Properties of pectinesterase immobilized on glycophase-coated controlled-pore glass

Pectinesterase was immobilized on a porous glass support, which surface was covered with glyceryl residues. The parameters of the immobilization were characterized with respect to the coupling method used as well as the support pore size. Chemical modification level resulted an important parameter in determining the activity of the immobilized derivative. Attachment of the enzyme through thiol groups gave the best results, whereas a nominal pore size of 20 nm seemed to be the most suitable for the demethoxylating activity of the enzyme on citrus pectin. Optimum conditions for activity as well as the inhibition constant for polygalacturonic acid did not change on immobilization, as the Michaelis constant did. Fluorescence spectra revealed a partial unfolding of the enzyme tertiary structure when immobilized.     

Catalytic biofunctional membranes containing site-specifically immobilized enzyme arrays: a review

Biofunctional membranes normally involve the random immobilization of biomolecules to porous, polymeric membranes, often through the numerous lysine residues on the protein. In this process, bioactivity is significantly decreased largely due to different orientations of the biomolecule with respect to the membrane or to multiple point attachment. To circumvent this difficulty, while still taking advantage of the immobilization of biomolecules, site-specific immobilization of the biomolecule with the active (or binding) site directed away from the membrane is essential. In this review, we summarize our efforts involving biophysical and bioanalytical chemistry and chemical engineering, together with molecular biology, to develop and characterize such site-specifically membrane immobilized catalytic enzyme bioreactors. Site-directed mutagenesis, gene fusion technology, and post-translational modification methods are employed to effectuate the site-specific membrane immobilization. Electron paramagnetic resonance, in conjunction with active-site specific spin labels, kinetic analyses, and membrane properties are used to characterize these systems. Biofunctional membranes incorporating site-specifically immobilized biomolecules provide greater efficiency of biocatalysis, bioseparations, and bioanalysis.     

Immobilization of the enzyme L-asparaginase of E. coli on polysaccharides. V. Preparation and properties of polymeric conjugates based on water-soluble CM-cellulose differing by the quantitative amounts of polymer

A precedure has been developed for the chemical binding of L-asparaginase to soluble CM-cellulose which permits final immobilization products with different degrees of modification to be obtained. Some physicochemical and immunological properties of the samples obtained have been studied.     

Optimization and characterization of immobilized E. coli for engineered thermostable xylanase excretion and cell viability

There are many parameters that may have influenced the properties of cell during immobilization process. Particularly, the immobilization methods, carrier materials, and enzyme loading amount that have been proved to be important for immobilization process. The physiological responses of microorganisms are depending on the immobilization technique used. Typical alterations to the micro-environment of the immobilized cell involved the altered water activity, presence of ionic charges, cell confinement and modified surface tension. In this study, the graphene oxide was selected as a suitable carrier for immobilization process of recombinant E.coli and adsorption was chosen as an appropriate method to improve the production of engineered thermostable xylanase. High level production of thermostable xylanase by immobilized recombinant cell in the 5 L bioreactor was studied by using optimum research surface methodology (RSM) conditions was studied. The immobilization of E. coli onto nanoparticle matrix manages to improve the cell performance by improving the protein expression, reduced the occurrences of cell lysis as well as improved the plasmid stability of the host cell. Thus, immobilization contributes a physical support for both whole cells as well as enzymes to develop a better operative achievement system for industrialized fields and give rise to the biological advancement existing enzyme for instance xylanase.     

Acetylcholinesterase Immobilization on Polyacrylamide/Functionalized Multi-walled Carbon Nanotube Nanocomposite Nanofibrous Membrane

In this work, polyacrylamide/multi-walled carbon nanotubes (MWCNT) solution is electrospun to nanocomposite nanofibrous membranes for acetylcholinesterase enzyme immobilization. A new method for enzyme immobilization is proposed, and the results of analysis show successful covalent bonding of enzymes on electrospun membrane surface besides their non-covalent entrapment. Fourier transform infrared spectroscopy, mechanical and thermal investigations of nanofibrous membrane approve successful cross-linking and enzyme immobilization. The enzyme relative activity and kinetic on both pure and nanocomposite membranes is investigated, and the results show proper performance of designed membrane to even improve the enzyme activity followed by immobilization compared to free enzyme. Scanning electron microscopy images show nanofibrous web of 3D structure with a low shrinkage and hydrogel structure followed by enzyme immobilization and cross-linking. Moreover, the important role of functionalized carbon nanotubes on final nanofibrous membrane functionality as a media for enzyme immobilization is investigated. The results show that MWCNT could act effectively for enzyme immobilization improvement via both physical (enhanced fibers’ morphology and conductivity) and chemical (enzyme entrapment) methods.

二氧化硅纳米与微米颗粒作为固定化酶载体的生物效应

分别将二氧化硅纳米颗粒(SiNPs)与微米颗粒(SiMPs)作为固定化载体, 选择多聚酶牛肝过氧化氢酶(CAT)和单体酶辣根过氧化物酶(HRP)作为酶模型, 通过考察酶固定化后在酶活回收率、热稳定性、 酶促反应最适温度以及酶在水-有机溶剂混合体系中催化能力的变化, 对载体与酶所产生的生物效应差异进行了系统研究. 酶活回收率结果表明, SiNPs显示出比SiMPs优越的对酶无选择性的高生物亲和性, 而SiMPs则能使固定于其上的酶热稳定性大幅度提高, 且二者都能使固定化酶在有机相中的稳定性得到明显增强. 但酶促反应最适温度的变化结果表明, 对不同类型的酶所产生的生物效应则表现出无规律性.     

SBS改性沥青机理研究进展

介绍了沥青的特性、苯乙烯-丁二烯-苯乙烯三嵌段共聚物(SBS)的性能,分析了SBS与基质沥青之间的溶胀性和相容性问题,着重论述了SBS改性沥青机理的研究进展,指出机理主要分为物理共混和化学改性两类:物理共混——SBS微粒受到沥青组分中油分的作用发生溶胀而均匀分散在沥青中,SBS与沥青之间没有发生化学作用,只是一种分子间作用力;化学改性——加入添加剂使沥青和SBS之间发生加成、交联或接枝等化学反应,形成较强的共价键或离子键,改善沥青的化学性质。提出化学改性是提高SBS改性沥青路用性能的重要手段。     

高分子对酶、抗体、DNA的修饰、固定化及其生物医学应用

为发展适于生物医用的生物功能高分子材料,本实验室近年来研究了可溶性高分子对L-天冬酰胺酶的修饰、纳米磁性高分子微粒对酶或抗体的固定化、亚微米高分子微球固定化碱性磷酸酶及其在DNA检测中的应用、高分子微球固定化酶的合成与性能、酶在导电高分子膜上的固定化及生物传感器制备等. 本文对此进行简要总结.     

生物催化在绿色化学和新药开发中的应用

近年来生物技术领域有了突破性进展,如: 公共基因数据库(GenBank)和蛋白质数据库(PDB)中序列的指数增长,高效基因克隆和表达平台的建立,可有效改进生物催化剂专一性、选择性和稳定性的酶定向进化技术的应用。这些进展使生物催化在化学合成中日趋重要[1]。本文综述了生物催化在如下领域的成功应用：在药物生产中用于开发经济的化学酶法合成工艺,在绿色化学领域中最大程度的减少废物的产生和危险试剂的应用,在天然化学领域中对天然产物进行修饰以发现具有更好生物活性的新药物。     

Physical and chemical adsorption of Mucor javanicus lipase on SBA-15 mesoporous silica. Synthesis, structural characterization, and activity performance

In this work a sample of SBA-15 mesoporous silica was synthesized and characterized by TEM, XRD, and N2 adsorption. The sample had a high value of specific surface area (1007 m2 g(-1)) and total pore volume (2.1 cm3 g(-1)). The pore diameter was 67 angstroms, so it was large enough to accommodate protein molecules inside the channels. Immobilization by physical adsorption of a commercial lipase preparation from Mucor javanicus was performed at different pH values (pH 5-8). pH 6 gave the highest lipase loading and hydrolytic activity of the corresponding biocatalyst. Chemical modification of the SBA-15 via glutardialdehyde allowed also the enzyme immobilization through chemical adsorption. This preparation was active toward tributyrin hydrolysis. On the contrary, very low activity toward triolein hydrolysis was observed. The reduction of the size of the channels due the immobilization process has been suggested as a possible explanation.     

iBodies: Modular Synthetic Antibody Mimetics Based on Hydrophilic Polymers Decorated with Functional Moieties

Antibodies are indispensable tools for biomedicine and anticancer therapy. Nevertheless, their use is compromised by high production costs, limited stability, and difficulty of chemical modification. The design and preparation of synthetic polymer conjugates capable of replacing antibodies in biomedical applications such as ELISA, flow cytometry, immunocytochemistry, and immunoprecipitation is reported. The conjugates, named “iBodies”, consist of an HPMA copolymer decorated with low‐molecular‐weight compounds that function as targeting ligands, affinity anchors, and imaging probes. We prepared specific conjugates targeting several proteins with known ligands and used these iBodies for enzyme inhibition, protein isolation, immobilization, quantification, and live‐cell imaging. Our data indicate that this highly modular and versatile polymer system can be used to produce inexpensive and stable antibody substitutes directed toward virtually any protein of interest with a known ligand.     

一种简化的重氮化法制备固定化酶的载体合成方法

重氮化法是固定化酶时常用的一种方法。用多孔玻璃等无机物作载体时,一般是先用g-氨丙基三乙氧基硅烷与多孔玻璃等载体反应[1,2],生成烷基胺玻璃,然后与对硝基苯酰氯反应,产物经过还原,生成带有芳胺的衍生物,最后进行重氮化。本文通过烷基胺与对氨基苯甲酸反应,直接生成芳胺的衍生物,比常用的方法缩短了一步。通过在新合成的载体上对木瓜蛋白酶进行固定化,研究了固定化条件对酶活力回收的影响,最适固定化条件如下:pH为7.0,时间为6h,酶量为240mg/g载体,并比较了固定化酶和溶液酶的有关性质,考察了固定化酶的操作稳定性。结果表明,用这种方法合成的载体固定化酶,其对热稳定性、操作稳定性及产率都比较理想。     

Glucose oxidase–magnetite nanoparticle bioconjugate for glucose sensing

Immobilization of bioactive molecules on the surface of magnetic nanoparticles is of great interest, because the magnetic properties of these bioconjugates promise to greatly improve the delivery and recovery of biomolecules in biomedical applications. Here we present the preparation and functionalization of magnetite (Fe₃O₄) nanoparticles 20 nm in diameter and the successful covalent conjugation of the enzyme glucose oxidase to the amino-modified nanoparticle surface. Functionalization of the magnetic nanoparticle surface with amino groups greatly increased the amount and activity of the immobilized enzyme compared with immobilization procedures involving physical adsorption. The enzymatic activity of the glucose oxidase-coated magnetic nanoparticles was investigated by monitoring oxygen consumption during the enzymatic oxidation of glucose using a ruthenium phenanthroline fluorescent complex for oxygen sensing. The glucose oxidase-coated magnetite nanoparticles could function as nanometric glucose sensors in glucose solutions of concentrations up to 20 mmol L^–1. Immobilization of glucose oxidase on the nanoparticles also increased the stability of the enzyme. When stored at 4°C the nanoparticle suspensions maintained their bioactivity for up to 3 months.     

Affinity Covalent Immobilization of Glucoamylase onto ρ-Benzoquinone Activated Alginate Beads: I. Beads Preparation and Characterization

ρ-Benzoquinone-activated alginate beads were presented as a new carrier for affinity covalent immobilization of glucoamylase enzyme. Evidences of alginate modification were extracted from FT-IR and thermal gravimetric analysis and supported by morphological changes recognized through SEM examination. Factors affecting the modification process such as ρ-benzoquinone (PBQ) concentration, reaction time, reaction temperature, reaction pH and finally alginate concentration, have been studied. Its influence on the amount of coupled PBQ was consequently correlated to the changes of the catalytic activity and the retained activity of immobilized enzyme, the main parameters judging the success of the immobilization process. The immobilized glucoamylase was found kept almost 80% of its native activity giving proof of non-significant substrate, starch, diffusion limitation. The proposed affinity covalent immobilizing technique would rank among the potential strategies for efficient immobilization of glucoamylase enzyme.     

Two-Step covalent immobilization of enzymes as a way for study of effects influencing catalytic activity

Isothiocyanatopropyl derivatives of trypsin and pepsin obtained by treatment with 3isothiocyanato-1-propyl isocyanate (in the first step) can be covalently bound to carriers having amino groups. Besides all the general principles valid for reactions of isothiocyanates with amines, prerequisite for the satisfactory coupling properties of isothiocyanatopropyl derivatives are (a) a sufficiently high modification degree with isothiocyanate, and (b) a concentration excess of NH₂ carrier groups. With respect to the coupling properties, nonspecific sorptions and the catalytic properties of the immobilized trypsin isothiocyanatopropy l derivatives, the better carrier appears to be the cross-linked polyethy leneimine. Isothiocyanatopropyl, 3-isothiocyanato-bromopropyl, and isothiocyanatopenty l derivatives of leucine, insulin, and albumin also have good coupling properties. The mode in which the amidolytic and caseinolytic activity of isothiocyanatopropyl derivatives of trypsin was influenced after its immobilization on polyethyleneimine clearly indicated that the functional groups of the enzyme did not participate in the coupling to the carrier. Moreover, no intermolecular reactions were observed during the immobilization process. On the other hand, the conformational changes of the protein molecule are important, since they probably influence the changes in the catalytic properties of modified enzymes after their immobilization.     

Enzyme immobilization by adsorption: a review

Endowed with unparalleled high catalytic activity and selectivity, enzymes offer enormous potential as catalysts in practical applications. These applications, however, are seriously hampered by enzymes’ low thermal and chemical stabilities. One way to improve these stabilities is the enzyme immobilization. Among various tested methods of this process that make use of different enzyme-carrier interactions, immobilization by adsorption on solid carriers has appeared most common. According to these findings, in this review we present a comparative analysis of the literature reports on the recent trends in the immobilization of the enzymes by adsorption. This thorough study was prepared in order to provide a deeper understanding of the process. Both carriers, carrier modifiers and procedures developed for effective adsorption of the enzymes are discussed. The review may thus be helpful in choosing the right adsorption scheme for a given enzyme to achieve the improvement of its stability and activity for a specific application.     

聚乙烯亚胺改性聚丙烯腈中空膜固定脂肪酶

聚丙烯腈是富腈基的高分子聚合物,易修饰改性,广泛应用于膜分离应用.我们以聚丙烯腈中空膜为载体,采用化学法交联聚乙烯亚胺并固定脂肪酶,固定过程中引入海藻酸钠,用CaCl_2进行后处理,得到固定化脂肪酶PAN-PEI-SA/E-CaCl_2载酶量为31.70(mg enzyme)/(g support),酶活为50.20 U/(g support),15次重复使用可保留58.77%的酶活,与游离酶相比耐酸性和耐温性有所提高,相同条件下与Nov 435相比,酶活更高,这表明最终得到的固定化脂肪酶有良好的工业应用前景.     

Immobilization of Fungal β-Glucosidase on Silica Gel and Kaolin Carriers

β-Glucosidase is a key enzyme in the hydrolysis of cellulose for producing feedstock glucose for various industrial processes. Reuse of enzyme through immobilization can significantly improve the economic characteristics of the process. Immobilization of the fungal β-glucosidase by covalent binding and physical adsorption on silica gel and kaolin was conducted for consequent application of these procedures in large-scale industrial processes. Different immobilization parameters (incubation time, ionic strength, pH, enzyme/support ratio, glutaric aldehyde concentration, etc.) were evaluated for their effect on the thermal stability of the immobilized enzyme. It was shown that the immobilized enzyme activity is stable at 50 °C over 8 days. It has also been shown that in the case of immobilization on kaolin, approximately 95% of the initial enzyme was immobilized onto support, and loss of activity was not observed. However, covalent binding of the enzyme to silica gel brings significant loss of enzyme activity, and only 35% of activity was preserved. In the case of physical adsorption on kaolin, gradual desorption of enzyme takes place. To prevent this process, we have carried out chemical modification of the protein. As a result, after repeated washings, enzyme desorption from kaolin has been reduced from 75 to 20–25% loss.     

有机磷农药酶生物传感器研究进展

酶生物传感器（EBS）以简单、廉价、易于微型化等优势成了有机磷农药（OPs）传统分析方法的最佳替代品。本文从识别OPs的酶及识别机理、电化学EBS、酶的固定化技术、高分子材料的酶固定载体不同角度综述了有机磷农药酶生物传感器研究近况,并重点介绍了一次性丝网印刷酶电极。     

Immobilization and Stabilization of Xylanase by Multipoint Covalent Attachment on Agarose and on Chitosan Supports

Xylanases have important applications in industry. Immobilization and stabilization of enzymes may allow their reuse in many cycles of the reaction, decreasing the process costs. This work proposes the use of a rational approach to obtain immobilized commercial xylanase biocatalysts with optimized features. Xylanase NS50014 from Novozymes was characterized and immobilized on glyoxyl-agarose, agarose-glutaraldehyde, and agarose-amino-epoxy support and on differently activated chitosan supports: glutaraldehyde-chitosan, glyoxyl-chitosan, and epoxy-chitosan. Two different chitosan matrices were tested. The best chitosan derivative was epoxy-chitosan-xylanase, which presented 100% of immobilization yield and 64% of recovered activity. No significant increase on the thermal stability was observed for all the chitosan-enzyme derivatives. Immobilization on glyoxyl-agarose showed low yield immobilization and stabilization degrees of the obtained derivative. The low concentration of lysine groups in the enzyme molecule could explain these poor results. The protein was then chemically modified with ethylenediamine and immobilized on glyoxyl-agarose. The new enzyme derivatives were 40-fold more stable than the soluble, aminated, and dialyzed enzyme (70 °C, pH 7), with 100% of immobilization yield. Therefore, the increase of the number of amine groups in the enzyme surface was confirmed to be a good strategy to improve the properties of immobilized xylanase.