【作者】 张茜；

【导师】 石艳；

【作者基本信息】 厦门大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 葡萄糖氧化酶（Glucose Oxidase,EC 1.1.3.4,简称GOD）在食品、医药及生物等领域有着广泛的应用。以尼崎青霉菌（Penicillium amagasakiense）为材料,采用发酵生产葡萄糖氧化酶,探讨不同时间、温度、转速、Ca²⁺、吐温80等对菌丝体和发酵液产酶的影响。24℃、200rpm、发酵六天、0.3mol/L的Ca²⁺、3%的吐温80的条件下,菌丝体和发酵液中总酶活力达到最大。发酵6天后,离心得菌丝体经破壁、缓冲液抽提,获得胞内葡萄糖氧化酶的粗酶液;发酵液即为胞外葡萄糖氧化酶的粗酶液。胞外葡萄糖氧化酶粗酶液浓缩后,采用DEAE-32离子交换柱层析、Sephacryl S-200分子筛凝胶过滤柱层析纯化,得到比活力为比活力达472 U/mg,纯化了29.7倍的的电泳单一纯的酶制剂,该酶的亚基分子量约为75 kD。该酶催化化葡萄糖反应的最适pH值为5.6,最适温度40℃,在pH 7.0,37℃条件下测得动力学,米氏常数K_m值为122.6 mmol/L,V_m为25.52μmol/（L·min）。酶的热稳定性研究表明:该酶在pH 5.5-8.5区间和在温度低于50℃下稳定。化学修饰剂对酶活力的影响表明咪唑基、吲哚基、精氨酸胍基是该酶的活性功能基团,而蛋白质分子中巯基、二硫键、赖氨酸的ε-氨基、蛋氨酸侧链的硫醚基和丝氨酸残基与酶活力无关。溴代乙酸（BrAc）、NBS的抑制机理为非竞争性类型,而乙酰丙酮其抑制机理表现为竞争性类型。它们的抑制常数分别为26.5、19.2、7.35 mmol/L。金属离子对酶活力的影响表明金属离子(Li⁺、Na⁺、K⁺、Ca²⁺、Mn²⁺)对酶活力也没有影响。Al³⁺和Zn²⁺对GOD具有轻微的激活作用;Ba²⁺、Co²⁺、Cd²⁺对酶有轻微抑制作用;Cu²⁺、Ag⁺对该酶的抑制机理表现为竞争性类型,Hg²⁺的抑制机理表现为非竞争性类型。其抑制常数（K₁）分别为10.78μmmol/L,0.065μmol/L和83.1μmol/L。有机溶剂对酶活力的影响表明甲醇、乙醇、丙醇、乙二醇、丙二醇、丙三醇对酶基本没有影响。甲醛对酶也都具有强烈的抑制作用,其半抑制浓度(IC₅₀)分别为81.25 mmol/L。甲醛的抑制机理表现为竞争性类型,其抑制常数（K₁）为30 mmol/L。变性剂对酶活力的影响:EDTA对酶基本上没有影响,脲对该酶有轻微的抑制作用。盐酸胍、SDS对酶有强烈的抑制作用,其抑制机理表现为竞争性类型,其抑制常数（K₁）分别为1.75、57.15 mmol/L。更多还原

【Abstract】 Glucose oxidase（GOD, EC 1.1.3.4） has great application in the fields of food、medicine and biology etc. This research established a set of method to extract and purity the glucose oxidase from Penicillium amagasakiense. We research different time、temperature、stirring speed and different concentration of Ca²⁺ Tween 80 on the GOD activity units from fermentation liquor. Under the consdition of 24℃、200rpm、fermentation for 6 days、0.3 mol/L of Ca²⁺、3% of Tween 80, the total GOD activity from fermentation liquor are to maximize。After 6 days fermentation, the mycelium was destroyed to withdraw the intracellular glucose oxidase with buffer solution, while the fermentation liquor was the extracellular glucose oxidase. Then fermentation liquor by the concentration, 50mmol/L Tris-HCl buffer （pH 8.0） dialysis to pH 8.0, the chromatography on DEAE-32, then by gel filtration through Sephacryl S-200. The purified enzyme was a single band on polyacrylamide gel electrophoresis and the specific activity was determined to be 472 U/mg. The subunit of enzyme was determined to be 75 kD.The optimum pH is 5.6, and the optimum temperature is 40℃for the oxidase of glucose. Under the condition of pH 7.0, 37℃, Mchaelis-Menten contant （Km） is 122.6 mmol/L, and the maximum velocity （Vm） is 25.52μmol/（L·min）. The enzyme is stable in range of pH from 5.5 to 8.5 and in temperature below 50℃. The enzyme （GOD） was modified respectively by several chemical modification reagents, The results showed that the residues of histidine, tryptophan and L-arginine guanidine were necessary for the enzyme activity, while the residues of mercapto, disulfide bond, Sulfide, serine deformity, carboxyl of acidic amino acids lysine were not necessary for the enzyme activity. The inactivation effects in chemical modification reagents （BrAc、NBS、acetyl acetone） have been studied using the kinetic method of Lineweaver-Burk plot. The inhibition constants（K₁） of BrAc, NBS and acetyl acetone are 26.5、19.2、7.35 mmol/L. The effects of several metal ions on the enzyme activity had been studied. The results show that: Cl^-、Br^-、I^- and Li⁺、Na⁺、K⁺、Ca²⁺、Mn²⁺ had no effects on the activity of the enzyme, white Ba²⁺、Co²⁺、Cd²⁺、have a slight inhibitory effect on the activity of the enzyme. The inhibition constants （K₁） of Cu²⁺、Ag⁺ and Hg²⁺ are 210.78μmmol/L, 0.065μmol/ and 83.1μmol/L. The effects of several organic solvent on the enzyme activity had been studied. The results show that: methanol, ethanol, propanol, glycol, propylene glycol and glycerol had no effects on the activity of the enzyme. Formaldehyde inhibit the enzyme activity in different degree with the inhibitor concentration leading to 50% of enzyme activity lost(IC₅₀) were estimated to be 81.25 mmol/L, The inhibition constants（K₁） of formaldehyde is 30 mmol/L. The effects of several denaturant on the enzyme activity had been studied. The results show that: EDTA、Urea have a slight inhibitory effect on the activity of the enzyme. The inhibition constants （K₁） of Gu·HCl、SDS are 1.75 mmol/L and 57.15 mmol/L.更多还原