【作者】 陈宏惠；

【导师】 黄一帆； 黄小红；

【作者基本信息】 福建农林大学 ， 基础兽医学， 2007， 硕士

【摘要】 以中华绒螯蟹（Eriocheir sinensis）内脏为材料，通过0.01mol·L^-1 Tris-HCI缓冲液(pH 7.5含0.2mol·L^-1 NaCl，5％乙醇)抽提处理，硫酸铵分级盐析、DEAE-Cellulose（DEAE-32）离子交换柱、Sephadex G-100分子筛、第二次DEAE-32离子交换柱3次层析纯化，获得比活力为4490.79U／mg的电泳单一纯N-乙酰-β-D-氨基葡萄糖苷酶（NAGase，EC3.2.1.52）。该酶亚基分子量分别为121.21、98.63和73.48 kD，等电点为4.5。以对-硝基苯-N-乙酰-β-D-氨基葡萄糖苷（pNP-NAG）为底物，测得该酶在0.2mol·L^-1磷酸缓冲液（pH5.6）中催化水解pNP-NAG的最适pH为5.5，最适温度为45℃；该酶在低于45℃，pH 4.9-9.3范围内稳定。在pH5.6的条件下，酶水解pNP-NAG的米氏常数K_m为0.357mmol·L^-1，最大反应速度V_max为10.409umol·L^-1·min^-1，反应的活化能为76.50KJ／mol。研究几种有机溶剂对该酶活力的影响。结果表明：甲醇和乙醇对酶有先扬后抑作用，正丙醇、异丙醇、甲醛、丙酮、苯酚和二甲亚砜对酶的活性均有不同程度的抑制作用。甲醇、乙醇和异丙醇对酶的抑制类型都为可逆竞争性抑制类型，它们对酶的抑制常数K_I分别为3.66mol·L^-1、0.36mol·L^-1和0.48mmol·L^-1。甲醛、苯酚和二甲亚砜对酶的抑制类型属于可逆非竞争性抑制类型，它们对酶的抑制常数K_I分别为0.288mol·L^-1、0.268mmol·L^-1和0.276 mol·L^-1。正丙醇、丙酮对酶的抑制类型为可逆混合型抑制类型，正丙醇对酶的抑制常数K_I与K_IS分别为3.14mmol·L^-1和9.35mmol·L^-1，丙酮对酶的抑制常数K_I与K_IS分别为0.195mol·L^-1和0.620 mol·L^-1。戊二醛对酶活力没有影响。研究金属离子对该酶的影响，结果表明：Na⁺对酶有激活作用，Ba²⁺、Ag⁺对酶活力有先扬后抑作用。Li⁺和K⁺对酶有微弱的抑制作用，Mg²⁺、Cu²⁺、pb²⁺、Zn²⁺、Mn²⁺和Al³⁺对酶活力有不同程度的抑制作用。Mg²⁺、Cu²⁺和Zn²⁺对酶的抑制均表现为可逆非竞争性抑制类型，它们对酶的抑制常数K_I分别为0.704mol·L^-1、1.25mmol·L^-1和8.10mmol·L^-1。Ag⁺对酶的抑制作用为可逆反竞争性抑制类型，Ag⁺对结合酶（ES）的抑制常数K_I为204.508mmol·L^-1。pb²⁺对酶的抑制表现为可逆混合型抑制，pb²⁺对酶的抑制常数K_I与K_IS分别为10.44mmol·L^-1和2.217mol·L^-1。Ca²⁺对酶活力没有显著影响。研究12种氨基酸对该酶的影响。结果表明：在特定浓度范围内，L-Gly、L-Val、L-Thr、L-Ala、L-Phe和L-Ser等氨基酸对酶活力没有显著影响，而L-Gln、L-Met、L-Pro、L-Arg、L-His和L-Lys对河蟹NAGase有不同程度的抑制作用。L-Pro对酶的抑制表现为可逆非竞争性类型，L-Pro对酶的抑制常数K_I为702.87mmol·L^-1。L-Arg、L-His和L-Lys对酶的抑制均表现为可逆反竞争性类型，它们对酶的抑制分别为1.682mmol·L^-1、85.81mmol·L^-1和1.517mmol·L^-1。水体中的富营养化产生的SO₃^2-对该酶活力有抑制作用，抑制类型为可逆反竞争性类型，50₃^2-对酶的抑制常数K_I为253.995mmol·L^-1。NO₂^-在0-10mmol·L^-1范围内对酶活力有激活作用。研究过氧化氢对该酶的影响，结果表明，过氧化氢对酶有抑制作用，抑制类型为可逆反竞争性类型，过氧化氢对结合酶（Es）的抑制常数K_I为136.78mmol·L^-1。研究尿素对酶的影响，结果表明，尿素对酶活力有抑制作用，尿素对酶的抑制属于可逆混合型抑制类型，尿素对酶的抑制常数K_I与K_IS分别为379.02mmol·L^<sub>-1和1.182mol·L^-1。更多还原

【Abstract】 N-Acetyl-β-D-glucosaminidase（NAGase,EC3．2．1．52） was purified from rivercrab （Eriocheir sinensis）, by extraction with 0．01mol·L^-1 Tris-HCl buffer （pH7．5） containing 0．2 mol·L^-1 NaCl and 5 percent ethanol, then ammonium sulfate fraction,and then chromatography on DEAE-cellulose （DEAE-32） , Sephades G-100 andDEAE-32．The purified enzyme was a single band on polyacrylamide gelelectrophoresis with specific activity to be 4490．79U/mg. The subunit molecularweight were determined to be 121．21, 98．63 and 73．48 kD. The pI value wasdetermined to be 4．5 by isoelectric focusing.The optimum pH and optimumtemperature of the enzyme for the hydrolysis of p-Nitrophenyl-N-Acetyl-β-D-gluco-saminide （pNP-NAG） were investigated to be at pH5．5 and at 45℃, respectively.The result of the stability showed that the enzyme was stable at the pH range from 4．9to 9．3 and at the temperature below 45℃. The kinetic behavior of the enzyme in thehydrolysis of pNP-NAG followed Michaelis-Menten kinetics with K_m of0．357mmol·L and V_m of 10．409umol·L^-1·min^-1 at pH5．6 and 37℃, and theactivation energy was determined to be 76．50KJ/mol.The effects of different organic solwents on activity of the enzyme wereinvestigated. The results indicated that methanol and alcohol activated the enzymeat low concentrations, but they inhibited it at high concentration. Propanal,isopropanal, formaldehyde, acetone, phenol and SMOS inhibited the enzyme in varydegree. Methanol, alcohol and isopropanal were reversible competitive inhibitors.Theinhibition constants of free enzyme（K₁） of methanol, alcohol and isopropanal were0．288mol·L^-1 , 0．36mol·L^-1 and 0．48mmol·L^-1 , respectively. Formaldehyde, phenoland SMOS were reversible non-competitive inhibitors. The inhibition constants offree enzyme （K₁） of formaldehyde, phenol and SMOS were 0．288mol·L^-1 ,0．268mmol·L^-1 and 0．288mol·L^-1 , respectively. Propanal and acetone werereversible mixed-type inhibitors. The inhibition constants of free enzyme（K₁） andenzyme-substrate complex(K_IS) of propanal were determined to be 3．14mmol·L^-1 and 9．35mmol·L^-1，respectively. The inhibition constants of free enzyme （K₁） andenzyme-substrate complex(K_IS) of acetone were determined to be 0．195 mol·L^-1 and0．620 mol·L^-1 , respectively. Glutaraldehyde had no influence on the activity of theenzyme.The effects of metal ions on the enzyme were studied.The results showed thatNa⁺ activated the enzyme activity. Ba²⁺ and Ag⁺ activated the enzyme at low concentrations, but they inhibited it at high concentrations. Li⁺ and K⁺ ions inhibitedthe enzyme slightly. Mg²⁺, Cu²⁺, Pb²⁺, Zn²⁺, Mn²⁺ and Al³⁺ inhibited the enzyme.Mg²⁺, Cu²⁺ and Zn²⁺ were reversible non-competitive inhibitors. The inhibitionconstants of free enzyme （K₁） of Mg²⁺, Cu²⁺ and Zn²⁺ were 0．704 mol·L^-1 , 1．25mmol·L^-1 and 8．10mmol·L^-1 , respectively. Ag⁺ was reversible un-competitiveinhibitor. The inhibition constants（K₁） was 204．508mmol·L^-1 . Pb²⁺ was reversiblemixed-type inhibition.The inhibition constants of free enzyme（K₁） andenzyme-substrate complex(K_IS) of Pb²⁺ were determined to be 10．44mmol·L^-1 and 2．217mmol·L^-1 , respectively. Ca²⁺ had no influence on the activityof the enzyme.The effects of 12 kinds of amino acids on the enzyme were studied. L-Gly,L-Val, L-Thr, L-Ala, L-Phe and L-Ser had no influence on the activity of theenzyme in range of the certain concentrations. L-Gln, L-Met, L-Pro, L-Arg, L-Hisand L-Lys inhibited the enzyme in vary degree. L-Pro was reversible non-competitiveinhibitor. The inhibition constants of free enzyme （K₁） of L-Pro was 702．87mmol·L^-1 .L-Arg, L-His and L-Lys were reversible un-competitive inhibitors. The inhibitionconstants of free enzyme （K₁） of L-Arg, L-His and L-Lys were 1．682mmol·L^-1 ,85．81mmol·L^-1 and 1．517 mmol·L^-1 , respectively.The SO-3^2- which was brought because of the eutrophication could inhibit theenzyme activity obviously.lt was reversible un-competitive inhibitor. The inhibitionconstants of free enzyme（K₁） of SO₃^2- was 253．995mmol·L^-1 . The NO₂^-activatedthe enzyme activity when its concentration was in the range from 0 mmol·L^-1 to10mmol·L^-1 .The effect of hydrogen peroxide on the enzyme was studied.The inhibitionmechanism of hydrogen peroxide was reversible un-competitive mechanism.Theinhibition constants of free enzyme（K₁） of hydrogen peroxide was determined to be136．78mmol·L^-1 .Urea was reversible mixed-type inhibitor. The inhibition constants of freeenzyme（K₁） and enzyme-substrate complex(K_IS) of urea were determined to be379．02mmol·L^-1 and 1．182 mol·L^-1 , respectively.更多还原