磁性纤维素纳米晶固定化酶的制备及应用研究
发布时间：2018-08-24 08:50
【摘要】：酶是高效的生物催化剂,在许多的工业领域都具有巨大的应用前景。然而,由于游离酶存在价格昂贵、操作稳定性低等缺点,其在工业上的应用受到了一定的限制。使用新型的酶载体和新的固定化方法对酶进行固定化是解决上述问题的有效途径。纤维素纳米晶(NCC)是近年来兴起的天然高分子纳米材料,具有作为酶载体的潜力,由于其在水溶液中稳定分散,难以从反应体系中快速分离。因此,研发制备均一稳定磁性纤维素纳米晶(MNCC)的方法,并将其作为酶载体通过新型酶固定化技术制备高催化活性、高稳定性的固定化酶催化剂具有重要的意义。二肽是一类重要的生物活性肽,在医药、食品、化妆品等领域有重要的应用。在众多二肽合成方法中,酶法是一种绿色环保的方法。但是,酶法制备存在以下问题:水相反应副产物多产率低、有机相反应对酶有不同程度的失活作用,酶操作稳定性差。因此,研究新型绿色介质——深度共熔溶剂中的固定化酶催化二肽合成反应是一个很有意义的课题。本文的主要研究成果如下:本研究通过共沉淀-静电自组装法以及共沉淀-交联法制备了两个系列的磁性纤维素纳米晶(分别为MNCC-A和MNCC-B)。结果表明,MNCC呈现棒状形貌,Fe3O4 MNP较均匀地分布在MNCC表面。MNCC同时具有NCC和Fe3O4的特征衍射峰,但Fe3O4的峰强度明显减弱。与MNCC-A相比,通过共沉淀-交联法制备的MNCC-B使用环氧氯丙烷替代三聚磷酸钠作为分子间交联剂,使MNCC-B的最高磁饱和强度比MNCC-A有所提高(10.1 emu/g v.s 6.9 emu/g)。在制备过程中增加铁盐的量或者减少壳聚糖的量可有效提高材料的磁饱和强度。通过机理分析表明:(1)Fe3O4与壳聚糖之间、壳聚糖与NCC之间的静电相互作用是共沉淀-静电自组装法制备MNCC-A的驱动力;(2)NCC与壳聚糖之间的静电相互作用以及环氧氯丙烷对NCC和壳聚糖的交联作用是共沉淀-交联法制备MNCC-B的两个阶段的主要驱动力。结果表明,两种制备MNCC的方法是可行的。为了考察MNCC酶载体对酶固定化的效果,本研究通过传统活化交联法将木瓜蛋白酶固定化在MNCC-B-3表面得到PA-c-MNCC。在最优条件下得到的固定化酶相对酶活回收率为74.5%,MNCC蛋白负载量为8.9 mg/g。PA-c-MNCC的最适pH为7.0(游离酶为6.0),最适温度为60 oC(与游离酶一致)。PA-c-MNCC的反应表观活化能(Ea)与游离酶相比有所降低,最适底物浓度比游离酶显著降低。PA-c-MNCC的pH稳定性、热稳定性、储存稳定性、有机溶剂耐受性等均优于游离酶。为了进一步提高酶在mncc载体的负载量以及酶活回收率,本研究通过沉淀-交联法将一种国产木瓜蛋白酶固定化在mncc-b-3表面得到pa@mncc。沉淀-交联法的最优的固定化条件为:4oc下将4.5mgmncc分散在0.5mlph7.0pbs中,向其中加入1.5mg木瓜蛋白酶。在搅拌下加入7.5ml乙醇,随后加入1.9wt%戊二醛在-10oc下交联2h,所得pa@mncc的酶载量为333mg/gmncc载体,酶活回收率约为80.1%。pa@mncc的最适ph为7.0(游离酶为6.5),最适温度为75oc(比游离酶提高5oc)。pa@mncc具有较游离酶低的温度敏感性以及较高的底物亲和性。此外,pa@mncc的ph稳定性、温度稳定性、储存稳定性、有机溶剂耐受性相比游离酶均有显著提高。通过固定化酶与游离酶的二级结构含量对比研究表明,酶在经过沉淀-交联法固定化在mncc载体后,酶结构中-螺旋含量增加、无规卷曲含量减少,-折叠和-转角结构无明显变化。因此,固定化酶稳定性的增加可能固定化后-螺旋含量的增加所导致的结构刚性增加。利用沉淀-交联法对5种常见的酶进行固定化,结果表明沉淀-交联法固定化酶具有一定的普适性。结果表明,沉淀-交联法是一种有效、可行的酶固定化方法。利用gromacs分子动力学模拟软件,对三种磁性纤维素纳米晶的典型片段与木瓜蛋白酶相互作用进行分子动力学模拟。结果表明,酶与mncc之间存在相互作用,使得体系势能下降、体系越稳定,酶与mncc间的平均距离减小。此外,pa与cellulose-so4片段以及cellulose-chitosan片段以静电作用为主,pa与cellulose片段以氢键作用为主。pa-mncc相互作用过程酶结构稳定性的增加可能由于:(1)mncc与木瓜蛋白酶分子之间形成的氢键部分替代了木瓜蛋白酶与水分子之间形成的氢键;(2)木瓜蛋白酶活性中心中his159-asn175氨基酸残基对的距离显著减小。此外,研究了pa-mncc模拟相互作用前后酶二级结构含量的变化,结果表明,相互作用后α-螺旋含量增加,无规卷曲减少。本文首次报道了含氯化胆碱/尿素des介质中游离及固定化木瓜蛋白酶催化苄氧羰基-丙谷二肽(z-ala-gln)合成反应。结果表明,固定化酶pa@mncc催化z-ala-gln的最优条件为:含水量16.6%,反应温度50oc,亲核试剂gln与酰基供体z-ala-ome摩尔比2.5-3,tea浓度560mm,在该条件下z-ala-gln产率为71.5%;此外,固定化酶在进行10次操作循环后仍然有80%左右的剩余酶活。以上述反应体系为基础,研究了苄氧羰基-肌肽(z-ala-his)在氯化胆碱/尿素des介质中的酶促合成反应。研究表明,在最优反应条件下z-ala-his的产率约为68.4%;此外,本研究对比了不同氢键供体的氯化胆碱类des中酶催化z-ala-his的产率。结果表明,对比三种醇类des中的Z-Ala-His合成产率可知,在粘度大的DES中,酶催化Z-Ala-His的产率较低。基于PA@MNCC酶催化剂和深度共熔溶剂新型反应介质的酶促二肽合成方法是可行的。本研究不仅丰富了固定化酶相关领域的理论知识,还提供了酶法制备二肽的行之有效的新途径。
[Abstract]:Enzymes are efficient biocatalysts and have great application prospects in many industrial fields. However, the application of free enzymes in industry is limited due to their high cost and low operational stability. Cellulose nanocrystals (NCC) are natural polymer nanomaterials that have been developed in recent years. They have potential as enzyme carriers. Because of their stable dispersion in aqueous solution, they are difficult to be separated from the reaction system quickly. Therefore, the method of preparing homogeneous stable magnetic cellulose nanocrystals (MNCC) is developed and used as enzyme carriers through novel methods. Dipeptides are a kind of important bioactive peptides, which have important applications in medicine, food, cosmetics and other fields. Among many synthetic methods of dipeptides, enzymatic method is a green environmental protection method. In this paper, the main research results are as follows: coprecipitation-static method is used to study the synthesis of dipeptides catalyzed by immobilized enzymes in deep eutectic solvents, a new green medium. Two series of magnetic cellulose nanocrystals (MNCC-A and MNCC-B) were prepared by electro-self-assembly method and co-precipitation-crosslinking method. The results showed that MNCC had rod-like morphology and Fe_3O_4 MNP was uniformly distributed on the surface of CNC. MNCC had both characteristic diffraction peaks of NCC and Fe_3O_4, but the peak strength of Fe_3O_4 was obviously weakened. The highest magnetic saturation strength of MNCC-B is higher than that of MNCC-A (10.1 emu/g v.s 6.9 emu/g) by using epichlorohydrin instead of sodium tripolyphosphate as intermolecular crosslinking agent. The magnetic saturation strength of the material can be improved effectively by increasing the amount of ferric salt or reducing the amount of chitosan in the preparation process. The results showed that: (1) between Fe3O4 and chitosan, the electrostatic interaction between chitosan and NCC was the driving force for the preparation of MNCC-A by coprecipitation-electrostatic self-assembly method; (2) The electrostatic interaction between NCC and chitosan and the cross-linking between epichlorohydrin and NCC were the main driving forces for the two stages of the preparation of MNCC-B by coprecipitation-cross-linking method. The results showed that the two methods of preparing MNCC were feasible. In order to investigate the effect of MNCC enzyme carrier on enzyme immobilization, PA-c-MNCC was obtained by Immobilizing Papain on the surface of MNCC-B-3 by traditional activation cross-linking method. Under the optimal conditions, the relative enzyme activity recovery of the immobilized enzyme was 74.5%, and the MNCC protein loading was 8.9 mg/g.PA. The optimum pH of PA-c-MNCC was 7.0 (free enzyme 6.0), and the optimum temperature was 60 oC (consistent with free enzyme). The apparent activation energy (Ea) of PA-c-MNCC was lower than that of free enzyme, and the optimum substrate concentration was significantly lower than that of free enzyme. PA @ MNCC was obtained by immobilizing a domestic papain on the surface of mncc-b-3 by precipitation-crosslinking method. the optimum immobilization conditions of the precipitation-crosslinking method were as follows: 4.5m gmncc was dispersed in 0.5mlph 7.0 PBS at 4oc, and 1.5mg papain was added to it. PA @ MNCC was prepared by adding 7.5 ml ethanol and 1.9 wt% glutaraldehyde at - 10 OC for 2 h. The yield of PA @ MNCC was 333 mg / gmncc. The optimum pH of PA @ MNCC was 7.0 (free enzyme was 6.5), and the optimum temperature was 75 OC (5 OC higher than free enzyme). PA @ MNCC had lower temperature sensitivity of free enzyme and higher substrate affinity. In addition, the pH stability, temperature stability, storage stability and organic solvent tolerance of PA @ MNCC were significantly improved compared with that of free enzyme. the results showed that after immobilized on MNCC carrier by precipitation-crosslinking method, the content of helix in enzyme structure increased and the content of random curl decreased. Therefore, the stability of immobilized enzymes may increase with the increase of helix content. Five kinds of common enzymes were immobilized by precipitation-crosslinking method. The results showed that the precipitation-crosslinking method had a certain universality. The molecular dynamics simulation of the interaction between three typical fragments of magnetic cellulose nanocrystals and papain was carried out by using GROMACS molecular dynamics simulation software. The results showed that there was an interaction between the enzyme and mncc, resulting in the decrease of potential energy of the system, the more stable the system, and the interaction between the enzyme and mncc. In addition, the electrostatic interaction between PA and cellulose-so4 fragment and Cellulose-chitosan fragment was dominant, while the hydrogen bonding between PA and cellulose fragment was dominant. The increase of enzyme structure stability during the interaction between pa-mncc may be due to: (1) the hydrogen bond formed between MNCC and papain molecules partially replaced papain and water. Hydrogen bonding between molecules; (2) the distance between his 159-asn 175 amino acid residues in papain activity center decreased significantly. in addition, the changes of enzyme secondary structure content before and after pa-mncc interaction were studied. the results showed that the content of alpha-helix increased and the random curl decreased after pa-mncc interaction. Benzyloxycarbonyl-propylglutathione (z-ala-gln) synthesis was catalyzed by free and immobilized papain in urea des medium. The results showed that the optimum conditions for the synthesis of z-ala-gln catalyzed by immobilized enzyme PA @ MNCC were as follows: water content 16.6%, reaction temperature 50 oc, molar ratio of nucleophilic reagent Gln to acyl donor z-ala-ome 2.5-3, tea concentration 560 mm. The yield of the immobilized enzyme was 71.5%. In addition, about 80% of the enzyme activity remained after 10 cycles of operation. Based on the above reaction system, the enzymatic synthesis of benzyloxycarbonyl-carnosine (z-ala-his) in the medium of choline chloride/urea DES was studied. The yield of z-ala-his catalyzed by enzymes from Chlorocholine DES with different hydrogen bond donors was compared. The results showed that the yield of Z-Ala-His catalyzed by enzymes was lower in the high viscosity DES than in the three alcohols. This study not only enriched the theoretical knowledge of immobilized enzymes, but also provided an effective new way to prepare dipeptides by enzymatic method.
【学位授予单位】：华南理工大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q814.2
[Abstract]:Enzymes are efficient biocatalysts and have great application prospects in many industrial fields. However, the application of free enzymes in industry is limited due to their high cost and low operational stability. Cellulose nanocrystals (NCC) are natural polymer nanomaterials that have been developed in recent years. They have potential as enzyme carriers. Because of their stable dispersion in aqueous solution, they are difficult to be separated from the reaction system quickly. Therefore, the method of preparing homogeneous stable magnetic cellulose nanocrystals (MNCC) is developed and used as enzyme carriers through novel methods. Dipeptides are a kind of important bioactive peptides, which have important applications in medicine, food, cosmetics and other fields. Among many synthetic methods of dipeptides, enzymatic method is a green environmental protection method. In this paper, the main research results are as follows: coprecipitation-static method is used to study the synthesis of dipeptides catalyzed by immobilized enzymes in deep eutectic solvents, a new green medium. Two series of magnetic cellulose nanocrystals (MNCC-A and MNCC-B) were prepared by electro-self-assembly method and co-precipitation-crosslinking method. The results showed that MNCC had rod-like morphology and Fe_3O_4 MNP was uniformly distributed on the surface of CNC. MNCC had both characteristic diffraction peaks of NCC and Fe_3O_4, but the peak strength of Fe_3O_4 was obviously weakened. The highest magnetic saturation strength of MNCC-B is higher than that of MNCC-A (10.1 emu/g v.s 6.9 emu/g) by using epichlorohydrin instead of sodium tripolyphosphate as intermolecular crosslinking agent. The magnetic saturation strength of the material can be improved effectively by increasing the amount of ferric salt or reducing the amount of chitosan in the preparation process. The results showed that: (1) between Fe3O4 and chitosan, the electrostatic interaction between chitosan and NCC was the driving force for the preparation of MNCC-A by coprecipitation-electrostatic self-assembly method; (2) The electrostatic interaction between NCC and chitosan and the cross-linking between epichlorohydrin and NCC were the main driving forces for the two stages of the preparation of MNCC-B by coprecipitation-cross-linking method. The results showed that the two methods of preparing MNCC were feasible. In order to investigate the effect of MNCC enzyme carrier on enzyme immobilization, PA-c-MNCC was obtained by Immobilizing Papain on the surface of MNCC-B-3 by traditional activation cross-linking method. Under the optimal conditions, the relative enzyme activity recovery of the immobilized enzyme was 74.5%, and the MNCC protein loading was 8.9 mg/g.PA. The optimum pH of PA-c-MNCC was 7.0 (free enzyme 6.0), and the optimum temperature was 60 oC (consistent with free enzyme). The apparent activation energy (Ea) of PA-c-MNCC was lower than that of free enzyme, and the optimum substrate concentration was significantly lower than that of free enzyme. PA @ MNCC was obtained by immobilizing a domestic papain on the surface of mncc-b-3 by precipitation-crosslinking method. the optimum immobilization conditions of the precipitation-crosslinking method were as follows: 4.5m gmncc was dispersed in 0.5mlph 7.0 PBS at 4oc, and 1.5mg papain was added to it. PA @ MNCC was prepared by adding 7.5 ml ethanol and 1.9 wt% glutaraldehyde at - 10 OC for 2 h. The yield of PA @ MNCC was 333 mg / gmncc. The optimum pH of PA @ MNCC was 7.0 (free enzyme was 6.5), and the optimum temperature was 75 OC (5 OC higher than free enzyme). PA @ MNCC had lower temperature sensitivity of free enzyme and higher substrate affinity. In addition, the pH stability, temperature stability, storage stability and organic solvent tolerance of PA @ MNCC were significantly improved compared with that of free enzyme. the results showed that after immobilized on MNCC carrier by precipitation-crosslinking method, the content of helix in enzyme structure increased and the content of random curl decreased. Therefore, the stability of immobilized enzymes may increase with the increase of helix content. Five kinds of common enzymes were immobilized by precipitation-crosslinking method. The results showed that the precipitation-crosslinking method had a certain universality. The molecular dynamics simulation of the interaction between three typical fragments of magnetic cellulose nanocrystals and papain was carried out by using GROMACS molecular dynamics simulation software. The results showed that there was an interaction between the enzyme and mncc, resulting in the decrease of potential energy of the system, the more stable the system, and the interaction between the enzyme and mncc. In addition, the electrostatic interaction between PA and cellulose-so4 fragment and Cellulose-chitosan fragment was dominant, while the hydrogen bonding between PA and cellulose fragment was dominant. The increase of enzyme structure stability during the interaction between pa-mncc may be due to: (1) the hydrogen bond formed between MNCC and papain molecules partially replaced papain and water. Hydrogen bonding between molecules; (2) the distance between his 159-asn 175 amino acid residues in papain activity center decreased significantly. in addition, the changes of enzyme secondary structure content before and after pa-mncc interaction were studied. the results showed that the content of alpha-helix increased and the random curl decreased after pa-mncc interaction. Benzyloxycarbonyl-propylglutathione (z-ala-gln) synthesis was catalyzed by free and immobilized papain in urea des medium. The results showed that the optimum conditions for the synthesis of z-ala-gln catalyzed by immobilized enzyme PA @ MNCC were as follows: water content 16.6%, reaction temperature 50 oc, molar ratio of nucleophilic reagent Gln to acyl donor z-ala-ome 2.5-3, tea concentration 560 mm. The yield of the immobilized enzyme was 71.5%. In addition, about 80% of the enzyme activity remained after 10 cycles of operation. Based on the above reaction system, the enzymatic synthesis of benzyloxycarbonyl-carnosine (z-ala-his) in the medium of choline chloride/urea DES was studied. The yield of z-ala-his catalyzed by enzymes from Chlorocholine DES with different hydrogen bond donors was compared. The results showed that the yield of Z-Ala-His catalyzed by enzymes was lower in the high viscosity DES than in the three alcohols. This study not only enriched the theoretical knowledge of immobilized enzymes, but also provided an effective new way to prepare dipeptides by enzymatic method.
【学位授予单位】：华南理工大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q814.2
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