Kenjiro Yazawa

Recent Advances in Chemoenzymatic Peptide Syntheses

Chemoenzymatic peptide synthesis is the hydrolase-catalyzed stereoselective formation of peptide bonds. It is a clean and mild procedure, unlike conventional chemical synthesis, which involves complicated and laborious protection-deprotection procedures and harsh reaction conditions. The chemoenzymatic approach has been utilized for several decades because determining the optimal conditions for conventional synthesis is often time-consuming. The synthesis of poly- and oligopeptides comprising various amino acids longer than a dipeptide continues to pose a challenge owing to the lack of knowledge about enzymatic mechanisms and owing to difficulty in optimizing the pH, temperature, and other reaction conditions. These drawbacks limit the applications of the chemoenzymatic approach. Recently, a variety of enzymes and substrates produced using recombinant techniques, substrate mimetics, and optimal reaction conditions (e.g., frozen aqueous media and ionic liquids) have broadened the scope of chemoenzymatic peptide syntheses. In this review, we highlight the recent advances in the chemoenzymatic syntheses of various peptides and their use in developing new materials and biomedical applications.

Relationships between physical properties and sequence in silkworm silks.

Silk has attracted widespread attention due to its superlative material properties and promising applications. However, the determinants behind the variations in material properties among different types of silk are not well understood. We analysed the physical properties of silk samples from a variety of silkmoth cocoons, including domesticated Bombyx mori varieties and several species from Saturniidae. Tensile deformation tests, thermal analyses, and investigations on crystalline structure and orientation of the fibres were performed. The results showed that saturniid silks produce more highly-defined structural transitions compared to B. mori, as seen in the yielding and strain hardening events during tensile deformation and in the changes observed during thermal analyses. These observations were analysed in terms of the constituent fibroin sequences, which in B. mori are predicted to produce heterogeneous structures, whereas the strictly modular repeats of the saturniid sequences are hypothesized to produce structures that respond in a concerted manner. Within saturniid fibroins, thermal stability was found to correlate with the abundance of poly-alanine residues, whereas differences in fibre extensibility can be related to varying ratios of GGX motifs versus bulky hydrophobic residues in the amorphous phase.

Influence of Water Content on the β-Sheet Formation, Thermal Stability, Water Removal, and Mechanical Properties of Silk Materials

Silk, which has excellent mechanical toughness and is lightweight, is used as a structural material in nature, for example, in silkworm cocoons and spider draglines. However, the industrial use of silk as a structural material has garnered little attention. For silk to be used as a structural material, its thermal processability and associated properties must be well understood. Although water molecules influence the glass transition of silk, the effects of water content on the other thermal properties of silks are not well understood. In this study, we prepared Bombyx mori cocoon raw fibers, degummed fibers, and films with different water contents and then investigated the effects of water content on crystallization, degradation, and water removal during thermal processing. Thermal gravimetric analyses of the silk materials showed that water content did not affect the thermal degradation temperature but did influence the water removal behavior. By increasing the water content of silk, the water molecules were removed at lower temperatures, indicating that the amount of free water in silk materials increased; additionally, the glass transition temperature decreased with increasing water plasticization. Differential scanning calorimetry and wide-angle X-ray scattering of the silk films also suggested that the water molecules in the amorphous regions of the silk films acted as a plasticizer and induced β-sheet crystallization. The plasticizing effect of water was not detected in silk fibers, owing to their lower amorphous content and mobility. The structural and mechanical characterizations of the silk films demonstrated the silk film prepared at RH 97% realized both crystallinity and ductility simultaneously. Thus, the thermal stability, mechanical, and other properties of silk materials are regulated by their water content and crystallinity.

The Benzyl Ester Group of Amino Acid Monomers Enhances Substrate Affinity and Broadens the Substrate Specificity of the Enzyme Catalyst in Chemoenzymatic Copolymerization

The chemoenzymatic polymerization of amino acid monomers by proteases involves a two-step reaction: the formation of a covalent acyl-intermediate complex between the protease and the carboxyl ester group of the monomer and the subsequent deacylation of the complex by aminolysis to form a peptide bond. Although the initiation with the ester group of the monomer is an important step, the influence of the ester group on the polymerization has not been studied in detail. Herein, we studied the effect of the ester groups (methyl, ethyl, benzyl, and tert-butyl esters) of alanine and glycine on the synthesis of peptides using papain as the catalyst. Alanine and glycine were selected as monomers because of their substantially different affinities toward papain. The efficiency of the polymerization of alanine and glycine benzyl esters was much greater than that of the other esters. The benzyl ester group therefore allowed papain to equally polymerize alanine and glycine, even though the affinity of alanine toward papain is substantially higher. The characterization of the copolymers of alanine and glycine in terms of the secondary structure and thermal properties revealed that the thermal stability of the peptides depends on the amino acid composition and resultant secondary structure. The current results indicate that the nature of the ester group drastically affects the polymerization efficiency and broadens the substrate specificity of the protease.

Papain-Catalyzed Synthesis of Polyglutamate Containing a Nylon Monomer Unit

Peptides have the potential to serve as an alternative for petroleum-based polymers to support a sustainable society. However, they lack thermoplasticity, owing to their strong intermolecular interactions. In contrast, nylon is famous for its thermoplasticity and chemical resistance. Here, we synthesized peptides containing a nylon unit to modify their thermal properties by using papain-catalyzed chemoenzymatic polymerization. We used l-glutamic acid alkyl ester as the amino acid monomer and nylon 1, 3, 4, and 6 alkyl esters as the nylon unit. Papain catalyzed the copolymerization of glutamic acid with nylon 3, 4, and 6 alkyl esters, whereas the nylon 1 unit could not be copolymerized. Other proteases used in this study, namely, bromelain, proteinase K, and Candida antarctica lipase (CALB), were not able to copolymerize with any nylon units. The broad substrate specificity of papain enabled the copolymerization of l-glutamic acid with a nylon unit. The peptides with nylon units demonstrated different thermal profiles from that of oligo(l-glutamic acid). Therefore, the resultant peptides with various nylon units are expected to form fewer intermolecular hydrogen bonds, thus altering their thermal properties. This finding is expected to broaden the applications of peptide materials and chemoenzymatic polymerization.

Real-time Monitoring of Intermediates Reveals the Reaction Pathway in the Thiol-Disulfide Exchange between Disulfide Bond Formation Protein A (DsbA) and B (DsbB) on a Membrane-immobilized Quartz Crystal Microbalance (QCM) System

Background: A device of QCM can be used in the transient kinetics of oxidation of a pair of cysteine residues in DsbA by DsbB. Results: The obtained kinetic parameters indicate that the two pairs of cysteine residues in DsbB are important. Conclusion: The reaction pathway of almost all DsbA oxidation processes would proceed through the stable intermediate. Significance: The transient kinetics of the reaction intermediate is important. Disulfide bond formation protein B (DsbBS-S,S-S) is an inner membrane protein in Escherichia coli that has two disulfide bonds (S-S, S-S) that play a role in oxidization of a pair of cysteine residues (SH, SH) in disulfide bond formation protein A (DsbASH,SH). The oxidized DsbAS-S, with one disulfide bond (S-S), can oxidize proteins with SH groups for maturation of a folding preprotein. Here, we have described the transient kinetics of the oxidation reaction between DsbASH,SH and DsbBS-S,S-S. We immobilized DsbBS-S,S-S embedded in lipid bilayers on the surface of a 27-MHz quartz crystal microbalance (QCM) device to detect both formation and degradation of the reaction intermediate (DsbA-DsbB), formed via intermolecular disulfide bonds, as a mass change in real time. The obtained kinetic parameters (intermediate formation, reverse, and oxidation rate constants (kf, kr, and kcat, respectively) indicated that the two pairs of cysteine residues in DsbBS-S,S-S were more important for the stability of the DsbA-DsbB intermediate than ubiquinone, an electron acceptor for DsbBS-S,S-S. Our data suggested that the reaction pathway of almost all DsbASH,SH oxidation processes would proceed through this stable intermediate, avoiding the requirement for ubiquinone.

Morphological and mechanical properties of flexible resilin joints on damselfly wings (Rhinocypha spp.)

Resilin functions as an elastic spring that demonstrates extraordinary extensibility and elasticity. Here we use combined techniques, laser scanning confocal microscopy (LSCM) and scanning electron microscopy (SEM) to illuminate the structure and study the function of wing flexibility in damselflies, focusing on the genus Rhinocypha. Morphological studies using LSCM and SEM revealed that resilin patches and cuticular spikes were widespread along the longitudinal veins on both dorsal and ventral wing surfaces. Nanoindentation was performed by using atomic force microscopy (AFM), where the wing samples were divided into three sections (membrane of the wing, mobile and immobile joints). The resulting topographic images revealed the presence of various sizes of nanostructures for all sample sections. The elasticity range values were: membrane (0.04 to 0.16 GPa), mobile joint (1.1 to 2.0 GPa) and immobile joint (1.8 to 6.0 GPa). The elastomeric and glycine-rich biopolymer, resilin was shown to be an important protein responsible for the elasticity and wing flexibility.

Combination of Amorphous Silk Fiber Spinning and Postspinning Crystallization for Tough Regenerated Silk Fibers

An artificial spinning system using regenerated silk fibroin solutions is adopted to produce high-performance silk fibers. In previous studies, alcohol-based agents, such as methanol or ethanol, were used to coagulate silk dope solutions, producing silk fiber with poor mechanical properties compared with those of native silk fibers. The alcohol-based coagulation agents induce rapid β-sheet crystallization of the silk molecules, which inhibits subsequent alignment of the β-sheet crystals. Here, we induce gradual β-sheet formation to afford adequate β-sheet alignment similar to that of native silk fiber. To this aim, we developed an amorphous silk fiber spinning process that prevents fast β-sheet formation in silk molecules by using tetrahydrofuran (THF) as a coagulation solvent. In addition, we apply postdrawing to the predominantly amorphous silk fibers to induce β-sheet formation and orientation. The resultant silk fibers showed a 2.5-fold higher extensibility, resulting in 1.5-fold tougher silk fibers compared with native Bombyx mori silk fiber. The amorphous silk fiber spinning process developed here will pave the way to the production of silk fibers with desired mechanical properties.

Two Birds with One Stone: Spontaneous Size Separation and Growth Inhibition of Femtosecond Laser-Generated Surfactant-Free Metallic Nanoparticles via ex Situ SU-8 Functionalization

Laser ablation in liquids (LAL) offers a facile technique to develop a large variety of surfactant-free nanomaterials with high purity. However, due to the difficulty in the control of the particle synthesis process, the as-prepared nanomaterials always have a broad size distribution with a large polydispersity (σ). Surfactant-free properties can also cause problems with particle growth, which further increases the difficulty in size control of the colloids. Therefore, searching for strategies to simultaneously unify the sizes of colloids and inhibit particle growth has become significantly important for LAL-synthesized nanomaterials to be extensively used for biological, catalytic, and optical applications, in which fields particle size plays an important role. In this work, we present a facile way to simultaneously realize these two goals by ex situ SU-8 photoresist functionalization. Ag nanoparticles (NPs) synthesized by femtosecond laser ablation of silver in acetone at laser powers of 300 and 600 mW were used as starting materials. The synthesized Ag NPs have a broad size distribution between 1 and 200 nm with an average size of ca. 5.9 nm and σ of 127–207%. After ex situ SU-8 functionalization and 6 months storage, most particles larger than 10 nm become aggregates and precipitate, which makes the size distribution narrow with an average diameter of 4–5 nm and σ of 48–78%. The precipitation process is accompanied by the decrease in colloid mass from the initial ∼0.2 to 0.10–0.11 mg after ex situ SU-8 functionalization and 6 months colloid storage. Morphology analysis indicates that ex situ SU-8 functionalization inhibits the particle growth into polygonal nanocrystals. Radical polymerization of SU-8 on Ag NPs is considered to be the reason for both spontaneous size separation and growth inhibition phenomena. Benefiting from Ag NPs embedment and acetone dissolution, the glass-transition temperature of SU-8 photoresist increased from 314 to 331 °C according to thermogravimetric analysis. The universality of ex situ SU-8 functionalization-induced growth inhibition and size separation behaviors is further proved using the Au colloids generated by LAL in acetone. This work is expected to provide a new route for better size control of LAL-synthesized colloids via ex situ photoresist functionalization, although a half of colloidal mass is wasted due to radical polymerization-induced colloidal precipitation.

Probing Multiple Binding Modes of DNA Hybridization: A Comparison between Single-Molecule Observations and Ensemble Measurements

Interactions between biomolecules are generally analyzed by ensemble measurements, assuming that the interactions occur in a single binding manner. However, such interactions may occur via multiple binding modes. We investigated the kinetics of DNA hybridization as a multiple dynamic model of biomolecular interactions. Two kinetic analyses were performed with a single-molecule observation using total internal reflection fluorescence microscopy (TIRFM) and with ensemble measurements using a quartz-crystal microbalance (QCM) biosensor. We observed the DNA hybridization of 8 and 12 bp DNAs with random sequences and dA12–dT12 and calculated the kinetic parameters, including the dissociation rate constant (koff). Hybridization of 8 bp DNA proceeded mainly via a single binding mode. However, hybridization of 12 bp DNA indicated at least two different binding modes and dA12–dT12 hybridization showed multiple binding modes. For the multiple binding interactions, the kinetic parameters obtained from TIRFM and QCM were different because kinetic parameters obtained from QCM indicate average number of molecules, whereas those from TIRFM indicate average association time. The present study revealed the details of multiple interactions, which can be utilized for better understanding of not only DNA hybridization but also biomolecular interaction mechanisms.