Nucleoside disaccharides are essential glycosides that naturally occur in specific living organisms. This study developed an enhanced UDP-glucose regeneration system to facilitate the in vitro multienzyme synthesis of nucleoside disaccharides by integrating it with nucleoside-specific glycosyltransferases. The system utilizes maltodextrin and polyphosphate as cost-effective substrates for UDP-glucose supply, catalyzed by α-glucan phosphorylase (αGP) and UDP-glucose pyrophosphorylase (UGP). To address the low activity of known polyphosphate kinases (PPKs) in the UDP phosphorylation reaction, a sequence-driven screening identified RhPPK with high activity against UDP (>1000 U/mg). Computational design further led to the creation of a double mutant with a 2566-fold increase in thermostability at 50 °C. The enhanced UDP-glucose regeneration system increased the production rate of nucleoside disaccharide synthesis by 25-fold. In addition, our UDP-glucose regeneration system is expected to be applied to other glycosyl transfer reactions.

Acidic xylanase PjxA from Penicillium janthinellum MA21601, with good eosinophilic and enzymatic activity, is an excellent candidate for xylan degradation to achieve effective utilization of biomass materials. However, the low thermal stability of PjxA has become a major bottleneck in its application. In this study, the flexible sites of PjxA were identified and rigidified through computational simulations of structure and sequence analysis combined with folding free energy calculations. Finally, a combined mutase PjxA-DS was constructed by rational integration of the two single mutants S82N and D45N. Compared to PjxA, PjxA-DS showed a 115.11-fold longer half-life at 50 °C and a 2.02-fold higher specific enzyme activity. Computer simulation analysis showed that S82N and D45N acted synergistically to improve the thermostability of PjxA. The stabilization of the N-terminus and the active center of PjxA, the increase in surface positive charge and hydrophilicity are the main reasons for the improved thermostability and catalytic activity of PjxA. Rigidification of the flexible site is an effective method for improving the thermostability of enzymes, S82N and D45N can be used as effective targets for the thermostability engineering modification of GH11 acidic xylanase.

The L-isoleucine-4-dioxygenase converts L-isoleucine (Ile) into(2S,3R,4S)-4-(OH)-isoleucine (4-HIL), a naturally occurring hydroxyl amino acid, which is a promising compound for drug and functional food development. Here, a novel L-isoleucine-4-dioxygenase (RaIDO) from Rahnella aquatilis was cloned, expressed and characterized, as one of only a few reported L-isoleucine-4-dioxygenases. RaIDO showed high catalytic efficiency with Ile as the substrate, as well as good stability. HPLC-MS and NMR confirmed that RaIDO converts Ile into (2S,3R,4S)-4-(OH)-isoleucine. Further, structural analysis of RaIDO revealed key active site residues, including H159, D161 and H212. The RaIDO enzyme showed an optimal reaction temperature range of 30°C-45°C, with the highest catalytic activity observed at 40°C. Additionally, the enzyme exhibited an optimal pH of 8.0. Thus, the novel L-isoleucine-4-dioxygenase (RaIDO) has high catalytic efficiency and good stability, making it a strong candidate for industrial applications.

In recent years, driven by ever-increasing application of energetic materials in deep-seated mineral resource exploitation and aerospace engineering, the mining of advanced safe energetic materials with significant thermal stability has drawn widespread publicity. Here, a tricyclic bridged energetic compound 2-amino-4,6-bis(3,5-diamino-4-nitropyrazol-1-yl)-1,3,5-triazine (NPX-03) was prepared using simple synthetic route. Furthermore, an interesting highly thermostable nitrogen-rich perchlorate, NPX-03·2HClO4, was prepared by the self-assembly reaction of NPX-03 and HClO4, displaying a thermal decomposition peak temperature of 375.9 °C. Moreover, NPX-03·2HClO4 exhibits good detonation velocity (D = 8187 m s-1) and insensitivity (IS = 50 J, FS > 360 N), thereby being promising candidates for advanced insensitive high-energy materials.

Molecular photoswitches provide interesting tools to reversibly control various biological functions with light. Thanks to its small size and easy introduction into the biomolecules, azobenzene derivatives have been widely employed in the field of photopharmacology. All visible-light switchable azobenzenes with controllable thermostability are highly demanded. Based on the reported tetra-o-chloroazobenzenes, we synthesized push-pull systems, by introducing dialkyl amine and nitro groups as strong electron-donating and electron-withdrawing groups on the para-positions, and then transformed to push-push systems by a simple reduction step. The developed push-pull and push-push tetra-o-chloroazobenzene derivatives displayed excellent photoswitching properties, as previously reported. The half-life of the Z-isomers can be tuned from milliseconds for the push-pull system to several hours for the push-push system. The n-π* and π-π* transitions have better resolution in the push-push molecules, and excitation at different wavelengths can tune the E/Z ratio at the photostationary state. For one push-pull molecule, structure and absorption spectra obtained from theoretical calculations are compared with experimental data, along with data on the push-push counterpart.

Methanogenic archaea are chemolithotrophic prokaryotes that can reduce carbon dioxide with hydrogen gas to form methane. These microorganisms make a significant contribution to the global carbon cycle, with methanogenic archaea from anoxic environments estimated to contribute > 500 million tons of global methane annually. Archaeal methanogenesis is dependent on the methanofurans; aminomethylfuran containing coenzymes that act as the primary C1 acceptor molecule during carbon dioxide fixation. Although the biosynthetic pathway to the methanofurans has been elucidated, structural adaptations which confer thermotolerance to Mfn enzymes from extremophilic archaea are yet to be investigated. Here we focus on the methanofuran biosynthetic enzyme MfnB, which catalyses the condensation of two molecules of glyceralde-3-phosphate to form 4‑(hydroxymethyl)-2-furancarboxaldehyde-phosphate. In this study, MfnB enzymes from the hyperthermophile Methanocaldococcus jannaschii and the mesophile Methanococcus maripaludis have been recombinantly overexpressed and purified to homogeneity. Thermal unfolding studies, together with steady-state kinetic assays, demonstrate thermoadaptation in the M. jannaschii enzyme. Molecular dynamics simulations have been used to provide a structural explanation for the observed properties. These reveal a greater number of side chain interactions in the M. jannaschii enzyme, which may confer protection from heating effects by enforcing spatial residue constraints.

Enteroviruses (EVs and RVs) are prevalent worldwide and cause various diseases in humans, of which the VP1-pocket is a target of antivirals, with a lipid molecule as a pocket factor to stabilize the virion. However, the characterization of the structure of the VP1-pocket in EVs is poor. Here, we compared the published capsid crystals of EVs and RVs and proposed a structural framework for the VP1-pocket: Frame 1-4, which is located at the CD loop, GH loop, and C-terminus, presenting with an outward opening appearance or not. The non-outward viral strains-CVB3, Echo 11, RV-A81, and RV-B70-are more thermally stable, with a breakpoint temperature (B.T.) of 51~62 °C for genome releasing, which is 4~10 °C higher than its outward temperature of 41~47 °C, and infectivity preservation when treated at 50 °C for 3 min. Its outward versus non-outward opening is correlated significantly with the B.T. for genome release (r = -0.90; p = 0.0004) and infectivity (r = -0.82, p = 0.0039). The energy of Frames 1, 2, and 4, including Van der Waals attractive and repulsive interactions and hydrogen bonds, showed significant correlations with the B.T. (r = -0.67, 0.75, and -0.8; p = 0.034, 0.013, and 0.006, respectively). These characters of the VP1-pocket could be predictors for virion thermostability and aid in the development of vaccines or antivirals.

The improvement of enzyme thermostability often accompanies the decreased activity due to the loss of the key regions\' flexibility. As a representative structure, unlocking the potential of loop dynamics will not only provide new ideas for stabilization strategies, but also help to deepen the understanding of the relationship between enzyme structural dynamics and function. In this study, a creative \"hook loop dynamics engineering\" (HLoD) strategy was successfully proposed for simultaneously improving the thermostability and maintaining activity of the model enzyme, Candida Antarctica lipase B. A small and smart mutant library involving five key residues located at the \"hook loop\" was meticulously identified and systematically investigated and thus yielded a five-point multiple mutant M1 (L147S/T244P/S250P/T256D/N292D), demonstrating a remarkable 7.0-fold increase in thermostability at 60 °C compared to the wild-type (WT). Furthermore, the activity of M1 remained comparable to that of WT, effectively transcending the barrier of activity-stability trade-off. Molecular dynamics simulations revealed that the precise regulation of hook loop dynamics via intermolecular interactions, such as salt bridges and hydrogen bonding, curbed the excessive flexibility of the pivotal regions α5 and α10 at high temperatures, thus driving the substantial enhancement of the thermostability of M1. Refining the dynamics of the flexible region via HLoD, which transcended the barrier of activity-stability trade-off, exhibited to be a robust and potentially universal strategy for designing enzymes with outstanding thermostability and activity.

BACKGROUND: D-Allulose is one of the most well-known rare sugars widely used in food, cosmetics, and pharmaceutical industries. The most popular method for D-allulose production is the conversion from D-fructose catalyzed by D-allulose 3-epimerase (DAEase). To address the general problem of low catalytic efficiency and poor thermostability of wild-type DAEase, D-allulose biosensor was adopted in this study to develop a convenient and efficient method for high-throughput screening of DAEase variants.
RESULTS: The catalytic activity and thermostability of DAEase from Caballeronia insecticola were simultaneously improved by semi-rational molecular modification. Compared with the wild-type enzyme, DAEaseS37N/F157Y variant exhibited 14.7% improvement in the catalytic activity and the half-time value (t1/2) at 65°C increased from 1.60 to 27.56 h by 17.23-fold. To our delight, the conversion rate of D-allulose was 33.6% from 500-g L-1 D-fructose in 1 h by Bacillus subtilis WB800 whole cells expressing this DAEase variant. Furthermore, the practicability of cell immobilization was evaluated and more than 80% relative activity of the immobilized cells was maintained from the second to seventh cycle.
CONCLUSIONS: All these results indicated that the DAEaseS37N/F157Y variant would be a potential candidate for the industrial production of D-allulose.

A major obstacle to the use of whey protein in protein-enriched sports beverages is the heat-induced gelation of the protein in the presence of salt. In this study, whey protein soluble aggregates (WPSAs) with high tolerance to NaCl and heat were successfully generated by preheating whey protein isolate (WPI) at a low concentration (1 % w/v) and pH 8.5. The suspension of WPSAs (5 % w/v) with 100 mM NaCl maintained clarity, transparency, and good flowability even after 30 min of heating at 100 °C. However, suspensions prepared by untreated WPI turned into milky white gels. WPSAs had a reduced Zeta potential at pH 7 compared to WPI, making them more resistant to the electrostatic screening caused by NaCl. Additionally, WPSAs exhibited reduced sensitivity to heat treatment due to a more compact structure achieved through preheating modification. In light of these findings, a straightforward and effective method was presented for regulating the heat and ionic strength tolerance of whey protein aggregates.