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.

Characterization of a novel thermostable alkaline lipase derived from a compost metagenomic library and its potential application in the detergent industry.

Using composted soil samples, a metagenomic library consisting of 36,000 clones was constructed. Then, a novel lipase, Lip54q, which belongs to the VIII family of lipolytic enzymes, was identified from the metagenomic library by functional screening. To explore the enzymatic properties of Lip54q, lip54q was heterologous expressed in Escherichia coli with a high expression level of recombinant protein up to 720 mg/L. The recombinant enzyme showed the highest activity (28,160 U/mg) against a C10 substrate at pH 9.0 and 47°C, and was stable at temperatures ≤50°C and pH 8.0-11.0. Of particular interest, the surfactants, Tween-20, Tween-80 and Tritonx-100, exhibited strong promoting effects on Lip54q activities regardless of whether low concentrations (0.1%) or high concentrations (10%) were used. Application studies of Lip54q using six commercial detergents indicated that the enzyme had strong tolerance and immersion resistance to all six detergents. The results of oil-stain removal experiments suggested that addition of the enzyme to various commercial detergents could significantly improve the abilities of these detergents to remove oil-stains. Furthermore, the results of a molecular docking analysis of Lip54q showed that both the C10 substrate and linoleic acid molecules could form hydrogen bond interactions with the catalytic amino acids, Ser-268, Glu-168, and Asp-192, in the catalytic center of the enzyme, and the hydrogen bond distances were shorter. The electrostatic attraction between the enzyme and the substrate formed by the hydrogen bond with a shorter distance is stronger, which is conducive to the formation of a more stable complex between the enzyme and the substrate, thus increasing the activity of the enzyme to such substrate. These results 1ay a good foundation for application of this enzyme in the detergent industry in the future.