Chemical Bonding and Dynamic Structural Fluxionality of a Boron-Based Na5B7 Sandwich Cluster.

Doping alkali metals into boron clusters can effectively compensate for the intrinsic electron deficiency of boron and lead to interesting boron-based binary clusters, owing to the small electronegativity of the former elements. We report on the computational design of a three-layered sandwich cluster, Na5B7, on the basis of global-minimum (GM) searches and electronic structure calculations. It is shown that the Na5B7 cluster can be described as a charge-transfer complex: [Na4]2+[B7]3-[Na]+. In this sandwich cluster, the [B7]3- core assumes a molecular wheel in shape and features in-plane hexagonal coordination. The magic 6π/6σ double aromaticity underlies the stability of the [B7]3- molecular wheel, following the (4n + 2) Hückel rule. The tetrahedral Na4 ligand in the sandwich has a [Na4]2+ charge-state, which is the simplest example of three-dimensional aromaticity, spherical aromaticity, or superatom. Its 2σ electron counting renders σ aromaticity for the ligand. Overall, the sandwich cluster has three-fold 6π/6σ/2σ aromaticity. Molecular dynamics simulation shows that the sandwich cluster is dynamically fluxional even at room temperature, with a negligible energy barrier for intramolecular twisting between the B7 wheel and the Na4 ligand. The Na5B7 cluster offers a new example for dynamic structural fluxionality in molecular systems.

Chemical bonding and dynamic structural fluxionality of a boron-based Al2B8 binary cluster: the robustness of a doubly 6π/6σ aromatic [B8]2- molecular wheel.

Despite the isovalency between Al and B elements, Al-doping in boron clusters can deviate substantially from an isoelectronic substitution process. We report herein on a unique sandwich di-Al-doped boron cluster, Al2B8, using global structural searches and quantum chemical calculations. The cluster features a perfectly planar B8 molecular wheel, with two isolated Al atoms symmetrically floating above and below it. The two Al atoms are offset from the center of the molecular wheel, resulting in a C 2v symmetry for the cluster. The Al2B8 cluster is shown to be dynamically fluxional even at far below room temperature (100 K), in which a vertical Al2 rod slides or rotates freely within a circular rail on the B8 plate, although there is no direct Al-Al interaction. The energy barrier for intramolecular rotation is only 0.01 kcal mol-1 at the single-point CCSD(T) level. Chemical bonding analysis shows that the cluster is a charge-transfer complex and can be formulated as [Al]+[B8]2-[Al]+. The [B8]2- molecular wheel in sandwich cluster has magic 6π/6σ double aromaticity, which underlies the dynamic fluxionality, despite strong electrostatic interactions between the [Al]+, [B8]2-, and [Al]+ layers.

Boron Oxide B5O6 - Cluster as a Boronyl-Based Inorganic Analog of Phenolate Anion.

Boron oxide clusters have structural richness and exotic chemical bonding. We report a quantum chemical study on the binary B5O6 - cluster, which is relatively oxygen-rich. A global structural search reveals planar C 2v (1A1) geometry as the global minimum structure, featuring a heteroatomic hexagonal B3O3 ring as its core. The three unsaturated B sites are terminated by two boronyl (BO) groups and an O- ligand. The B5O6 - cluster can be faithfully formulated as B3O3(BO)2O-. This structure is in stark contrast to that of its predecessors, C s B5O5 - and T d B5O4 -, both of which have a tetrahedral B center. Thus, there exists a major structural transformation in B5O n - series upon oxidation, indicating intriguing competition between tetrahedral and heterocyclic structures. The chemical bonding analyses show weak 6π aromaticity in the B5O6 - cluster, rendering it a boronyl analog of phenolate anion (C6H5O-) or boronyl boroxine. The calculated vertical detachment energy of B5O6 - cluster is 5.26 eV at PBE0, which greatly surpasses the electron affinities of halogens (Cl: 3.61 eV), suggesting that the cluster belongs to superhalogen anions.

Optimized expression, purification and characterization of a family 11 xylanase (AuXyn11A) from Aspergillus usamii E001 in Pichia pastoris.

BACKGROUND: Xylanases have attracted much attention because of their potential applications. Unfortunately, the commercialization of xylanases is limited by their low catalytic activities. The aim of this study was to improve the activity of a xylanase by optimization of the expression conditions and to investigate its characterization. RESULTS: The activity of recombinant AuXyn11A (reAuXyn11A), a family 11 xylanase from Aspergillus usamii E001 expressed in Pichia pastoris GS115, reached 912.6 U mL⁻¹ under the optimized conditions, which was 2.14 times as high as that expressed using the standard protocol. After the endogenous 18-aa propeptide had been processed in P. pastoris, reAuXyn11A (188-aa mature peptide) was secreted and purified with a specific activity of 22 714 U mg⁻¹. It displayed maximum activity at pH 5 and 50 °C and was stable in the pH range 4-8 and at a temperature of 45 °C or below. Its activity was not significantly affected by most metal ions and EDTA. Xylooligosaccharides ranging from xylobiose (X2) to xylohexaose (X6) were produced from insoluble corncob xylan by reAuXyn11A. CONCLUSION: Its high specific activity and good enzymatic properties suggest that reAuXyn11A is a potential candidate for applications in industrial processes.

Fusing a carbohydrate-binding module into the Aspergillus usamii ß-mannanase to improve its thermostability and cellulose-binding capacity by in silico design.

The AuMan5A, an acidophilic glycoside hydrolase (GH) family 5 ß-mannanase derived from Aspergillus usamii YL-01-78, consists of an only catalytic domain (CD). To perfect enzymatic properties of the AuMan5A, a family 1 carbohydrate-binding module (CBM) of the Trichoderma reesei cellobiohydrolase I (TrCBH I), having the lowest binding free energy with cellobiose, was selected by in silico design, and fused into its C-terminus forming a fusion ß-mannanase, designated as AuMan5A-CBM. Then, its encoding gene, Auman5A-cbm, was constructed as it was designed theoretically, and expressed in Pichia pastoris GS115. SDS-PAGE analysis displayed that both recombinant AuMan5A-CBM (reAuMan5A-CBM) and AuMan5A (reAuMan5A) were secreted into the cultured media with apparent molecular masses of 57.3 and 49.8 kDa, respectively. The temperature optimum of the reAuMan5A-CBM was 75°C, being 5°C higher than that of the reAuMan5A. They were stable at temperatures of 68 and 60°C, respectively. Compared with reAuMan5A, the reAuMan5A-CBM showed an obvious decrease in K m and a slight alteration in V max. In addition, the fusion of a CBM of the TrCBH I into the AuMan5A contributed to its cellulose-binding capacity.

Expression of a family 10 xylanase gene from Aspergillus usamii E001 in Pichia pastoris and characterization of the recombinant enzyme.

A cDNA gene (Auxyn10A), which encodes a mesophilic family 10 xylanase from Aspergillus usamii E001 (abbreviated to AuXyn10A), was amplified and inserted into the XhoI and NotI sites of pPIC9K(M) vector constructed from a parent pPIC9K. The recombinant expression vector, designated pPIC9K(M)-Auxyn10A, was transformed into Pichia pastoris GS115. All P. pastoris transformants were spread on a MD plate, and then inoculated on geneticin G418-containing YPD plates for screening multiple copies of integration of the Auxyn10A. One transformant expressing the highest recombinant AuXyn10A (reAuXyn10A) activity of 368.6 U/ml, numbered as P. pastoris GSX10A4-14, was selected by flask expression test. SDS-PAGE assay demonstrated that the reAuXyn10A was extracellularly expressed with an apparent M.W. of 39.8 kDa. The purified reAuXyn10A displayed the maximum activity at pH 5.5 and 50 °C. It was highly stable at a broad pH range of 4.5-8.5, and at a temperature of 45 °C. Its activity was not significantly affected by EDTA and several metal ions except Mn(2+), which caused a strong inhibition. The K(m) and V(max), towards birchwood xylan at pH 5.5 and 50 °C, were 2.25 mg/ml and 6,267 U/mg, respectively. TLC analysis verified that the AuXyn10A is an endo-ß-1,4-D-xylanase, which yielded a major product of xylotriose and a small amount of xylose, xylotetraose, and xylopentose from birchwood xylan, but no xylobiose.

Engineering hyperthermostability into a mesophilic family 11 xylanase from Aspergillus oryzae by in silico design of N-terminus substitution.

A mesophilic xylanase from Aspergillus oryzae CICC40186 (abbreviated to AoXyn11A) belongs to glycoside hydrolase family 11. The thermostability of AoXyn11A was significantly improved by substituting its N-terminus with the corresponding region of a hyperthermostable family 11 xylanase, EvXyn11(TS) . The suitable N-terminus of AoXyn11A to be replaced was selected by the comparison of B-factors between AoXyn11A and EvXyn11(TS) , which were generated and calculated after a 15 ns molecular dynamic (MD) simulation process. Then, the predicted hybrid xylanase (designated AEx11A) was modeled, and subjected to a 2 ns MD simulation process for calculating its total energy value. The N-terminus substitution was confirmed by comparing the total energy value of AEx11A with that of AoXyn11A. Based on the in silico design, the AEx11A was constructed and expressed in Pichia pastoris GS115. After 72 h of methanol induction, the recombinant AEx11A (reAEx11A) activity reached 82.2 U/mL. The apparent temperature optimum of reAEx11A was 80°C, much higher than that of reAoXyn11A. Its half-life was 197-fold longer than that of reAoXyn11A at 70°C. Compared with reAoXyn11A, the reAEx11A displayed a slight alteration in K(m) but a decrease in V(max).