Preparation and characterization of alkaline phosphatase,horseradish peroxidase,and glucose oxidase conjugates with carboxylated carbon nano-onions

Carbon nanomaterials have emerged as suitable supports for enzyme immobilization and stabilization due to their inherently large surface area, high electrical conductivity, chemical stability, and mechanical strength. In this paper, carbon nano-onions (CNOs) were used as supports to immobilize alkaline phosphatase, horseradish peroxidase, and glucose oxidase. CNOs were first functionalized by oxidation to generate carboxylic groups on the surface followed by the covalent linking of using a soluble carbodiimide as coupling agent. The CNO–enzyme conjugates were characterized by transmission electron microscopy and Raman spectroscopy. Thermogravimetric analysis revealed a specific enzyme load of ～0.5?mg of protein per milligram of CNO. The immobilized enzymes showed enhanced storage stability without altering the optimum pH and temperatures. These properties make the prepared nanobiocatalyst of potential interest in biosensing and other biotechnological applications.     

Covalent functionalization of multiwalled carbon nanotubes with a low molecular weight chitosan

Covalent functionalization of shortened multiwalled carbon nanotubes (MWNTs) with a natural low molecular weight chitosan (LMCS) was accomplished by a nucleophilic substitution reaction. Amino and primary hydroxyl groups of the LMCS contributed mainly to the formation of MWNT-LMCS conjugates. The LMCS content in the MWNT-LMCS is approximately 58 wt %, and approximately four molecular chains of the LMCS are attached to 1000 carbon atoms of the nanotube sidewalls. Most interestingly, the amorphous packing structure of the LMCS changed dramatically when it attached to the MWNTs. The MWNTs might induce the crystalline character of the LMCS. As a novel derivative of MWNTs, the MWNT-LMCS is soluble in dimethylformamide, dimethyl acetamide, dimethylsulfoxide, and acetic acid aqueous solution. The confirmation of the chitosan-based covalent functionalization route might lead to further studies aiming for potential applications in catalysis and environmental protection.     

Polyphenoloxidases immobilized in organic gels: Properties and applications in the detoxification of aromatic compounds

Gelatine gels originate from water in oil microemulsions in which the ternary system consists of isooctane/ sulfosuccinic acid bis [2-ethyl hexyl] ester/water; the solubilization of gelatin in the water pool of these microemulsions transforms them into viscous gels in which it is possible to cosolubilize various reactive molecules. These gels were used to immobilize two phenoloxidases, a laccase from Trametes versicolor and a tyrosinase from mushroom. The best balance between gel retention and catalytic activity was reached at a gelatine concentration of 2.5% (w/v) in the case of tyrosinase, while laccase immobilization was independent of gelatine concentration. Both enzymes kept the same optimum pH as the corresponding soluble controls, while a partial loss of activity was observed when they were immobilized. Immobilized enzymes showed an increased stability when incubated for several days at 4 degrees C with a very low release from the gels in the incubation solutions. The immobilization of tyrosinase and of laccase enhanced stability to thermal inactivation. Furthermore, gel-entrapped tyrosinase was almost completely preserved from proteolysis: more than 80% of the activity was maintained, while only 25% of the soluble control activity was detected after the same proteolytic treatments. A column packed with gel-immobilized tyrosinase was used to demonstrate that enzymes immobilized with this technique may be reused several times in the same reaction without loosing their efficiency. Finally, gel-entrapped tyrosinase and laccase were capable of removing naturally occurring and xeno-biotic aromatic compounds from aqueous suspensions with different degrees of efficiency. (c) 1995 John Wiley & Sons, Inc.     

Immobilization of D-ribulose-1,5-bisphosphate carboxylase/oxygenase: a step toward carbon dioxide fixation bioprocess

Immobilization of D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from spinach leaves is described. This enzyme enables the fixation of carbon dioxide on a five-carbon sugar D-ribulose-1,5-bisphosphate (RuBP). Two different immobilization methods were employed: dicyclohexylcarbodiimide coupling on nylon membrane matrix and dimethylpimelimidate immobilization on protein A agarose. The reusability of immobilized enzymes, coupling efficiency, and temperature-activity relationship of soluble and immobilized Rubisco are presented. The immobilization imparted greater thermal and storage stability. The thermal deactivation rates of the immobilized enzymes were considerably lower than those of the soluble enzyme.     

β-d-Galactosidases immobilized on soluble matrices: Kinetics and stability

Three β-d-galactosidases (β-d-galactoside galactohydrolase, EC 3.2.1.23) from different origins have been immobilized on sucrose-polyacrolein and sucrose sulphate-polyacrolein. This gave enzyme conjugates insoluble in the immobilization medium but which could be made soluble by reduction with sodium borohydride before use. The optimum conditions for both copolymer synthesis and the immobilization reaction were investigated. I.r. and ¹³C n.m.r. spectroscopy were used to follow the sulphation and the copolymerization reaction. The characteristics of the enzyme conjugates were compared with those of the free enzymes: the V_max values of the enzyme conjugates were lower than those of the corresponding free enzymes, whilst the K_m values were similar. The thermal stability of the enzyme conjugates depended on the enzyme origin, while their pH stability was in all cases higher than that of the free enzymes. These data suggest some advantages in using enzyme immobilization supports which can be made soluble after separation of the immobilized enzyme without altering the enzyme characteristics.     

微生物嗜盐酶的研究进展

嗜盐酶一般来自于嗜盐菌,它的主要特点是严格依赖体系中一定的盐离子浓度,可以在高盐环境中维持其结构稳定,并且能够抵抗高温、p H和有机溶剂存在下的变性,因此在高盐、水/有机和非水介质环境的催化中具有重要的应用价值。本综述从盐对嗜盐酶活性和稳定性的影响、金属离子和有机溶剂对嗜盐酶的影响几个方面介绍了嗜盐酶的特点。在总结蛋白质数据库(PDB)中已有嗜盐酶的结构和特点的基础上,对嗜盐酶的嗜盐机制进行了分析,认为嗜盐酶不同于非嗜盐酶的特点在于盐桥和氢键明显增多,含有一些特殊的盐离子结合位点并且常以低聚体的形式存在,表面酸性氨基酸含量明显增多。最后对嗜盐酶的分子改造和应用进行了简要的介绍。     

Poly(2-oxazoline)s terminated with 2,2′-imino diacetic acid form noncovalent polymer–enzyme conjugates that are highly active in organic solvents

A great limitation for the usability of free enzymes in organic solvents is their insolubility in these media. Some surfactants are capable of solubilizing enzymes in such media, but they are hard to remove. Covalent modification of enzymes with polymers has led to polymer–enzyme conjugates (PECs) that are soluble in organic solvents, but the process is quite elaborate. Poly(2-oxazoline)s (POx) with the end group 2,2′-imino diacetic acid were shown to form reversible, nano-sized noncovalent aggregates with enzymes. These PECs give clear solutions in organic solvents. The enzymes lysozyme, horseradish peroxidase (HRP), laccase, α-chymotrypsin (CT), catalase, and alcohol dehydrogenase could be solubilized in chloroform and toluene at concentrations of up to 2 mg protein/ml. Laccase, HRP, and CT were shown to survive the transfer into the organic medium and back to water in their active form. The distribution coefficient of the proteins between water and the organic solvent was shown to be dependent on the nature of the POx backbone. All three biocatalysts exhibit greatly enhanced activity in the respective organic solvent.     

Thermal stabilization of amylolytic enzymes by covalent coupling to soluble polysaccharides

The immobilization of pullulanase and beta-amylase on soluble polysaccharides (dextrans and amylose) has been carried out. The method used for coupling the enzymes to the carbohydrate support involves limited periodate oxidation of the polysaccharide followed by reductive alkylation with sodium cyanoborohydride or borohydride. The influence of the degree of functionalization of the carbohydrate, the incubation time, the nature of the reducing agent and, for the dextrans studied, their molecular weight, on the properties of the conjugate were studied. We have observed an apparent correlation between the molecular weight of the glycoprotein conjugates formed and their thermal stability, resistance to urea denaturation and their kinetic parameters. By selecting the proper experimental conditions leading to conjugates with maximum thermal stabilities, it has also been shown that beta-amylase conjugates can hydrolyze starch at a temperature 20 degrees C higher than the corresponding value for the native enzyme. This result demonstrates that conjugation may result in modified enzymes leaving a high operational stability at elevated temperatures. We suggest that the immobilization method presented in this article represents an approach to the stabilization of enzymes employed at an industrial level, which may be of general application.     

Immobilization Increases the Stability and Reusability of Pigeon Pea NADP<Superscript>+</Superscript> Linked Glucose-6-Phosphate Dehydrogenase

Immobilization of enzymes is valuably important as it improves the stability and hence increases the reusability of enzymes. The present investigation is an attempt for immobilization of purified glucose-6-phosphate dehydrogenase from pigeon pea on different matrix. Maximum immobilization was achieved when alginate was used as immobilization matrix. As compared to soluble enzyme the alginate immobilized enzyme exhibited enhanced optimum pH and temperature. The alginate immobilized enzyme displayed more than 80% activity up to 7 continuous reactions and more than 50% activity up to 11 continuous reactions.     

Amphiphilic hollow carbonaceous microsphere-encapsulated enzymes: Facile immobilization and robust biocatalytic properties

This study reports a general strategy for the encapsulation of various enzymes in amphiphilic hollow carbonaceous microspheres (CMs). We found that enzymes could be spontaneously encapsulated in the interior cavity of the CMs via hydrophobic interactions. Due to strong hydrophobic interactions and robust confinement, leaching of the physically adsorbed enzymes is substantially restricted. As a novel immobilization matrix, the CMs display many significant advantages. They are capable of encapsulating a wide range of proteins/enzymes of different sizes, which can then be used in both aqueous and organic media and retain high activity, stability, and excellent reusability. Moreover, CMs could be considered as efficient microreactors that provide a favorable microaqueous environment for enzymes in organic systems. Therefore, this doubly effective and simple immobilization approach can be easily expanded to many other enzymes and has great potential in a variety of enzyme applications.     

Layer-by-layer assembled carbon nanotubes for selective determination of dopamine in the presence of ascorbic acid

Multilayer films of shortened multi-walled carbon nanotubes (MWNTs) are homogeneously and stably assembled on glassy carbon (GC) electrodes using layer-by-layer (LBL) method based on electrostatic interaction of positively charged poly(diallyldimethylammonium chloride) (PDDA) and negatively charged shortened MWNTs. The assembled MWNT multilayer films were studied with respect to the electrocatalytic activity toward ascorbic acid (AA) and dopamine (DA) and were further applied for selective determination of DA in the presence of AA. Scanning electron microscopy (SEM) used for characterization of MWNT films indicates that the assembled MWNTs are almost in a form of small bundles or single nanotubes on the electrodes. Cyclic voltammetric results with assembled MWNT electrode indicate that the strategy based on the LBL method for assembling the MWNT multilayer films on substrate well retains the electrochemical catalytic activity of the MWNTs toward AA and DA, offering some advantages particularly attractive for analytical applications, such as the form of MWNTs assembled on the substrate, i.e., small bundles or single tubes, homogeneity and stability of the as-assembled MWNT films. These features make the assembled MWNTs relatively potential for selective and sensitive determination of DA in the presence of AA.     

Immobilization of amyloglucosidase from SSF of Aspergillus niger by crosslinked enzyme aggregate onto magnetic nanoparticles using minimum amount of carrier and characterizations

Immobilizations of enzymes are done for operational stability, recovery and re-use of the enzymes and easy separation of products. Amyloglucosidase (AMG) obtained from solid state fermentation (SSF) of Aspergillus niger was directly immobilized by novel technique of crosslinked enzyme aggregate onto magnetic nanoparticles. AMG was covalently linked to the magnetic nanoparticle (MNP) to form a monolayer of AMG (MNP–AMG), followed by crosslinked aggregates with free AMG (which was not immobilized) to yield MNP with high enzyme loading (MNP–AMG_n). Under optimized conditions, very high recovery (92.8%) of enzyme activity was obtained in MNP–AMG_n using 14 times less carrier compared to the quantity of carrier required by conventional method. MNP–AMG_n showed enhanced affinity for substrate, thermal stability, storage stability and reusability.     

多级孔金属-有机框架固定化酶的研究进展

金属-有机框架(metal-organic frameworks, MOFs)作为酶固定化的优良载体,为生物催化反应提供优越的物理和化学保护。近年来,多级孔金属-有机框架(hierarchical porous metal-organic frameworks, HP-MOFs)由于其独特的结构优势,在固定化酶方面显示出更大的潜力。到目前为止,已经开发了各类具有原生多级孔或缺陷多级孔的HP-MOFs用于酶的固定化研究,并且使得固定化酶在催化活性、稳定性和重复利用性等方面得到了显著增强。本文系统总结了HP-MOFs用于固定化酶的各种策略,介绍了HP-MOFs固定化酶(enzyme@HP-MOFs)在催化合成、生物传感、生物医药等领域的最新应用进展。最后,讨论并展望了HP-MOFs固定化酶这一领域所面临的挑战和机遇。     

Organosoluble enzyme conjugates with poly(2-oxazoline)s via pyromellitic acid dianhydride

The use of enzymes in organic solvents offers a great opportunity for the synthesis of complex organic compounds and is therefore in focus of current research. In this work we describe the synthesis of poly(2-methyl-1,3-oxazoline) (PMOx) and poly(2-ethyl-1,3-oxazoline) (PEtOx) enzyme conjugates with hen-egg white lysozyme, RNase A and α-chymotrypsin using a new coupling technique. The POXylation was carried out reacting pyromellitic acid dianhydride subsequently with ethylenediamine terminated POx and then with the NH?-groups of the respective enzymes. Upon conjugation with the polymers, RNase A and lysozyme became fully soluble in DMF (1.4 mg/ml). These are the first examples of fully POXylated proteins, which become organosoluble. The synthesized enzyme conjugates were characterized by SDS-PAGE, isoelectric focusing, dynamic light scattering and size exclusion chromatography, which all indicated the full POXylation of the enzymes. The modified enzymes even partly retained their activity in water. With α-chymotrypsin as example we could demonstrate that the molecular weight of the attached polymer significantly influences the activity.     

Synthesis of Protein-Inorganic Nanohybrids with Improved Catalytic Properties Using Co<Subscript>3</Subscript>(PO<Subscript>4</Subscript>)<Subscript>2</Subscript>

In the present study, a method for easy and rapid synthesis of lipase nanohybrids was evaluated using cobalt chloride as an encapsulating agent. The synthesized nanohybrids exhibited higher activity (181%) compared to free lipase and improved catalytic properties at higher temperature and in harsh conditions. The nanohybrids retained 84% of their residual activity at 25 °C after 10 days. In addition, these nanohybrids also exhibited high storage stability and reusability. Collectively, the synthesis of carrier-free immobilized biocatalysts was performed rapidly within 24 h at 4 °C. Their high reusability and catalytic activities highlight the broad applicability of this method for catalysis in organic and aqueous media.     

Preparation and characterization of heparinized multi-walled carbon nanotubes

The characteristics of heparinized multiwalled carbon nanotubes (MWNTs) were investigated in terms of the activated partial thromboplastin time (APTT) to verify the heparin activity, a carbazole assay was done to measure the content of the immobilized heparin, and the octanol-water partition coefficient was assessed to determine the lipophilicity. Two heparin-immobilized MWNTs were prepared to evaluate their differences. The first preparation method involved polymer-coated MWNTs with heparin indirectly center-point-attached. In the second approach, heparin was directly end-point-attached through its reducing end onto acid-treated MWNTs. The blood compatibility of MWNTs to which heparin was end-point-attached through its reducing end was greatly enhanced compared to that of the MWNTs onto which heparin was center-point-attached. The APTT and carbazole assay results demonstrated that heparinized MWNTs prepared through end-point attachment result in prolonged plasma-based anticoagulant activity. The blood compatibility of MWNTs heparinized by end-point attachment was not decreased up to the fourth pasteurization. Heparinized MWNTs were also studied using octanol-water partition, which should be useful for exploring heparinized MWNTs as drug carriers including delivery systems. The results of octanol/water partition on the design of heparinized MWNTs prepared by end-point attachment with a specific binding can facilitate the design of drug delivery carriers with high blood compatibility.     

Glucose biosensor based on electrodeposition of platinum nanoparticles onto carbon nanotubes and immobilizing enzyme with chitosan-SiO(2) sol-gel

A novel amperometric biosensor, based on electrodeposition of platinum nanoparticles onto multi-walled carbon nanotube (MWNTs) and immobilizing enzyme with chitosan-SiO(2) sol-gel, is presented in this article. MWNTs were cast on the glass carbon (GC) substrate directly. An extra Nafion coating was used to eliminate common interferents such as acetaminophen and ascorbic acids. The morphologies and electrochemical performance of the modified electrodes have been investigated by scanning electron microscopy (SEM) and amperometric methods, respectively. The synergistic action of Pt and MWNTs and the biocompatibility of chitosan-SiO(2) sol-gel made the biosensor have excellent electrocatalytic activity and high stability. The resulting biosensor exhibits good response performance to glucose with a wide linear range from 1 microM to 23 mM and a low detection limit 1 microM. The biosensor also shows a short response time (within 5s), and a high sensitivity (58.9 microAm M(-1)cm(-2)). In addition, effects of pH value, applied potential, rotating rate, electrode construction and electroactive interferents on the amperometric response of the sensor were investigated and discussed in detail.     

Stability of the insoluble form of uridine kinase coupled to zn2+ or pb2+ ions.

Partially purified calf brain uridine kinase precipitated by bivalent metal cations has been compared with the soluble enzyme fraction regarding its stability in the presence of inactivating factors. The freeze-dried preparations of uridine kinase precipitaated by Pb2+ or Zn2+ ions, althouth enzymatically highly active, are insoluble in aqueous solutions. The activity of metal-insolubilized enzymes disappears during their preincubation in acidic media or in the presence of silver ions. Also trypsin, chymotrypsin and cathepsin B1 caused decreases in enzyme activity. However, fractions which have been precipitated by metal ions and freeze-dried are stable at high temperatures, whereas the activity of soluble uridine kinase is completely lost. Both unheated metal-ion precipitated uridine kinase preparations and those heated at 100 degrees C are equally sensitive to the feedback inhibition by CTP.     

Selective tumor targeting by enhanced permeability and retention effect. Synthesis and antitumor activity of polyphosphazene-platinum (II) conjugates

Nanosized polyphosphazene-platinum (II) conjugates with a wide range of molecular weight from 24,000 to 115,000 were synthesized to study their tumor selectivity by enhanced permeability and retention (EPR) effect and their antitumor activity. It has been found from biodistribution study that the present polyphosphazene-Pt(II) conjugates exhibit high tumor selectivity by EPR effect with the tumor to tissue ratio (TTR) from 3.6 to 13 depending on the molecular size. These polymer conjugates have shown excellent in vivo antitumor activity against both murine and human cancer cell lines. In particular, xenograft trials of the conjugates have shown outstanding tumor inhibition effect on the stomach cancer cell line, YCC-3, which is one of the least responsive to the anticancer agents currently in clinical use, although the reason is not clearly explainable yet. The high in vivo activity seems to be attributed to the controlled-release of the antitumor active platinum (II) moiety, [GlyGluPt(dach] (dach=trans-(+/-)-1,2-diaminocyclohexane) from the phosphazene backbone by degradation in aqueous solution.     

Comparative study of stability of soluble and cell wall invertase from Saccharomyces cerevisiae

Yeast Saccharomyces cerevisiae is the most significant source of enzyme invertase. It is mainly used in the food industry as a soluble or immobilized enzyme. The greatest amount of invertase is located in the periplasmic space in yeast. In this work, it was isolated into two forms of enzyme from yeast S. cerevisiae cell, soluble and cell wall invertase (CWI). Both forms of enzyme showed same temperature optimum (60°C), similar pH optimum, and kinetic parameters. The significant difference between these biocatalysts was observed in their thermal stability, stability in urea and methanol solution. At 60°C, CWI had 1.7 times longer half-life than soluble enzyme, while at 70°C CWI showed 8.7 times longer half-life than soluble enzyme. After 2-hr of incubation in 8?M urea solution, soluble invertase and CWI retained 10 and 60% of its initial activity, respectively. During 22?hr of incubation of both enzymes in 30 and 40% methanol, soluble invertase was completely inactivated, while CWI changed its activity within the experimental error. Therefore, soluble invertase and CWI have not shown any substantial difference, but CWI showed better thermal stability and stability in some of the typical protein-denaturing agents.