【作者】 陈欣颖；

【导师】 黄小红；

【作者基本信息】 福建农林大学 ， 动物遗传育种与繁殖， 2010， 硕士

【摘要】 N-乙酰-β-D-氨基葡萄糖苷酶在昆虫和甲壳动物角质层的转化和转移及营养消化的生理过程中发挥了重要作用。本研究采用硫酸铵分级沉淀从克氏原螯虾内脏分离出粗酶制剂后再经前后2次DEAE-32离子交换柱层析和SephadexG-100分子筛柱层析获得比活力为6540.24 U·mg-1的聚丙烯酰胺凝胶电泳纯N-乙酰-β-D-氨基葡萄糖苷酶纯酶制剂。测得该酶分子的总分子量为118.66 kDa,等电点为5.15 ,每个酶分子中含有约400余个氨基酸残基;以对-硝基苯-N-乙酰-β-D-氨基葡萄糖(pNP-β-D-GlcNAc)为底物,研究酶催化底物水解的反应动力学,结果表明,酶的最适温度为40℃,最适pH为5.5。该酶在50℃以下处理30 min,酶活力稳定,高于55℃,酶稳定性较差, 75℃酶完全失活;在pH 3.6~5.9范围内较稳定,而在pH>6.2和pH<3.6下失活加快;酶催化底物反应的活化能为114.89 kJ·mol-1。以对-硝基苯-N-乙酰-β-D-氨基葡萄糖(pNP-β-D-GlcNAc)和p-硝基苯基-N-乙酰-β-D-氨基半乳糖苷(pNP-β-D-GaNAc)为底物,研究酶促反应动力学符合米氏双曲线方程,测得以pNP-β-D-GlcNAc为底物时最大反应速度Vm为10.87μmol·L-1min-1,米氏常数Km为0.46 mmol·L-1;以pNP-β-D-GaNAc为底物时最大反应速度Vm为39.40μmol·L-1min-1,米氏常数Km为1.04 mmol·L-1。金属离子对克氏原螯虾NAGase的效应试验表明:在特定浓度范围内, Li+ ,K+对酶活力几乎没有影响; Na+、Ba2+、Ca2+、Co2+则对酶有不同程度的激活作用;Mn2+、Mg2+、Pb2+、Ag+对酶有先扬后抑的作用;Zn2+、Hg2+、Cu2+、Al3+等重金属离子对酶都有不同程度的抑制作用。在一定浓度范围内,金属离子对克氏原螯虾NAGase的抑制作用强度从大到小依次为:Hg+ > Cu2+ > Al3+ > Zn2+ > Ag+ > Pb2+ > Mn2+ > Mg2+。Mn2+、Ag+、Zn2+、Al3+、Hg+对克氏原螯虾NAGase的抑制均表现为可逆反竞争性机制,其抑制常数KI分别为0.41 mmol·L-1、1.08 mmol·L-1、1.22 mmol·L-1、0.87 mmol·L-1、0.069 mmol·L-1。Mg2+和Pb2+对克氏原螯虾NAGase的抑制表现分别为竞争性和非竞争性,其抑制常数KI分别为1.59 mmol·L-1和0.41 mmol·L-1。氨基酸对克氏原螯虾NAGase的效应试验表明:在特定浓度范围内,非极性氨基酸L-Ala、L-Phe、L-Met、L-Pro,极性氨基酸L-Gly、L-Thr、L-Ser、L-Gln对克氏原螯虾NAGase活力没有显著影响,碱性氨基酸L-Arg、L-His、L-Lys对克氏原螯虾NAGase有不同程度的抑制作用。L-Arg、L-His、L-Lys对克氏原螯虾NAGase的IC50为45mmol·L-1、130mmol·L-1、110mmol·L-1。碱性氨基酸L-Arg、L-His、L-Lys对克氏原螯虾NAGase的抑制均表现为可逆非竞争性,其KI分别为10.76 mmol·L-1、226.12 mmol·L-1、157.70 mmol·L-1。研究不同生长阶段克氏原螯虾各器官NAGase活力变化。结果表明:克氏原螯虾肝胰腺NAGsase活力随着年龄的增大逐期递减。酶活力I期(2~3月龄)与II期(5~6月龄)比较降低趋势差异显著,III期(9~10月龄)和II期比较有降低趋势,但差异不明显;克氏原螯虾性腺和壳膜NAGase活力变化规律一致,都是逐期递减,但I期和II期递减趋势不明显,到了III期后有了明显的下降。此外,比较同一生长期克氏原螯虾各器官NAGsase活力大小顺序为:肝胰腺>性腺>壳膜。比较研究不同生长阶段克氏原螯虾肝胰腺、性腺及甲壳膜三个器官NAGase的理化性质,发现克氏原螯虾肝胰腺NAGase在I、II、III期的最适温度都是35℃;性腺NAGase在I、II期最适温度为35℃,III期为40℃;甲壳膜NAGase在I期最适温度为40℃,II、III期均为35℃。克氏原螯虾肝胰腺NAGase在I、III期的最适pH都是5.5,II期为5.2;性腺NAGase在I期最适pH为5.5,II、III期为5.2;甲壳膜NAGase在I、II期的最适pH均为4.5,在III期为5.5。更多还原

【Abstract】 β-N-Acetyl-D-glucosaminidase play an important role in the physiological process the conversion and transformation of the cuticle of insects ,crustaceans and nutrition digestion.Theβ-N-Acetyl-D-glucosaminidase was purified from Procambarus clarkii by ammonium sulfate fractionation, twice tchromatography on DEAE-cellulose (DEAE-32) and Sephadex G-100.The purified enzyme showed a single band on polyacrylamide gel electrophoresis and isoelectric focusing electrophoresis,and the specific activity was 6540.24 U·mg -1.The molecular weight of the whole enzyme was 118.66kD, consisted of 364 amino acid residues.Its isoelectric point is 5.15. The optimum pH and optimum temperature of the enzyme for the hydrolysis of p-nitrophenyl-N acetyl-β-D-glucosaminide (pNP-β-D-GlcNAc)were found at pH 5.5 and at 40°C, respectively. The study of its stability showed that the enzyme was stable in the pH range from 3.6 to 5.9 and at temperatures below 50°C. The activation energy was 114.89kJ·mol-1.The kinetic behavior of the enzyme in the hydrolysis of pNP-β-D-GlcNAc followed Michaelis–Menten kinetics with Km of 0.46 mmol·L-1 and Vm of 10.87μmol·L-1·min -1 ,and the kinetic behavior of the enzyme in the hydrolysis of pNP-β-D-GaNAc followed Michaelis–Menten kinetics with Km of 1.04 mmol·L-1 and Vm of 39.40μmol·L-1·min -1 .The effects of metal ions on the enzyme were studied. The results showed that Li2+、K+ had not any effect on enzyme activity in the tested range of concentration. Na+、Ba2+、Ca2+、Co2+ could increase the activity of enzyme at different concentration. Mn2+、Mg2+、Pb2+、Ag+ increase enzyme activity at first then inhibit it. Zn2+、Hg2+、Cu2+、Al3+ were significantly reduce enzyme activity to different degrees. The intensity of inhibition effects of metal ions on the enzyme from big to small in turn: Hg+ > Cu2+ > Al3+ > Zn2+ > Ag+ > Pb2+ > Mn2+ > Mg2+. Mn2+、Ag+、Zn2+、Al3+、Hg+ were reversible anticompetitive inhibitor . The KI were 0.41 mmol·L-1、1.08 mmol·L-1、1.22 mmol·L-1、0.87 mmol·L-1、0.07 mmol·L-1 respectively. Mg2+ was revealed to be a reversible non-competitive inhibitor with a KI of 1.59 mmol?L-1.Pb2+ was revealed to be a reversible competitive inhibitor with a KI of 26.17 mmol?L-1. The effects of amino acids on the enzyme were studied. The results showed that L-Ala、L-Phe、L-Met、L-Pro、L-Gly、L-Thr、L-Ser、L-Gln had not any effect on enzyme activity in the tested range of concentration. L-Arg、L-His、L-Lys reduced the activity of enzyme at different concentration.The IC50 of L-Arg、L-His、L-Lys on the enzyme in turn: 45mmol·L-1、130mmol·L-1、110mmol·L-1. L-Arg、L-His、L-Lys were revealed to be a reversible competitive inhibitor with KI of 10.76mmol?L-1、226.12 mmol?L-1、.157.70 mmol?L-1.We studied the variations of activity ofβ-N-Acetyl-D-glucosaminidase from different organs of Procambarus clarkii in different growth stages. The result s showed that the enzyme activities of the hepatopancreas of Procambarus clarkii in different growth stages are variational.The activity of enzyme from the hepatopancreas of Procambarus clarkii was decreased one by one stage,there was a significant decreasing in stage I(2~3moon’s age) and stage II(5~6 moon’s age)、stage III(9~10 moon’s age).But had no significantly decreased in stageII and stage III . The activity of enzyme of the gonad were consistent with variation of shell membrane: decreasing one by one stage ,but there were no significantly decreased between in stageII and stageIII,significantly decreased in stageIII . In addition, the results showed that the enzyme activities of the variations organs of Procambarus clarkii from big to small in turn:The hepatopancreas> The gonad >The shell membrane.Studied some basic properties ofβ-N-Acetyl-D-glucosaminidase from different organs of Procambarus clarkii in different growth stages. The result s showed that the optimum temperature of the enzyme from the hepatopancreas of Procambarus clarkii is 35℃in all stage; the optimum temperature of the activity of enzyme of the gonad were 35℃in stageI、stageII.but 40℃in stageIII.The optimum temperature of the activity of enzyme of of shell membrane were 35℃in stageII、stageIII.but 40℃in stageI. The optimum pH of the enzyme from the hepatopancreas were 5.5 in stageI、stageIII. but 5.2 in stageII;The optimum pH of the activity of enzyme of the gonad were 5.2 in stageII、stageIII,but 5.5 in stageI; The optimum pH of the activity of enzyme of of shell membrane were 4.5 in stageI、stageII,but5.5 in stageIII.更多还原