Engineering enzyme specificity using computational design of a defined-sequence library

Engineered biosynthetic pathways have the potential to produce high-value molecules from inexpensive feedstocks, but a key limitation is engineering enzymes with high activity and specificity for new reactions. Here, we developed a method for combining structure-based computational protein design with library-based enzyme screening, in which inter-residue correlations favored by the design are encoded into a defined-sequence library. We validated this approach by engineering a glucose 6-oxidase enzyme for use in a proposed pathway to convert D-glucose into D-glucaric acid. The most active variant, identified after only one round of diversification and screening of only 10,000 wells, is approximately 400-fold more active on glucose than is the wild-type enzyme. We anticipate that this strategy will be broadly applicable to the discovery of new enzymes for engineered biological pathways.     

Using conformationally locked nucleosides to calibrate the anomeric effect: Implications for glycosyl bond stability

Steric and electronic parameters, such as the anomeric effect (AE) and gauche effect play significant roles in steering the North⇆South equilibrium of nucleosides in solution.Two isomeric oxa-bicyclo[3.1.0]hexane nucleosides that are conformationally locked in either the North or the South conformation of the pseudorotational cycle were designed to study the consequences of having the AE operational or not, independent of other parameters. The rigidity of the system allowed the orientation of the orbitals involved to be set in ‘fixed’ relationships, either antiperiplanar where the AE is permanently ‘on’, or gauche where the AE is impaired. The consequences of these two alternatives were subjected to high-level calculations and measured experimentally by X-ray crystallography, hydrolytic stability of the glycosyl bond, and pK_a values.     

Tunable luminescence and energy transfer process between Tb3+ and Eu3+ in GYAG:Bi3+, Tb3+, Eu3+ phosphors

A series of Eu³⁺ ions co-doped (Gd_0.9Y_0.1)₃Al₅O₁₂:Bi³⁺, Tb³⁺ (GYAG) phosphors have been synthesized by means of solvothermal reaction method. The XRD pattern of GYAG phosphor sintered at 1500 °C confirms their garnet phase. The luminescence properties of these phosphors have been explored by analyzing their excitation and emission spectra along with their decay curves. The excitation spectra of the GYAG:Bi³⁺, Tb³⁺, Eu³⁺ phosphors consists of broad bands in the shorter wavelength region due to 4f⁸ → 4f⁷5d¹ transition of Tb³⁺ ions overlapped with 6s² → 6s¹6p¹ (¹S₀ → ³P₁) transition of Bi³⁺ ions and the charge transfer band of Eu³⁺–O^2?. The present phosphors exhibit green and red colors due to ⁵D₄ → ⁷F₅ transition of Tb³⁺ ions and ⁵D₀ → ⁷F₁ transition of Eu³⁺ ions, respectively. The emission was shifted from green to red color by co-doping with Eu³⁺ ions, which indicate that the energy transfer probability from Tb³⁺ to Eu³⁺ ions are dependent strongly on the concentration of Eu³⁺ ions.     

Continuous Ethanol Production from Cassava Through Simultaneous Saccharification and Fermentation by Self-Flocculating Yeast Saccharomyces Cerevisiae CHFY0321

In this study, a fermentor consisting of four linked stirred towers that can be used for simultaneous saccharification and fermentation (SSF) and for the accumulation of cell mass was applied to the continuous production of ethanol using cassava as the starchy material. For the continuous process with SSF, the pretreated cassava liquor and saccharification enzyme at total sugar concentrations of 175 g/L and 195 g/L were continuously fed to the fermentor with dilution rates of 0.014, 0.021, 0.031, 0.042, and 0.05 h⁻¹. Considering the maximum saccharification time, the highest volumetric productivity and ethanol yield were observed at a dilution rate of 0.042 h⁻¹. At dilution rates in the range of 0.014 h⁻¹ to 0.042 h⁻¹, high production rates were observed, and the yeast in the first to fourth fermentor showed long-term stability for 2 months with good performance. Under the optimal culture conditions with a feed sugar concentration of 195 g/L and dilution rate of 0.042 h⁻¹, the ethanol volumetric productivity and ethanol yield were 3.58 g/L∙h and 86.2%, respectively. The cell concentrations in the first to fourth stirred tower fermentors were 74.3, 71.5, 71.2, and 70.1 g dry cell/L, respectively. The self-flocculating yeast, Saccharomyces cerevisiae CHFY0321, developed by our group showed excellent fermentation results under continuous ethanol production.     

Thermodynamic Investigation of Molecular Interactions in 1,3-Dioxolane or 1,4-Dioxane+Benzene or Toluene+Formamide or +<Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-Dimethylformamide Ternary Mixtures at 308.15 K and Atmospheric Pressure

Excess molar volumes, V^E₁₂₃V^{\mathrm{E}}_{123} of 1,3-dioxolane or 1,4-dioxane (1) + benzene or toluene (2) + formamide or + N,N-dimethylformamide (3) ternary mixtures at 308.15 K and at atmospheric pressure have been determined dilatometrically over the entire composition range. The excess molar volumes data of these ternary systems were fitted to the Redlich–Kister equation. The data have been analyzed in terms of Graph theory (model) to understand the nature and strength of molecular interactions existing in these mixtures. It has been observed that V^E₁₂₃V^{\mathrm{E}}_{123} values predicted by Graph theory compare well with their corresponding experimental values.     

Excess thermodynamic properties, and for the binary systems {1,2-dichloropropane + 2-alkoxyethanols} at T = 298.15 K

This article presents the experimental data of and , obtained at T = 298.15 K and atmospheric pressure, for four binary systems composed of 1,2-dichloropropane (1,2-DCP) and four 2-alkoxyethanols. The 2-alkoxyethanols are 2-methoxyethanol (2-ME), 2-ethoxyethanol (2-EE), 2-propoxyethanol (2-PE) and 2-butoxyethanol (2-BE). The of the mixture has been shown positive for 2-ME, ‘s-shaped’ for all remaining systems, being negative at low and positive at high mole fraction of 1,2-DCP. The values for all binary mixtures are also shown both positive at low and negative at high mole fraction of 1,2-DCP. The experimental results of and were fitted to Redlich–Kister equation to correlate the composition dependence of both excess properties. In this work, data were also used to test the suitability of thermodynamic models (Wilson, NRTL, and UNIQUAC equations) based on local-composition theory. The results have been qualitatively discussed in terms of the polarity, self-association, and hydrogen bond among molecules.     

A Chemical Inhibitor of the Skp2/p300 Interaction that Promotes p53‐Mediated Apoptosis

Skp2 is thought to have two critical roles in tumorigenesis. As part of the SCF^Skp2 ubiquitin ligase, Skp2 drives the cell cycle by mediating the degradation of cell cycle proteins. Besides the proteolytic activity, Skp2 also blocks p53‐mediated apoptosis by outcompeting p53 for binding p300. Herein, we exploit the Skp2/p300 interaction as a new target for Skp2 inhibition. An affinity‐based high‐throughput screen of a combinatorial cyclic peptoid library identified an inhibitor that binds to Skp2 and interferes with the Skp2/p300 interaction. We show that antagonism of the Skp2/p300 interaction by the inhibitor leads to p300‐mediated p53 acetylation, resulting in p53‐mediated apoptosis in cancer cells, without affecting Skp2 proteolytic activity. Our results suggest that inhibition of the Skp2/p300 interaction has a great potential as a new anticancer strategy, and our Skp2 inhibitor can be developed as a chemical probe to delineate Skp2 non‐proteolytic function in tumorigenesis.     

Mechanical properties of cellulose nanomaterials studied by contact resonance atomic force microscopy

Quantification of the mechanical properties of cellulose nanomaterials is key to the development of new cellulose nanomaterial based products. Using contact resonance atomic force microscopy we measured and mapped the transverse elastic modulus of three types of cellulosic nanoparticles: tunicate cellulose nanocrystals, wood cellulose nanocrystals, and wood cellulose nanofibrils. These modulus values were calculated with different contact mechanics models exploring the effects of cellulose geometry and thickness on the interpretation of the data. While intra-particle variations in modulus are detected, we did not observe a measureable difference in modulus between the three types of cellulose particles. Improved practices and experimental complications for the characterization of cellulosic nanomaterials with atomic force microscopy are discussed.