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温敏P(NIPAM-co-AAc)微球的制备及其在蛋白质组学样品处理中的应用
Alternative TitlePreparation of Poly(N-isopropylacylamide-co-acrylic acid) microspheres and its application in proteolytic digestion
司天保
Subtype硕士
Thesis Advisor马光辉
2011-06-02
Degree Grantor中国科学院研究生院
Degree Discipline材料学
Keywordp(Nipam-co-aac)微球 膜乳化 羧基分布 孔结构 酶固定化
Abstract蛋白质组学样品制备过程中,溶液酶解法存在酶解时间长、自降解、低效率等问题。本论文尝试通过将酶固定在聚(异丙基丙烯酰胺-co-丙烯酸)[P(NIPAM-co-AAc)]微球上,以期克服溶液酶解法存在的上述问题。首先,采用膜乳化法制备了尺寸均一、大小可控的不同丙烯酸含量的P(NIPAM-co-AAc)微球,然后对微球进行了表征,最后将胰蛋白酶固定在微球上并将载酶微球用于酶解模型蛋白,结果如下: 1) 使用膜乳化法,以环己烷和氯仿混合溶液为油相,得到均一乳液液滴,然后在室温条件下通过外加四甲基二乙胺(TEMED)引发聚合反应,制备了一系列不同丙烯酸含量尺寸均一的P(NIPAM-co-AAc)微球。结果表明,在压力 2 kPa,环己烷氯仿体积配比7:3,第一阶段过膜搅拌速度 140 r/min,第二阶段反应搅拌速度 170 r/min的条件下,可以制备粒径均一,大小可控,重复性较好的P(NIPAM-co-AAc)微球。激光粒度仪表征结果显示,其20 ℃水溶液中其平均粒径为20 μm,CV值21.6%。 2) 对P(NIPAM-co-AAc)微球的最低临界溶解温度(LCST)进行了表征。结果显示,随着丙烯酸含量在P(NIPAM-co-AAc)微球中逐渐增加,LCST逐渐减小;随着pH的提高,微球的LCST逐渐增大,转变温度范围变宽。电导滴定结果显示,所有不同丙烯酸含量的P(NIPAM-co-AAc)微球电导滴定曲线均为4阶梯形状;随着丙烯酸含量的增加,微球外层和内层包埋羧基含量逐渐增加,但是外层羧基占总羧基含量的百分比呈现先增大,然后趋于不变的趋势,在丙烯酸为15%达到相对最大值。 3) 使用真空、冷冻干燥、超临界干燥法对得到的P(NIPAM-co-AAc)微球进行干燥。结果显示,三种干燥方法中,只有超临界干燥法可以有效保护微球球形及其表面形貌。SEM、CLSM和孔隙度测量结果显示,在不加丙烯酸的PNIPAM微球内部存在大量的大孔,在不同丙烯酸含量P(NIPAM-co-AAc)微球内部存在大量2 nm-50 nm的狭缝孔。研究表明,随着丙烯酸含量的增加,微球内部介孔和微孔百分比逐渐增大,而大孔百分比逐渐减小,这导致微球内部平均孔径、比表面积呈现随丙烯酸含量提高而减小的趋势。 4) 将丙烯酸含量7.5% P(NIPAM-co-AAc)微球用于胰蛋白酶固定,并将载酶微球用于模型蛋白酶解。结果显示,当酶浓度固定为2.7 mg/ml时,在pH 5.5和25 °C条件下,反应时间为2 hr,碳二亚胺(EDC) 浓度为0.45 mg/ml时,酶固载量和活性回收率分别达到 615 mg/g和81.2 % 的相对最优值。对固定化胰蛋白酶储存稳定性考查结果显示,4 °C条件下,30天内酶活保留率达98%。当酶与底物BSA比例为1:50时,总离子流量图结果显示,自由胰蛋白酶和固定化酶均可很好地酶解BSA。匹配度搜索结果显示,两者均成功鉴别出BSA。当固定化胰蛋白酶与BSA比例为1:25时,仍然可以有效鉴别BSA,表明固定化胰蛋白酶可以有效降低胰蛋白酶自解,而文献报道游离胰蛋白酶在这个比例下由于酶自解严重,已经无法有效鉴别模型蛋白,这一结果使进一步提高酶浓度,缩短酶解时间成为可能。
Other AbstractProteolytic digestion is an important step in protein identification. Traditional methods of protein digestion require extended incubation times with severely autolysis and low efficiency. In this dissertation, enzyme was immobilized on poly(N-Isopropyl acrylamide-co-acrylic acid) [P(NIPAM-co-AAc)] microspheres to solve the above problems in traditional proteolytic digestion. Firstly, monodispersed P(NIPAM-co-AAc) microspheres with different AAc content were prepared by membrane emulsification technology. Then, the micospheres were characterized with different methods. Finally, trypsin was immobilized on the microspheres, and the trypsin-microsphere was used in the following proteolytic digestion. Follows are the details of the dissertation: 1) Temperature-/pH- responsive monodispersed poly(N-Isopropyl acrylamide-co- acrylic acid) [P(NIPAM-co-AAc)] microspheres were successfully prepared by Shirasu Porous Glass (SPG) membrane emulsification technology at room temperature with N,N,N′N′-tetramethylethylenediamine (TEMED) as accelerator and cyclohexane/ trichloromethane mixture as oil phase. The P(NIPAM-co-AAc) microspheres with controllable diameter can be reproducibly obtained with cyclohexane/trichloromethane mixture (volume rate 7:3) as oil phase and two stage stirring speeds (the first stage 140 r/min, and the second stage 170 r/min) under the membrane emulsification pressure of 2 kPa. 2) The temperature-/pH- responsive behaviors of the prepared microspheres were characterized. It was confirmed that the LCST (Low critical solution temperature) of the P(NIPAM-co-AAc) microspheres decreased with the increase of AAc content in the low pH (pH =1), and it became higher when the pH of the medium was increased. Conductometric titration was used to determine the distribution of the carboxyl in the microspheres. Four-step titration curves of all the P(NIPAM-co-AAc) microspheres with different acrylic acid content were obtained. With the increase of the AAc content, the amount of the exterior carboxyl and the embeded carboxyl both incease gradually, but the percentage of the exterior carboxyl increase firstly, then keeps at a fixed value. The highest percentage of the exterior carboxyl was obtained with 15 wt% acrylic acid in the microspheres. 3) The microspheres were dried by supercritical drying (SCD), lyophilization and vacuum drying, separately. It was observed that only SCD was an effective drying method to maintain the morphologies of the microspheres. Scanning electron microscopy (SEM), confocal laser scanning microscope (CLSM), and porosity analyzer results showed that the P(NIPAM-co-AAc) microspheres contained abundant slit-like mesoporous structure with pore size from 2 nm-50 nm. The increase of AAc wt% would increase the percentage of mesopores and micropores in the microspheres. Meanwhile, the average pore size, pore volume, and surface area decreased with increasing AAc content in the recipes. 4) Trypsin was covalently immobilized onto the P(NIPAM-co-AAc) microspheres in a high yield. The result showed that when the concentration of trypsin and carbodiimide (EDC) was 2.7 mg/m and 0.45 mg/ml at the pH 5.5 in the room temperature the highest trypsin loading and activity yield were 615 mg/g and 81.2 % respectively. The residual activity was 98% after the immobilized trypsin was stored at 4 °C within 30 days. Total ion chromatogram (TIC) of BSA treated by trypsin at trypsin/BSA ratio of 1:50 showed that BSA was successfully dissociated by free and P(NIPAM-co-AAc) microsphere-immobilized trypsin. The BSA was successfully identitied by data matching. Specially, when the immobilized-trypsin/BSA ratio was 1:25 BSA still can be successfully dissociated and identitied. However, with this ratio of free-trypsin/BSA, the BSA can not be identitied for the severely autolysis of trypsin. which indicated that autolysis of trypsin can be reduced by immobilization.
Pages79
Language中文
Document Type学位论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/1679
Collection研究所(批量导入)
Recommended Citation
GB/T 7714
司天保. 温敏P(NIPAM-co-AAc)微球的制备及其在蛋白质组学样品处理中的应用[D]. 中国科学院研究生院,2011.
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