日本鳗鲡肠道N-乙酰-β-D-氨基葡萄糖苷酶的分离纯化及酶学性质  

PURIFICATION OF N-ACETYL-β-D-GLUCOSAMINIDASE FROM INTESTINE OF ANGUILLA JAPONICA AND ITS ENZYMATIC CHARACTERISTICS

作　　者：林建城 胡建辉 吴钦端 LIN Jian-Cheng;HU Jian-Hui;WU Qin-Duan(Fujian Provincial Key Laboratory of Ecology-toxicological Effects&Control for Emerging Contaminants,Key Laboratory Ecological Environment and Information Atlas(Putian University),College of Environmental and Biological Engineering,Putian University,Putian 351100,China)

机构地区：[1]莆田学院环境与生物工程学院,福建省新型污染物生态毒理效应与控制重点实验室,生态环境及其信息图谱福建省高等学校重点实验室(莆田学院),莆田351100

出　　处：《水生生物学报》2022年第9期1301-1309,共9页Acta Hydrobiologica Sinica

基　　金：福建省自然科学基金(2018J01467);莆田市科技计划项目(2021ZP09)资助。

摘　　要：为了探讨日本鳗鲡(Anguilla japonica)N-乙酰-β-D-氨基葡萄糖苷酶(EC3.2.1.52,NAGase)的分离纯化及其酶学性质,通过硫酸铵沉淀分级分离、Sephadex G-100分子筛凝胶柱层析和DEAE-32离子交换柱层析纯化NAGase,经聚丙烯酰胺凝胶电泳(PAGE)和SDS-PAGE鉴定酶的纯度、测定酶蛋白亚基分子质量;以对-硝基苯-N-乙酰-β-D-氨基葡萄糖为底物,研究NAGase催化反应的动力学参数,探讨其酶学性质。结果表明:日本鳗鲡肠道NAGase纯酶制剂比活力为2517.40 U/mg,酶蛋白亚基分子质量为69.98 kD,酶的最适pH、最适温度、米氏常数K_(m)和最大反应速度V_(max)分别为6.0、60℃、0.336 mmol/L和7.634μmol/(L·min);酶在pH 4.8—7.2较稳定,在温度60℃以下具有较好的热稳定性,在65℃以上酶迅速失活。Mg^(2+)、Ca^(2+)、Mn^(2+)、Cu^(2+)和Fe^(3+)对NAGase表现出不同程度的激活作用,Na^(+)、Li+和Ba^(2+)对酶活力几乎没有影响,Zn^(2+)、Fe^(2+)、Pb^(2+)和Hg^(2+)对酶活力有不同程度的抑制作用,Hg^(2+)对酶活力抑制作用最强,1.0μmol/L Hg^(2+)可使酶活力丧失83.69%。化学修饰法研究表明,精氨酸胍基不是日本鳗鲡NAGase的必需基团,而赖氨酸ԑ-氨基、半胱氨酸巯基、组氨酸咪唑基、丝氨酸羟基和色氨酸吲哚基是酶的必需基团,二硫键是NAGase活性所必需的。综上所述,实验采用的日本鳗鲡肠道NAGase分离纯化方案有效可行,酶活力易受环境中酸碱度、温度和金属离子的影响,且与其他不同动物来源的NAGase具有相似的必需基团。In order to investigate the purification and its enzymatic characteristics of N-acetyl-β-D-glucosaminidase(EC3.2.1.52,NAGase)from intestinal tract of Japanese eel,Anguilla japonica,NAGase was purified by extraction with ammonium sulfate fractionation,then chromatographed on Sephadex G-100 followed by DEAE-cellulose(DE-32)columns.The purified enzyme was determined to be homogeneous by polyacrylamide gel electrophoresis(PAGE)and SDS-PAGE.The kinetic parameters of NAGase for the hydrolysis of pNP-β-D-GlcNAc(enzyme substrate)and en-zymatic characteristics were also determined.The specific activity of the purified enzyme was 2517.40 U/mg.The mo-lecular weight of enzyme was 69.98 kD.The optimum pH and optimum temperature of the enzyme were 6.0 and 60℃,respectively.The K_(m) value was 0.336 mmol/L and the V_(max) value was 7.634μmol/(L·min),respectively.The enzyme was stable with pH of 4.8 to 7.2 and temperature of 4-60℃.The enzyme lost its activity rapidly when temperature>65℃.The effects of metal ions on the enzyme were also studied.Mg^(2+),Ca^(2+),Mn^(2+),Cu^(2+)and Fe^(3+)showed diffe-rent degrees of activation effects on the NAGase.Na^(+),Li+and Ba^(2+)had no influence on enzyme activity.Zn^(2+),Fe^(2+),Pb^(2+)and Hg^(2+)showed various degrees of inhibitory effects on the NAGase.Hg^(2+)inhibited the enzyme the most,and the enzyme activity decreased by 83.69%when its concentration reached 1.0μmol/L.The essential groups of the NAGase were investigated using chemical modification method.The results demonstrated that essential groups of NAGase in-cluded lysine'sԑ-amidogen group,cysteine's sulfhydryl group,histidine's imidazolyl group,serine's hydroxyl group and tryptophan's indole group,while guanidyl of arginine was not an essential group of enzyme.Disulfide bond was essen-tial for the catalytic activity of the enzyme.In conclusion,the purification scheme of NAGase from intestine of An-guilla japonica was effective and feasible.The activity of enzyme was affected easily by acidity-alkalinity,temperature an

关 键 词：分离纯化 酶学性质 必需基团 N-乙酰-Β-D-氨基葡萄糖苷酶 日本鳗鲡 

分 类 号：Q556.2[生物学—生物化学]