磁性壳聚糖的制备及在脂肪酶固定的应用

采用共沉淀法制备磁性壳聚糖微球,戊二醛做交联剂把脂肪酶固定在磁性壳聚糖载体上,分别使用扫描电子显微镜、傅立叶红外光谱仪、X射线衍射仪、振动样品磁强计、热重分析仪等对磁性壳聚糖微球进行表征分析,并检测了固定化酶的特性.结果表明,制备的磁性壳聚糖性能良好,与游离酶相比,固定化酶的最适p H,最适温度均有提高,固定化酶的热稳定性,对变性剂的耐受力,重复使用性都有较大幅度提高.     

氧化铁磁性纳米粒子固定化酶*

纳米粒子作为酶固定化的载体,当其具有磁性时,制备的固定化酶易于从反应体系中分离和回收,操作简便;并且利用外部磁场可以控制磁性材料固定化酶的运动方式和方向,替代传统的机械搅拌方式,提高固定化酶的催化效率。在众多纳米材料中,氧化铁因其在磁性、催化等多方面的良好特性而倍受瞩目。本文对近年来各种氧化铁磁性纳米粒子固定化酶,尤其是固定化脂肪酶和蛋白酶的制备方法及其应用做了较为详细的阐述,对这些氧化铁磁性纳米粒子固定化酶的优缺点和发展前景进行了讨论。     

α，ω─二羧基聚乙二醇磁性毫微粒固定化碱性蛋白酶的研究

本文用共沉淀法制备了平均直径为３８４纳米的α，ω─二羧基聚乙二醇磁性毫微粒．碱性蛋白酶通过吸附交联法被固定于磁性毫微粒．研究了制备过程中的吸附时间、给酶量、戊二醛浓度、ｐＨ和离子强度对磁性固定化酶活力及酶固定化率的影响．比较了磁性蛋白酶磁性固定化酶与自由酶的酶学性质，磁性固定化酶的最适温度有改变，但热稳定性显著提高；磁性固定化酶的最适ｐＨ向酸性方向移动了１．０个ＰＨ单位。     

α,ω—二羧基聚乙二醇磁性毫微粒固定化碱性蛋白酶的研究

本文用共沉淀法制备了平均直径为３８４纳米的α，ω－二羧基聚乙二醇磁性毫微粒，碱性蛋白酶通过吸附交联法被固定于磁性毫微粒，研究了制备研究中的吸附时间，给酶量，戊二醛浓度，ｐＨ和离子强度对磁性固定化酶活力及酶固定化率的影响。比较了碱性蛋白酶磁性固定化酶与自由酶的酶学性质，磁性固定化酶的最适温度没有改变，但热稳定性显著使高；磁性固定化酶的最适ｐＨ向酸性方向移动了１．０个ｐＨ单位。     

磁性纳米氮化铝颗粒固定化β-葡萄糖苷酶的性质

以戊二醛为交联剂,研究了磁性纳米氮化铝颗粒固定化β-葡萄糖苷酶的条件及固定化酶的结构特征,并考察了固定化酶的某些酶学性质.结果表明,在4.5 ml磁性纳米氮化铝颗粒悬液(100 mg/ml)中加入0.5 ml戊二醛溶液(2%)超声波分散后,加入5 ml β-葡萄糖苷酶溶液(50 mg/ml),于20℃,pH 5.0和100 r/min条件下固载3.5 h,酶蛋白和酶活回收率分别为82.6%和78.4%.固定化β-葡萄糖苷酶的结构松散,不改变酶的结构特征.与游离酶相比,固定化酶对对硝基苯基-β-D-葡萄糖苷水解反应的最佳反应温度有所降低,最佳反应pH值有所升高,而米氏常数Km值有所增大,且具有良好的贮存稳定性和操作稳定性,表明磁性纳米氮化铝颗粒适合作为β-葡萄糖苷酶的固定化载体.     

基于聚多巴胺的磁性纳米生物催化剂高效催化二氢杨梅素酰化

脂肪酶是一种三酰基甘油水解酶,目前广泛用于油脂化学、食品、有机合成和生物医药等领域.但是,游离脂肪酶在有机反应体系中容易失活,难以从反应体系中回收,导致其循环利用困难和生产成本增加.因此,需要对游离脂肪酶进行固定化,提高酶的稳定性和重复使用性,使其能够大规模用于工业生产.
　　磁性四氧化三铁纳米粒子(MNPs)具有其超顺磁性和大比表面积等性质,但MNPs需表面修饰才能进一步应用.近年来,仿生矿化法制备的聚多巴胺纳米材料受到人们关注.在仿生矿化过程中,单体多巴胺经自聚合作用后形成聚多巴胺,该反应活性高,能对各类有机和无机纳米材料进行表面修饰.而且,聚多巴胺表层中的活性基团能与含有氨基和巯基的生物大分子发生迈克尔加成或席夫碱反应,从而将生物大分子固定在材料表面.
　　本文利用聚多巴胺表面修饰MNPs,对所得聚多巴胺表面修饰的四氧化三铁纳米粒子(PD-MNPs)进行了结构表征.结果表明, PD-MNPs尺寸在14 nm左右.同时,成功将黑曲霉脂肪酶(ANL)固定在PD-MNPs上,结果显示在pH=8、固定化时间为12 h条件下,酶负载量为138 mg/g,酶活回收率达到83.6%,而且固定化酶的pH稳定性及热稳定性、储藏稳定性都优于游离酶.动力学研究表明,固定化酶Km值(63.2 mmol/L)低于游离酶(74.5 mmol/L),固定化酶的底物亲和性增强.进一步研究了固定化酶和游离酶在乙腈、二甲基亚砜、乙醇和[HMIm]BF4这四种溶剂中的溶剂耐受性,结果显示固定化酶的耐受性均强于游离酶.采用红外光谱对游离酶和固定化酶二级结构的分析表明,游离黑曲霉脂肪酶经固定化后,α-螺旋和β-折叠含量分别增加了0.84%和2.74%,使得固定化后α-螺旋和β-折叠中存在的氢键能够更好地保持酶结构刚性,避免因结构改变而引起酶失活,增强了固定化酶在溶剂中的耐受性.
　　二氢杨梅素是一种具有类黄酮结构的天然产物,具有抗氧化、抗菌、抗肿瘤和保护肝脏等作用,但其脂溶性很差,很难透过细胞膜被人体吸收.本课题组曾首次以乙酸乙烯酯为酰基供体,采用游离脂肪酶生物催化方法成功将二氢杨梅素酰化.本文考察了PD-MNPs固定化脂肪酶在二氢杨梅素酰化反应中的应用.结果表明,与游离酶相比,固定化酶在反应介质二甲基亚砜中的耐受性更强,反应48 h后其催化二氢杨梅素酰化的转化率接近80%,明显好于游离酶(69%).固定化酶催化二氢杨梅素酰化的最适底物摩尔比、温度和酶量分别为10：1(乙酸乙烯酯：二氢杨梅素)、45oC,和40 U.此外,固定化酶在外界磁场作用下能迅速从反应混合物中分离,从而可回收利用,在重复使用10次后,其活性仍保持在初始活性的55%以上,具有良好的工业应用前景.     

基于聚多巴胺的磁性纳米生物催化剂高效催化二氢杨梅素酰化（英文）

脂肪酶是一种三酰基甘油水解酶,目前广泛用于油脂化学、食品、有机合成和生物医药等领域.但是,游离脂肪酶在有机反应体系中容易失活,难以从反应体系中回收,导致其循环利用困难和生产成本增加.因此,需要对游离脂肪酶进行固定化,提高酶的稳定性和重复使用性,使其能够大规模用于工业生产.磁性四氧化三铁纳米粒子(MNPs)具有其超顺磁性和大比表面积等性质,但MNPs需表面修饰才能进一步应用.近年来,仿生矿化法制备的聚多巴胺纳米材料受到人们关注.在仿生矿化过程中,单体多巴胺经自聚合作用后形成聚多巴胺,该反应活性高,能对各类有机和无机纳米材料进行表面修饰.而且,聚多巴胺表层中的活性基团能与含有氨基和巯基的生物大分子发生迈克尔加成或席夫碱反应,从而将生物大分子固定在材料表面.本文利用聚多巴胺表面修饰MNPs,对所得聚多巴胺表面修饰的四氧化三铁纳米粒子(PD-MNPs)进行了结构表征.结果表明,PD-MNPs尺寸在14 nm左右.同时,成功将黑曲霉脂肪酶(ANL)固定在PD-MNPs上,结果显示在pH=8、固定化时间为12 h条件下,酶负载量为138 mg/g,酶活回收率达到83.6%,而且固定化酶的pH稳定性及热稳定性、储藏稳定性都优于游离酶.动力学研究表明,固定化酶Km值(63.2 mmol/L)低于游离酶(74.5 mmol/L),固定化酶的底物亲和性增强.进一步研究了固定化酶和游离酶在乙腈、二甲基亚砜、乙醇和[HMIm]BF_4这四种溶剂中的溶剂耐受性,结果显示固定化酶的耐受性均强于游离酶.采用红外光谱对游离酶和固定化酶二级结构的分析表明,游离黑曲霉脂肪酶经固定化后,α-螺旋和β-折叠含量分别增加了0.84%和2.74%,使得固定化后α-螺旋和β-折叠中存在的氢键能够更好地保持酶结构刚性,避免因结构改变而引起酶失活,增强了固定化酶在溶剂中的耐受性.二氢杨梅素是一种具有类黄酮结构的天然产物,具有抗氧化、抗菌、抗肿瘤和保护肝脏等作用,但其脂溶性很差,很难透过细胞膜被人体吸收.本课题组曾首次以乙酸乙烯酯为酰基供体,采用游离脂肪酶生物催化方法成功将二氢杨梅素酰化.本文考察了PD-MNPs固定化脂肪酶在二氢杨梅素酰化反应中的应用.结果表明,与游离酶相比,固定化酶在反应介质二甲基亚砜中的耐受性更强,反应48 h后其催化二氢杨梅素酰化的转化率接近80%,明显好于游离酶(69%).固定化酶催化二氢杨梅素酰化的最适底物摩尔比、温度和酶量分别为10:1(乙酸乙烯酯:二氢杨梅素)、45 ℃,和40 U.此外,固定化酶在外界磁场作用下能迅速从反应混合物中分离,从而可回收利用,在重复使用10次后,其活性仍保持在初始活性的55%以上,具有良好的工业应用前景.     

壳聚糖固定化乳酸脱氢酶的制备及酶学性质研究

以磁性壳聚糖作为载体,戊二醛作为交联剂,对乳酸脱氢酶(LDH)进行固定化.固定化的最适条件为:戊二醛浓度6%,pH值7.5,酶的偶联时间2 h.对游离及固定化LDH酶学性质的研究表明,酶促反应的最适pH值为9.2,最适温度分别为37℃和50℃,对乳酸的表观米氏常数分别为1.6 mmol/L和0.9 mmol/L.游离酶和固定化酶在40℃放置150 min后,其活力分别为最初的56.5%和76.1%.固定化酶在4℃贮存4周后,活力仍保留50%以上.固定化酶在室温下与底物重复反应6次后,活力仍保留60%以上,说明固定化酶具有较好的热稳定性、贮存稳定性和复用性.     

壳聚糖固定化碱性脂肪酶的研究

以蟹壳为原料提取壳聚糖,用戊二醛作交联剂,将碱性脂肪酶固定于壳聚糖上。同时探讨了一定量干壳聚糖载体与交联剂浓度、给酶量等关系的最适固定化酶条件,并对固定化酶的热稳定性、操作稳定性、米氏常数、最适温度、离子强度的影响以及使用半衰期等理化性质进行探讨。     

氨化大孔球状聚氯乙烯固定化木瓜蛋白酶的研究

以氨化大孔球状聚氯乙烯为载体,采用戊二醛载体交联的方法,将木瓜蛋白酶进行了固定化。以酪蛋白为底物,测定了固定化酶的活力回收。研究了固定化条件对固定化酶活力回收的影响。同时,对所得固定化酶的性质,如温度-活力关系、pH-活力关系、热稳定性以及重复使用性进行了考察。结果表明,所得固定化木瓜蛋白酶具有较好的稳定性和重复使用性。     

猪胰脂肪酶的固定化及其在有机相中催化丁酸甲酯和正丁醇的酯交换反应研究

考察了大孔苯乙烯－二乙烯苯交联共聚物的交联度、致孔剂量及胺化试剂对载体固定化猪胰脂肪酶的影响。选择出一种最佳载体对猪胰脂肪酶进行固定化，对比了自由酶和固定化酶在有机相中催化丁酸甲酯和正丁醇的酯交换反应。结果表明，酶经固定化后催化反应活力比自由酶提高近１倍。     

树枝状大分子聚酰胺-胺用于氨基酰化酶的固定化研究

以树枝状大分子修饰的硅胶为载体,戊二醛为交联剂,对氨基酰化酶进行固定化研究。考察了树枝状大分子的代数、戊二醛浓度、反应温度与时间对氨基酰化酶固定化效果的影响,并且考察了该固定化酶的最佳酶解条件。结果表明,随着树枝状大分子代数的增加,固定化的酶量随之增大,同时,固定后的酶仍然保持较高的活性。     

Activity of Candida rugosa lipase immobilized on gamma-Fe2O3 magnetic nanoparticles

We report the stability and enzymatic activity of Candida rugosa Lipase (E.C.3.1.1.3) immobilized on gamma-Fe2O3 magnetic nanoparticles. The immobilization strategies were either reacting the enzyme amine group with a nanoparticle surface acetyl, or amine groups. In the former, the enzyme was attached through a C=N bond, while in the latter it was connected using glutaraldehyde. AFM images show an average particle size of 20 +/- 10 nm after deconvolution. The enzymatic activity of the immobilized lipase was determined by following the ester cleavage of p-nitrophenol butyrate. The covalently immobilized enzyme was stabile and reactive over 30 days.     

Covalent attachment of microbial lipase onto microporous styrene-divinylbenzene copolymer by means of polyglutaraldehyde

A novel method for immobilization of Thermomyces lanuginosus lipase onto polyglutaraldehyde-activated poly(styrene-divinylbenzene) (STY-DVB), which is a hydrophobic microporous support has been successfully developed. The copolymer was prepared by the polymerization of the continuous phase of a high internal phase emulsion (polyHIPE). The concentrated emulsion consists of a mixture of styrene and divinylbenzene containing a suitable surfactant and an initiator as the continuous phase and water as the dispersed phase. Lipase from T. lanuginosus was immobilized covalently with 85% yield on the internal surface of the hydrophobic microporous poly(styrene-divinylbenzene) copolymer and used as a biocatalyst for the transesterification reaction. The immobilized enzyme has been fully active 30 days in storage and retained the activity during the 15 repeated batch reactions. The properties of free and immobilized lipase were studied. The effects of protein concentration, pH, temperature, and time on the immobilization, activity, and stability of the immobilized lipase were also studied. The newly synthesized microporous poly(styrene-divinylbenzene) copolymer constitutes excellent support for lipase. It given rise to high immobilization yield, retains enzymatic activity for 30 days, stable in structure and allows for the immobilization of large amount of protein (11.4mg/g support). Since immobilization is simple yet effective, the newly immobilized lipase could be used in several application including oil hydrolysis, production of modified oils, biodiesel synthesis, and removal of fatty acids from oils.     

SYNTHESIS OF POLY(5,5-DIMETHYL-1,3-DIOXAN-2-ONE)BY LIPASE-CATALYZED RING-OPENING POLYMERIZATION

Porcine pancreas lipase (PPL) and PPL immobilized on narrow distributed micron-sized glass beads wereemployed successfully for the ring-opening polymerization of 5, 5-dimethyl-1, 3-dioxan-2-one (DTC) for the first time.Different polymerization conditions such as enzyme concentration and reaction temperature were studied. Immobilized PPLexhibits higher activity than native PPL. Along wth the increasing enzyme concentration, the molecular weigh of resultingPDTC decreases. PPL immobilized on narrow distributed micron-sized glass beads has outstanding recyclability. For thethird recycle time, immobilized PPL exhibits the highest catalytic activity and with high activity even after the fifth recyletime for the synthesis of PDTC. The ~1H-NMR spectra indicate that decarboxylation does not occur during the ring-openingpolymerization.     

Covalent immobilization of glucose oxidase onto new modified acrylonitrile copolymer/silica gel hybrid supports

New polymer/silica gel hybrid supports were prepared by coating high surface area of silica gel with modified acrylonitrile copolymer. The concentrations of the modifying agent (NaOH) and the modified polymer were varied. GOD was covalently immobilized on these hybrid supports and the relative activity and the amount of bound protein were determined. The highest relative activity and sufficient amount of bound protein of the immobilized GOD were achieved in 10% NaOH and 2% solution of modified acrylonitrile copolymer. The influence of glutaraldehyde concentration and the storage time on enzyme efficiency were examined. Glutaraldehyde concentration of 0.5% is optimal for the immobilized GOD. It was shown that the covalently bound enzyme (using 0.5% glutaraldehyde) had higher relative activity than the activity of the adsorbed enzyme. Covalently immobilized GOD with 0.5% glutaraldehyde was more stable for four months in comparison with the one immobilized on pure silica gel, hybrid support with 10% glutaraldehyde and the free enzyme. The effect of the pore size on the enzyme efficiency was studied on four types of silica gel with different pore size. Silica with large pores (CPC-Silica carrier, 375 A) presented higher relative activity than those with smaller pore size (Silica gel with 4, 40 and 100 A). The amount of bound protein was also reduced with decreasing the pore size. The effect of particle size was studied and it was found out that the smaller the particle size was, the greater the activity and the amount of immobilized enzyme were. The obtained results proved that these new polymer/silica gel hybrid supports were suitable for GOD immobilization.     

Functionalized ionic liquid modified mesoporous silica SBA-15: a novel, designable and efficient carrier for porcine pancreas lipase

A series of functionalized ionic liquid modified mesoporous silicas SBA-15 (FIL-SBA) were synthesized by modulating the loading and cation/anion ratio of the functionalized ionic liquid (FIL). The prepared materials FIL-SBA were used as a novel carrier system to immobilize porcine pancreas lipase (PPL). Enzymatic activity and reusability of the immobilized enzyme were investigated using the triacetin hydrolysis reaction. The combined advantages of the nano-sized pore diameter, large surface area and high pore volume of SBA-15, and the tunable properties of the FIL for enzymes immobilized in FIL-SBA gave a maximum improvement of 570% in relative activity, with 63% retention of initial activity after five cycles of use. Carriers and immobilized enzymes were characterized using nitrogen adsorption, small-angle X-ray diffraction (SXRD), Fourier transform infrared (FT-IR), elemental analysis, nuclear magnetic resonance (NMR), scanning and transmission electron microscopy (SEM and TEM). It was shown that the introduction of FIL influenced the catalytic behavior of PPL significantly by changing the structure and surface properties of the carriers.     

Identification of Antioxidant and Anti-α-amylase Components in Lotus (Nelumbo nucifera,Gaertn.) Seed Epicarp

Nano-sized Fe₃O₄ was synthesized by chemical co-precipitation and subsequently modified with 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde to introduce aldehyde group on its surface. With the help of “interface activation” by adding sucrose esters-11 as surfactant, lipase from Rhizopus oryzae was successfully immobilized onto the carrier with great enhancement of activity. The hydrolysis activity of immobilized enzyme were 9.16 times and 31.6 times of free enzyme when p-nitrophenol butyrate and p-nitrophenol palmitate were used as substrates. The thermo-stability of immobilized enzyme was also enhanced compared to free enzyme. The immobilized enzyme was successfully applied in synthesis of 1,3-diacyglycerols (1,3-DAG). The specific esterification activity of immobilized enzyme was about 1.5 times of the free enzyme. The immobilized enzyme showed good region-selectivity towards 1,3-diacyglycerols and retained nearly 80% of its activity after reused for 60 times, revealing a good industrial application prospect.

     

Immobilization of a lactase onto a magnetic support by covalent attachment to polyethyleneimine-glutaraldehyde-activated magnetite

A magnetic immobilized lactase has been prepared using magnetite as the magnetic material. Magnetite was functionalized by treatment with polyethyleneimine and crosslinked with glutaraldehyde. Lactase was then covalently coupled to the activated magnetic matrix via the aldehyde groups. The conditions for optimal immobilization of enzyme are described. Eighty percent of the lactase activity was lost on immobilization and is thought to be owing to the orientation of enzyme binding to the matrix. The amount of protein coupled was 80% of that applied. The maximum lactase activity retained on the matrix following immobilization was 360 U/g matrix. The immobilized lactase showed optimal activity at pH 4.5 and 65 degrees C. The immobilized lactase was more heat stable than the free enzyme, and retained 83% of its original activity after 14 d at 55 degrees C. Galactose competitively inhibited the immobilized lactase preparation (Ki 20 m/M). The presence of high initial concentrations of galactose (10% w/v) did not prevent total hydrolysis of lactose. Glucose and calcium ions were activators of the immobilized enzyme. The immobilized enzyme hydrolyzed high concentrations of lactose (up to 25% w/v) to completion within 4-6 h in a stirred batch reactor at 55 degrees C. There was no evidence of substrate inhibition at high substrate concentrations. The efficiency of hydrolysis of lactose by the immobilized lactase was better than that of the free enzyme. The magnetic immobilized lactase was demonstrated to be suitable for use in the enzymatic hydrolysis of both pure, and cheese whey permeate, lactose.