Engineering the thermal stability of a polyphosphate kinase by ancestral sequence reconstruction to expand the temperature boundary for an industrially applicable ATP regeneration system.

ATP-dependent energy-consuming enzymatic reactions are widely used in cell-free biocatalysis. However, the direct addition of large amounts of expensive ATP can greatly increase cost, and enzymatic production is often difficult to achieve as a result. Although a polyphosphate kinase (PPK)-polyphosphate-based ATP regeneration system has the potential to solve this challenge, the generally poor thermal stability of PPKs limits the widespread use of this method. In this paper, we evaluated the thermal stability of a PPK from Sulfurovum lithotrophicum (SlPPK2). After directed evolution and computation-supported design, we found that SlPPK2 is very recalcitrant and cannot acquire beneficial mutations. Inspired by the usually outstanding stability of ancestral enzymes, we reconstructed the ancestral sequence of the PPK family and used it as a guide to construct three heat-stable variants of SlPPK2, of which the L35F/T144S variant has a half-life of more than 14 h at 60°C. Molecular dynamics simulations were performed on all enzymes to analyze the reasons for the increased thermal stability. The results showed that mutations at these two positions act synergistically from the interior and surface of the protein, leading to a more compact structure. Finally, the robustness of the L35F/T144S variant was verified in the synthesis of nucleotides at high temperature. In practice, the use of this high-temperature ATP regeneration system can effectively avoid byproduct accumulation. Our work extends the temperature boundary of ATP regeneration and has great potential for industrial applications.IMPORTANCEATP regeneration is an important basic applied study in the field of cell-free biocatalysis. Polyphosphate kinase (PPK) is an enzyme tool widely used for energy regeneration during enzymatic reactions. However, the thermal stability of the PPKs reported to date that can efficiently regenerate ATP is usually poor, which greatly limits their application. In this study, the thermal stability of a difficult-to-engineer PPK from Sulfurovum lithotrophicum was improved, guided by an ancestral sequence reconstruction strategy. The optimal variant has a 4.5-fold longer half-life at 60°C than the wild-type enzyme, thus enabling the extension of the temperature boundary for ATP regeneration. The ability of this variant to regenerate ATP was well demonstrated during high-temperature enzymatic production of nucleotides.

Mining unique cysteine synthetases and computational study on thoroughly eliminating feedback inhibition through tunnel engineering.

L-cysteine is an essential component in pharmaceutical and agricultural industries, and synthetic biology has made strides in developing new metabolic pathways for its production, particularly in archaea with unique O-phosphoserine sulfhydrylases (OPSS) as key enzymes. In this study, we employed database mining to identify a highly catalytic activity OPSS from Acetobacterium sp. (AsOPSS). However, it was observed that the enzymatic activity of AsOPSS suffered significant feedback inhibition from the product L-cysteine, exhibiting an IC50 value of merely 1.2 mM. A semi-rational design combined with tunnel analysis strategy was conducted to engineer AsOPSS. The best variant, AsOPSSA218R was achieved, totally eliminating product inhibition without sacrificing catalytic efficiency. Molecular docking and molecular dynamic simulations indicated that the binding conformation of AsOPSSA218R with L-cys was altered, leading to a reduced affinity between L-cysteine and the active pocket. Tunnel analysis revealed that the AsOPSSA218R variant reshaped the landscape of the tunnel, resulting in the construction of a new tunnel. Furthermore, random acceleration molecular dynamics simulation and umbrella sampling simulation demonstrated that the novel tunnel improved the suitability for product release and effectively separated the interference between the product release and substrate binding processes. Finally, more than 45 mM of L-cysteine was produced in vitro within 2 h using the AsOPSSA218R variant. Our findings emphasize the potential for relieving feedback inhibition by artificially generating new product release channels, while also laying an enzymatic foundation for efficient L-cysteine production.

Study of the anomalous sorption behavior of CO2 into poly(methyl methacrylate) films in the vicinity of the critical pressure and temperature using a quartz crystal microbalance (QCM).

The anomalous solubility maximum of CO2 in polymer thin films in the vicinity of the critical temperature and pressure has not yet been clearly understood when the quartz crystal microbalance (QCM) technique has been used to determine the micromass change. In this study, the adsorption of CO2 on the surface of bare polished and unpolished crystals at different pressures and temperatures was investigated using the QCM technique to illustrate why a plot of the true frequency shift as a function of temperature and pressure can intuitively exhibit the adsorption behavior of CO2 on bare crystals. The sorption of CO2 into a PMMA film at different temperatures, pressures, and PMMA film thicknesses was also investigated. An accurate solubility for CO2 in the PMMA film could be obtained by an improved data correction method from the linear relation between the true frequency shift and the polymer film mass, and the anomalous solubility maximum could be corrected by this method. The mechanism of nonabsorbed CO2 transitorily staying in the interspace between the PMMA film and the crystal surface can be explained by the morphology change of the PMMA film. The assumption of "passerby CO2" was satisfactorily confirmed to explain the anomalous sorption behavior of CO2 into PMMA films in the vicinity of the CO2 critical temperature and pressure, and this assumption could be valid for other CO2-polymer thin film systems.

Interfacial tension in phase-separated aqueous cationic/anionic surfactant mixtures.

Interfacial tensions in two aqueous phase-separated cationic/anionic surfactant mixtures, CTAB/AS and 12-3-12/AS, without and with NaBr added were determined by the spinning drop method at 318.15 K. CTAB, 12-3-12 and AS are the abbreviations for cetyltrimethylammonium bromide, 1,3-propanediyl-bis(dodecyldimethylammonium bromide) and sodium dodecyl sulfonate, respectively. The interfacial tension sigma was found to be in the range of 0.06-21 microNm(-1). Toward a better understanding of the influence of the concentration difference between the separated phases in aqueous two-phase systems (ATPS) to interfacial tension, compositions of equilibrium phases were determined by elemental analysis coupled with material balance and electroneutrality. The investigation indicates that the concentration differences of surfactant ions between the separated phases and the adsorption of surfactant ions at the interface are the decisive factors determining the magnitude of interfacial tension.

[Solubility behavior of four diastereomeric salts and two amino acids in near-critical CO2].

AIM: To establish a suitable condition for extraction of phenylalanine (Phe), 5-hydroxytryptophan (5-OH-Trp) and four diastereomeric salts, (1R,2S)-ephedrine- (2S,3S)-tartaric acid, (1R,2S)-ephedrine-(2R,3R)-tartaric acid, (1S,2S)-pseudoephedrine-(2S,3S)-tartaric acid, (1S,2S)-pseudoephedrine- (2R,3R)-tartaric acid in supercritical fluid extraction and to assess the solubilities of Phe, 5-OH-Trp and the four diastereomeric salts in CO2. METHODS: Single-pass method and HPCE. RESULTS: The solubilities of Phe, 5-OH-Trp and the four diastereomeric salts in CO2 were determined over temperature and pressure ranges of 25-50 degrees C and 6.32-34.03 MPa respectively. The experimental results showed that the solubilities of Phe, 5-OH-Trp and the four diastereomeric salts do not increase with density of CO2. There existed a maximum in the critical region of CO2. CONCLUSION: The dramatically high solubilities in the pressure of 6.32-7.78 MPa show a critical behavior, which can be explained by critical characteristic through thermodynamics analysis. The results suggest that the separation of Phe, 5-OH-Trp and the four diastereomeric salts is more efficient in critical region of CO2.

[Edge influence of soil moisture at farmland-grassland boundary in agriculture-pasturage ecotone of northern China].

In the agriculture-pasturage ecotone of Northern China, a typical zone with linear boundary of cropland and grassland was chosen to investigate its soil moisture regime, and the moving split-window technique was adopted to study the edge influence of soil moisture at the boundary. The results showed that the edge influence was 10 m, from 6 m within grassland and 4 m within cropland, and was categorized as the acute change type boundary. Accordingly, the farmland-grassland landscape boundary could be divided into three functional zones, i.e., grassland zone, farmland zone, and compositional ecotone zone. Soil moisture content varied abruptly in the ecotone zone, but presented linear distribution in both grassland zone and farmland zone. The average soil moisture content in grassland was about 1 g x g(-1) higher than that in farmland, which was mainly caused by the decreased capillary moisture capacity of farmland. Owing to the different vegetation cover, farmland and grassland had different transpiration and evaporation, which led to the diverse soil moisture regime, making soil water potential changed and water movement from one ecosystem to another possible.

Spontaneous crystallization at the air-water interface: an unusual feature of gemini surfactant with a rigid spacer.

An unusual feature which involves spontaneous crystallization at the air-water interface from aqueous solution was reported for a water-soluble gemini surfactant with xylyl spacer, (p-phenylenedimethylene) bis(dodecyldimethylammonium) dibromide. Polarizing microscope, in situ confocal microscopic Raman spectroscopy, and powder XRD were used to characterize the structure of the crystal and investigate the driving force for nucleation. It was inferred that, besides the surface enrichment of amphiphiles and the intra- and intermolecular interaction of alkyl chains, the pi-pi stacking interaction of benzene rings plays an extraordinary role in promoting nucleation and stabilizing crystal structure. A mechanism for constructing supramolecular architectures in situ at the air-water interface directly from aqueous solution via water-soluble amphiphiles with groups favorable for pi-pi stacking interaction was proposed.