【作者】 翁亚军；

【导师】 周小华；

【作者基本信息】 重庆大学 ， 应用化学， 2004， 硕士

【摘要】 反胶束萃取具有使用条件温和,萃取效率高,成本低,溶剂可重复利用,不引起蛋白质和酶变性等许多优点,用于分离和纯化蛋白质等生物大分子具有巨大的应用前景。本论文运用该技术进行了从粗胰酶中分离纯化胰蛋白酶的研究,对反胶束中胰蛋白酶STM图像进行了探讨,验证了在AOT反胶束中用胰凝乳蛋白酶催化合成乙酰氨基苯丙酰基-丁膦酸酰胺的反应。观察AOT/异辛烷反胶束中胰蛋白酶的STM形貌,发现该酶的构象发生了变化,卷曲程度增加,空间结构更紧密,相对于水中“刚性”增强,处于“部分活化”状态。由于处于“相对刚性”结构,其π-π共轭程度减弱,反胶束中紫外光谱、荧光光谱相对于水中均发生了蓝移。用生物纯胰蛋白酶进行的反胶束萃取初步试验结果指出:① 胰蛋白酶萃取率与pH值关系经非线性方程拟合为型,对0.1mol/L AOT/异辛烷,,;对0.05mol/L AOT/异辛烷,,;对0.01mol/L AOT/异辛烷,,。② 在5.2>pH>4.8区间,由于大量H+竞争AOT的极性头部,随着pH降低萃取率降低;在10>pH>5.3区间,萃取率随pH值降低而增加,因为pI-pH差越大,萃取蛋白的推动力越大。增大表面活性剂浓度,可以适当减小pH对萃取率的影响,但酶失活增加。③ 试验表明:用AOT/异辛烷反胶束萃取生物纯胰蛋白酶,其AOT最佳浓度为0.05mol/L,萃取最适pH为5.2～5.3,反萃取最适pH为9.9～10。④ 各种离子对萃取率影响大小是:Ca2+<Na+<K+;增大相同离子的离子强度,反胶束内表面的双电层变薄,减弱了蛋白质与反胶束内表面之间的静电吸引,萃取率减小。⑤试验表明:最佳萃取离子强度为0.1mol/L CaCl2;最佳反萃取离子强度为1mol/L KCl。用AOT/异辛烷反胶束进行了粗胰酶中胰蛋白酶的分离纯化,系统研究了各种因素对胰蛋白酶萃取率和比活力的影响。① 萃取及反萃取时间均对胰蛋白酶活力有影响,最佳萃取与反萃取时间均为5min。② 正交试验结果表明:本体相浓度、萃取与反萃取相比也影响胰蛋白酶活力,最佳纯化条件是本体相浓度为1mg/mL,萃取与反萃取相比均为1:1。③ 在此条件下,总萃取率22.7%,胰蛋白酶纯化倍数为3.6,胰蛋白酶比活力达到78.9BAEE U/mg,比活力纯化倍数为3.1,失活率13.9%。④ 将回收的反胶束再次用于萃取试验,胰蛋白酶总萃取率达到19.8%,比活力纯化倍数为2.8。⑤对AOT/Span-40混合反胶束试验结果表明,总萃取率为16.5%,胰蛋白酶比活力纯化倍数为2.9,分别比AOT/异辛烷反胶束体系低6.2%和6.5%。 <WP=5>反胶束酶系统能同时增溶亲水性底物和疏水性底物,而且蛋白水解酶在反胶束中具有催化合成酰胺键的特性。本文验证了在0.1mol/L AOT反胶束中,40℃,pH值9.40条件下,α-胰凝乳蛋白酶催化α-N-乙酰苯丙氨酸乙酯与α-氨基丁膦酸合成含膦二肽的反应。实验结果表明:通过检测游离氨基与邻苯二甲醛反应产生的荧光,得到反应30min、60min后游离氨基比反应前显著降低,因而证实乙酰氨基苯丙酰基-丁膦酸酰胺被合成。更多还原

【Abstract】 Reversed micellar extraction of biological macromolecule, especially proteins, has a promising profit, for the mild operational condition , high extraction yield, low cost and solvent recycling. The dissertation focuses on the research of extraction and purification of trypsin in raw pancreatin, probing into the STM image of trypsin in AOT-isooctane reversed micelles and validation synthesis of AcPheBPA (BPA:Butyl Phosphonic acid) catalysed by α-chymotrypsin.The STM image shows that the conformation of trypsin in reversed micelles is changed and in a partly active state, for increased curly degree, compacted dimensional configuration and enhanced rigidity in contrast with that in water. The conjugated degree of π-π bonds in trypsin is weakened so that ultraviolet and fluorescent spectra are blue-shifted, owing to the rigid state in reversed micelles.Firstly, pH and ionic strength were discussed with biological purified trypsin. ① The relation of pH and extraction rate is fitted no-linear curve . To 0.1mol/L AOT-isooctane, , ; To 0.05mol/L AOT, , ; To 0.01mol/L AOT, , ; ② when pH is in [5.2, 4.8], extraction rate decreases with pH because H+ competes with trypsin for combining to AOT polar terminal; when pH is in [5.3, 10.0], extraction rate increases with difference of pI-pH for more impetus; When AOT concentration is increased, the effect of pH on extraction rate drops but inactivation rate rises. ③ The optimum AOT concentration is 0.05 mol/L, and the optimum pH for extraction is 5.2~5.3 and 9.9~10.0 for reextraction. ④ The effect of different cations on extraction is Ca2+<Na+<K+. When ionic strength of the cation is increased, the double ionization layer becomes thinner and extraction rate decreases for electrostatic force weakened. ⑤ The optimum ionic strength for extraction is 0.1mol/L CaCl2 and 1mol/L KCl for reextraction.Then other factors affecting trypsin extraction rate and activity were discussed in AOT-isooctane reversed micellar extration and purification of trypsin in raw pancreatin. ① Time of extraction or reextraction has effects on extraction rate and activity of trypsin. The studying results show that the optimum time is 5 min for both. ② Pancreatin concentration in bulk water together with extraction and reextraction phase ratio was optimized by the orthogonal tests, and the results show that the optimum concentration is 1mg/mL and the optimum phase ratio is 1:1 for both. ③ Trypsin was <WP=7>purified under the optimum condition and the results show that total extraction rate is 22.7%, trypsin specific activity is 3.1 times of that raw material, protein of trypsin is 3.6 times of that, and inactivation rate of trypsin is 13.9%. ④ The recycled reversed micelles were reused and the results show total extraction rate is 19.8% and trypsin specific activity is 2.8 times. ⑤ With AOT/Span-40 mixed reversed micelles, total extraction rate is 16.5% and trypsin specific activity is 2.9 times, which are 6.2% and 6.5% lower than sole AOT reversed micellar system respectively.Reversed micelles can solubilize hydrophile and lipophile substrates. And it was found that proteolytic enzyme in reversed micelles can act as synthetase. Phosphoryl Dipeptide was synthesized by AcPheOEt and (1-Aminobutyl)Phosphonic acid in AOT-isooctane reversed micelles at 40℃ and pH 9.4, which is catalysed by α-chymotrypsin. The amino group combined with OPA was examined by fluorescence, and fluorescent intensity is decreased after synthesis for 30 or 60 min. So the synthesis of AcPheBPA is validated.更多还原