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Polyethylene terephthalate (PET) is a major component of plastic waste. Enzymatic PET hydrolysis is the most ecofriendly recycling technology. The biorecycling of PET waste requires the complete depolymerization of PET to terephthalate and ethylene glycol. The history of enzymatic PET depolymerization has revealed two critical issues for the industrial depolymerization of PET: industrially available PET hydrolases and pretreatment of PET waste to make it susceptible to full enzymatic hydrolysis. As none of the wild-type enzymes can satisfy the requirements for industrialization, various mutational improvements have been performed, through classical technology to state-of-the-art computational/machine-learning technology. Recent engineering studies on PET hydrolases have brought a new insight that flexibility of the substrate-binding groove may improve the efficiency of PET hydrolysis while maintaining sufficient thermostability, although the previous studies focused only on enzymatic thermostability above the glass transition temperature of PET. Industrial biorecycling of PET waste is scheduled to be implemented, using micronized amorphous PET. Next stage must be the development of PET hydrolases that can efficiently degrade crystalline parts of PET and expansion of target PET materials, not only bottles but also textiles, packages, and microplastics. This review discusses the current status of PET hydrolases, their potential applications, and their profespectal goals. Key points: • PET hydrolases must be thermophilic, but their operation must be below 70 °C • Classical and state-of-the-art engineering approaches are useful for PET hydrolases • Enzyme activity on crystalline PET is most expected for future PET biorecycling [ABSTRACT FROM AUTHOR]

The enzymatic recycling of polyethylene terephthalate (PET) can be a promising approach to tackle the problem of plastic waste. The thermostability and activity of PET-hydrolyzing enzymes are still insufficient for practical application. Pretreatment of PET waste is needed for bio-recycling. Here, we analyzed the degradation of PET films, packages, and bottles using the newly engineered cutinase Cut190. Using gel permeation chromatography and high-performance liquid chromatography, the degradation of PET films by the Cut190 variant was shown to proceed via a repeating two-step hydrolysis process; initial endo-type scission of a surface polymer chain, followed by exo-type hydrolysis to produce mono/bis(2-hydroxyethyl) terephthalate and terephthalate from the ends of fragmented polymer molecules. Amorphous PET powders were degraded more than twofold higher than amorphous PET film with the same weight. Moreover, homogenization of post-consumer PET products, such as packages and bottles, increased their degradability, indicating the importance of surface area for the enzymatic hydrolysis of PET. In addition, it was required to maintain an alkaline pH to enable continuous enzymatic hydrolysis, by increasing the buffer concentration (HEPES, pH 9.0) depending on the level of the acidic products formed. The cationic surfactant dodecyltrimethylammonium chloride promoted PET degradation via adsorption on the PET surface and binding to the anionic surface of the Cut190 variant. The Cut190 variant also hydrolyzed polyethylene furanoate. Using the best performing Cut190 variant (L136F/Q138A/S226P/R228S/D250C-E296C/Q123H/N202H/K305del/L306del/N307del) and amorphous PET powders, more than 90 mM degradation products were obtained in 3 days and approximately 80 mM in 1 day. Key points: The increased surface area of PET promotes its hydrolysis by Cut190, supporting the surface erosion mechanism by a two-step process (endo-type scission of a polymer chain and exo-type hydrolysis of depolymerized fragments). Dodecyltrimethylammonium chloride functioned as a binding module between Cut190 and PET surface, showing the higher hydrolysis rate at 65 °C than at 70 °C in the absence of the detergent. The best performing Cut190 variant produced more than 90 mM degradation products at 63 °C in 3 days and approximately 80 mM at 65 °C in one day, using amorphous PET powders. Additionally, polyethylene furanoate was highly hydrolyzed at 63 °C in 3 days by the Cut190 variant. [ABSTRACT FROM AUTHOR]

RNA helicases are enzymes that generally unwind double-stranded RNA using ATP hydrolysis energy, mainly involved in RNA metabolism, transcription, translation, and mRNA splicing. While the helicase core is crucial for RNA unwinding activity, N- and C-terminal extensions of specific helicases may contain an intrinsically disordered region for electrostatic interaction, resulting in the formation of droplets in the cytoplasm. However, how the disordered region of the RNA helicase contributes to RNA unwinding and dissociation remains unclear. Here, we focused on Bombyx mori Vasa, which unwinds truncated target transposon RNAs from the piRNA-induced silencing complex piRISC. In this study, we used single-molecule techniques to visualise how Vasa dynamically interacts with piRISC and investigate how Vasa oligomerization is involved in the process of piRNA amplification, named the ping-pong pathway. We found that Vasa's oligomerization is required during these processes in vitro and in vivo, and that Vasa triggers the dissociation of truncated RNA in heterogeneous pathways. Our single-molecule results suggest that oligomerized Vasa guides the timing of the process regulating overall dissociation efficiency. Kinoshita et al. use single molecule imaging to show that the N-terminal extension of BmVasa, a germ-specific DEAD box RNA helicase, is necessary for protein oligomerization and the dissociation of target RNA from the Siwi-piRISC complex. The authors conclude that oligomerized Vasa guides the timing of the regulation of overall dissociation efficiency. [ABSTRACT FROM AUTHOR]

We conducted a comparative analysis of the effects of beryllium fluoride (BeFx) on protein folding mediated by the α- and β-subunit homooligomers (α16mer or β16mer) from the hyperthermophilic archaeum Thermococcus strain KS-1. BeFx inhibited the ATPase activities of both α16mer and β16mer with equal efficiency. This indicated that BeFx replaces the γ-phosphate of chaperonin-bound ATP, thereby forming a stable chaperonin–ADP–BeFx complex. In the presence of ATP and BeFx, both of the two chaperonin subunits mediated green fluorescent protein (GFP) folding. Gel filtration chromatography revealed that the refolded GFP was retained by both chaperonins. Protease digestion and electron microscopic analyses showed that both chaperonin–ADP–BeFx complexes of the two subunits adopted a symmetric closed conformation with the built-in lids of both rings closed and that protein folding took place in their central cavities. These data indicated that basic protein folding mechanisms of α16mer and β16mer are likely similar although there were some apparent differences. While β16mer-mediated GFP refolding in the presence of ATP–BeFx that proceeded more rapidly than in the presence of ATP alone and reached a twofold higher plateau than that achieved with AMP–PNP, the folding mediated by the α16mer that proceeded with much lower yields. A mutant of α16mer, trapα, which traps the unfolded and partially folded substrate protein, did not affect the ATP–BeFx-dependent GFP folding by β16mer but it suppressed that mediated by α16mer to the level of spontaneous folding. These results suggested that β16mer differed from the α16mer in nucleotide binding affinity or the rate of nucleotide hydrolysis. [ABSTRACT FROM AUTHOR]

Chaperonin is a double ring-shaped oligomeric protein complex, which captures a protein in the folding intermediate state and assists its folding in an ATP-dependent manner. The chaperonin from a hyperthermophilic archaeum, Thermococcus sp. strain KS-1, is a group II chaperonin and is composed of two distinct subunits, α and β. Although these subunits are highly homologous in sequence, the homo-oligomer of the β-subunit is more thermostable than that of the α-subunit. To identify the region responsible for this difference in thermostability, we constructed domain-exchange mutants. The mutants containing the equatorial domain of the β-subunit were more resistant to thermal dissociation than the mutants with that of the α-subunit. Thermostability of a β-subunit mutant whose C-terminal 22 residues were replaced with those of the α-subunit decreased to the comparable level of that of the α-subunit homo-oligomer. These results indicate that the difference in thermostability between α- and β-subunits mainly originates in the C-terminal residues in the equatorial domain, only where they exhibit substantial sequence difference. [ABSTRACT FROM AUTHOR]

The nucleosome including H2A.B, a mammalian-specific H2A variant, plays pivotal roles in spermatogenesis, embryogenesis, and oncogenesis, indicating unique involvement in transcriptional regulation distinct from canonical H2A nucleosomes. Despite its significance, the exact regulatory mechanism remains elusive. This study utilized solid-state nanopores to investigate DNA unwinding dynamics, applying local force between DNA and histones. Comparative analysis of canonical H2A and H2A.B nucleosomes demonstrated that the H2A.B variant required a lower voltage for complete DNA unwinding. Furthermore, synchronization analysis and molecular dynamics simulations indicate that the H2A.B variant rapidly unwinds DNA, causing the H2A-H2B dimer to dissociate from DNA immediately upon disassembly of the histone octamer. In contrast, canonical H2A nucleosomes unwind DNA at a slower rate, suggesting that the H2A-H2B dimer undergoes a state of stacking at the pore. These findings suggest that nucleosomal DNA in the H2A.B nucleosomes undergoes a DNA unwinding process involving histone octamer disassembly distinct from that of canonical H2A nucleosomes, enabling smoother unwinding. The integrated approach of MD simulations and nanopore measurements is expected to evolve into a versatile tool for studying molecular interactions, not only within nucleosomes but also through the forced dissociation of DNA-protein complexes. Research combining molecular dynamics simulations and solid-state nanopore measurements reveals distinct disassembly pathways that may explain the distinct properties and roles of H2A.B nucleosomes in spermatogenesis, embryogenesis, and oncogenesis. [ABSTRACT FROM AUTHOR]