【作者】 龚伟；

【导师】 宋锡瑾；

【作者基本信息】 浙江大学 ， 化学工艺， 2006， 硕士

【摘要】 本文首次阐述了一种新型固定化酶——“刻录酶”的制备方法。和普通固定化酶相比,首先,它在制备过程中加入了配体诱导酶构象;其次,最终制备得到的刻录酶构象是刚性化的。文中对刻录酶的整个制备流程进行了摸索,重点讨论了它在水相和有机相中的催化活性、稳定性以及在有机相中的吸附配体能力,最后利用SEM对刻录酶载体进行了宏观表征。 以聚乙二醇200(400)二(甲基)丙烯酸酯4种不同的功能单体和三羟甲基丙烷三甲基丙烯酸酯为聚合原料,脂肪酶为模板分子,加入配体月桂酸对脂肪酶进行活性构象诱导,再通过紫外光引发聚合以及无水有机溶剂索式抽提月桂酸后,获得了4种锁定脂肪酶活性构象的聚合物,即刻录脂肪酶聚合物。在以无水丙酮为洗脱剂时获得了最高86.31%的月桂酸洗脱率,且脂肪酶的洗脱量保持在一个较小范围。当单体和交联剂以质量比为2:1,紫外光照聚合时间为45s时,所得四种刻录酶都表现出了优异的抗溶胀稳定性,尤其在正庚烷和水中为最佳。这些都基本符合对“刻录酶”载体性能的预期设想。 论文对刻录酶在水相和有机相的催化特性进行了探索。水相催化采用橄榄油水解法,发现四种刻录酶都具有较好的活性和稳定性。它们在水相中最佳催化时间为15min,活性最大在Da(聚乙二醇400二丙烯酸酯为单体的刻录酶)上获得(6.07U/g),活性收率高达171%;同时经过比较,发现含有甲基的单体聚合时容易形成超支化聚合物,增大反应时的传质阻力,造成其活性和活性收率都大大降低:四种刻录酶在强酸(pH=2.42)、强碱(pH=12.00)和高温(80℃)下均可以分别保持它们初始活性的75%、50%和70%以上,其中Da在160℃左右完全失活,这是因为载体在153℃开始热不稳定造成的;用沉淀变性剂和溶解变性剂分别浸泡刻录酶30d后,四种刻录酶仍然可以表现出它们初始活性的50%和45%以上。这说明脂肪酶构象的高度刚性化对它在水相催化时的活性和稳定性都是非常有利的。有机相催化选用月桂酸和正丁醇的正庚烷酯化反应体系,以活性最大的Da为考察对象,发现它在有机相催化中活性较为低下。40℃时催化24h活性为15.30U/g,升温至50℃可以小幅度提升Da活性为16.67U/g,而此时活性仅为固体酶粉的46.88%;但刻录酶优异的稳定性在有机相中继续得以体现。经过5更多还原

【Abstract】 The concept of a novel immobilized enzyme (called "recorded enzyme") is first described by this dissertation. Compared to the ordinary immobilized enzyme, a ligand is added into preparation process to enduce enzyme conformation and the final enzyme conformation is rigid. Then its preparation process and the catalytic characteristic in water and organic solvent were investigated, SEM was utilized to straight token the state of recorded enzyme in the end.The first part of this dissertation emphasizes on exploring the process of recorded enzyme preparation. Lipase and lauric acid were added into a copolymer system, which contained four different kinds of PEG200(400)-di(meth)acrylate ester and trimethylolpropane trimethacrylate. The polymerization was initiated by ultraviolet irradiation and lauric acid was extracted by neat organic solvent. Then four recorded lipase polymers of locking lipase conformation could be obtained. When actone was selected for washout solvent, the maximal extraction rate of 86.31% can obtained, as well as the leak of lipase was keep in a small range. When function monomer and crosslinked agent were mixed by the scale of 2:1 (mass scale) and irradiation time was 45s, four recoreded lipases shown the excellent resistant to swelling behavior in six common solvents, especially in water and heptane, which is full consistent with the anticipant assumption for recored lipase carrier.The second part presents the catalytic characterization of recorded lipase in water and organic solvent. The activity of four recoreded lipases in water was obtained by olive oil hydrolyzation. Theirs optimization reaction time was 15 min, and the best activity (6.07 U/g) was attained by Da (PEG400-diacrylate as function monomer), Its activity yield was 171%. At the same time, the results shown that the monomer containing methyl could easily form super branch polymer, which will increase the mass-transfer barriers lead to the decrease of activity and yield. In addition, theirsstability were enhanced greatly. Recorded lipases were able to retain more than 75 %> 50 % and 70 % of their initial activities under the circumstance of strong acid(pH=2.40^ strong alkaline(pH= 12.00) and high temperature(80°C), respectively. Moreover, Because of the thermal unstable of carrier from 153°C, Da inactivate around 160°C. After immersing recorded lipases into precipitation denaturants for 30 days, recorded lipases were able to retain at least 50 % of their initial activities. All these illuminate the rigidity conformation is advantageous for the activity and stability when the catalytic reaction occur in water. The activity in organic solvent (heptane) was measured by esterify reaction of lauric acid and n-butanol. Da was selected for model for its high activity. The ativity was 14.46 U/g for 24h at 40 °C and activity yield was only 33.72%. Rising temperature could up activity and yield to 16.42 U/g and 43.52%, respectively. In the further research displayed that added the pore-causing solvent (toluene) to reaction system for decrease mass-transfer barriers was no sense, because toluene would make lipase inactivate. But add water into heptane was able to enhance enzyme activity. When the content of water was l%(v/v), the recorded lipase activity reach the best activity 38.23 U/g after catalyzing 24h, which was 2.64 times compared to anhydrous system. If continued to increase water content, the esterify activity decrease sharply and inactivate at the content of 10%(v/v). All these further shown that the flexibility exploded by water was crucial to the lipase activiy. The best activity and stability can be obtained by keep the lipase active conformation and increase some flexibility at the same time. The reaction mechanism of recorded lipase according to the Ping-Pong theory, the equation of,. . ... , 20.390[A][B]kinetics could be express by v =3It was found that Da also has adsorptive ability to lauric acid. Its equilibrium adsorption amount was 30.22(mg-g"1) at 40°C, which was 1.35 times compared to 30 °C, and its absorption yield was 70.46%. The adsorption behavior accorded with Langmuir law, its kL-0.024(mg>g"1-min’1); The pore-causing solvent (toluene) was advantageous for the adsorption amount to 40.04(mg-g"1) and yield to 93.35%, here kL=0.033(mg-g"1-min’1). But water was unamiable to adsorption process. When thewater content was only 0.5% (v/v) at 40°C, the adsorption amount decrease about 1 times, when up to 5%(v/v), there are no adsorption ability exist. This phenomenon is ascribed to the water alter the rigidity action point lead to weaken the adsorption.The last part refers to the direct token of the surface of Da by SEM. Some exiguous power existed in truncation surface and pore interior. Its size was ten or some tens times of lipase molecular. But these powers can not be found in carrier surface. It was speculated that these powers may be the polycoin of Upases.更多还原