磁性纳米粒子的制备及脂肪酶的固定化

建立了以纳米级磁性粒子为载体固定化脂肪酶的方法,优化了脂肪酶的固定化条件,考察了固定化酶的性质. 制备的磁性载体平均粒径20 nm,具有超顺磁性,分散和再分散效果好. 固定化酶的最适吸附时间为60 min,酶用量:载体量为1:1,固定化酶的酶活达到718 U/g. 结果表明,经纳米磁性粒子固定化后,脂肪酶得到活化,固定化酶比活为游离酶的1.8倍. 同时,固定化脂肪酶的pH稳定性显著提高.     

黏土吸附法固定化脂肪酶的初步研究

为研究天然黏土为载体固定化脂肪酶的可行性,采用羟基化、硅烷化处理,对黏土进行改性,并以此为载体吸附固定化脂肪酶,探讨黏土固定化脂肪酶的条件对酶活及蛋白吸附量的影响,优化固定化脂肪酶条件。研究结果表明:黏土经羟基化、硅烷化改性处理后能显著提高固定化酶活和蛋白固定量,其中硅烷化改性最优;载体固定脂肪酶最优条件为:加酶量50 mg/g,载体粒径180—250μm,pH值为4.0,固定化温度25℃,固定化时间2.0 h;与游离酶相比,固定化酶显示出更广的pH值适应性。黏土固定化脂肪酶重复使用10批次后,仍能保留76.85%的初始活力。以天然黏土为载体固定化脂肪酶,具有较好的实际可应用性及操作稳定性,在较低pH值条件下应用具有一定优势。     

以壳聚糖-戊二醛为载体柔性固定假丝酵母脂肪酶candida rugosa lipase

以壳聚糖-戊二醛(Chitosan-GA)为载体固定假丝酵母脂肪酶candida rugosa lipase(CRL)最高酶活可达240μ·g~(-1)。Chitosan-GA固定化酶循环使用4次后保留了20%左右的初始酶活力。固定化酶在水相中保存185d后保留了40%以上初始酶活力。固定化酶在有机溶剂(正庚醇)中浸泡120h后固定化酶酶活保留32.2%,而游离酶只保留原始酶活的8.5%。Chitosan-GA载体固定化脂肪酶在水相和有机相中的保存稳定性都好于游离酶。     

大孔载体固定化脂肪酶

用自制大孔载体固定化脂肪酶,对固定化条件进行了优化,比较了固定化酶与游离酶的酶学参数. 结果表明,酶粉与载体质量比为1:1、固定化温度在20~25℃之间、固定化时间1.5 h的条件下,所得固定化酶的酶活最高. 固定化酶的最适pH为8.5,最适温度为40℃,其热稳定性、操作稳定性都比游离酶高,4℃下保存7 d后,酶活仍剩余94%.     

三维有序大孔硅材料载体固定化脂肪酶研究

以聚苯乙烯胶体晶体为模板制备三维有序大孔硅材料(3DOM-SiO_2),以其作为载体来固定脂肪酶。分别考察了脂肪酶加入量、反应体系pH、固定反应时间对固定化效果的影响。结果表明,3DOM-SiO_2材料固定脂肪酶的最佳酶液加入量为200 mL/g,固定化最适宜pH为7.0,最佳反应时间为5 h。固定化的脂肪酶在催化性能上与游离脂肪酶相比优势明显,最适宜反应温度提高到40℃左右,并且酶活随温度变化率低,热稳定性明显提高;脂肪酶固定化后对pH的敏感度降低,适应范围更宽,催化反应的最适pH为8.0;固定化脂肪酶重复使用8次后,相对酶活保持在62%。由此可见,3DOMSiO_2材料是固定脂肪酶的优良载体,在酶固定化领域应用前景广阔。     

假丝酵母产脂肪酶在壳聚糖和硅胶上的固定化研究

本实验分别壳聚糖和氨基化后的硅胶作为固定化载体,用戊二醛作为交联剂,将脂舫酶共价固定化于载体上,壳聚糖的最佳固定化条件是戊二醛浓度2．5％,给酶量为4125U／g,氨基化硅胶的最佳固定化条件为戊二醛浓度2％,最佳给酶量在3456U／g,固定化酶活为2952U／g,最佳酶活表现率为85％。两种方法所得的固定化酶的耐温性和储藏稳定性都有了显著提高。氨基化硅胶固定化酶在50℃下保温3小时后仍保留有14％的酶活。结果显示假丝酵母产脂肪酶固定于弱亲水性载体上要比固定于强亲水性载体上效果更好。     

琼脂包埋法固定化α-淀粉酶特性的研究

以琼脂作为载体材料,采用包埋法固定化α-淀粉酶,并对其特性进行了研究.结果表明,该固定化酶最适pH 值为7.5、最适温度为55～ 58℃,具有较好的贮存稳定性和反应稳定性,18 d后该固定化酶的残余活力仍保留原酶活的71.6%左右,重复使用7次,酶活力下降不大,其酶活回收率达到78.8%.     

树脂吸附法固定Candida rugosa脂肪酶

Candida rugosa脂肪酶具有优良的催化性能,对其进行固定化可以很方便地实现酶的回收和再利用。采用南开大学化工厂生产的4种阴离子交换树脂和4种大孔吸附树脂为载体,对来源于Candida rugosa的脂肪酶进行了吸附固定化,结果表明,以大孔吸附树脂AB-8为载体的固定酶比活性最高。固定化酶制备过程中缓冲液的最适宜pH值为7.2,最佳固定化时间为1 h,载体和酶的最佳质量配比为10∶1。与游离酶相比,固定化后酶活损失大约30%,但稳定性平均约提高60%。     

冰胶印迹聚合物固定化脂肪酶的制备及催化性能

利用冰冻凝胶(cryogel,简称冰胶)印迹聚合物实现了脂肪酶的固定化.在脂肪酶存在的条件下,以过硫酸铵/亚硫酸氢钠为引发剂,由丙烯酰胺、N,N-亚甲基双丙烯酰胺、丙烯酸、烯丙胺共聚而得到印迹聚合物固定化酶.通过催化三油酸甘油酯与甲醇的酯交换反应,发现冰胶固定化脂肪酶、常规凝胶固定化脂肪酶、游离脂肪酶具有相似的催化性能.冰胶固定化酶与相应的凝胶固定化酶显示出类似的稳定性,而传质方面则优于常规凝胶固定化酶,因此冰胶印迹聚合物固定化有望成为一种具有吸引力的酶固定化方法.     

溶胶-凝胶法固定化Candida sp.99-125脂肪酶

利用正硅酸甲酯(TMOS)和丙基三甲氧基硅烷(PTMS)为复合硅源,以PEG(MW=20000)为稳定剂,以HCl为催化剂,经过溶胶-凝胶过程包埋假丝酵母99-125脂肪酶. 研究得到最适的固定化条件为：PTMS与TMOS的摩尔比4: 1, R值(水与硅源的摩尔比)20, 给酶量(酶占硅源的质量百分数)3.71%, PEG与酶的质量比(1~1.5):1, 硅源水解时间35 min. 在该条件下,固定化脂肪酶的最高酯化活力是游离酶最高酯化活力的2.02倍. 固定化脂肪酶在100℃保温2 h后酶活仍维持为59.1%,固定化酶催化特定酯化反应,经过8批连续反应96 h后酶活维持不变.     

Synthesis of Core/Shell Magnetic Porous Microspheres for Lipase Immobilization

Magnetic porous hydrophobic microspheres were prepared by modified suspension copolymerization of methacrylate (MMA) and divinylbenzene (DVB) in the presence of oleic acid coated magnetite (Fe₃O₄), and the microspheres were used as biocarrier for the lipase immobilization. The results showed that the magnetic microspheres possessed spherical shape, core/shell structure, porous structure and high magnetic content, and the size and structure of magnetic microspheres had no significant changes after enzyme binding. The particle average size of microspheres was 66 μm, the magnetic content of microspheres was up to 31%, and the magnetization saturation values of the core/shell magnetic microspheres were measured at 300 K to be 11.02 emu g⁻¹. Lipase was immobilized on the magnetic porous carrier at up to 16.30 mg/g carrier. Activity and enantioselectivity of the immobilized lipase for the synthesis of R-HMPC acetate were investigated, indicating an interfacial activation of the enzyme after immobilization. Moreover, the pH dependency and operational stability of the immobilized lipase were studied, and they possess high stability and can be reused for ten cycles with loss 10% activity.     

磁性微粒对青霉素G酰化酶的固定化研究

将无机磁性粒子Fe3O4与有机材料海藻酸钠结合起来制成一种复合的磁性微粒,并将其进行表面修饰,通过化学共价法来固定青霉素G酰化酶。通过扫描电镜等对微粒进行形态学观察,并用傅立叶红外图谱表征微粒表面修饰基团。酶学性质研究表明,该微粒固定化的青霉素G酰化酶的最适pH值为7.5,最适温度为40℃。固定化酶与底物的亲和力有所降低,但是稳定性显著提高。重复催化研究结果表明,固定化酶具有比游离酶更广泛的温度及pH值适用范围,并且具有良好的热稳定性、可循环使用性和贮存稳定性。     

Stability of β‐galactosidase immobilized on composite microspheres of artemisia seed gum and chitosan

In this work, composite microspheres were prepared by using artemisia seed gum and chitosan as a source. The composite microspheres have activated aldehyde groups by using glutaraldehyde. β‐Galactosidase was covalently bound on these activated microspheres. The properties of the immobilized enzyme were investigated and compared with those of the free enzyme, for which o‐nitrophenol β‐D ‐galactopyranoside (ONPG) was chosen as a substrate. The results showed that the pH and thermal stability of the immobilized β‐galactosidase were higher than those of the soluble one. Apart from these, the Michaelis constant K_m was evaluated for the immobilized β‐galactosidase and the soluble enzyme. The immobilized β‐galactosidase exhibited better environmental adaptability and reusability than the soluble one. POLYM. COMPOS., 29:9–14, 2008. © 2007 Society of Plastics Engineers     

Immobilization of lipase on magnetic porous microspheres

开发了以磁性多孔微粒作为载体固定化脂肪酶的方法,进行了载体的FTIR、XRD、SEM、TEM、BET、TGA和VSM等测定与分析,考察了固定化时间、酶载量和缓冲液pH值等因素对固定化酶在有机相中催化烯丙醇酮转酯化反应性能的影响。结果表明,制备的磁性微粒是以Fe₃O₄为磁核,呈现多孔,比表面积12.16 m²/g,平均孔径为171.7 nm,磁铁含量38%并为超顺磁性;在酶与载体质量比为1∶1、pH值8.0及固定化时间6 h制得固定化酶的效果最佳,固定化酶的活力回收率可达240%。以其作为载体制备获得固定化酶操作稳定性得到显著提高,重复利用30批次后残余活力为74.5%,而游离酶7批次后仅为37.1%。     

Immobilization of lipase on poly(N-vinyl-2-pyrrolidone-co-2-hydroxyethyl methacrylate) hydrogel for the synthesis of butyl oleate

Lipase from Candida rugosa was immobilized by entrapment on poly(N-vinyl-2-pyrrolidone-co-2-hydroxyethyl methacrylate) [poly(VP-co-HEMA)] hydrogel, cross-linked with ethylene glycol dimethacrylate (EDMA). The immobilized enzyme was used in the esterification of oleic acid with butanol in hexane. The activities of the immobilized enzyme preparations and the leaching of the enzyme from the hydrogel supports with respect to composition were investigated. The thermal, solvent, and storage stability of the immobilized preparations also were determined. Increasing the percentage VP from 0 to 90, which corresponds to the increase in the hydrophilicity of the hydrogels, increased the activity of the immobilized enzyme. Lipase immobilized onto VP(%):HEMA(%), 90:10 hydrogel had the highest activity. Increasing the hydrophobicity of the hydrogel (increasing the percentage HEMA) seemed to decrease leaching of the enzyme from the support. Immobilized lipase on 100% HEMA hydrogel indicated highest entrapment and lowest leaching by hexane washing. The lipase immobilized on VP(%):HEMA(%), 50:50 hydrogel showed highest thermal, solvent, and storage stability compared to lipase immobilized on other hydrogel compositions as well as the native lipase.     

在PVDF-SiO2复合微球上的脂肪酶固定化

采用相转移法制备了包裹SiO2的聚偏氟乙烯(PVDF)复合微球,用其固定化假丝酵母脂肪酶(CRL). 结果表明,纯PVDF球、PVD-SiO2复合球用作酶固定化载体,对CRL的吸附量都较大. 随着PVDF-SiO2复合球中SiO2含量增多,其酶吸附量和活力回收率都有提高. PVDF与SiO2质量比为1:1的复合球的酶活力回收率最高,可达50.98%. 固定化CRL后,PVDF-SiO2复合球比SiO2更易实现固液分离,且对底物溶液无污染.     

Characterization and application of immobilized lipase enzyme on different radiation grafted polymeric films: Assessment of the immobilization process using spectroscopic analysis

Lipase has been immobilized onto different films, polypropylene and poly(tetrafluoroethylene‐perfluroro‐propyl vinyl ether) using glutalaradehyde as a crosslinker. Differential scanning calorimetery, Fourier transform infrared spectroscopic, x‐ray diffraction, and scanning electron microscopy measurements were carried out to confirm the structure of the polymer films as well as the immobilization process of the enzyme onto the polymeric carrier. The activity and stability of the resulting biopolymers produced by lipase have been compared to those for the native lipase. The experimental results showed that the optimum temperature and pH were 40°C and 8.0, respectively. The activity of the immobilized lipases varied with lipase concentration and with the yield of grafting. Subjecting the immobilized enzymes to a dose of γ‐radiation of (0.5–10 Mrad) showed complete loss in the activity of the free enzyme at a dose of 5 Mrad. A leakage of the enzyme from the irradiated membranes was not observed in the repeated batch enzyme reactions. The operational stability of the free and immobilized lipase in n‐hexane showed that the immobilized enzyme was much more stable than the free one. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 90: 155–167, 2003     

PREPARATION OF PVA/CHITOSAN LIPASE MEMBRANE REACTOR AND ITS APPLICATION TO SYNTHESIS OF MONOGLYCERIDE

PVA/ Chitosan (CS) composite membrane was studied in this paper, which could be used for enzyme processing of fat and oils. The parameters such as concentration of lipase, pH, and cross-linking agent as well as metal ions, which influence the immobilization of lipase in membrane, were optimized. The immobilized lipase was 0.66 u/cm2 and the recovery of immobilized lipase activity was 24%. The membrane reactor could be used to synthesize monoglyceride (MG) with many batches.     

Enzyme immobilization: Part 1. Modified bentonite as a new and efficient support for immobilization of Candida rugosa lipase

Immobilization of Candida rugosa lipase onto modified and unmodified bentonites is described. The effect of hydrophilic or hydrophobic nature of the support, the reuse efficiency, and kinetic behavior of immobilized lipase were studied. The modified bentonite with monolayer surfactant (BMS), was the best support, for immobilization. The activity of the immobilized enzyme was examined under varying experimental conditions. The effect of various factors such as concentration of enzyme solution, pH and temperature, stirring and various thermodynamic parameters were also evaluated. The activity of lipase on Na-bentonite, on BMS and on bentonite with bilayer surfactant (BBS) at the optimum pH was 7.2%, 56.6% and 3.6%, respectively. The adsorption isotherm was modelled by the Langmuir equation. The amounts of immobilized lipase on Na-bentonite, BMS and BBS at the highest activity were 42.6%, 61.2% and 28.3%, respectively. The effect of substrate concentration on enzymatic activity of the free and immobilized enzymes showed a good fit to the Michaelis–Menten plots. The immobilized enzyme exhibited an activity comparable to the free enzyme after storage at 30 °C. The thermal stability of free and immobilized lipase were also studied.     

α‐Amylase immobilized by Fe3O4/poly(styrene‐co‐maleic anhydride) magnetic composite microspheres: Preparation and characterization

Fe₃O₄/poly(styrene‐co‐maleic anhydride) core–shell composite microspheres, suitable for binding enzymes, were prepared using magnetite particles as seeds by copolymerization of styrene and maleic anhydride. The magnetite particles were encapsulated by polyethylene glycol, which improved the affinity between the magnetite particles and the monomers, thus showing that the size of the microspheres, the amount of the surface anhydrides, and the magnetite content in the composite are highly dependent on magnetite particles, comonomer ratio, and dispersion medium used in the polymerization. The composite microspheres, having 0.08–0.8 μm diameter and containing 100–800 μg magnetite/g microspheres and 0–18 mmol surface‐anhydride groups/g microsphere, were obtained. Free α‐amylase was immobilized on the microspheres containing reactive surface‐anhydride groups by covalent binding. The effects of immobilization on the properties of the immobilized α‐amylase [magnetic immobilized enzyme (MIE)] were studied. The activity of MIE and protein binding capacity reached 113,800 U and 544.3 mg/g dry microspheres, respectively. The activity recovery was 47.2%. The MIE had higher optimum temperature and pH compared with those of free α‐amylase and showed excellent thermal, storage, pH, and operational stability. Furthermore, it can be easily separated in a magnetic field and reused repeatedly. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 95: 328–335, 2005