(191al) Homologous Constitutive Expression of Halophilic and Acidophilic ?-Glucosidases in Marine Aspergillus Niger Zjube-1

Abstract: β-glucosidase (BGL) is widely applied in biofuel industry, as a part of cellulase cocktail to catalyze the hydrolysis of the β-1,4 linkages that join two glucose molecules in a cellulose polymer. The hydrolysis step by BGL is generally recognized as the major limiting step in cellulose degradation and the BGLs with prominent enzymatic properties are of great importance for the efficient utilization of lignocellulosic biomass.

In this study, two bgl genes (bgl1 and bgl2) were cloned from the genome of marine Aspergillus niger ZJUBE-1. Then two bgl expression cassettes driven by gpdA promoter were respectively transformed into marine A. niger for homologous constitutive expression. For few protein was secreted during fermentation with original strain at non-induced condition, the constitutive expressed BGLs (BGL1 and BGL2) were dominated in the fermantation broth. Conveniently, the purification of BGLs was accomplished by ultrafiltration and anion exchange chromatography and the purity was up to 95%. Subsequently, the study of enzymatic properties showed that BGL1 and BGL2 exhibited maximum activity at pH 3.0-4.0 and pH 3.5-4.5, respectively, suggesting these two BGLs were acidophilic, especially for BGL1. Notably, the activity of BGL2 increased by 44% in the presence of 4 M NaCl compared with the activity in the absence of NaCl, suggesting BGL2 was halophilic. Furthermore, the thermostability of BGL2 improved in the presence of NaCl. Incubated at 60 °C for 2 h, BGL2 in 4 M NaCl maintained 81 % activity compared with 22% activity in the absence of NaCl. At last, the key amino acid residues involved binding and catalysis were determined by homology modeling to these two BGLs. Hypothetically, the difference of asparagine residues in substrate-binding pocket changed the properties of hydrogen bonds between pocket and substrate, and then endowed the halophilism to BGL2.

Marine microorganisms are the treasury of enzymes with special properties, especially the halophilic enzymes. The halophilic BGL2 obtained from marine A. niger can be applied in some catalytic processes with high salt concentration and the structure study on BGL2 can provide the basis for other halophilic enzymes.

Keywords: β-glucosidase, marine Aspergillus niger, homologous constitutive expression, acidophilic, halophilic, homology modeling