磁性纖維素納米晶固定化酶的制備及應(yīng)用研究
發(fā)布時間：2018-08-24 08:50
【摘要】：酶是高效的生物催化劑,在許多的工業(yè)領(lǐng)域都具有巨大的應(yīng)用前景。然而,由于游離酶存在價格昂貴、操作穩(wěn)定性低等缺點,其在工業(yè)上的應(yīng)用受到了一定的限制。使用新型的酶載體和新的固定化方法對酶進行固定化是解決上述問題的有效途徑。纖維素納米晶(NCC)是近年來興起的天然高分子納米材料,具有作為酶載體的潛力,由于其在水溶液中穩(wěn)定分散,難以從反應(yīng)體系中快速分離。因此,研發(fā)制備均一穩(wěn)定磁性纖維素納米晶(MNCC)的方法,并將其作為酶載體通過新型酶固定化技術(shù)制備高催化活性、高穩(wěn)定性的固定化酶催化劑具有重要的意義。二肽是一類重要的生物活性肽,在醫(yī)藥、食品、化妝品等領(lǐng)域有重要的應(yīng)用。在眾多二肽合成方法中,酶法是一種綠色環(huán)保的方法。但是,酶法制備存在以下問題:水相反應(yīng)副產(chǎn)物多產(chǎn)率低、有機相反應(yīng)對酶有不同程度的失活作用,酶操作穩(wěn)定性差。因此,研究新型綠色介質(zhì)——深度共熔溶劑中的固定化酶催化二肽合成反應(yīng)是一個很有意義的課題。本文的主要研究成果如下:本研究通過共沉淀-靜電自組裝法以及共沉淀-交聯(lián)法制備了兩個系列的磁性纖維素納米晶(分別為MNCC-A和MNCC-B)。結(jié)果表明,MNCC呈現(xiàn)棒狀形貌,Fe3O4 MNP較均勻地分布在MNCC表面。MNCC同時具有NCC和Fe3O4的特征衍射峰,但Fe3O4的峰強度明顯減弱。與MNCC-A相比,通過共沉淀-交聯(lián)法制備的MNCC-B使用環(huán)氧氯丙烷替代三聚磷酸鈉作為分子間交聯(lián)劑,使MNCC-B的最高磁飽和強度比MNCC-A有所提高(10.1 emu/g v.s 6.9 emu/g)。在制備過程中增加鐵鹽的量或者減少殼聚糖的量可有效提高材料的磁飽和強度。通過機理分析表明:(1)Fe3O4與殼聚糖之間、殼聚糖與NCC之間的靜電相互作用是共沉淀-靜電自組裝法制備MNCC-A的驅(qū)動力;(2)NCC與殼聚糖之間的靜電相互作用以及環(huán)氧氯丙烷對NCC和殼聚糖的交聯(lián)作用是共沉淀-交聯(lián)法制備MNCC-B的兩個階段的主要驅(qū)動力。結(jié)果表明,兩種制備MNCC的方法是可行的。為了考察MNCC酶載體對酶固定化的效果,本研究通過傳統(tǒng)活化交聯(lián)法將木瓜蛋白酶固定化在MNCC-B-3表面得到PA-c-MNCC。在最優(yōu)條件下得到的固定化酶相對酶活回收率為74.5%,MNCC蛋白負載量為8.9 mg/g。PA-c-MNCC的最適pH為7.0(游離酶為6.0),最適溫度為60 oC(與游離酶一致)。PA-c-MNCC的反應(yīng)表觀活化能(Ea)與游離酶相比有所降低,最適底物濃度比游離酶顯著降低。PA-c-MNCC的pH穩(wěn)定性、熱穩(wěn)定性、儲存穩(wěn)定性、有機溶劑耐受性等均優(yōu)于游離酶。為了進一步提高酶在mncc載體的負載量以及酶活回收率,本研究通過沉淀-交聯(lián)法將一種國產(chǎn)木瓜蛋白酶固定化在mncc-b-3表面得到pa@mncc。沉淀-交聯(lián)法的最優(yōu)的固定化條件為:4oc下將4.5mgmncc分散在0.5mlph7.0pbs中,向其中加入1.5mg木瓜蛋白酶。在攪拌下加入7.5ml乙醇,隨后加入1.9wt%戊二醛在-10oc下交聯(lián)2h,所得pa@mncc的酶載量為333mg/gmncc載體,酶活回收率約為80.1%。pa@mncc的最適ph為7.0(游離酶為6.5),最適溫度為75oc(比游離酶提高5oc)。pa@mncc具有較游離酶低的溫度敏感性以及較高的底物親和性。此外,pa@mncc的ph穩(wěn)定性、溫度穩(wěn)定性、儲存穩(wěn)定性、有機溶劑耐受性相比游離酶均有顯著提高。通過固定化酶與游離酶的二級結(jié)構(gòu)含量對比研究表明,酶在經(jīng)過沉淀-交聯(lián)法固定化在mncc載體后,酶結(jié)構(gòu)中-螺旋含量增加、無規(guī)卷曲含量減少,-折疊和-轉(zhuǎn)角結(jié)構(gòu)無明顯變化。因此,固定化酶穩(wěn)定性的增加可能固定化后-螺旋含量的增加所導(dǎo)致的結(jié)構(gòu)剛性增加。利用沉淀-交聯(lián)法對5種常見的酶進行固定化,結(jié)果表明沉淀-交聯(lián)法固定化酶具有一定的普適性。結(jié)果表明,沉淀-交聯(lián)法是一種有效、可行的酶固定化方法。利用gromacs分子動力學(xué)模擬軟件,對三種磁性纖維素納米晶的典型片段與木瓜蛋白酶相互作用進行分子動力學(xué)模擬。結(jié)果表明,酶與mncc之間存在相互作用,使得體系勢能下降、體系越穩(wěn)定,酶與mncc間的平均距離減小。此外,pa與cellulose-so4片段以及cellulose-chitosan片段以靜電作用為主,pa與cellulose片段以氫鍵作用為主。pa-mncc相互作用過程酶結(jié)構(gòu)穩(wěn)定性的增加可能由于:(1)mncc與木瓜蛋白酶分子之間形成的氫鍵部分替代了木瓜蛋白酶與水分子之間形成的氫鍵;(2)木瓜蛋白酶活性中心中his159-asn175氨基酸殘基對的距離顯著減小。此外,研究了pa-mncc模擬相互作用前后酶二級結(jié)構(gòu)含量的變化,結(jié)果表明,相互作用后α-螺旋含量增加,無規(guī)卷曲減少。本文首次報道了含氯化膽堿/尿素des介質(zhì)中游離及固定化木瓜蛋白酶催化芐氧羰基-丙谷二肽(z-ala-gln)合成反應(yīng)。結(jié)果表明,固定化酶pa@mncc催化z-ala-gln的最優(yōu)條件為:含水量16.6%,反應(yīng)溫度50oc,親核試劑gln與�；wz-ala-ome摩爾比2.5-3,tea濃度560mm,在該條件下z-ala-gln產(chǎn)率為71.5%;此外,固定化酶在進行10次操作循環(huán)后仍然有80%左右的剩余酶活。以上述反應(yīng)體系為基礎(chǔ),研究了芐氧羰基-肌肽(z-ala-his)在氯化膽堿/尿素des介質(zhì)中的酶促合成反應(yīng)。研究表明,在最優(yōu)反應(yīng)條件下z-ala-his的產(chǎn)率約為68.4%;此外,本研究對比了不同氫鍵供體的氯化膽堿類des中酶催化z-ala-his的產(chǎn)率。結(jié)果表明,對比三種醇類des中的Z-Ala-His合成產(chǎn)率可知,在粘度大的DES中,酶催化Z-Ala-His的產(chǎn)率較低�；赑A@MNCC酶催化劑和深度共熔溶劑新型反應(yīng)介質(zhì)的酶促二肽合成方法是可行的。本研究不僅豐富了固定化酶相關(guān)領(lǐng)域的理論知識,還提供了酶法制備二肽的行之有效的新途徑。
[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.
【學(xué)位授予單位】：華南理工大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：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.
【學(xué)位授予單位】：華南理工大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q814.2
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