Abstract: The gene (PsHex) encoding β-N-acetylglucosaminidase from the marine bacterium Pseudoalteromonas sp. DL-6 was cloned into the pET-28a(+) vector and then successfully heterologously expressed in Escherichia coli BL21(DE3). The expressed protein was purified through nickel-nitrilotriacetic acid affinity chromatography. The results showed that PsHex encoded 876 amino acids, and the predicted molecular mass and pI were 99.46 kD and 5.65, respectively. Phylogenetic analysis and multiple sequence alignments showed that PsHex belonged to the glycosyl hydrolase 20 (GH20) family. The optimal temperature and pH for the recombinant enzyme were 30 ℃ and 8.5, respectively, its specific activity was 11.30 U/mg and it displayed the highest stability at 20 ℃ and pH 7.5. This enzyme could degrade various substrates, including agarose, chitosan, soluble starch and carrageenan and could be activated by metal ions (Na+, Mg2+, Fe3+, K+, and Ca2+), DL-dithiothreitol, tween-80, ethylenediaminetetraacetic acid and β-mercaptoethanol. The Michaelis constant (Km), catalytic rate constant (Kcat), maximum reaction rate (vmax) and Kcat/Km values toward the substrate 4-nitrophenyl 2-acetamido-2-deoxy-β-d-glucopyranoside were 2.523 mol/L, 79.035 min-1, 2.081 U/mg and 31.326, respectively. This study provides a theoretical reference for the enzymatic degradation of chitin to produce N-acetylglucosamine.

Key words: β-N-acetylhexosaminidase; Pseudoalteromonas; purification and expression; enzymatic properties