title: Enzymatic characterizations and activity regulations of N-acetyl-β-D-glucosaminidase from the spermary of Nile tilapia (Oreochromis niloticus).
abstract: N-Acetyl-β-D-glucosaminidase (NAGase) is proved to be correlated with reproduction of male animals. In this study, enzymatic characterizations of NAGase from spermary of Nile tilapia (Oreochromis niloticus) were investigated in order to further study its reproductive function in fish. Tilapia NAGase was purified to be PAGE homogeneous by the following techniques: (NH4)2SO4 fractionation (40-55%), DEAE-cellulose (DE-32) ion exchange chromatography, Sephadex G-200 gel filtration and DEAE-Sephadex (A-50). The specific activity of the purified enzyme was 4100 U/mg. The enzyme molecular weight was estimated as 118.0 kD. Kinetic studies showed that the hydrolysis of p-nitrophenyl-N-acetyl-β-D-glucosaminide (pNP-NAG) by the enzyme followed Michaelis-Menten kinetics. The Michaelis-Menten constant (Km) and maximum velocity (Vm) were determined to be 0.67 mM and 23.26 μM/min, respectively. The optimum pH and optimum temperature of the enzyme for hydrolysis of pNP-NAG was to be at pH 5.7 and 55°C, respectively. The enzyme was stable in a pH range from 3.3 to 8.1 at 37°C, and inactive at temperature above 45°C. The enzyme activity was regulated by the following ions in decreasing order: Hg(2+) > Zn(2+) > Cu(2+) > Pb(2+) > Mn(2+). The IC50 of Cu(2+), Zn(2+) and Hg(2+) was 1.23, 0.28, and 0.0027 mM, respectively. However, the ions Li(+), Na(+), K(+), Mg(2+) and Ca(2+) had almost no influence on enzyme activity. In conclusion, the enzymatic characterizations of NAGase from tilapia were special to the other animals, which were correlated with its living habit; besides, CuSO4 and ZnSO4 should used very carefully as insecticides in tilapia cultivation since they both had strong regulations on the enzyme.
authors.affiliation: University Key Lab for Integrated Chinese Traditional and Western Veterinary Medicine and Animal Healthcare in Fujian Province, Fujian Agriculture and Forestry University, Fuzhou 350002, China.
authors.forename: Wei-Ni
authors.initials: WN
authors.lastname: Zhang
authors.affiliation: University Key Lab for Integrated Chinese Traditional and Western Veterinary Medicine and Animal Healthcare in Fujian Province, Fujian Agriculture and Forestry University, Fuzhou 350002, China.
authors.forename: Ding-Ping
authors.initials: DP
authors.lastname: Bai
authors.affiliation: University Key Lab for Integrated Chinese Traditional and Western Veterinary Medicine and Animal Healthcare in Fujian Province, Fujian Agriculture and Forestry University, Fuzhou 350002, China.
authors.forename: Yi-Fan
authors.initials: YF
authors.lastname: Huang
authors.affiliation: University Key Lab for Integrated Chinese Traditional and Western Veterinary Medicine and Animal Healthcare in Fujian Province, Fujian Agriculture and Forestry University, Fuzhou 350002, China.
authors.forename: Chong-Wei
authors.initials: CW
authors.lastname: Hu
authors.affiliation: School of Life Sciences, Xiamen University, Xiamen 361005, China.
authors.forename: Qing-Xi
authors.initials: QX
authors.lastname: Chen
authors.affiliation: University Key Lab for Integrated Chinese Traditional and Western Veterinary Medicine and Animal Healthcare in Fujian Province, Fujian Agriculture and Forestry University, Fuzhou 350002, China. Electronic address: xhhuang138@hotmail.com.
authors.forename: Xiao-Hong
authors.initials: XH
authors.lastname: Huang
mesh_terms: D000118:Acetylglucosaminidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D023681:Cichlids; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D008297:Male; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D013696:Temperature
chemical_list: D008670:Metals; D000118:Acetylglucosaminidase
keywords: Activity regulation; IC(50); K(m); Kinetics; Michaelis–Menten constant; N-Acetyl-β-d-glucosaminidase; N-acetyl-β-d-glucosaminidase; NAGase; PAGE; PBS; Reproduction; Tilapia; V(m); inhibitor concentrations leading to 50% activity loss; maximum velocity; p-nitrophenyl; p-nitrophenyl-N-acetyl-β-d-glucosaminide; pNP; pNP-NAG; polyacrylamide gel electrophoresis; sodium phosphate buffer