以中国鲎(Tachypleus tridentatus)内脏为材料,分别通过30%和80%饱和度(NH₄)_2SO₄沉淀分级分离、葡聚糖凝胶柱Sephadex G-200层析、以及离子交换柱DEAE-32层析,从而获得N-乙酰-β-D-氨基葡萄糖苷酶(EC3.2.1.52,NAGase),经PAGE检定达到电泳纯,酶的比活力为505.21 U/mg。SDS-PAGE显示一谱带,测得该酶蛋白亚基分子量为121.5kDa,等电聚焦电泳法测得酶的等电点pI为6.01。以pNP-β-D-GlcNAc为底物,研究NAGase催化反应的动力学参数。结果表明:中国鲎NAGase的最适pH、最适温度、K_m和V_max分别为5.4和55℃、0.421 mmol/L和13.158μmol/L·min^-1;酶在pH=4.5⁷.0范围内较稳定,在20~50℃之间具有较好的热稳定性。酶在276nm波长处有紫外吸收峰,在232.2nm波长的内源荧光激发下,酶内源荧光发射光谱峰在330.9nm波长处。Na⁺,K⁺和Li⁺对NAGase活力没有影响,Ca²⁺、Ba²⁺和Co²⁺对NAGase有不同程度的激活作用,Co²⁺对NAGase的激活作用较大,5mmol/L Co²⁺能使NAGase活力提高41.67%;Mg²⁺、Cu²⁺、Zn²⁺、Mn²⁺、Fe³⁺、Al³⁺、Pb²⁺、Ag⁺、Cd²⁺和Hg²⁺等10种金属离子对NAGase活力有不同程度的抑制作用,5mmol/L Cd²⁺可使酶活力丧失85.60%,而Hg²⁺对酶的抑制作用最强,0.1mmol/L Hg²⁺可使酶活力丧失90.10%。EDTA对酶活力没有影响,推断中国鲎NAGase属于非金属酶类。

Chitinase was composed of endochitinase,exochitinase,andβ-N-acetyl-D-glucosaminidase(EC3.2.1.52,NAGase).Chitin was cleavaged into monomer and oligomers ofβ-N-acetylglucosamine by NAGase,thenNAGase hydrolyzed chitobiose into monomer.NAGase had many functions in arthropod.NAGase not only degraded chitin in food,but also played important roles in the molting and hatching processes.Horseshoe Crab(Tachypleustridentatus)was an important marine species.Tachypleustridentatus needed no more than six times molting during embryonic development and thirteen to fourteen times molting in its life cycle.Therefore,the appropriate level of NAGase activity was a benefit to the molting cycle,especially during embryonic development.Marine environmental factors,such as temperature,pH,metal ions and others pollutants might influence the NAGase activity,which would regulate the animal's molting.Now,the purpose of this study was to discuss the purification of NAGase from the viscera of Tachypleustridentatus and its enzymatic characteristics.The NAGase was purified from the viscera of Tachypleustridentatus by 30%and 80%ammonium sulfate fractionation and chromatography on Sephadex G-200 and DEAE-cellulose(DE-32).The purified enzyme preparation was homogeneous as judged by polyacrylamide gel electrophoresis(PAGE)and SDS-PAGE.The specific activity of purified enzyme was 505.21U/mg.The molecular weight of enzyme protein subunit was determined to be 121.5kDa.The pI value was calculated to be 6.01 by isoelectric focusing.The optimal pH value was5.4and the optimal temperature was 55 ℃.The NAGase was stable at a temperature range from 20 ℃to 50 ℃ and in the pH range of 4.5to 7.5.The activity of NAGase also had 21.3% at temperature60 ℃for one hour treatment.The result suggested that the thermal stability of NAGase in Tachypleustridentatus was better than other species.In addition,the activity of NAGase was 19.1% at pH 10.0for one hour treatment.The result suggested that NAGase was not stable at alkaline environment.The enzyme followed typical Michaelis-Menten kinetics for the hydrolysis ofρ-nitrophenyl-N-acetyl-β-D-glucosaminide(pNP-β-D-GlcNAc).The K_mand V_max values were determined to be 0.421 mmol/L and13.158μmol/L·min^-1,respectively.The peak wavelength of UV absorption of the NAGase was276 nm.When the excitation wavelength was 232.2nm,the peak wavelength of fluorescence emission spectra of the NAGase was 330.9nm.The effect of metal ions on the NAGase was studied.Metal ions Na⁺,K~+and Li~+had no effect on the enzyme activity.Ca²⁺and Ba²⁺activated the enzyme slightly.5mmol/L Co²⁺led enzyme activity to increase by 41.67%.Mg²⁺、Cu²⁺、Zn²⁺、Mn²⁺、Fe³⁺、Al ³⁺、Pb²⁺、Ag⁺、Cd²⁺and Hg²⁺showed various degrees of inhibitory effects on the enzyme.5mmol/L of Cd²⁺inhibited the activity of enzyme by 85.60% when 0.1mmol/L of Hg²⁺inhibited the activity of enzyme by90.10%.EDTA had no effect on the enzyme activity indicating the NAGase was nonmetallic enzyme.