Cloning, Expression, Characterization And Site Mutagenesis Of A β-Glucosidase From A Deep-Sea Bacterium, Aeromonas Sp.HC11e-3

P-glucosidase is of great use in various industrial area, for instance, during cellulose degrading, it works with exo-cellulase and endocellulase together aiming to transform cellulose into glucose; in the food industry, it can hydrolyze aryl glucosidic compounds to liberate flavor reagents as a result to enhance flavor in juice or tea; in the pharmaceutical industry, it can hydrolyze glycosidic precursor in Chinese medicine to prepare active ingredients. In the mean time, the transglycosyl activity of P-glucosidase renders it a very good prospect in area of synthesizing glucosidic active compounds, such as arkyl-glucoside of surfactants activity and gentian-polysaccharides as prebiotic. So cloning and development of novel glucosidases are of great significance for the relative industrial application.In this research, one strain Aeromonas sp. HC11e-3 with high P-glucosidase activity is screened from the marine bacteria stored in our lab. After constructing genomic library using shotgun method, a P-glucosidase gene, with a length of 2382bp, encoding 793 amino acids, designated BglA is cloned from this bacterium. Homologous comparing shows that the enzyme belongs to glycosyl hydrolase family 3, exhibiting 45% identity with glucosidase from Thermotoga neapolitana, and no more than 30% identity with other biochemically characterizedβ-glucosidases.After cloning the enzyme into pGEX-6p-1 vector and optimizing the expressing condition, the enzyme showed very good expression in E.coli BL21 (DE3) at 0.1mM IPTG and 18 degrees. The enzyme is purified using glutathione affinity purification system, which later, exhibits the best activity at pH 6,55℃. It hydrolyzed aryl glucoside specially and displayed the optimal activity toward synthesized pNPG, and plant originated glucoside, as well as slight activity toward pNPX and pNPGal. The enzyme can tolerate various chemical reagents and metal ions very well. Ca2+, Mn2+, Zn2+, Ba2+, Pb2+, Sr2+ can activate the enzyme activity whereas SDS, EDTA, DTT showing slight inhibition to the enzyme activity. Meanwhile, at the glucose concentration of 0.5M, the enzyme remains obvious activity, indicating that the enzyme has somewhat good tolerance to glucose. Overall, the enzyme is of easy expression, low identity, strong substrate specialty and great tolerance toward chemical reagents. These features make the glucosidase be capable of using in suitable industrial areas.By homologous comparison and structural modeling, the active sites of this enzyme are determined to be aspartate 288 and glutamate 495. Site-directed mutagenesis of these amino sites into Ala by overlapping extension, it is found that the mutants D288A and E495A show 820 fold and 4920 fold decreased activity respectively, confirming that Asp288 and Glu495 involved in the activity of the enzyme hydrolysis. Such results provide the basis for functioanl analysis of other glucosidases in glycosyl hydrolase family 3.