Determining electrospun morphology from the properties of protein-polymer solutions.

Integrating natural macromolecules, e.g. proteins, is a progressive trend in the fabrication of biocompatible sub-micrometer fibers with tunable diameters using the electrospinning technique. The correlation between solution properties and electrospun morphology is critical; it is quite clear for synthetic linear polymer solutions but remains uncertain for solutions with protein. Here, we report the determination of electrospun morphology in protein-polymer solutions of poly(ethylene oxide) (PEO) and zein, a storage protein from corn. The viscosity of the zein/PEO mixed solutions can be well described using the Lederer-Roegiers equation and decreases with the increase of the fraction of zein. The surface tension sharply decreases above a critical concentration at the saturation of the interfacial monolayer. Correspondingly, the different electrospun morphologies-from bead, coexisting bead and fiber, to fiber and ribbon-were mapped onto a ternary phase diagram and a viscosity contour plot. Such coupling provides a clear way to determine the electrospun morphology from solution properties. The occurrence of electrospun fibers partially follows two empirical rules, while the critical point revealed from surface tension has the best approximation. The diameters of electrospun fibers were found to have a scaling relationship against concentration, zero-shear viscosity and surface tension of solutions. These scaling exponents were compared with those from typical polymer solutions. The analysis suggests that aqueous ethanol gives different solvent qualities to zein and PEO solutions, resulting in the irregular shape in the phase diagram that correlates solution properties and electrospun morphologies.

Synthesis and Characterization of Polyethylene/Starch Nanocomposites: A Spherical Starch-Supported Catalyst and In Situ Ethylene Polymerization.

In the present article, a novel spherical starch-supported vanadium (V)-based Ziegler-Natta catalyst was synthesized. The active centers of the obtained catalyst well dispersed in the starch through the SEM-EDX analysis. The effects of reaction conditions on ethylene polymerization were studied. The synthesized catalyst exhibited high activity toward ethylene polymerization in the presence of ethylaluminium sesquichloride (EASC) cocatalyst. Interestingly, the fiber shape PE was obtained directly during the polymerization process.

Conserved residues Glu and Phe at substrate binding groove of α-1,6-glucanases modulate branch of the product.

Understanding what amino acids in α-1,6-glucanases target α-1,6 glycosidic bonds of polysaccharides is timely and important for generating products with branch structure. With this objective, we investigated 330 sequences from seven subfamilies to excavate amino acids for recognition or catalysis of α-1,6 glycosidic bonds. Computational analysis identified two amino acids, E343 and W521, trigger α-1,6 glycosidic bond specificity of enzymes. To explore the effect of E343 and W521 on the product structure, several engineered mutants were studied in our research. Product structural analysis showed that the ratio of amylose and amylopectin is obviously different. The catalytic mechanism revealed that the bulky aromatic side chain is a trigger that controls the ratio of branch glucans. The E148 acts as a proton donor to regulate the generation of branched structures in the product during transglycosidation of the glucan branching enzyme (GBE).

Bifunctional Mutant of Oligo-1,6-glucosidase for the Production of Glucose from Glucose Mother Liquor.

Glucose mother liquor (GML) is a byproduct of the glucose (G1) crystallization process. However, the presence of maltooligosaccharides and isomaltooligosaccharides within GML imposes limitations on its reutilization. Furthermore, the high concentration of G1 in GML leads to product inhibition of G1-producing enzymes. To overcome these challenges, a variant enzyme called V219A was developed through genetic mutation. The V219A exhibits the ability to hydrolyze both maltooligosaccharides and isomaltooligosaccharides. Product inhibition kinetics showed that the IC50 value of V219A was 7 times higher than that of the wild type. Upon subjecting primary, secondary, and tertiary GML to treatment with V219A, the G1 content exhibited notable increases, reaching 96.88, 95.70, and 90.46%, respectively. These significant findings not only establish an innovative and environmentally conscious approach for G1 production from GML but also provide a promising strategy for enzyme construction that caters to the demands of industrial-scale production.

Conserved residues at the family and subfamily levels determine enzyme activity and substrate binding in glycoside hydrolase family 13.

Site-directed mutagenesis is a valuable strategy for modifying enzymes, but the lack of understanding of conserved residues regulating glycosidase function hinders enzyme design. We analyzed 1662 enzyme sequences to identify conserved amino acids in maltohexaose-forming amylase at both family and subfamily levels. Several conserved residues at the family level (G37, P45, R52, Y57, D101, V103, H106, G230, R232, D234, E264, H330, D331, and G360) were found, mutations of which resulted in reduced enzyme activity or inactivation. At the subfamily level, several conserved residues (L65, E67, F68, D111, E114, R126, R147, F154, W156, F161, G163, D165, W218H, V342, W345, and F346) were identified, which primarily facilitate substrate binding in the enzyme's active site, as shown by molecular dynamics and kinetic assays. Our findings provide critical insights into conserved residues essential for catalysis and can inform targeted enzyme design in protein engineering.

The amino acid on the top of the active groove allosterically modulates product specificity of the 1,4-α-glucan branching enzyme.

The 1,4-α-glucan branching enzymes (GBEs, EC 2.4.1.18) catalyse the formation of α-1,6 branching points in starch, presenting several potential applications in modifying starch. Previous study proved that W285 is considered to act as a "switch" to stop extension of substrates in the structure of GBE from Cyanothece sp. (cceBE). In the structure of GBE from Rhodothermus obamensis STB05 (RoGBE), the amino acid 160 site is structurally similar to the W285 in cceBE. In order to explore the role of this site in RoGBE, several engineered mutants individually substituted with Arg, Phe and Ala at G160 were studied in our research. The results show that substitution with Arg and Phe increased branching activity significantly, and the ratio of short glucan chains among all oligosaccharides increased. Finally, we proposed that the G160 is a 'door model' to elucidate introduced mutagenesis that triggers and controls the length of binding glucan chains of starch.