for methane-based refinery systems and discuss future prospects for this technology.

In vivo residue-specific dopa-incorporated engineered mussel bioglue with enhanced adhesion and water resistance.

Misaminoacylation of 3,4-dihydroxyphenylalanine (Dopa) molecules to tRNA(Tyr) by endogenous tyrosyl-tRNA synthetase allowed the quantitative replacement of tyrosine residues with a yield of over 90 % by an in vivo residue-specific incorporation strategy, to create, for the first time, engineered mussel adhesive proteins (MAPs) in Escherichia coli with a very high Dopa content, close to that of natural MAPs. The Dopa-incorporated MAPs exhibited a superior surface adhesion and water resistance ability by assistance of Dopa-mediated interactions including the oxidative Dopa cross-linking, and furthermore, showed underwater adhesive properties comparable to those of natural MAPs. These results propose promising use of Dopa-incorporated engineered MAPs as bioglues or adhesive hydrogels for practical underwater applications.

Comparison of Ecklonia cava, Ecklonia stolonifera and Eisenia bicyclis for phlorotannin extraction.

Phlorotannins are polyphenols of marine algae, particularly brown seaweed, having multiple biological activities. A reverse phase-high performance liquid chromatography method was developed for rapid and routine quantification of two major phlorotannins, dieckol and phlorofucofuroeckol-A (PFE-A), from boiling water- and organic solvent-extracts of brown seaweeds Ecklonia cava, E. stolonifera and Eisenia bicyclis. The regression equations for dieckol and PFE-A were as follows: the concentration (mg ml(-1)) = 16.56 x peak height (cm) + 0.44, and the concentration = 20.60 x peak height (cm) + 0.11, with correlation coefficients of 0.996 and 0.999, respectively. Compared to organic solvent extraction, the recovery yield of dieckol from boiling water extracts of E. cava, E. stolonifera and E. bicyclis was 86%, 93%, and 98%, respectively. The recovery yield of PFE-A was 74%, 86% and 62%, respectively. Antioxidant activity was detected in each E. bicyclis water extract (91%), followed by E. stolonifera (90%) and E. cava (74%). Dieckol and PFE-A showed almost 9- and 7-fold stronger antioxidant activity than the standard butylhydroxytoluene, and 6-and 4-fold greater than L-ascorbic acid in molar concentration, respectively.

Characterization of the N-oxygenase AurF from Streptomyces thioletus.

AurF catalyzes the N-oxidation of p-aminobenzoic acid to p-nitrobenzoic acid in the biosynthesis of the antibiotic aureothin. Here we report the characterization of AurF under optimized conditions to explore its potential use in biocatalysis. The pH optimum of the enzyme was established to be 5.5 using phenazine methosulfate (PMS)/NADH as the enzyme mediator system, showing ~10-fold higher activity than previous reports in literature. Kinetic characterization at optimized conditions give a Km of 14.7 ± 1.1 µM, a kcat of 47.5 ± 5.4 min(-1) and a kcat/Km of 3.2 ± 0.4 µM(-1)min(-1). PMS/NADH and the native electron transfer proteins showed significant formation of the p-hydroxylaminobenzoic acid intermediate, however H2O2 produced mostly p-nitrobenzoic acid. Alanine scanning identified the role of important active site residues. The substrate specificity of AurF was examined and rationalized based on the protein crystal structure. Kinetic studies indicate that the Km is the main determinant of AurF activity toward alternative substrates.

In vitro evolution of glutathione S-transferase using a plasmid display system based on the GAL4 DNA-binding domain.

Enzyme characteristics, such as thermal stability and catalytic activity, can be improved in a targeted manner. However, the screening of target mutants is time-consuming and requires highly experimental downstream efforts. Here, we describe a simple strategy based on plasmid display and limited proteolysis for the rapid and easy screening of highly stable and active mutants from a library. When glutathione S-transferase was used as a model enzyme, the resulting mutants obtained in the first round of screening were approximately two- to sevenfold more thermostable than the wild-type enzyme at 50 °C, with similar enzyme activity. This methodology is therefore powerful for the in vitro enrichment and screening of thermostable and active mutants. It can reduce downstream experimental effort and can create a high-quality library using relatively simple steps.

A comparative study on the bulk adhesive strength of the recombinant mussel adhesive protein fp-3.

Mussel adhesive protein (MAP) type 3 (fp-3) is considered one of the key components for mussel adhesion. However, its bulk adhesive strength has not been characterized due to its availability in limited quantities. In the present work, a feasible production (~47 mg l(-1)) of recombinant fp-3 was achieved, and its bulk adhesive strength was measured for the first time; ~0.57 MPa for the unmodified form and ~0.94 and ~2.28 MPa for the 3,4-dihydroxy-L-phenylalanine (DOPA)-modified form, having a 9.6% yield without and with oxidant treatment, respectively. Furthermore, values for the bulk adhesive strength of several DOPA-modified recombinant MAPs were compared. The maximum adhesive strength of DOPA-modified fp-3 after oxidant treatment was stronger than that of type 5 (fp-5), which has a 6.2% modification yield, and was comparable to that of hybrid types fp-131 and fp-151, which have similar yields (~5%). The strong bulk adhesive property of recombinant fp-3 demonstrates its potential use as a promising bioadhesive.

In vivo modification of tyrosine residues in recombinant mussel adhesive protein by tyrosinase co-expression in Escherichia coli.

BACKGROUND: In nature, mussel adhesive proteins (MAPs) show remarkable adhesive properties, biocompatibility, and biodegradability. Thus, they have been considered promising adhesive biomaterials for various biomedical and industrial applications. However, limited production of natural MAPs has hampered their practical applications. Recombinant production in bacterial cells could be one alternative to obtain useable amounts of MAPs, although additional post-translational modification of tyrosine residues into 3,4-dihydroxyphenyl-alanine (Dopa) and Dopaquinone is required. The superior properties of MAPs are mainly attributed to the introduction of quinone-derived intermolecular cross-links. To solve this problem, we utilized a co-expression strategy of recombinant MAP and tyrosinase in Escherichia coli to successfully modify tyrosine residues in vivo. RESULTS: A recombinant hybrid MAP, fp-151, was used as a target for in vivo modification, and a dual vector system of pET and pACYC-Duet provided co-expression of fp-151 and tyrosinase. As a result, fp-151 was over-expressed and mainly obtained from the soluble fraction in the co-expression system. Without tyrosinase co-expression, fp-151 was over-expressed in an insoluble form in inclusion bodies. The modification of tyrosine residues in the soluble-expressed fp-151 was clearly observed from nitroblue tetrazolium staining and liquid-chromatography-mass/mass spectrometry analyses. The purified, in vivo modified, fp-151 from the co-expression system showed approximately 4-fold higher bulk-scale adhesive strength compared to in vitro tyrosinase-treated fp-151. CONCLUSION: Here, we reported a co-expression system to obtain in vivo modified MAP; additional in vitro tyrosinase modification was not needed to obtain adhesive properties and the in vivo modified MAP showed superior adhesive strength compared to in vitro modified protein. It is expected that this co-expression strategy will accelerate the use of functional MAPs in practical applications and can be successfully applied to prepare other Dopa/Dopaquinone-based biomaterials.

Biomineralization-based conversion of carbon dioxide to calcium carbonate using recombinant carbonic anhydrase.

Recently, as a mimic of the natural biomineralization process, the use of carbonic anhydrase (CA), which is an enzyme catalyzing fast reversible hydration of carbon dioxide to bicarbonate, has been suggested for biological conversion of CO(2) to valuable chemicals. While purified bovine CA (BCA) has been used in previous studies, its practical utilization in CO(2) conversion has been limited due to the expense of BCA preparation. In the present work, we investigated conversion of CO(2) into calcium carbonate as a target carbonate mineral by using a more economical, recombinant CA. To our knowledge, this is the first report of the usage of recombinant CA for biological CO(2) conversion. Recombinant α-type CA originating in Neisseria gonorrhoeae (NCA) was highly expressed as a soluble form in Escherichia coli. We found that purified recombinant NCA which showed comparable CO(2) hydration activity to commercial BCA significantly promoted formation of solid CaCO(3) through the acceleration of CO(2) hydration rate, which is naturally slow. In addition, the rate of calcite crystal formation was also accelerated using recombinant NCA. Moreover, non-purified crude recombinant NCA also showed relatively significant ability. Therefore, recombinant CA could be an effective, economical biocatalyst in practical CO(2) conversion system.