中华绒螯蟹(Eriocheir sinensis)N-乙酰-β-D-氨基葡萄糖苷酶的分离纯化及性质的初步研究
PRELIMINARY STUDIES ON ISOLATION, PURIFICATION AND SOME PROPERTIES OF THE β-N-ACETYL-D-GLUCOSAMINIDASE FROM ERIOCHEIR SINENSIS
-
摘要: 本研究以中华绒螯蟹内脏为材料,经过硫酸铵沉淀分级分离、两次DEAE-32离子交换柱层析和Sephadex G-100分子筛柱层析纯化,获得比活力为4490.79U/mg、纯化倍数为28.07倍的聚丙烯酰胺凝胶电泳纯的N-乙酰-β-D-氨基葡萄糖苷酶制剂。酶分子中各亚基的分子量分别为121.219、8.63和73.48 kD,等电点为4.5。以对-硝基苯-N-乙酰-β-D-氨基葡萄糖为底物,进行酶催化底物水解的反应动力学研究,结果表明:酶催化底物反应的最适pH为5.5,最适温度为45℃。该酶在pH4.9-9.3区域或40℃以下处理30min,酶活力保持稳定。酶促反应动力学符合米氏双曲线方程,测得米氏常数Km为0.357 mmol/L,最大反应速度Vm为10.41μmol/L.min。酶催化pNP-β-D-GlcNAc反应的活化能为76.50kJ/mol。金属离子对酶的效应试验表明:Mg2+、Ca2+和Ba2+对酶活力没有影响。Na+对酶有激活作用,Li+、K+、Zn2+、Hg2+、Pb2+、Cu2+和Al3+对酶活力表现出不同程度的抑制作用。
-
关键词:
- 中华绒螯蟹 /
- N-乙酰-β-D-氨基葡萄糖苷酶 /
- 分离纯化 /
- 动力学 /
- 稳定性
Abstract: A β-N-Acetyl-D-glucosaminidase (EC3.2.1.30) was purified from the viscera of Edocheir sinensis by ammonium sulfate fractionation,chromatography on DEAE-32, Sephadex G-100 and DEAE-32. The purified enzyme preparation was homogeneous as judged by polyacrylamide gel electrophoresis. The specific activity of the enzyme was 4490.79U/rag. The molecular weight of the subunits was determined to be 121.21,98.63 and 73.48kD,respectively. The pI value was calculated to be 4.5 by isoelectric focusing. The optimum temperature and pH of the enzyme for the hydrolysis of p-nitrophenyl-N-acetyl-β-D-glucosaminide (pNP-NAG) were determined to be at 45℃and at pHS.5,respectively. The enzyme was stable in the pH ranges of 4.9 to 9.3 under 37℃ and at temperatures below 40℃. The enzyme follows typical Michaelis-Menten kinetics for the hydrolysis of pNP-β-D-GlcNAc.The Km and Vm values were determined to he 0.357mmo/L and 10.41μmol/L min at pH5.6 and 37℃, respectively.The activation energy of the enzyme for the hydrolysis of pNP-β-D-GlcNAc was to be 76.50kJ/mol.The effects of metal ions on the enzyme were studied. Mg2+, Ca2+ and Ba2+ had not influenced the enzyme activity. Na+ activated the enzyme, while, Li+, K+, Zn2+, Hg2+, Pb2+, Cu2+, and Al3+ showed various degrees of inhibitory effects on the enzyme. -
-
[1] Leake J R, Read D J. Chit in as a nitrogen source for mycorrhizal fungi[J]. Mycological. Res., 1990, 94: 993)995
[2] Austin P R. Chitin, chitoson and relat ed enzymes[M]. New York:Academic Press. 1984
[3] Keyhani N O, Roseman S. The chitin cat abolic cascade in the Marine bacterium Vibrio f urnissii[J]. J. Bi ol. Chem., 1996, 271 (52):33425)33432
[4] Broadway R M, Will iams D L, Kaln W C, et al. Part ial charact erization of chit inolytic enzymes from Streptomyces albidof lavus[J]. Letters in Applied Microbiology, 1995, 20: 271)276
[5] Funke B, Spindler K D. Charact erizat ion of chit inase from the Brine Shrimp Artemia[J]. Comp. Biochem. Physiol., 1989, 94B (4): 691)695
[6] Kono M, Matsui T, Shimizu C, Koga D. Purificat ions and some properties of chitinase from the liver of a Prawn, Penaeus japonicus[J].Agri c. Biol. Chem., 1990, 54(8): 2145)2147
[7] Huang X H, Chen Q X, You M S, Wang J, Guan X. Isolation, purification and partial characterizat ion of the B-N-Acet y-l D-glu-cosaminidase from the pupae of H elicoverpa armigera[J], Acta Ento-mologica Sinica, 2005, 48 (4): 498)502[黄小红, 陈清西, 尤民生, 王君, 关雄. 棉铃虫N-乙酰-B-D-氨基葡萄糖苷酶的分离纯化及性质的初步研究. 昆虫学报, 2005, 48(4): 498)502]
[8] Lowry O H, Rosebrough N J, Farr A L. Protein measurement with theFolin phenol reagent[J]. J. Bi ol. Chem., 1951, 193: 265)269
[9] Li J W, Xiao N G, Yu L Y, et al. Principle and method of biochemistry[M]. Beijing: University of Beijing Press. 1997[李建武, 萧能庚,余瑞元, 等. 生物化学实验原理和方法. 北京:北京大学出版社. 1997]
[10] Arnould C, Jeuniaux C. Les enzymes hydrolytiques du syst eme digest if ches les Crustaces Pagurides[J]. Cash Biol, 1982, 23: 89)103
[11] Spindler K D. Init ial characterization of chitinase and chitobiase from the integument of Drosophlia hydei[J]. Insect Biochem., 1976, (6): 663)667
[12] Koga D, Nakashima M, Matsukura T, et al. Purifications and some properties ofB-N-Acetyl-D-glucosaminidase from alimentary canal of the silk-worm Bombyx mori[J]. Agric. Biol. Chem., 1986, 50(9): 2357)2368
[13] Sabrowski R, Buchholz F. Alaborat ory study on digest ive processes in the Antarctic krill, Euphausia superba, with Special regard to chitinolytic enzymes[J]. Polar Biol., 1999, 21: 295)304
[14] Tsou C L. Kinet ics of substrat e reaction during irreversible modification of enzyme actirity[J]. Adv. Enzymol. Relat. Areas Mol. Biol.,1988, 61: 381)436
[15] Koga D, Hoshika H, M atsushit a M, et al. Purif ication and characterization of bat -aN-acetyl hexosaminidase from the l iver of a prawn, Penaeus j aponicus[J]. Bi osci. BioChem, 1996, 60(2): 194)199
[16] Spindler K D, Buchholz F. Part ial charact erization of chitin degrading enzymes from two euphauaiids, Euphausia superba and Meganyc-tiphanes norvegica[J]. Polar. Biol., 1988, (9): 115)122
[17] Espie P J, Ro J C. Charact erization of chito biase from Daphnia magna and its relat ion to chit in flux[J]. Physiol. Zool., 1995, 68: 727)748
[18] Sun S L, Yu Z N, Jia X C. Advance in study and application on chitinase produced by mi crobes[J]. Journal of Mi crobiol ogy, 2002, 22(5): 47)50[孙胜利, 喻子牛, 贾新成. 微生物产几丁质酶的研究和应用进展. 微生物学杂志, 2002, 22(5): 47)50]
[19] Huang X H, Chen Q X, Wang J, et al. Isolat ion, purifi cation and some propert ies of the chit inase from Bacillus thuri ngiensi s[J]. Chin J Appl Environ Biol, 2004, 10 (6): 771)773[黄小红, 陈清西, 王君,等. 苏云金芽孢杆菌几丁质酶的分离纯化及酶学性质. 应用与环境生物学报,2004, 10(6): 771)773]
[20] Chen S, Li D C, Liu K Q, et al. Purificat ion, properties ant ifungal activity of on chit inase produced by Tal aromycesf lavus[J]. Forest Pest and Disease, 2003, 22(3): 6)9[陈述, 李多川, 刘开启, 等. 黄蓝状菌一种几丁质酶的纯化、性质及抗菌活性. 中国森林病虫,2003, 22(3): 6)9]
[21] Ohtakara A, Mitsutomi M, Uchida Y. Purification and some propert ies of chitinase from Vibro sp.[J]. J. Ferment. Technol., 1979, 57(3): 169)177
[22] Koga D, Shimazaki C, Yamamot o K, et al.B-N-Acet y-l D-glu-cosaminidases from integument of the silkworm, Bombyx mori: comparative biochemist ry w ith t he pupal alimentary canal enzyme[J].Agri c. Biol. Chem., 1987, 51(6): 1679)1681
[23] Xie X L, Chen Q X, Lin J C, Wang Y. Purificat ion and some prope-rties ofB-N-Acetyl-D-glucosaminidase from Prawn (Penaeus vannamei)[J]. Marine Biol ogy, 2004, 146(1): 143)148
[24] Chen Q X, Zheng W Z, Lin J Y, et al. Ef fect of metal ions on the activity of green crab (Scylla serrata) alkaline phosphat ase[J]. Int J Biochem Cell Bi ol, 2000, 32(8): 879)885
[25] Wang J, Huang X H, Chen Q X. Effects of met al ions on t he activity of B-N-A cet yl-D-glucosaminidase from the pupae of H eli coverpa armigera[J]. Journal of Xiamen University, 2005, 44(6): 843)846[王君, 黄小红,陈清西,金属离子对棉铃虫N-乙酰-B-D-氨基葡萄糖苷酶活力的影响. 厦门大学学报, 2005,44(6): 843)846]
计量
- 文章访问数: 927
- HTML全文浏览量: 1
- PDF下载量: 520
下载: