功能性磁性纳米颗粒的制备及其在食品体系中的应用研究
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摘要
由于功能性磁性纳米颗粒同时具有磁性核体材料的磁响应性和表面壳体材料的功能性,可以方便、快捷地在外加磁场的作用下从反应体系中分离纯化目标生物分子,因此,在生物工程、生物医药和食品工业等领域显示出广阔的应用前景。目前,探索磁性纳米颗粒的功能化修饰以及将这些纳米颗粒应用于复杂的生物体系是磁性纳米颗粒重要的研究方向。本论文针对蛋白质分离纯化及酶固定化研究中所面临的热点难点问题,开展了一系列研究工作,制备了新型的功能性磁性纳米颗粒,并将其应用于生物体系中,取得了一些创新性研究成果,主要研究内容和取得的研究成果如下:
1.以聚乙二醇6000(PEG 6000)作为分散剂,羧甲基壳聚糖(CM-CTS)作为表面壳体材料,采用化学共沉淀方法,制备得到了功能性的超顺磁性纳米颗粒,简写为Fe_3O_4 (PEG+CM-CTS);Fe_3O_4 (PEG+CM-CTS)大小均一、粒径大约在15 nm左右,同时具有良好的水分散特性;以功能性Fe_3O_4 (PEG+CM-CTS)纳米颗粒为载体,通过静电相互作用,对水溶液及蛋清中的溶菌酶(Lyz)进行了分离纯化,经SDS?PAGE电泳对其纯度鉴定,结果表明:采用本研究制备所得的磁性载体纯化蛋清中的Lyz,所得Lyz电泳显示为单带,具有较高的纯度;由于其具有高效、低成本、高吸附容量以及易于分离等优点,Fe_3O_4 (PEG+CM-CTS)纳米颗粒在蛋清中Lyz的分离纯化应用中明显优于传统的溶菌酶分离纯化技术。
2.采用傅里叶变换红外光谱(FTIR)技术、圆二色光谱(CD)技术和荧光光谱技术为表征手段,对Lyz在Fe_3O_4 (PEG+CM-CTS)纳米颗粒表面吸附以及洗脱过程中的构象变化进行了较为全面的分析,研究结果表明:超顺磁性Fe_3O_4 (PEG+CM-CTS)纳米颗粒可以使Lyz分子构象变得更加紧密;Lyz与Fe_3O_4 (PEG+CM-CTS)纳米颗粒之间生成了稳定的复合物,其可能作用位点在Trp 62和Trp108位点处;磷酸盐缓冲溶液PBS (20 mmol/L, pH 8.0, 0.2 mol/L NaCl), PBS (20 mmol/L, pH 8.0, 0.5 mol/L NaCl)对Lyz构象的影响较小,相比于自然状态下的Lyz,其活性保留率较高,分别为92.4%和89.5%,而醋酸缓冲溶液洗脱得到的Lyz其构象变化较大,活性保留率仅为24.7%;由于Fe_3O_4 (PEG+CM-CTS)纳米颗粒粒径较小,相比于同类材料粒径为微米级的颗粒相比,对蛋白质的构象具有较好的稳定作用,为Fe_3O_4 (PEG+CM-CTS)纳米颗粒在生物技术如酶的固定化和蛋白质的分离纯化等领域的应用奠定了坚实基础。
3.此外,通过静电相互作用,胰蛋白酶成功固定化于Fe_3O_4 (PEG+CM-CTS)纳米颗粒表面,并将此固定化胰蛋白酶成功应用于蛋白质的快速酶解研究。FTIR和荧光光谱数据表明:Fe_3O_4 (PEG+CM-CTS)纳米颗粒可有效阻止胰蛋白酶分子结构的展开;相比于游离胰蛋白酶,固定化胰蛋白酶与底物的亲和力略有所降低,然而,固定化胰蛋白酶可以成功实现牛血清白蛋白(BSA)的快速、有效酶解,酶解操作过程简单,可以在外加磁场的作用下对固定化酶进行方便、快速地分离、定位及导向,同时,酶解时间大大缩短,从传统的12 hr(采用游离胰蛋白酶进行酶解)缩短到现在的15 min(固定化胰蛋白酶);以上结论显示:试验中制备的固定化胰蛋白酶有望在蛋白质组学研究中发挥重要作用,预计Fe_3O_4 (PEG+CM-CTS)纳米颗粒作为胰蛋白酶的固定化载体将具有巨大的应用潜能。
4.研究中,分别以Cu (II)、Fe (III)和Zn (II)三种金属离子修饰Fe_3O_4 (PEG+CM-CTS)纳米颗粒制备得到了三种固定化金属亲和磁性纳米颗粒(IMANs),简写为IMAN @ Cu (II)、IMAN @ Fe (III)和IMAN @ Zn (II);制备所得的IMANs可以方便地用于Lyz的吸附研究;IMAN @ Cu (II)、IMAN @ Fe (III)和IMAN @ Zn (II)纳米颗粒对Lyz的最大吸附容量分别为185.19 mg/g、232.56 mg/g和200 mg/g;荧光光谱结果表明:IMAN @ Fe (III)和IMAN @ Zn (II)磁性载体可以最大限度的保持Lyz的构象稳定性,而不会导致Lyz的变性,而从IMAN @ Cu (II)纳米颗粒上洗脱得到的Lyz其构象发生了一定程度的变化。
5.首次应用IMAN @ Fe (III)纳米颗粒作为分离纯化载体,对蛋黄中的卵黄高磷蛋白磷酸肽(PPPs)进行分离、富集研究,研究结果表明:IMAN @ Fe (III)纳米颗粒对PPPs的富集显示出较高特异性(N/P可由提纯前的39.49降低到5.52),具有操作简便、成本低廉,生产周期短,明显优于传统的富集方法,显示出较高的磷酸化肽选择性富集能力;此外,研究中制备所得的IMAN @ Fe (III)纳米颗粒不仅适用于蛋黄中PPPs的富集,同时势必在磷酸肽富集的蛋白质组学研究中发挥重要作用。
总之,本论文针对蛋白质分离纯化、酶固定化以及PPPs的富集方面的热点难点问题,以新型的功能性磁性纳米颗粒为基础,为解决蛋清中Lyz的分离纯化,蛋黄中PPPs的富集,胰蛋白酶的固定化及其快速酶解提供了新颖有效的研究技术。
Since it possesses the magnetic responsively, as well as the functionality, the functional magnetic nanoparticles can be convenient, fast separation and purification of the target biomolecules from reaction system with the help of magnets, it has extensive application prospect in biotechnology, biomedicine, food industry, and so on. At present, exploring the functionalization of magnetic nanoparticles and its practical application is the important research direction for magnetic nanoparticles. This dissertation aimed at finding out new techniques in protein separation and enzyme immobilization which using the prepared novel magnetic nanoparticles as Nano carriers, the practical bio-system was chosen as the application system, and obtained some innovative research results. The main research results are as follows:
1. Using polyethylene glycol 6000 (PEG 6000) as dispersant, the functional superparamagnetic nanoparticles which conjugated with carboxymethyl chitosan (CM-CTS) were developed (shorted as Fe_3O_4 (PEG + CM-CTS)); the obtained Fe_3O_4 (PEG + CM-CTS) with uniform size, its diameter was about 15 nm, furthermore, the dispersing behavior of Fe_3O_4 (PEG+CM-CTS) nanoparticles has obviously been improved in comparison with the naked Fe_3O_4; On this basis, through electrostatic interaction, the functional Fe_3O_4 (PEG+CM-CTS) nanoparticles was used as a novel magnetic absorbing carrier for the separation and purification of lysozyme from the aqueous solution and chicken egg white respectively, the purity of lysozyme which purified from chicken egg white was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS?PAGE), it indicated that: The lysozyme which purified from chicken egg white in a single step had higher purity; Considering that the Fe_3O_4 (PEG+CM-CTS) nanoparticles possesses the advantages of high efficiency, cost-effectiveness and excellent binding of a larger amount of lysozyme and easier separation from the reaction system, thus this type of superparamagnetic nanoparticles would bring advantages to the conventional separation techniques of lysozyme from chicken egg white.
2. An fundamental understanding of the conformational behaviors of lysozyme during the process of adsorption and desorption has been studied using Fourier transform infrared spectroscopy (FTIR), circular dichroic (CD) and fluorescence spectroscopy, and it indicated that: on the surface of Fe_3O_4 (PEG+CM-CTS) nanoparticles, the lysozyme would adopt a more compact conformation state; a complex between lysozyme and the Fe_3O_4 (PEG+CM-CTS) nanoparticles has been formed, Trp 62 and Trp108 mol/Light be the possible site of binding of lysozyme to Fe_3O_4 (PEG+CM-CTS) nanoparticles; Lysozyme which desorbed by PBS (20 mmol/L, pH 8.0, 0.2 mol/L NaCl) and PBS (20 mmol/L, pH 8.0, 0.5 mol/L NaCl) retain high fraction of its native structure with negligible effect on its activity, and about 92.4% and 89.5% activity were retained upon desorption from nanoparticles, however, lysozyme desorbed by acetic acid solution (0.2 mol/L, pH 4.0) showed significant conformational changes, only about 24.7% activity were retained; Due to the small diameter, the Fe_3O_4 (PEG+CM-CTS) magnetic nanoparticles has favorable properties to ensure highest level of conformational stabilization of protein compared to the larger particles of the same material, so it may find useful applications in biotechnology ranging from enzyme immobilization to protein purification.
3. The immobilized trypsin which absorbed on the surface of the Fe_3O_4 (PEG+CM-CTS) nanoparticles was prepared and applied for fast and efficient proteolysis. FTIR and fluorescence spectroscopy data demonstrated that the magnetic Fe_3O_4 (PEG+CM-CTS) nanoparticles were capable of improving conformation stability of trypsin and preventing the trypsin unfolding; the immobilized trypsin, compared with native trypsin, has less affinity to the substrate, however, the immobilized trypsin could be used for fast and efficient Bovine Serum Albumin (BSA) digestion under very facile processes, thanks to the easy manipulation of the magnetic nanoparticles, as well as the greatly reduced digestion time (from 12 hr to 15 min); These results are expected to open up a new possible application of the trypsin-Fe_3O_4 (PEG+CM-CTS) conjugates in proteome research as well as a great potential use of such Fe_3O_4 (PEG+CM-CTS) nanoparticles as a superior nanosupport for the immobilization of trypsin.
4. In the research, Fe_3O_4 (PEG+CM-CTS) nanoparticles were treated with Cu (II), Fe (III) and Zn (II) ions solutions to obtain immobilized metal affinity magnetic nanoparticles (IMANs) (short as IMAN @ Cu (II), IMAN @ Fe (III) and IMAN @ Zn (II)). The as-prepared IMANs were conveniently applied for lysozyme adsorption; The maximum equilibrium adsorption capacity of IMAN @ Cu (II), IMAN @ Fe (III) and IMAN @ Zn (II) nanoparticles to lysozyme were calculated to be 185.19 mg/g, 232.56 mg/g and 200 mg/g, respectively; Fluorescence analysis demonstrated that IMAN @ Zn (II) and IMAN @ Fe (III) nanoparticles may not cause the conformational change and denaturation for the lysozyme, but the lysozyme which desorbed from IMAN @ Cu (II) underwent some subtle change in comparison with the original lysozyme.
5. IMAN @ Fe (III) were conveniently applied for phosvitin phosphopeptides (PPPs) enrichment for the first time, the research indicated that: IMAN @ Fe (III) nanoparticles showed greater specificity for PPPs enrichment (N/P can be reduced from 39.49 to 5.52); Since the separation process is convenient, economic and efficient, this type of IMAN @ Fe (III) nanoparticles would bring advantages to the conventional separation techniques of PPPs from chicken egg yolk; In addition, the chosen IMAN @ Fe (III) nanoparticles not only showing potential application in PPPs separation from chicken egg yolk, but also in phosphopeptides enrichment in proteomics research.
In summary, the main contribution of the dissertation is that we initially synthesized the functional magnetic nanoparticles and successfully utilized them for protein separation and enzyme immobiliztation; we aimed at exploring and finding out new techniques in lysozyme separation from chicken egg white, PPPs enrichment from chicken egg yolk, trypsin immobilization and its efficient proteolysis.
1.以聚乙二醇6000(PEG 6000)作为分散剂,羧甲基壳聚糖(CM-CTS)作为表面壳体材料,采用化学共沉淀方法,制备得到了功能性的超顺磁性纳米颗粒,简写为Fe_3O_4 (PEG+CM-CTS);Fe_3O_4 (PEG+CM-CTS)大小均一、粒径大约在15 nm左右,同时具有良好的水分散特性;以功能性Fe_3O_4 (PEG+CM-CTS)纳米颗粒为载体,通过静电相互作用,对水溶液及蛋清中的溶菌酶(Lyz)进行了分离纯化,经SDS?PAGE电泳对其纯度鉴定,结果表明:采用本研究制备所得的磁性载体纯化蛋清中的Lyz,所得Lyz电泳显示为单带,具有较高的纯度;由于其具有高效、低成本、高吸附容量以及易于分离等优点,Fe_3O_4 (PEG+CM-CTS)纳米颗粒在蛋清中Lyz的分离纯化应用中明显优于传统的溶菌酶分离纯化技术。
2.采用傅里叶变换红外光谱(FTIR)技术、圆二色光谱(CD)技术和荧光光谱技术为表征手段,对Lyz在Fe_3O_4 (PEG+CM-CTS)纳米颗粒表面吸附以及洗脱过程中的构象变化进行了较为全面的分析,研究结果表明:超顺磁性Fe_3O_4 (PEG+CM-CTS)纳米颗粒可以使Lyz分子构象变得更加紧密;Lyz与Fe_3O_4 (PEG+CM-CTS)纳米颗粒之间生成了稳定的复合物,其可能作用位点在Trp 62和Trp108位点处;磷酸盐缓冲溶液PBS (20 mmol/L, pH 8.0, 0.2 mol/L NaCl), PBS (20 mmol/L, pH 8.0, 0.5 mol/L NaCl)对Lyz构象的影响较小,相比于自然状态下的Lyz,其活性保留率较高,分别为92.4%和89.5%,而醋酸缓冲溶液洗脱得到的Lyz其构象变化较大,活性保留率仅为24.7%;由于Fe_3O_4 (PEG+CM-CTS)纳米颗粒粒径较小,相比于同类材料粒径为微米级的颗粒相比,对蛋白质的构象具有较好的稳定作用,为Fe_3O_4 (PEG+CM-CTS)纳米颗粒在生物技术如酶的固定化和蛋白质的分离纯化等领域的应用奠定了坚实基础。
3.此外,通过静电相互作用,胰蛋白酶成功固定化于Fe_3O_4 (PEG+CM-CTS)纳米颗粒表面,并将此固定化胰蛋白酶成功应用于蛋白质的快速酶解研究。FTIR和荧光光谱数据表明:Fe_3O_4 (PEG+CM-CTS)纳米颗粒可有效阻止胰蛋白酶分子结构的展开;相比于游离胰蛋白酶,固定化胰蛋白酶与底物的亲和力略有所降低,然而,固定化胰蛋白酶可以成功实现牛血清白蛋白(BSA)的快速、有效酶解,酶解操作过程简单,可以在外加磁场的作用下对固定化酶进行方便、快速地分离、定位及导向,同时,酶解时间大大缩短,从传统的12 hr(采用游离胰蛋白酶进行酶解)缩短到现在的15 min(固定化胰蛋白酶);以上结论显示:试验中制备的固定化胰蛋白酶有望在蛋白质组学研究中发挥重要作用,预计Fe_3O_4 (PEG+CM-CTS)纳米颗粒作为胰蛋白酶的固定化载体将具有巨大的应用潜能。
4.研究中,分别以Cu (II)、Fe (III)和Zn (II)三种金属离子修饰Fe_3O_4 (PEG+CM-CTS)纳米颗粒制备得到了三种固定化金属亲和磁性纳米颗粒(IMANs),简写为IMAN @ Cu (II)、IMAN @ Fe (III)和IMAN @ Zn (II);制备所得的IMANs可以方便地用于Lyz的吸附研究;IMAN @ Cu (II)、IMAN @ Fe (III)和IMAN @ Zn (II)纳米颗粒对Lyz的最大吸附容量分别为185.19 mg/g、232.56 mg/g和200 mg/g;荧光光谱结果表明:IMAN @ Fe (III)和IMAN @ Zn (II)磁性载体可以最大限度的保持Lyz的构象稳定性,而不会导致Lyz的变性,而从IMAN @ Cu (II)纳米颗粒上洗脱得到的Lyz其构象发生了一定程度的变化。
5.首次应用IMAN @ Fe (III)纳米颗粒作为分离纯化载体,对蛋黄中的卵黄高磷蛋白磷酸肽(PPPs)进行分离、富集研究,研究结果表明:IMAN @ Fe (III)纳米颗粒对PPPs的富集显示出较高特异性(N/P可由提纯前的39.49降低到5.52),具有操作简便、成本低廉,生产周期短,明显优于传统的富集方法,显示出较高的磷酸化肽选择性富集能力;此外,研究中制备所得的IMAN @ Fe (III)纳米颗粒不仅适用于蛋黄中PPPs的富集,同时势必在磷酸肽富集的蛋白质组学研究中发挥重要作用。
总之,本论文针对蛋白质分离纯化、酶固定化以及PPPs的富集方面的热点难点问题,以新型的功能性磁性纳米颗粒为基础,为解决蛋清中Lyz的分离纯化,蛋黄中PPPs的富集,胰蛋白酶的固定化及其快速酶解提供了新颖有效的研究技术。
Since it possesses the magnetic responsively, as well as the functionality, the functional magnetic nanoparticles can be convenient, fast separation and purification of the target biomolecules from reaction system with the help of magnets, it has extensive application prospect in biotechnology, biomedicine, food industry, and so on. At present, exploring the functionalization of magnetic nanoparticles and its practical application is the important research direction for magnetic nanoparticles. This dissertation aimed at finding out new techniques in protein separation and enzyme immobilization which using the prepared novel magnetic nanoparticles as Nano carriers, the practical bio-system was chosen as the application system, and obtained some innovative research results. The main research results are as follows:
1. Using polyethylene glycol 6000 (PEG 6000) as dispersant, the functional superparamagnetic nanoparticles which conjugated with carboxymethyl chitosan (CM-CTS) were developed (shorted as Fe_3O_4 (PEG + CM-CTS)); the obtained Fe_3O_4 (PEG + CM-CTS) with uniform size, its diameter was about 15 nm, furthermore, the dispersing behavior of Fe_3O_4 (PEG+CM-CTS) nanoparticles has obviously been improved in comparison with the naked Fe_3O_4; On this basis, through electrostatic interaction, the functional Fe_3O_4 (PEG+CM-CTS) nanoparticles was used as a novel magnetic absorbing carrier for the separation and purification of lysozyme from the aqueous solution and chicken egg white respectively, the purity of lysozyme which purified from chicken egg white was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS?PAGE), it indicated that: The lysozyme which purified from chicken egg white in a single step had higher purity; Considering that the Fe_3O_4 (PEG+CM-CTS) nanoparticles possesses the advantages of high efficiency, cost-effectiveness and excellent binding of a larger amount of lysozyme and easier separation from the reaction system, thus this type of superparamagnetic nanoparticles would bring advantages to the conventional separation techniques of lysozyme from chicken egg white.
2. An fundamental understanding of the conformational behaviors of lysozyme during the process of adsorption and desorption has been studied using Fourier transform infrared spectroscopy (FTIR), circular dichroic (CD) and fluorescence spectroscopy, and it indicated that: on the surface of Fe_3O_4 (PEG+CM-CTS) nanoparticles, the lysozyme would adopt a more compact conformation state; a complex between lysozyme and the Fe_3O_4 (PEG+CM-CTS) nanoparticles has been formed, Trp 62 and Trp108 mol/Light be the possible site of binding of lysozyme to Fe_3O_4 (PEG+CM-CTS) nanoparticles; Lysozyme which desorbed by PBS (20 mmol/L, pH 8.0, 0.2 mol/L NaCl) and PBS (20 mmol/L, pH 8.0, 0.5 mol/L NaCl) retain high fraction of its native structure with negligible effect on its activity, and about 92.4% and 89.5% activity were retained upon desorption from nanoparticles, however, lysozyme desorbed by acetic acid solution (0.2 mol/L, pH 4.0) showed significant conformational changes, only about 24.7% activity were retained; Due to the small diameter, the Fe_3O_4 (PEG+CM-CTS) magnetic nanoparticles has favorable properties to ensure highest level of conformational stabilization of protein compared to the larger particles of the same material, so it may find useful applications in biotechnology ranging from enzyme immobilization to protein purification.
3. The immobilized trypsin which absorbed on the surface of the Fe_3O_4 (PEG+CM-CTS) nanoparticles was prepared and applied for fast and efficient proteolysis. FTIR and fluorescence spectroscopy data demonstrated that the magnetic Fe_3O_4 (PEG+CM-CTS) nanoparticles were capable of improving conformation stability of trypsin and preventing the trypsin unfolding; the immobilized trypsin, compared with native trypsin, has less affinity to the substrate, however, the immobilized trypsin could be used for fast and efficient Bovine Serum Albumin (BSA) digestion under very facile processes, thanks to the easy manipulation of the magnetic nanoparticles, as well as the greatly reduced digestion time (from 12 hr to 15 min); These results are expected to open up a new possible application of the trypsin-Fe_3O_4 (PEG+CM-CTS) conjugates in proteome research as well as a great potential use of such Fe_3O_4 (PEG+CM-CTS) nanoparticles as a superior nanosupport for the immobilization of trypsin.
4. In the research, Fe_3O_4 (PEG+CM-CTS) nanoparticles were treated with Cu (II), Fe (III) and Zn (II) ions solutions to obtain immobilized metal affinity magnetic nanoparticles (IMANs) (short as IMAN @ Cu (II), IMAN @ Fe (III) and IMAN @ Zn (II)). The as-prepared IMANs were conveniently applied for lysozyme adsorption; The maximum equilibrium adsorption capacity of IMAN @ Cu (II), IMAN @ Fe (III) and IMAN @ Zn (II) nanoparticles to lysozyme were calculated to be 185.19 mg/g, 232.56 mg/g and 200 mg/g, respectively; Fluorescence analysis demonstrated that IMAN @ Zn (II) and IMAN @ Fe (III) nanoparticles may not cause the conformational change and denaturation for the lysozyme, but the lysozyme which desorbed from IMAN @ Cu (II) underwent some subtle change in comparison with the original lysozyme.
5. IMAN @ Fe (III) were conveniently applied for phosvitin phosphopeptides (PPPs) enrichment for the first time, the research indicated that: IMAN @ Fe (III) nanoparticles showed greater specificity for PPPs enrichment (N/P can be reduced from 39.49 to 5.52); Since the separation process is convenient, economic and efficient, this type of IMAN @ Fe (III) nanoparticles would bring advantages to the conventional separation techniques of PPPs from chicken egg yolk; In addition, the chosen IMAN @ Fe (III) nanoparticles not only showing potential application in PPPs separation from chicken egg yolk, but also in phosphopeptides enrichment in proteomics research.
In summary, the main contribution of the dissertation is that we initially synthesized the functional magnetic nanoparticles and successfully utilized them for protein separation and enzyme immobiliztation; we aimed at exploring and finding out new techniques in lysozyme separation from chicken egg white, PPPs enrichment from chicken egg yolk, trypsin immobilization and its efficient proteolysis.
引文
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