Purification and some properties of β-N-acetyl-D-glucosaminidase from prawn (Penaeus vannamei)

The β-N-acetyl-D-glucosaminidase (NAGase, EC 3.2.1.52) from prawn (Penaeus vannamei) was purified by extraction with 30% ethanol solution and ammonium sulfate fractionation, then chromatographed on Sephadex G-100 followed by DEAE-cellulose (DE-32) columns. The purified enzyme determined to be homogeneous by polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE. The specific activity of the purified enzyme was 1,560 U mg−1. Enzyme molecular weight was determined to be 105,000 Da; it contained two subunits of the same mass (45,000 Da). The pI value was calculated to be 4.8 by isoelectric focusing. The optimum pH and optimum temperature of the enzyme for the hydrolysis of pNP-β-D-GlcNAc (enzyme substrate) were determined to be pH 5.2 and 45°C, respectively. The behavior of the enzyme during hydrolysis of pNP-β-D-GlcNAc followed Michaelis–Menten kinetics, with Km=0.254 mM and Vm=9.438 μM min−1, at pH 5.2 and 37°C. The stability of the enzyme was investigated, and the results showed that the enzyme was stable in a pH range from 4.2 to 10.0 and at temperatures <40°C. The effects of metal ions on the enzyme were also studied. Li+, Na+ and K+ had no influence on enzyme activity. Mg2+, Ca2+ and Mn2+ activated the enzyme, while Ba2+, Zn2+, Co2+, Cd2+, Hg2+, Pb2+ Cu2+, Fe3+ and Al3+ showed various degrees of inhibitory effects on the enzyme.