【作者】 宣锋；

【导师】 宋锡瑾；

【作者基本信息】 浙江大学 ， 化学工艺， 2007， 硕士

【摘要】 本文以脂肪酶为研究对象，结合分子印迹和生物印迹两种技术制备双底物刻录脂肪酶。在制备过程中利用底物诱导酶构象，然后利用高交联度聚合物对该构象进行刻录，最终制备出的刻录酶构象是刚性化的。本文重点研究了双底物刻录酶在水相和有机相中的催化活性、稳定性以及有机相中吸附配体能力，并考察了影响吸附能力的一些因素。本文采用四种不同的功能单体（聚乙二醇200（400）二（甲基）丙烯酸酯）和三羟甲基丙烷三甲基丙烯酸酯为聚合原料，并加入底物月桂酸和正丁醇对脂肪酶进行活性构象诱导，再经紫外光引发聚合、抽提和干燥后，获得了四种刻录脂肪酶聚合物。其中，选用石油醚作为抽提溶剂，单体和交联剂的质量比为2：1，紫外光照聚合时间为45s。本文对制备得到的双底物刻录酶进行水相和有机相中催化活性的测定。四种双底物刻录脂肪酶在水相催化中表现出较高的活性和稳定性，其中双底物刻录酶聚合物Da具有最高水相催化活性（3.71U／g），而且还发现含有甲基的单体聚合时容易增大刻录脂肪酶聚合物的空间位阻，从而增大了反应时的传质阻力，造成其催化活性比不含甲基的单体为原料聚合成的双底物刻录脂肪酶的催化活性低（Ba＞Aa，Da＞Ca）。四种双底物刻录脂肪酶在pH=7.5和温度50℃～60℃时均表现出它们各自的最大催化活性，而且在强酸、强碱和高温环境下也可保持较高的催化活性；同样用沉淀变性剂和溶解变性剂分别浸泡20d后，它们的活性仍都保持在初始活性的50％以上。这表明双底物刻录脂肪酶聚合物在有变性剂存在的环境中也能保持较高的催化效率。有机相催化以选用月桂酸和正丁醇的正庚烷酯化反应体系，随着温度的升高，平衡催化转化率也随之提高，同时催化活性也逐步提高。但温度越高，平衡催化转化率和催化活性上升幅度越来越小。反应24h后，在60℃时，有机相双底物刻录酶聚合物Da的催化活性达到最大值（20.92U／g）。本文还对双底物刻录酶Da的吸附能力做了考察。发现Da对配体月桂酸和正丁醇显示出了一定的吸附能力，其中还对温度、溶剂、双底物刻录酶聚合物Da中的含水量以及Da的催化活性等因素对Da吸附性能的影响作了考察。结果发现升温有利于双底物刻录酶Da对配体吸附能力的上升，在50℃下它的平衡吸附量为37.55(mg·g^-1)，但随着温度的进一步上升，吸附量增加不明显，甚至出现吸附量下降的情况；溶剂是通过对聚合物和配体的分子作用来影响两者之间的选择性和亲和力的；双底物刻录酶聚合物Da中含水量的增加导致其吸附能力大大减小；失活的双底物刻录酶聚合物Da对配体的吸附能力明显下降：此外，双底物刻录酶聚合物上的空穴对配体来说并不是特定对应的，许多其它酸、醇类似物也能吸附到双底物刻录酶聚合物上。更多还原

【Abstract】 The article is studied in lipase and use two new technique-bioimprinting technique and molecular imprinting technique to prepare the double substrates recorded lipase. Lipase conformation can be induced by substrate to be an activated one in preparation process. Then the activated conformation was recorded using high cross linking degree polymer. And the final enzyme conformation is rigid. The double substrates recorded lipase’s catalytic characteristic in water and organic solvent, and its adsorption capacity were investigated.The dissertation emphasizes on exploring the process of double substrates recorded lipase preparation. Lipase, lauric acid and n-butyl alcohol were added into a copolymer system, which contained four different kinds of function monomer and trimethylolpropane trimethacrylate. The polymerization was obtained by ultraviolet irradiation and lauric acid, n-butyl alcohol were extracted by petroleum ether. The function monomer and crosslinked agent were mixed by the scale of 2:1（mass scale） and irradiation time was 45s.The dissertation studied the catalytic characterization of double substrates recorded lipases in water and organic solvent. The four double substrates recorded lipases presents some catalytic activity and stability. The best catalytic activity was attained by double substrates recorded lipase Da in water （3.71U/g）. And the results shown that the monomer containing methyl could easily increase steric hindrance of recorded lipase Da, which will increase the mass-transfer barriers lead to the decrease of the catalytic activity（Ba>Aa, Da>Ca）. The four double substrates recorded lipases present their the best catalytic activity at pH=7.5 and at 50℃～60℃. Moreover, they were able to retain higher catalytic activity under the circumstance of strong acid（pH=2.40）、 strong alkaline（pH=12.00） and high temperature, respectively. And after immersing the double substrates recorded lipases into precipitation denaturants for 20 days, the four double substrates recorded lipases were able to retain at least 50% of their initial activities. It illuminates the four double substrates recorded lipases can be also keep higher catalytic activity in precipitation denaturants. The activity in organic solvent （heptane） was measured by esterification of lauric acid and n-butyl alcohol. When rising the temperature, the activity and yield are also rising. After catalyzing 24h, Da’s best catalytic activity （20.92 U/g） was attained at 60℃ in organic solvent.The dissertation is also studied the adsorptive ability of the double substrates recorded lipase Da. It has found the Da has some adsorptive ability to lauric acid and n-butyl alcohol. The influence factors including temperature, solvent, Da’s catalytic activity is studied. When rising the temperature, the adsorption is also rising. Its equilibrium adsorption amount was 37.55(mg·g^-1) at 50℃.The solvent impact Da’s adsorptive ability through influence the interaction between the polymer and ligand. The non- activity Da’s adsorptive ability drops significantly. Moreover, the cavities created in the double substrates recorded lipase by lauric acid and n-butyl alcohol during impringting are not unique for these ligand. A dozen of lauric acid and n-butyl alcohol analogs can bind to the double substrates recorded lipase.更多还原