【作者】 王盼盼；

【导师】 李洪军；

【作者基本信息】 西南大学 ， 农产品加工及贮藏工程， 2009， 硕士

【摘要】 双水相萃取技术是近年来发展起来的一种新型生化分离技术,该体系由两种化学结构不同的亲水性聚合物或一种亲水性聚合物和无机盐在水中以适当的浓度形成互不相溶的两水相系统,利用组分在两相分配的差异而进行萃取分离的技术。双水相萃取技术具有生物相相溶性高,分离条件温和,步骤简单,易于放大,且不存在有机溶剂残留等优点,目前双水相萃取技术已被广泛用于酶、核酸、生长激素及各种植物中有效成分的萃取分离。脂肪酶（EC 3.1.1.3）是一类催化长链脂肪酸甘油酯水解为甘油和长链脂肪酸的生物催化剂。近年来,脂肪酶己被广泛应用于食品、洗涤业、有机合成、皮革和造纸等行业,脂肪酶在肉制品加工过程中和风味形成中也占有重要的地位,脂肪酶对肉制品风味的调控,工艺的改进及工业化生产均有重要的意义。本文以荣昌猪肉脂肪酶为研究对象,采用双水相萃取技术分离荣昌猪肉脂肪酶,建立了适宜的双水相萃取体系,并对影响萃取效果的各种因素和荣昌猪肉脂肪酶酶学特性进行了研究。本课题采用PEG/无机盐双水相萃取体系,研究了双水相成相物质及不同的操作条件对双水相萃取结果的影响。实验结果表明,双水相萃取荣昌猪肉脂肪酶的最优条件如下:（NH₄）₂SO₄浓度为12%,PEG分子量为2000,24%的PEG2000,pH=6.50条件下,粗酶液的添加量为10%,浓度12%的Na₂HPO₄反萃取脂肪酶时,脂肪酶的分配系数和回收率较高,将本实验结果直线放大,脂肪酶的分配系数、相体积比和脂肪酶的回收率均比较稳定。荣昌猪肉脂肪酶酶学特性主要实验结果如下:荣昌猪肉脂肪酶最适反应温度为37℃,该酶在37℃以下能保持比较稳定的酶活性;脂肪酶的最适pH为6.50,pH在4.00-7.00范围内有较好的酸碱稳定性;金属离子浓度为2mmol/L、5mmol/L时,Ba²⁺、Ca²⁺离子对脂肪酶有强烈的激活作用,Fe²⁺、Mn²⁺、Zn²⁺、Sn²⁺等离子对脂肪酶活性有抑制作用,K⁺、Na⁺、Mg²⁺无明显的激活或抑制作用:0.05%阳离子型表面活性剂CATB、阴离子型表面活性剂SDS和非离子型表面活性剂Trition-100对荣昌猪肉脂肪酶活性有一定的激活作用,非离子型表面活性剂Tween-80对荣昌猪肉脂肪酶活性有一定的抑制作用,而且随着表面活性剂质量浓度的提高,表面活性剂对脂肪酶的激活作用会转为抑制作用,而且会使得其抑制作用增大;2mmol/LCa²⁺使脂肪酶活性的热稳定性降低,45℃处理30min后,酶活力基本丧失,加入Ca²⁺使得酶活性活力提高,但是加入Ca²⁺后造成脂肪酶的热稳定性下降:以三油酸甘油酯为底物,荣昌猪肉脂肪酶在最适反应条件下动力学常数K_m和V_max分别为18.17mmol/L和588U/L。更多还原

【Abstract】 Aqueous two-phase extraction is a new bio-separation technique developed in recent years. Two hydrophilic polymer with different chemical constitution or hydrophilic polymer and inorganic salt do not dissolve mutually in suitable density. The object component could be separated by using aqueous two-phase extraction.The bio-separation technique has many advantages such as higher bio-compatibility, less separation procedures, milder operating condition, easier engineering amplification, less residue of organic solvents in the product and so on. It has been widely used to purify different enzymes, nucleic acids, hormone and bioactive component in plants.Lipase （EC 3.1.1.3） is a kind of biocatalysts to hydrolysis long-chain triglycerides to glycerol and long-chain fatty acids. Lipase is widely used in food, medicine, scour, leather and paper industry. The lipase holds an important status during the processing process. It has vital significance to meat product flavor regulation, craft improvement and production’s industrialization.Aqueous two-phase system was used in this paper to extracting the lipase of Rongchang pig. The influencing factors of extracting and properties of lipase were studied.In this experiment the extraction effects of several factors on Rongchang pig’s lipase were investigated. These factors included components of aqueous two-phase system and different operating conditions. The distribution coefficient and recovery rate are high when the density of （NH₄）₂SO₄ is 12%, PEG molecular weight is 2000 and density is 24%, pH value is 6.50, supplementary enzyme is 10%, counter-extract material Na₂HPO₄ density is 12%. The distribution coefficient and recovery rate are stable with enlarging this experimental result. And properties of the lipase from Rongchang pig are as follows.The lipase showed the highest activity at pH 6.5 and 37℃. It was stable under 37℃. The pH from 4.00 to 7.00 Rongchang pig’s lipase has good acid-base stability. When ion density is 2mmol/L and 5mmol/L Ba²⁺ and Ca²⁺ stimulated the enzyme activity significantly while Fe²⁺、Mn²⁺、Zn²⁺、Sn²⁺had inhibitory effect. But K⁺、Na⁺、Mg²⁺had no effect on the activity of lipase. The density of surfactant is 0.05%. Cationic surfactant CATB, anionic surfactant SDS and non-ionic surfactant Trition-100 stimulated the enzyme activity significantly. Non-ionic surfactant Tween-80 had an inhibitory effect. Along with increasing density of surfactant the active effect will convert into inhibitory effect. The inhibitory effect will be enhanced at the same time. The Ca²⁺ reduced thermal stability of lipase. The activity of lipase was almost lost under 45℃treatment. The enzyme activity enhanced but the thermal stability reduced with adding Ca²⁺.The values of K_m and V_max were 18.17mmol/L and 588U/L in optimum reaction conditions.更多还原