含环氧基亲水性固定化青霉素酰化酶共聚载体的合成与性能研究

环氧基团可以在温和条件下与酶分子的氨基反应使其固定于载体表面.选用含有活性环氧基团的甲基丙烯酸缩水甘油酯(GMA)和亲水性的N-乙烯吡咯烷酮(NVP)两种单体,以N,N′-亚甲基双丙烯酰胺(MBAA)为交联剂,甲醇水溶液作致孔剂,液体石蜡为主介质,通过反相悬浮聚合技术成功地合成了亲水性大孔GMA-NVP-MBAA三元共聚物载体(GNM).通过调节交联剂的用量和单体NVP与GMA的比例,可以调节载体的孔径、比表面积及在水中的溶胀性能.将巨大芽孢杆菌青霉素酰化酶共价偶联于平均孔径为16.5nm、表面环氧基含量为0.906mmol/g的GNM共聚物载体,制成固定化酰化酶,其表观活性高达625U/g,水解青霉素G钾盐的最适宜温度为50℃,pH值为8.0.固定化酶在4℃保存40d,活性保持不变.经3次使用后,活性达到稳定值(601U/g左右),再经12次使用,活性几乎保持不变.     

青霉素G酰化酶在含环氧基团的顺磁性聚合物微球上的固定化（英文）

青霉素G酰化酶(PGA)是一种重要的工业生物催化剂,常用于以青霉素G为底物生产7-氨基去乙酰氧基头孢烷酸(7-ADCA)和6-氨基青霉烷酸(6-APA)等半合成β-内酰胺类抗生素.然而,PGA较差的稳定性和可重复使用性能限制了其在工业上的广泛应用.因此,将PGA固定在固体载体上是很有必要的,可以形成一种可重复使用的高性能的多相催化剂.用于生物酶固定化的良好载体应具备以下条件:(1)载体表面具有可用于与生物酶多点结合的高密度的官能团;(2)载体具有较大的比表面积以固定更多的生物酶.通常情况下,可以通过减小载体的粒径来增加其比表面积,然而,小粒径的载体很难从反应混合液中分离出来,造成固定化酶回收使用困难.为了将聚合物微球的优异固定化性能与磁性纳米粒子的独特顺磁性结合起来,我们制备了一种含环氧基团的顺磁性聚合物微球作为PGA的固定化载体.但由于Fe_3O_4纳米颗粒具有较高的表面能,在反相悬浮聚合反应过程中容易团聚成大颗粒,从而导致制备的顺磁性聚合物微球的磁体含量、表面形貌和粒径分布存在差异.此外,Fe_3O_4纳米颗粒与聚合反应单体之间的相容性不好,使得部分磁性颗粒不能很好地包埋于聚合物微球内部,影响固定化酶的活性和操作稳定性.本文以N,N′–亚甲基双丙烯酰胺为交联剂,以甲基丙烯酸缩水甘油酯和烯丙基缩水甘油醚为功能性单体,用反相悬浮聚合方法在SiO_2包覆的Fe_3O_4纳米颗粒表面成功制备出含环氧基团的顺磁性聚合物微球.用SEM,FT-IR,XRD,VSM和低温氮气吸附等手段对含环氧基团的顺磁性聚合物微球进行了表征.研究了SiO_2对Fe_3O_4纳米颗粒的包覆和Fe_3O_4/SiO_2纳米颗粒的数量对于固定化酶的初始活性和操作稳定性的影响.SiO_2在反相悬浮聚合过程中发挥重要作用,用SiO_2对Fe_3O_4纳米颗粒进行亲水性改性,有效改善了Fe_3O_4纳米颗粒与聚合反应单体的相容性,将其引入反相悬浮聚合体系中,可以制备得到球形度好、粒径分布均匀和超顺磁性的含环氧基团的顺磁性聚合物微球,其中当Fe_3O_4/SiO_2纳米颗粒的质量比为7.5%时制备的含环氧基团的顺磁性聚合物微球具有最好的PGA固定化性能.PGA通过其活性非必需侧链基团–氨基与顺磁性聚合物微球表面的环氧基团的共价结合来制备顺磁性固定化酶,该固定化PGA的初始活性为430 U/g(wet),在外加磁场的作用下容易回收使用,重复使用10次后可保留99%的初始活性,具有良好的热稳定性和酸碱稳定性,具有较好的工业应用前景.     

磁性聚合物微球固定化青霉素G酰化酶制备光学纯(S)-2-氯苯甘氨酸

通过反相悬浮聚合法制备了超顺磁性环氧聚合物微球用于固定化青霉素G酰化酶,利用磁性固定化酶催化N-苯乙酰-(R,S)-2-氯苯甘氨酸进行不对称水解反应,制备出(S)-2-氯苯甘氨酸单一对映体。磁性固定化酶催化水解反应的适宜条件为:底物浓度100 mg·m L-1,反应温度和时间30℃和12 h,反应溶液p H 8.0。在此条件下,N-苯乙酰-(R,S)-2-氯苯甘氨酸的转化率为48.8%,产物(S)-2-氯苯甘氨酸的对映体过量值eep达99.4%。磁场下回收磁性固定化青霉素G酰化酶,重复使用6次,底物的转化率和产物的对映体过量值分别为47.8%和91.4%。     

亲水性交联聚合物载体的合成及其固定化青霉素酰化酶

选用含环氧基团的甲基丙烯酸缩水甘油酯(GMA)和亲水性的N-乙烯吡咯烷酮(NVP)单体,以N,N′-亚甲基双丙烯酰胺(MBAA)为交联剂,甲酰胺作致孔剂,通过反相悬浮聚合技术成功合成了一系列大孔、珠状GMA-NVP-MBAA三元共聚物载体.N-乙烯吡咯烷酮介入共聚物体系,使共聚物载体具有较强的亲水性,有利于青霉素酰化酶的固定化.通过调节交联剂的用量和单体NVP与GMA的比例,可以调节共聚物载体的孔结构与表面性能.用合成的平均孔径为15.7nm、表面环氧基含量1.11mmol·g-1亲水性珠状载体固定青霉素酰化酶,固定化酶水解青霉素G钾盐的活性达491U·g-1;在4℃保存30d,活性保持不变.经4次使用后活性达到稳定(444U·g-1),再经14次使用后,活性没有明显变化.     

亲水性含环氧基磁性聚合物微球的制备与性能表征

选择甲酰胺作磁性Fe3O4微晶的分散剂,通过设计反相悬浮聚合体系,合成了粒径分布窄、球状亲水性含环氧基磁性聚合物(MGM).利用扫描电子显微镜(SEM)、红外光谱(FT-IR)、X射线粉末衍射仪(XRD)、振动样品磁强计(VSM)和低温N2吸附以及化学分析方法对聚合物进行了性能表征.结果表明,合成的MGM呈球形,且粒度分布较窄,粒径为0.13~0.28 mm的粒子占91%;甲酰胺分散Fe3O4,微晶表面的亲水性进一步增强,单体甲基丙烯酸缩水甘油酯和N,N′-亚甲基双丙烯酰胺交联共聚生成的胶粒能够包埋Fe3O4微晶形成胶核,胶核聚集形成均匀、稳定的MGM微球.MGM中Fe3O4含量为6.17%时,比饱和磁化强度σs达6.5 emu/g;其比表面积、平均孔径和孔容分别为117.6 m2/g,15.6 nm和0.46 cm3/g,表面环氧基团含量为0.53 mmol/g.MGM借助自身的活性环氧基团在十分温和的条件下共价偶联青霉素酰化酶(penicillin G acylase EC 3.5.1.11,简称PGA),制备的固定化酶在37℃下催化水解青霉素G钾生成6-氨基青霉烷酸(6-APA)的表观活性达502IU/g,并且在使用过程中没有出现磁聚集现象.     

聚丙烯酸载体用于青霉素酰化酶的固定

以反应性单体丙烯酸和交联剂二乙烯基苯,以石油醚为致孔剂,通过悬浮聚合制备固定化酶的载体,并用于对青霉素酰化酶的固定。研究了丙烯酸与二乙烯基苯以不同摩尔比对青霉素酰化酶固定活性的影响,以及悬浮聚合时水油相比例的不同所合成的载体对固定化酶性能的影响。当丙烯酸和二乙烯基苯摩尔比为84.2:4时合成的载体固定青霉素酰化酶的酶活为2784U/g,而水油相比为2.75:1（丙烯酸和二乙烯基苯摩洋比为84.2:5）时固定青霉素酰化酶活达到2183U/g。固定青霉素酰化酶可使青霉素转化,得到半合成青霉素的中间体6-氨基青霉烷酸,由此可制成高效、广谱、服用方便的新青霉素。     

新型疏水性酶载体的合成及其性能研究

研究了聚丙烯酸酯类疏水性固定化酶载体(包括环氧基和胺基)的合成,旨在提高载体固载酶的性能。结果表明,所得载体的孔结构适合固定化青霉素酰化酶、D-氨基酸氧化酶、DL-7-ACA酰化酶、头孢菌素C酰化酶等。固定化D-氨基酸氧化酶的酶活与载体的含水量(与其孔结构相关)有密切的关系;载体的突出优点是固载酶时,酶的固定化效率可高达50%。     

含环碳酸酯基新型聚合物载体的合成及固定化葡萄糖淀粉酶研究

碳酸亚乙烯酯(VCA)分子中的反应性环碳酸酯基可于温和条件下与氨基发生反应形成稳定的氨基甲酸酯.利用这一性质,将含-NH₂基团的酶分子直接以σ键的形式固定于含有环碳酸酯基的聚合物载体上.本文通过反相悬浮聚合,以液体石蜡为介质,VCA为反应性单体,甲基丙烯酸-β-羟乙酯(HEMA)及丙烯酸羟丙酯(HPA)为亲水性共聚单体,合成出一系列交联树脂聚合物.以此聚合物为载体对葡萄糖淀粉酶进行固载实验,表现出良好的固定化性能.同时,固定化酶的稳定性也有所提高.     

介孔材料的修饰及固定青霉素酰化酶的稳定性研究

利用扩孔剂的作用合成出较大孔径(12 nm)的介孔材料SBA-15, 并进行表面氨基修饰, 以此为载体, 以戊二醛为交联剂, 对青霉素酰化酶进行组装固定, 并对固定化青霉素酰化酶(PGA)的稳定性进行了深入的研究. 实验结果表明, PGA与载体交联后仍保持活性. 热稳定性研究结果表明, 制备的固定化青霉素酰化酶在低于60 ℃时保持稳定; pH在6~11范围内保持稳定; 固定化酶重复使用10次之后, 仍具有高达90%的残留活力.     

原子转移自由基聚合制备聚甲基丙烯酸(N-羟基琥珀酰亚胺)酯及其嵌段共聚物

以CuCl/N-苄基-2-吡啶基甲亚胺(NBPM)/2-溴异丁酸乙酯(EBrIB)作为引发催化体系,使甲基丙烯酸(N-羟基琥珀酰亚胺)酯(MASI)进行ATRP聚合,得到的聚甲基丙烯酸(N-羟基琥珀酰亚胺)酯(PMASI)具有较高的单体转化率(90%)、较窄PDI(~1.10)和较高的分子量。在整个聚合过程中,较强的C—Cl键仍使聚合物的端基保持活性,有利于与第二单体甲基丙烯酸(N,N-二甲氨基)乙酯(DMAEMA)嵌段共聚形成结构明确的嵌段共聚物P(MASI-b-DMAEMA)。当MASI的链长较短时,P(DMAEMA40-b-MASI16)具有水溶性并可自组装成直径均匀的核-壳型微胶束,间接证明了聚合过程的可控特征。     

Immobilization of penicillin G acylase on poly[(glycidyl methacrylate)-co-(glycerol monomethacrylate)]-grafted magnetic microspheres

Poly[(glycidyl methacrylate)-co-(glycerol monomethacrylate)]-grafted magnetic microspheres were prepared by graft random copolymerization via ATRP from polymer microspheres with dispersed Fe(3)O(4) nanoparticles. Penicillin G acylase (PGA) was immobilized onto the polymer brush-grafted magnetic microspheres. The immobilized PGA prepared with initial glycidyl methacrylate/glycerol monomethacrylate ratios of 40/60 to 60/40 possessed higher catalytic activity than that prepared with higher proportions of glycidyl methacrylate in the initial monomer mixture. The immobilized PGA showed high thermal stability and enhanced tolerability to the pH variance.     

Covalent immobilization of antibody fragments on well-defined polymer brushes via site-directed method

Well-defined polymer brushes and block copolymer brushes consisting of 2-methacryloyloxyethyl phosphorylcholine (MPC) and glycidyl methacrylate (GMA) were prepared by surface-initiated atom transfer radical polymerization (ATRP). The polymer brushes were used for the immobilization of antibody fragments in a defined orientation. Pyridyl disulfide moieties were introduced to the polymer brushes via a reaction of epoxy groups in GMA units. Fab’ fragments were then immobilized onto these surfaces via a thiol-disulfide interchange reaction and the reactivity of antibodies with antigens was investigated. Antigen/antibody binding on the polymer brushes was more preferable than that on epoxysilane films as a control surface. Furthermore, the activity of the antibodies immobilized on the block copolymer brushes having biocompatible PMPC was greater than that on other surfaces that did not have PMPC in their structures.     

介孔材料MCFs的合成及组装青霉素酰化酶的性质研究

介孔材料由于具有纳米级规则孔道和巨大的比表面积而在催化、吸附及分离等方面存在较大的应用价值．近年来,由介孔分子筛如MCM-41和SBA-15州等组装功能性材料已成为研究的热点．酶作为高效催化剂有许多优点,但在溶液中易失活,使用后无法回收,有的酶在溶液中还存在自水解问题：将酶组装在介孔材料中制成固定化酶则可解决上述问题．目前已成功地将辣根过氧化物酶     

Preparation of magnetic microspheres with thiol-containing polymer brushes and immobilization of gold nanoparticles in the brush layer

Narrow-disperse magnetic microspheres were prepared by alkaline coprecipitation of Fe²⁺ and Fe³⁺ ions within poly(acrylic acid–divinylbenzene) microspheres that were prepared by distillation–precipitation copolymerization. Magnetic microspheres with polymer brushes that contain epoxy groups were prepared by graft copolymerization of glycidyl methacrylate and glycerol monomethacrylate via atom transfer radical polymerization (ATRP) from the magnetic microsphere surfaces. Subsequently, magnetic microspheres with thiol-containing polymer brushes were prepared by treating the epoxy group-containing magnetic microspheres with sodium hydrosulfide. Gold nanoparticles were immobilized in the brush layer of the thiol-containing magnetic microspheres through Au–S coordination. The catalytic activity of the gold nanoparticle-immobilized magnetic microspheres was investigated using the reduction of 4-nitrophenol to 4-aminophenol with sodium borohydride as a model reaction. The catalyst could be reused for over 10 cycles without noticeable loss of catalytic activity.     

Evaluation of a monolithic epoxy silica support for penicillin G acylase immobilization

In this work, a new type of penicillin G acylase (PGA)-based monolithic silica support was developed and evaluated for the chiral separation in HPLC. The preparation procedure consisted of two steps: preparation of an epoxy derivatized monolithic silica column and chemical modification of the epoxide groups with the enzyme chiral selector. The epoxy Silica-Rod column for the immobilization of PGA was prepared with the in situ modification process by using epoxy-silanes and the identification of the species bound to the surface was achieved by solid-state nuclear magnetic resonance. The enzyme was covalently immobilized to the surface of the derivatized monolithic column. The enantioselectivity and the performance of the developed column are discussed and compared to the corresponding experimental data obtained with a PGA-based microparticulate (5 microm) silica column.     

不同介孔材料固定青霉素酰化酶的稳定性研究

介孔材料由于具有在2～30nm之间可调的纳米级规则孔道、大比表面积和强吸附性能而成为固定化酶的优良载体．将酶固定于介孔材料的孔道中制备成的固定化酶与溶液酶相比,有易于与产物分离,并可回收和反复使用,可降低生产成本,减少酶的自水解和保持酶的活性．青霉素酰化酶（Penicillin acylase,PGA,EC．3．5．1．11）又称为青霉素酰胺酶或青霉素氨基水解酶,该酶属于球蛋白,分子量较大,由2个亚基组成：分子量为19500的含有侧链结合位点的亚基和分子量为60000的含有催化位点的亚基．     

Beaded reactive polymers 3 effect of triacrylates as crosslinkers on the physical properties of glycidyl methacrylate copolymers and immobilization of penicillin G acylase

Various glycidyl methacrylate (GMA) copolymers were synthesized by suspension polymerization, using pentaerythritol triacrylate (PETA), trimethylolpropane triacrylate (TMPTA), and trimethylolpropane trimethacrylate (TRIM) as crosslinking comonomers. These copolymers were evaluated for the immobilization of penicillin G acylase. Broad pore-size distribution that was observed was in the range 5-300 nm. Both surface area and pore volume increased with increase in the mole fraction of crosslinking comonomer (increasing crosslink density). The pore volume of the copolymers was more than doubled by including lauryl alcohol as porogen. Binding of penicillin G acylase (PGA) was quantitative on highly crosslinked copolymers. The expression of bound PGA was better on the relatively more hydrophilic GMA-TMPTA and GMA-PETA copolymer supports compared to the GMA-TRIM copolymers. Among the different copolymers studied, GMA-TMPTA copolymer 7411 exhibited highest activity of immobilized penicillin G acylase (167.4 IU/g) with 35.1% expression.     

Surface-initiated Atom-transfer Radical Polymerization from Polyimide Films and Their Anti-fouling Properties

A simple one-step method for the chloromethylation of polyimide (PI) under mild conditions was used to introduce benzyl chloride groups into PI film surface. Covalently tethered hydrophilic polymer brushes of poly(ethylene glycol) monomethacrylate (PEGMA) and glycidyl methacrylate (GMA) were prepared via surface initiated atom-transfer radical polymerization (ATRP) from the chloromethylated PI surfaces using benzyl chloride groups as the active ATRP initiators. A kinetics study indicated that the chain growth from the films was in agreement with a controlled process. The dormant chain ends of the grafted polymer on the PI films could reinitiate the consecutive surface-initiated ATRP to prepare surface-functionalized diblock copolymer brushes on the PI films. The modified surface was characterized by X-ray photoelectron spectroscopy (XPS) after each modification stage. Protein adsorption experiments indicated that the PI-P(PEGMA) membrane exhibited substantially improved anti-fouling properties compared to the pristine PI surface.     

Synthesis of random polyampholyte brushes by atom transfer radical polymerization

We report the synthesis of random polyampholyte brushes containing 2‐(dimethylamino)ethyl methacrylate (DMAEMA) and methacrylic acid (MAA). The preparation of polyampholyte brushes is performed by the “grafting from” strategy using surface‐initiated atom transfer radical polymerization (ATRP). The first step consists in the formation of the self‐assembled monolayer of the ATRP initiator. Secondly, the chains are grown from the surface by controlled/“living” radical polymerization. The random copolymer brushes and the corresponding homopolymers brushes containing 2‐(dimethylamino)ethyl methacrylate and tert‐butyl methacrylate (^tBuMA) are prepared. The last step is the deprotection of the ^tBuMA form to the MAA segment by in situ hydrolysis reaction. The annealed DMAEMA group can also be converted to the quenched form by in situ quaternization reaction. This results in the formation of “annealed” and “semiannealed” polyampholyte brushes. The “annealed” polyampholyte corresponds to the random copolymer that contains only annealed units, weak acid and weak base. The “semiannealed” polyampholyte consists of the mixture of annealed (weak acid) and quenched (quaternized segment) units. Polyampholyte brushes with various grafting densities are synthesized and carefully characterized using surface techniques such as ellipsometry and FTIR‐ATR. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 4305–4319, 2008