A robust affinity chromatography system based on ceramic monoliths coated with poly(amino acid)‐based polymeric constructs

Traditional chromatographic separation systems are disadvantaged by low flow rates, a high pressure drop across the column, low capacity and poor reusability. Searching for more efficient separation systems we introduced the use of a ceramic monolith as robust support in bioseparations. A coating consisting of l ‐asparagine as ligand, poly(l ‐lysine) as spacer arm and a commercial poly(ethylene acrylic acid) film forming copolymer network (Michem 4983‐40R) was developed as a coating for these ceramic monoliths. Poly(l ‐lysine) was synthesized by ring‐opening polymerization of ε‐trifluoroacetyl‐l ‐lysine N‐carboxyanhydride and coupled to a commercial film‐forming poly(ethylene acrylic acid) network. This construct was then ‘decorated’ with l ‐asparagine via the terminal amino functional groups of poly(L‐lysine) and coated onto the ceramic monolith to selectively bind l ‐asparaginase. Adsorption/elution experiments showed reversible binding between l ‐asparagine and l ‐asparaginase, and the subsequent release of l ‐asparaginase, and between 83% and 94% of the active enzyme was recovered by elution with d ‐asparagine and NaCl solutions. The functional activity of the eluted l ‐asparaginase was verified by a Nessler's assay. While traditional separation processes (adsorption and elution) using gel bead packings take many hours, the ceramic monolith system achieves the same of level of separation in about 1 h. This new system served as a proof of concept for its application in protein separation and purification. This work paves the way to a better understanding of the use of ceramic monoliths as stationary phase coated with a stable polymer construct for more robust and efficient supports in affinity chromatography. © 2020 Society of Industrial Chemistry     

Prediction and comparison of textural properties of magnetic copolymer supports for enzyme immobilization

Magnetic polymers supports have proven to be valuable materials for enzyme immobilization, as they allow recovering the catalyst by magnetic separation, precluding the need for costly and time-consuming separation steps. In this study, magnetic copolymer supports were synthesized using styrene (STY) and different crosslinking agents (divinylbenzene, ethylene glycol dimethacrylate, or triethylene glycol dimethacrylate) and initiators (azobisisobutyronitrile or benzoyl peroxide) and used to immobilize Candida antarctica lipase B (CALB). The aim was to obtain biocatalysts with high enzymatic activity and satisfactory morphological properties for use in biotransformation reactions. Two morphological properties known to influence the immobilization yield were taken into consideration, specific surface area, and swelling index. Experimental data were compared to the predictions of a model based on molar balance, method of moments, numerical fractionation, and elementary gel structures. The high correlation (R² = 0.9974) between experimental and predicted values demonstrated the suitability of the model for estimating the textural properties of enzyme supports. CALB was successfully immobilized, showing high hydrolytic activity (500–700 U g⁻¹) and good thermal stability at 50°C. CALB/STY-EGDMA-M was 14 times more stable than free CALB. The results confirm the efficiency of the immobilization method and the suitability of the copolymers for enzyme immobilization.     

葡聚糖磁性微球在生物医学领域的应用

葡聚糖磁性微球作为一种新型功能材料,在生物医学、细胞学和生物工程学等领域有着广泛的应用前景.着重介绍了葡聚糖磁性微球在固定化酶、靶向药物、细胞分离与免疫分析、临床诊断和治疗等领域的应用,并展望了今后的研究方向.     

Synthesis and Characterization of Asparaginase Bound Silver Nanocomposite Against Ovarian Cancer Cell Line A2780 and Lung Cancer Cell Line A549

Development of new strategies of drug delivery is essential for effective treatment of cancer. In the present work, nanobiocomposite of fungal asparaginase was produced by immobilizing with silver nanoparticles. Asparaginase bound silver nanoparticle has shown higher enzyme activity than crude asparaginase. The primary and secondary amine/amide functional groups were found responsible for binding of asparaginase to silver nanoparticles. The silver nanobiocomposite of asparaginase was found to have smooth surface and crystalline in nature. The size of the nanobiocomposites ranged from 60 to 80 nm. The cytotoxicity of silver nanobiocomposite of asparaginase was found to be higher than free asparaginase on ovarian cancer cell line. The silver nanobiocomposite of asparaginase showed better cytotoxicity against ovarian cancer cell line A2780 than lung cancer cell line A549. Thus the synthesized silver nanobiocomposite of asparaginase can be used as an effective anticancer agent against lung cancer.     

Synthesis and characterization of PMMA composites activated with starch for immobilization of L‐asparaginase

Poly (methyl methacrylate) (PMMA)–starch composites were prepared by emulsion polymerization technique for L‐asparaginase (L‐ASNase) immobilization as highly activated support. The hydroxide groups on the prepared composites offer a very simple, mild and firm combination for enzyme immobilization. The pure PMMA and PMMA‐starch composites were characterized as structural, thermal and morphological. PMMA‐starch composites were found to have better thermal stability and more hydrophilic character than pure PMMA. L‐ASNase was immobilized onto PMMA‐starch composites contained the different ratio of starch (1, 3, 5, and 10 wt %). Immobilized L‐ASNase showed better performance as compared to the native enzyme in terms of thermal stability and pH. K_m value of immobilized enzyme decreased approximately eightfold compared with the native enzyme. In addition to, immobilized L‐ASNase was found to retain 60% of activity after 1‐month storage period at 4 °C. Therefore, PMMA‐starch composites can be provided more advantageous in terms of enzymatic affinity, thermal, pH and storage stability as L‐ASNase immobilization matrix. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43421.     

大肠杆菌L- 天冬酰胺酶的分离纯化及其特性

目的 建立简单有效的L- 天冬酰胺酶分离纯化工艺，研究L-天冬酰胺酶的特性。方法 采用冷丙酮破壁处理，经稀碱液抽提、离子交换层析和亲和层析进行纯化。通过温度试验、pH试验、SDS－PAGE图谱、紫外吸收光谱等研究酶的特性。结果 建立了简单高效的纯化工艺，可使L-天冬酰胺酶的纯度达94．4％，比活为520u/mg蛋白，总收率为65．7％。L-天冬酰胺酶的相对分子质量分别为143900和166700，均为有抗癌活性的EC－2组分，在270nm有最大吸收。最适作用温度为40－50℃，最适作用州为8.0-9.0在40℃以下能保持稳定。结论建立了大肠杆菌L-天冬酰胺酶的分离纯化工艺，并研究了L-天冬酰胺酶的酶学特性。     

磁性微球在生物医学领域的最新进展

磁性微球作为一种新型功能材料,在生物医学工程等领域有着广泛的应用前景。首先介绍磁性微球的几种制备方法,包括喷雾干燥、热处理等物理方法,以及乳液聚合、自组装等化学方法。同时着重阐述了磁性微球在固定化酶、靶向药物、细胞分离、诊断等生物医学领域的应用。     

Immobilization of Pycnoporus sanguineus laccase by metal affinity adsorption on magnetic chelator particles

BACKGROUND: Immobilized enzymes provide many advantages over free enzymes including repeated or continuous reuse, easy separation of the product from reaction media, easy recovery of the enzyme, and improvement in enzyme stability. In order to improve catalytic activity of laccase and increase its industrial application, there is great interest in developing novel technologies on laccase immobilization. RESULTS: Magnetic Cu²⁺‐chelated particles, prepared by cerium‐initiated graft polymerization of tentacle‐type polymer chains with iminodiacetic acid (IDA) as chelating ligand, were employed for Pycnoporus sanguineus laccase immobilization. The particles showed an obvious high adsorption capacity of laccase (94.1 mg g^?1 support) with an activity recovery of 68.0% after immobilization. The laccase exhibited improved stability in reaction conditions over a broad temperature range between 45 °C and 70 °C and an optimal pH value of 3.0 after being adsorbed on the magnetic metal‐chelated particles. The value of the Michaelis constant (K_m) of the immobilized laccase (1.597 mmol L^?1) was higher than that of the free one (0.761 mmol L^?1), whereas the maximum velocity (V_max) was lower for the adsorbed laccase. Storage stability and temperature endurance of the immobilized laccase were found to increase greatly, and the immobilized laccase retained 87.8% of its initial activity after 10 successive batch reactions. CONCLUSION: The immobilized laccase not only can be operated magnetically, but also exhibits remarkably improved catalytic capacity and stability properties for various parameters, such as pH, temperature, reuse, and storage time, which can provide economic advantages for large‐scale biotechnological applications of laccase. Copyright © 2007 Society of Chemical Industry     

Surface analysis of PP/EVA blends modified with amino acids for biomedical applications

The copolymers poly(propylene-co-ethylene) (PP/E) and poly(ethylene-co-vinyl acetate) (EVA) and blends of these were modified to develop an artificial matrix which promotes the growth of endothelial cells. Covalent immobilization of amino acids or sequences of adhesion glycoproteins should trigger the formation of an endothelial cell monolayer onto the polymeric surface. Reactive functional groups were generated by saponifying the ester groups of the EVA component. Esterification with oxalylic or malonic dichlorides in the gas phase yielded the required monoesters and gave the best results for further immobilization of amino acids, while reaction with α,ω-dicarboxylic acid dichlorides in solution led to diester formation. Subsequently, various protected amino acids were immobilized via the carbodiimide method. Surface analytical methods like infrared spectroscopy using at tenuated total reflection (IR-ATR), X-ray photoelectron spectroscopy (XPS), and secondary ion mass spectrometry (SIMS) were used to prove the modification steps. The analytical results confirmed covalent side-chain generation in the upper surface region. © 1995 John Wiley & Sons, Inc.     

Immobilization of glucoamylase on magnetic poly(styrene) particles

Magnetic poly(styrene) particles including active groups were prepared for enzyme immobilization without any activation process. Glucoamylase, which is widely used in industry, was immobilized onto these particles. The effects of pH, buffer concentration, and temperature on immobilization were investigated; moreover, the effect of immobilization temperature on immobilized glucoamylase activity was determined for the hydrolysis of maltose. The acetate buffer with the concentration of 6 × 10⁻⁴ M at pH 4 and 20–30°C was found as the most suitable medium for the immobilization of the glucoamylase. The amount of bound protein is 8 mg/g particle with the immobilization yield of 70%. The maximum activity obtained with immobilized glucoamylase is approximately 70% of the free one. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 72: 69–73, 1999     

羧基修饰的超顺磁纳米粒子直接固定化罗伊氏乳杆菌

利用共沉淀法结合高锰酸钾氧化制备所得表面羧基修饰的超顺磁性纳米粒子吸附于罗伊氏乳酸杆菌表面,在磁场协助下实现细胞的固定化。吸附机理分析表明小尺寸相互作用和静电相互作用是磁性纳米粒子与细胞之间的主要作用。分别考察了菌体/磁性粒子质量比、pH、温度、时间等对固定化罗伊氏乳酸杆菌的影响,确定最佳固定条件为菌体与磁性纳米粒子相对质量比为2.25,在pH=3、温度25℃的条件下固定化0.5 h,可实现91%的细胞固定化。最后,对固定化后的细胞进行再培养,与游离细胞相比,两者表现出类似的代谢特征,证实细胞经固定化后仍具有活性。因此,羧基修饰的超顺磁性纳米粒子可成功用于细胞固定化,在不影响细胞活性的情况下,通过磁分离实现细胞的重复利用。     

生物提取用磁性微球的制备及其性能

磁性微球因其自身兼具高分子微球和磁性粒子的众多特性,广泛应用在细胞分离、固定化酶、靶向药物、分析检测、免疫测定等领域。本文通过化学共沉淀法制备了Fe3O4纳米粒子,并采用分散聚合法对其包覆,得到Fe3O4/SiO2磁性微球。研究结果表明,通过上述方法制备的磁性微球可以快速、有效的提取生物中的RNA,而且最佳的聚合反应温度为80℃。     

离子液体修饰超顺磁性纳米颗粒用于青霉素G酰化酶固定化(英文)

Functionalized ionic liquids containing ethyoxyl groups were synthesized and immobilized on magnetic silica nanoparticles(MSNP) prepared by two steps,i.e.,Fe3O4 synthesis and silica shell growth on the surface.This magnetic nanoparticle supported ionic liquid(MNP-IL) were applied in the immobilization of penicillin G acylase(PGA).The MSNPs and MNP-ILs were characterized by the means of Fourier transform infrared spectroscopy(FTIR),scanning electron microscopy(SEM),transmission electron microscopy(TEM),and vibrating sample magnetometer(VSM).The results showed that the average size of magnetic Fe3O4 nanoparticles and MSNPs were ~10 and ~90 nm,respectively.The saturation magnetizations of magnetic Fe3O4 nanoparticles and MNP-ILs were 63.7 and 26.9 A?m2?kg?1,respectively.The MNP-IL was successfully applied in the immobilization of PGA.The maximum amount of loaded enzyme was about 209 mg?g?1(based on carrier),and the highest enzyme activity of immobilized PGA(based on ImPGA) was 261 U?g?1.Both the amount of loaded enzyme and the activity of ImPGA are at the same level of or higher than that in previous reports.After 10 consecutive operations,ImPGA still main-tained 62% of its initial activity,indicating the good recovery property of ImPGA activity.The ionic liquid modified magnetic particles integrate the magnetic properties of Fe3O4 and the structure-tunable properties of ionic liquids,and have extensive potential uses in protein immobilization and magnetic bioseparation.This work may open up a novel strategy to immobilize proteins by ionic liquids.     

Preparation and application of magnetic microsphere carriers

Magnetic microsphere carriers have received considerable attention, primarily because of their wide applications in the fields of biomedicine and bioengineering. In this paper, preparation methods, surface modification and application of magnetic carriers are reviewed. Emphasis will be placed on recent biological and biomedical developments and trends such as enzyme immobilization, cell isolation, protein purification, target drugs and DNA separation.     

Glycosylated membranes: A promising biomimetic material

Development in the area of glycosylated membranes has been actively pursued in the past few years. This kind of promising biomimetic material is inspired by cell membranes. The recent surge of interest in these glycosylated membranes stems from their widespread number of applications to many areas in science and technology. With the glycosylation strategy, membrane separation properties, such as flux and antifouling, are greatly improved. Moreover, the ability to modulate biocompatibility, protein recognition, separation of biomolecules, enzyme immobilization, cell culture, and microorganisms capture are important in a variety of biological and medical applications. This review focuses on the recent progress in the preparation of these glycosylated membranes and highlights their applications. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 39658.     

油品固定化细胞脱硫研究进展

发展固定化技术实现油品的生物深度脱硫,可以提高生物催化剂的稳定性,简化油水分离工艺和生物催化剂的回收工艺,降低生物催化剂的生产成本,对促进油品生物深度脱硫技术的产业化发展具有重要意义。本文综述了固定化细胞油品生物脱硫方法及固定化反应器研究进展,指出了目前存在的问题及可能的解决方案,并对油品生物脱硫技术今后的发展方向进行了展望。     

Immobilization of β‐galactosidase from Escherichia coli onto modified natural silk fibers

A polymeric support based on the natural silk fibers was prepared and characterized for covalent immobilization of β‐galactosidase from Escherichia coli. The silk fibers were grafted using polyacrylonitrile in presence of benzophenone as a photo‐initiator. The grafted fibers were then activated by treatment with hydrazine hydrate followed by glyoxal cross‐linker. FTIR spectra, scanning electron microscope (SEM) in addition to the staining test derived from the Coomassie protein assay were utilized for investigation of the modification and immobilization steps. Also, the activity of both free and immobilized β‐galactosidase was evaluated as a function of the various important parameters like grafting percentage, pH, and temperature. In addition, the kinetic parameters K_m and v_max for both free and immobilized enzyme were anticipated using Michaelis–Menten equation. The results in this study indicated that the prepared modified woven silk fibers could be used effectively as a polymeric support for immobilization of β‐galactosidase. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 130: 2923–2931, 2013     

Development of polysulfone membranes for bacteria immobilization to remove phenol

We have investigated the feasibility of developing polysulfone (PS) membranes to partially immobilize Pseudomonas and to evaluate the inhibitory effect of phenol on immobilized Pseudomonas by monitoring their growths in partially immobilized cell and free-suspension systems. The polysulfone membranes used in this study were wet spun from 20 wt % of PS in 1-methyl-2-pyrrolidone (NMP) solvent using water as the bore fluid as well as the external coagulant. Scanning electron microscopy (SEM) characterization of the newly developed PS hollow fibers suggests that fiber cross-section consists of multilayer microporous structures useful for cell immobilization. Experiments were conducted using Pseudomonas bacteria to remove phenol with initial phenol concentrations of 300 mg/L and 1000 mg/L. In a free suspension (no membrane) system, it was observed that the bacteria were able to grow optimally at 300 mg/L of phenol and degraded phenol almost completely in about 26 h. However, neither cell growth nor phenol degradation occurred when initial concentration of phenol was increased to 1000 mg/L. In a cell-immobilized membrane system, the cell growth and phenol concentration profile in the medium were very similar to those obtained in a free-suspension culture if phenol concentration was 300 mg/L. However, when the initial phenol concentration was increased to 1000 mg/L, data obtained in a cell-immobilized membrane system was discernibly different from that obtained in the suspension culture. In the former case, phenol concentration decreased in the beginning of the test, indicating that the carbon source has been consumed and immobilized cells within the membrane had begun to multiply. As soon as the phenol concentration decreased to about 600 mg/L (at which concentration, substrate inhibition was not as severe as 1000 mg/L), partial immobilization occurred when some cells diffused out of the membrane into the medium and optical density became measurable in the medium. It was found that cell growth continued for the next 28 h, reaching a maximum optical density in the medium of 0.610 absorbance units, and phenol was also completely degraded. © 1998 John Wiley & Sons, Inc. J Appl Polym Sci 70: 2585–2594, 1998     

生物磁性分离研究进展(Ⅱ)磁性分离装置和生物技术应用

磁性分离技术在生物化学和生物医学领域有许多潜在的应用前景,本文主要介绍磁性分离装置和磁性分离在生物技术领域的应用进展情况,包括细胞分离、免疫检测、靶向药物、固定化酶及亲和分离等。     

磁性微球在生化领域中的应用

磁性微球作为一种新型功能材料,在生物工程等领域有着广泛的应用前景,着重介绍了磁性微球在生物分离、固定化酶、靶向药物等领域的应用。随着人们对磁性微球研究的不断深入,必将引起制备和应用技术的革命性进步,并且实现商业化具有广阔的前景。