Purification and some properties of beta-N-acetyl-D-glucosaminidase from prawn (Penaeus vannamei)
- 生命科学－已发表论文 
The beta-N-acetyl-D-glucosaminidase ( NAGase, EC 126.96.36.199) 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-beta-D-GlcNAc ( enzyme substrate) were determined to be pH 5.2 and 45 degreesC, respectively. The behavior of the enzyme during hydrolysis of pNP-beta-D-GlcNAc followed Michaelis - Menten kinetics, with K-m = 0.254 mM and V-m = 9.438 muM min(-1), at pH 5.2 and 37 degreesC. 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 &DEG;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.