意大利蜜蜂N-乙酰-β-D-氨基葡萄糖苷酶的分离纯化及性质分析  

Purification and characterization of N-acetyl-β-D-glucosaminidase from the Italian honey bee,Apis mellifera ligustica( Hymenoptera: Apidae)

作　　者：张伟妮[1] 胡崇伟[1] 张梦馨 韩威超 黄小红[1] ZHANG Wei-Ni;HU Chong-Wei;ZHANG Meng-Xin;HAN Wei-Chao;HUANG Xiao-Hong(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)

机构地区：[1]福建农林大学,中西兽医结合与动物保健福建省高校重点实验室,福州350002

出　　处：《昆虫学报》2018年第10期1153-1159,共7页Acta Entomologica Sinica

基　　金：国家自然科学基金项目(31572484)。

摘　　要：【目标】N-乙酰-β-D-氨基葡萄糖糖苷酶(NAGase)是一种重要的几丁质分解酶,能从N-乙酰葡萄糖苷的非还原端催化去除β-1,4-N-乙酰-D-氨基葡萄糖残基,参与了昆虫外骨骼的蜕皮过程。研究蜜蜂该酶的特征有助于阐明其在蜜蜂发育过程中的作用机制。【方法】采用40%-70%硫酸铵分级沉淀、DEAE-纤维素离子交换层析和葡聚糖G-100凝胶过滤层析的方法从意大利蜜蜂Apis mellifera ligustica幼虫体内分离纯化NAGase。以对-硝基苯-N-乙酰-β-D-氨基葡萄糖苷(pNP-NAG)为底物检测该酶的活力,用native PAGE和SDS-PAGE检测酶的纯度。IEF-PAGE测定该酶等电点。葡聚糖G-200凝胶过滤层析测定酶的总分子量。【结果】结果显示,纯化的NAGase酶的比活力为803. 09 U/mg,总分子量为77. 3 kD。结合SDS-PAGE表明该酶由两个具有相同分子量(39 k D)的亚基组成。该酶等电点为4. 8。酶水解底物pNP-NAG的过程遵循米氏方程,米氏常数(Km)和最大反应速度(Vm)分别为0. 11 mmol/L和17. 65μmol/L·min。该酶水解反应的最适pH和最适温度分别为pH 5. 5和60℃。酶催化pNP-NAG反应的活化能为64. 8 k J/mol。Pb2+,Cu2+,Zn2+和Al3+对该酶有不同程度的抑制作用。【结论】本研究描述了意大利蜜蜂NAGase的分离纯化方法及其理化性质,为进一步进行蜜蜂NAGase的结构解析和功能研究奠定基础。【Aim】N-acetyl-β-D-glucosaminidase(NAGase)is an important chitinase for degradation of chitin.It can cleave terminal N-acetyl-β-D-glucosamine residues from the nonreducing ends of N-acetyl-β-D-glucosides,participating in the ecdysis of exoskeletons in insects.Researching and characterizing this enzyme from honey bees may help to clarify its mechanism of action in the development of honey bees.【Methods】NAGase was purified from larvae of the Italian honey bee(Apis mellifera ligustica)by means of(NH 4)2SO 4 fractionation(40%-70%of saturation),DEAE-cellulose ion exchange chromatography,and Sephadex G-100 gel filtration.The enzyme activity was determined by using p-nitrophenyl-N-acetyl-β-D-glucosaminide(pNP-NAG)as the substrate.The purity of the enzyme was determined by native PAGE and SDS-PAGE.The isoelectric point(pI)of the enzyme was measured by IEF-PAGE,and its molecular weight was determined by Sephadex G-200 gel filtration.【Results】The specific activity of purified NAGase was 803.09 U/mg and the molecular weight was 77.3 kD.The enzyme was composed of two subunits with the same molecular weight of 39 kD.Its pI was determined to be 4.8.Results of kinetic analysis indicated that the enzyme in the hydrolysis of pNP-NAG followed Michaelis-Menten kinetics with the Michaelis-Menten constant(K m)of 0.11 mmol/L and the maximum velocity(V m)of 17.65μmol/L·min,respectively.The optimum pH and optimum temperature of the enzyme for hydrolysis of pNP-NAG was pH 5.5 and 60℃,respectively.And the activation energy of the enzyme for the hydrolysis of pNP-NAG was determined to be 64.8 kJ/mol.Pb 2+,Cu 2+,Zn 2+and Al 3+inhibited the enzyme activity in different degrees.【Conclusion】In this study the enzyme NAGase has been purified and characterized from A.mellifera ligustica,laying a foundation for further unveiling the structure and function of NAGase in bees.

关 键 词：意大利蜜蜂 N-乙酰-Β-D-氨基葡萄糖苷酶 纯化 性质分析 动力学 

分 类 号：Q556.2[生物学—生物化学]