Salt-tolerant and thermostable mechanisms of an endoglucanase from marine Aspergillus niger.

The cellulase cocktail of marine Aspergillus niger exhibited salt-tolerant and thermostable properties, which is of great potential in industrial application. In order to excavate the single tolerant cellulase components from complex cellulase cocktail, constitutive homologous expression was employed for direct obtainment of the endoglucanase (AnEGL). Enzymatic property study revealed that AnEGL exhibited a property of salt tolerance and a strong thermostability in high salinity environment. Significantly, its activity increased to 129% and the half-life at 65 °C increased to 27.7-fold with the presence of 4.5 M NaCl. Molecular dynamics simulation revealed that Na+ and Cl- could form salt bridges with charged residues, and then influenced the activity of loops and the stability of substrate binding pocket, which accounted for the salt tolerance and thermostability. Further, site-specific mutagenesis study proved that the residues Asp95 and Asp99 in the pocket were of great concern for the tolerant properties. The salt-tolerant and thermostable AnEGL was of great value in lignocellulosic utilization and the conjectural mechanisms were of referential significance for other tolerant enzymes.

Directed expression of halophilic and acidophilic ß-glucosidases by introducing homologous constitutive expression cassettes in marine Aspergillus niger.

The hydrolysis step by ß-glucosidase (BGL) is generally recognized as the major limiting step in cellulose degradation and the BGLs with prominent enzymatic properties are of great importance for efficient utilization of lignocellulosic biomass. In order to identify some salt-tolerant BGLs, two BGL genes were cloned from marine Aspergillus niger ZJUBE-1 genome. Then two bgl expression cassettes driven by gpdA promoter were respectively transformed into marine A. niger for homologous constitutive expression. Directed expression was achieved for the domination of target BGLs in fermentation broth. Conveniently, two BGLs were purified to homogeneity by two separation steps, ultrafiltration and anion exchange chromatography. The purified BGL1 and BGL2 showed maximum activity at pH 3.0-4.0 and 3.5-4.5, respectively, suggesting these two BGLs were relatively acidophilic, especially for BGL1. Besides, BGL1 was stable to most of metal ions, while BGL2 was sensitive to Cu2+, Fe3+ and Ag+. Most specially, BGL2 activity increased by 44% in the presence of 4 M NaCl, suggesting BGL2 was halophilic. Homology modeling revealed that longer loops and linkers as well as polymerous Glu492 may contribute to the halophilism of BGL2. At last, the medium for directed expression was optimized and the content as well as the purity of target protein was improved.