Molecular cloning and characterization of a novel bifunctional cellobiohydrolase/ß-xylosidase from a metagenomic library of mangrove soil.

A metagenomic library of mangrove soil samples consisting of approximately 11,000 clones was constructed, and a rare bifunctional cellobiohydrolase/ß-xylosidase Cbh2124 was identified by functional screening. Cbh2124 displayed the highest homology (56.43%) with a protein of the glycoside hydrolase 10 (GH10) family from Proteobacteria. Phylogenetic analysis confirmed that Cbh2124 belongs to the GH10 family. The recombinant enzyme showed a strong cellobiohydrolase activity and a relatively high ß-xylosidase activity, and its catalytic efficiency to the cellobiose substrate was as high as 1.27 × 105 s-1·mM-1, the highest efficiency among reported cellobiohydrolases. Of particular interest, some enzymatic properties of the ß-xylosidase activity of Cbh2124 were significantly different from those of the cellobiohydrolase activity. The optimal pH and temperature of the cellobiohydrolase activity of Cbh2124 was 6.4 and 36 °C, and the activity was essentially lost after treatment at 45 °C for 1 h. The optimal pH and temperature of the ß-xylosidase activity of Cbh2124 was 8.0 and 60 °C, and the residual activity was still over 90% after treatment at 80 °C for 6 h. The molecular docking results of the ß-xylosidase activity of Cbh2124 revealed the additional presence of catalytic amino acids Ser175 and Lys420, thus increasing the number of hydrogen bonds involved in the catalytic process, which possibly let to the improved thermostability compared with that of the cellobiohydrolase activity.

An uncharacterized protein from the metagenome with no obvious homology to known lipases shows excellent alkaline lipase properties and potential applications in the detergent industry.

A novel lipase, Lip486, which has no obvious homology with known lipases, was discovered using functional metagenomics technology. Phylogenetic tree analysis suggested that the enzyme belongs to a new subfamily called lipolytic enzyme family II. To explore the enzymatic properties, lip486 was expressed heterologously and efficiently in Escherichia coli. The recombinant enzyme displayed the highest activity on the substrate p-nitrophenyl caprate with a carbon chain length of 10, and its optimum temperature and pH were 53 °C and 8.0, respectively. The recombinant Lip486 showed good activity and stability in strong alkaline and medium-low-temperature environments. The results of compatibility and soaking tests showed that the enzyme had good compatibility with 4 kinds of commercial detergents, and an appropriate soaking time could further improve the enzyme activity. Oil stain removal test results for a cotton cloth indicated that the washing performance of commercial laundry detergent supplemented with Lip486 was further improved. In addition, as one of the smallest lipases found to date, Lip486 also has the advantages of high yield, good stability and easy molecular modification. These characteristics reflect the good application prospects for Lip486 in the detergent and other industries in the future.

Characterization and purification via nucleic acid aptamers of a novel esterase from the metagenome of paper mill wastewater sediments.

A new esterase gene est906 was identified from paper mill wastewater sediments via a function-based metagenomic approach. The gene encoded a protein of 331 amino acids, that shared 86% homology with known esterases. Based on the results of multiple sequence alignment and phylogenetic analysis, it was confirmed that Est906 contained a characteristic hexapeptide motif (G-F-S-M-G-G), which classified it as a lipolytic enzyme family V protein. Est906 displayed the highest hydrolysis activity to ρ-nitrophenyl caproate (C6), and its optimal temperature and pH were 54 °C and 9.5, respectively. Additionally, this enzyme had good stability under strong alkaline conditions (pH 10.0-11.0) in addition to moderate heat resistance and good tolerance against several metal ions and organic solvents. Furthermore, a specific nucleic acid aptamer (Apt1) bound to Est906 was obtained after five rounds of magnetic bead SELEX screening. Apt1 displayed high specific recognition and capture ability to Est906. In conclusion, this study not only identified a new esterase of family V with potential industrial application by metagenomic technology but also provided a new method to purify recombinant esterases via nucleic acid aptamers, which will facilitate the isolation and purification of target proteins in the future.

A gate-free monolayer WSe2 pn diode.

Interest in bringing p- and n-type monolayer semiconducting transition metal dichalcogenides (TMD) into contact to form rectifying pn diode has thrived since it is crucial to control the electrical properties in two-dimensional (2D) electronic and optoelectronic devices. Usually this involves vertically stacking different TMDs with pn heterojunction or, laterally manipulating carrier density by gate biasing. Here, by utilizing a locally reversed ferroelectric polarization, we laterally manipulate the carrier density and created a WSe2 pn homojunction on the supporting ferroelectric BiFeO3 substrate. This non-volatile WSe2 pn homojunction is demonstrated with optical and scanning probe methods and scanning photoelectron micro-spectroscopy. A homo-interface is a direct manifestation of our WSe2 pn diode, which can be quantitatively understood as a clear rectifying behavior. The non-volatile confinement of carriers and associated gate-free pn homojunction can be an addition to the 2D electron-photon toolbox and pave the way to develop laterally 2D electronics and photonics.