Enzymes immobilized in mesoporous silica: A physical–chemical perspective

Mesoporous materials as support for immobilized enzymes have been explored extensively during the last two decades, primarily not only for biocatalysis applications, but also for biosensing, biofuels and enzyme-controlled drug delivery. The activity of the immobilized enzymes inside the pores is often different compared to that of the free enzymes, and an important challenge is to understand how the immobilization affects the enzymes in order to design immobilization conditions that lead to optimal enzyme activity. This review summarizes methods that can be used to understand how material properties can be linked to changes in enzyme activity. Real-time monitoring of the immobilization process and techniques that demonstrate that the enzymes are located inside the pores is discussed by contrasting them to the common practice of indirectly measuring the depletion of the protein concentration or enzyme activity in the surrounding bulk phase. We propose that pore filling (pore volume fraction occupied by proteins) is the best standard for comparing the amount of immobilized enzymes at the molecular level, and present equations to calculate pore filling from the more commonly reported immobilized mass. Methods to detect changes in enzyme structure upon immobilization and to study the microenvironment inside the pores are discussed in detail. Combining the knowledge generated from these methodologies should aid in rationally designing biocatalyst based on enzymes immobilized in mesoporous materials.     

Use of the clark oxygen sensor with immobilized enzymes for determinations in flow systems

A small-volume cell has been constructed for amperometric flow measurements with a Clark oxygen sensor and its performance was tested. The Clark sensor can be combined with immobilized enzymes for determination of substances after enzymatic conversion during which oxygen is consumed or released. Two enzymes, glucose oxidase and tyrosinase, were used and two measuring techniques, employing the enzyme immobilized on the Clark sensor membrane and with the enzyme bound on a support in a preceding reactor, were tested and compared. It was found that, in the given system, measurement with the enzyme immobilized on the sensor membrane has better sensitivity, precision and response rate.     

ImmobilizedE. coli Alkaline Phosphatase

We have immobilized E.coli alkaline phosphatase (EC 3.1.3.1) by linking it covalently to sepharose 4B. This preparation has several advantages over the soluble enzyme. The immobilized enzyme is easily separable from other constituents in incubation mixtures. The immobilized enzyme can be reused repeatedly and is more stable than the soluble enzyme to heat treatment in the presence of 10 mM Mg²⁺. The insoluble and soluble phosphatases removed 75 and77%, respectively, of the inorganic phosphorus from casein. The immobilized enzyme inactivated two enzymes believed to be active in the phosphorylated state, acyl-CoA : cholesterol acyltransferase (ACAT) by 39% and NADPH-cytochrome P-450 reductase by 89%. The utility of immobilized alkaline phosphatase for studying the phosphorylation and dephosphorylation of soluble or membrane-bound enzymes and proteins is discussed.     

蛋白质组学中的固定化酶反应器制备的研究进展

固定化酶反应器作为蛋白质组学研究中"bottom-up"策略重要的组件,具有酶解快速、酶解效率高、酶稳定性和活性高、简单易操作、能够与多种检测方式联用等优点,对于发展高效快速的蛋白质组学分析方法具有重要意义。本文就固定化酶反应器的制备方法及其在蛋白质组学中的应用做简单的概述,着重介绍酶的固定方法、固定化酶的载体、用于固定的酶的种类。近几年固定化酶反应器的研究集中于提高固酶量、保持酶活性、增加酶解效率、减小非特异性吸附等方面。研究结果表明,采用纳米材料、整体材料等新型载体,提高载体亲水性,采用多酶同时酶解等方法能够有效改善固定化酶反应器的性能,提高蛋白质的鉴定效率。     

Selective removal of enzymes from substrate and products. An alternative to immobilization for enzymes acting on macromolecular or solid substrates

A new approach for the control and interruption of enzymatic reactions via selective enzyme immobilization has been developed. The technique was exemplified by the use of three model enzymes with the corresponding macromolecular substrates: α-amylase/starch, trypsin/ insoluble collagen, and alkaline phosphatase/plasmid DNA. Prior to incubation with its substrate, each enzyme was provided withde novo thiol-groups by a two-step reaction involvingN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) and DTT. The chemical modification was achieved such that at least 80% of the native enzyme activity was preserved in all cases. In order to interrupt rapidly the reactions in which the enzymes were used, the modified enzyme was immobilized by reaction via its thiol groups on a thiolsulfinate-agarose derivative. The gel-bound enzyme could then be easily removed from unreacted substrate and product by filtration or centrifugation. Comparative studies showed that the immobilized enzymes had much lower activities in the reactions studied than the corresponding soluble ones. The potential for enzyme reuse was also demonstrated with the a-amylase derivatives, which were quantitatively released and eluted in fully active form from the agarose. We have shown that it is possible to achieve practically complete enzyme immobilization in short times and thus to control the progress of the reactions. Because of its simplicity and high efficiency, this approach may represent an interesting alternative for biotechnological processes involving macromolecular or solid substrates.     

Can immobilized enzymes Be applicable for homogeneous reaction? A novel technique of acetylcholinesterase immobilization for assaying carbaryl pesticide

A novel technique of enzyme immobilization is developed for making the immobilized enzymes capable of further releasing for homogeneous reaction. Water-soluble poly(vinyl alcohol) was used to prepare enzyme-loaded membranes with immobilized acetylcholinesterase (AChE). When used, a piece of enzymecontaining membrane is put into the solution and dissolves quickly. The released AChE is mixed and interacts with substrates and carbaryl inhibitors. The catalytical activity and inhibition sensitivity of released enzyme are comparable to those of free AChE. The values of Michaelis constant and maximum reaction rate for released AChE are also very close to those for free AChE. The experimental conditions such as the concentrations of PVA and acetone, the time of enzymatic reaction and that of AChE inhibition by carbaryl pesticide were optimized. The relative inhibition of AChE activity increased with the carbaryl concentration ranging from 0.1 μg/L to 100 mg/L. When compared to free AChE in solution or solid powder, the prepared PVA-AChE membranes are advantageous with respect to storage and handling. The suggested technique of enzyme immobilization is suitable for the variety of applications, when the enzyme catalysed reactions allows for single-using of the active material and does not require further enzyme recovering.     

Assessment of immobilized PGA orientation via the LC-MS analysis of tryptic digests of the wild type and its 3K-PGA mutant assists in the rational design of a high-performance biocatalyst

The mutant penicillin G acylase (PGA) 3K-PGA contains three additional Lys residues on the surface opposite the active site. This protein was designed to selectively drive its immobilization on aldehyde supports. We describe here a modified bottom-up proteomic method to assess the orientation of the immobilized wild-type and mutant proteins to verify our hypothesis of a driven immobilization induced by the mutations introduced. Tryptic digestion of the immobilized enzymes followed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis of released peptides was performed. This protocol generated peptides from the most accessible surface areas of the immobilized protein, thus not directly bound to the solid support, providing direct evidence of the areas involved in the linkage to the solid matrix. The results obtained suggest that 72 % of the wild-type PGA is immobilized on aldehyde agarose mainly through the Lys residues on the same side of the active site, whereas 3K-PGA reacted with the same support preferentially through the additional Lys residues introduced by mutation on the opposite side. This demonstrates that the active site of the 3K-PGA faces mostly (63 %) toward the reaction medium, resulting in significantly improved accessibility to the substrates. This finding is supported by the catalytic properties of the immobilized biocatalysts. The two immobilized preparations were tested in the synthesis of mandelyl-7-aminocephalosporanic acid (mandelyl-7-ACA) by N-acylation of the β-lactam nucleus (7-aminocephalosporanic acid) with mandelic acid methyl ester: upon immobilization, the synthetic properties of wild-type PGA strongly decreased, whereas those of 3K-PGA were unaffected. Furthermore, the activity of 3K-PGA was not influenced by the physicochemical nature of the support used for immobilization (glyoxyl agarose or aldehyde Sepabeads) unlike that of wild-type PGA, whose active site is close to the matrix. The results obtained from the analytical characterization correlate well with those obtained by investigation of the synthetic properties of the immobilized enzymes both in the synthesis of mandelyl-7-ACA and in the preparative synthesis of cefazolin. This work highlights the effect exerted by site-directed mutagenesis on the orientation of PGA upon immobilization on solid matrices and suggests how protein engineering tools can be exploited in a synergistic fashion to rationally develop efficient biocatalysts.

纳米复合材料固定化酶的研究进展

载体材料的选择对固定化酶的性能有着至关重要的影响。纳米复合材料不仅具有纳米尺寸的特性,而且可以克服单一材料的不足,在固定化酶领域引起了广泛关注。本文就目前在固定化酶领域使用的纳米复合载体分类进行了系统的阐述,重点介绍了目前在固定化酶研究领域运用较为广泛的硅基纳米复合材料、碳基纳米复合材料和纳米纤维复合材料等材料的制备方法及不同材料对酶学性能的影响,并对这些纳米复合材料固定化酶发展前景进行了展望。     

纳米复合材料固定化酶的研究进展

载体材料的选择对固定化酶的性能有着至关重要的影响。纳米复合材料不仅具有纳米尺寸的特性,而且可以克服单一材料的不足,在固定化酶领域引起了广泛关注。本文就目前在固定化酶领域使用的纳米复合载体分类进行了系统的阐述,重点介绍了目前在固定化酶研究领域运用较为广泛的硅基纳米复合材料、碳基纳米复合材料和纳米纤维复合材料等材料的制备方法及不同材料对酶学性能的影响,并对这些纳米复合材料固定化酶发展前景进行了展望。     

Enzymes immobilized on montmorillonite: comparison of performance in batch and packed-bed reactors

Summary The enzymes a-amylase, invertase and glucoamylase were immobilized on acid activated montmorillonite using two techniques, viz. adsorption and covalent binding, and their activities were tested in a batch and packed-bed reactor and were compared. The packed-bed reactor showed an improved performance for all immobilized enzymes, which was attributed to lowering of diffusional restrictions to mass transfer. Lower activity in case of batch reactor for immobilized invertase was due to a combined effect of loss of native conformation of enzyme on account of immobilization and mass transfer resistances due to improper diffusion of substrate to the active site of enzyme. For immobilized glucoamylase, the packed-bed reactor demonstrated exceptionally high activity that was very close to the free enzyme. Covalently bound glucoamylase showed higher activity than the free enzyme.     

Immobilization of fumarase and malate dehydrogenase on agarose efficiency of activity as a function of the enzyme ratio

Two enzymes, L-malate dehydrogenase, L-malate: NAD⁺ oxidoreductase (EC 1.1.1.37) and fumarase, L-malate hydrolyase (EC 4.2.1.2) were immobilized on a (Sepharose 4B) resin by the cyanogen bromide method. Studies showed that the matrix-immobilized fumarase retains the same characteristics as the free enzyme, while the matrix-immobilized malate dehydrogenase has reduced activity. The activity of the coupled enzymes is more enzymeconcentration dependent than the free enzymes, and at a ratio of 0.3 (fumarase: malate dehydrogenase) the simultaneously coupled immobilized enzymes become a better catalytic system. Individually immobilized enzymes, mixed to form a coupled system, yielded the poorest catalytic action.     

Development of antimicrobial packaging materials with immobilized glucose oxidase and lysozyme

Packaging based on immobilization of antimicrobial enzymes provides a promising form of active packaging systems applicable in food processing. Glucose oxidase and lysozyme were immobilized by the Ugi reaction with cyclohexyl isocyanide and glutaraldehyde on polyamide and ionomer films partially hydrolysed by hydrochloric acid. The immobilization of the enzymes on the surface of films was confirmed by FT-IR spectroscopy and the films were characterized by the specific activity of the immobilized enzymes. The enzyme migration into model solutions and the effect of pH, temperature and storage time on the activity of immobilized enzyme were also evaluated. Immobilization of lysozyme onto polyamide and ionomer films resulted in the loss of enzyme activity. The polyamide and ionomer films with immobilized glucose oxidase inhibited the growth of bacteria Escherichia coli CNCTC 6859, Pseudomonas fluorescens CNCTC 5793, Lactobacillus helveticus CH-1, Listeria ivanovii CCM 5884 and Listeria innocua CCM 4030 on agar media.      

Selective detection in flow analysis based on the combination of immobilized enzymes and chemically modified electrodes

The combination of immobilized enzymes and amperometry to build selective detection devices in flow-injection analysis and liquid chromatography is described. The pros and cons of enzyme electrodes and of immobilized enzyme reactors are discussed. The paper concentrates on the use of immobilized dehydrogenases, oxidases, peroxidases, and on electrodes on which these enzyme reactions can be selectively followed. The work in the field by the authors is reviewed.     

Stabilization of glucose oxidase fromPenicillium vitale entrapped in high-concentration gels

Glucose ixidase fromPenicillium vitale was immobilized in a 2-hydroxyethyl methacrylate (HEMA) gel containing 0.3 to 2% of methacrylic acid (MAA) or MAA neutralized by allylamine (AA). Depending on the MAA quantity of MAA in the gel, the thermal irreversible inactivation(k _in) constants of the immobilized enzymes sharply decrease at gel concentrations higher than 29 to 50% at pH 5.8. A 220- to 250-fold decrease ofk _in was observed in 60 to 80% gel. The inactivation rate of enzyme in HEMA gel also decreases considerably under the action of urea. Over the range of pH 5.0 to 9.0 thek _in of the native enzyme depends on pH by a degree of 2.1, and of the immobilized enzyme, 0.3 to 0.55. Over the pH range of 5.2 to 5.7,k _in of the native and immobilized enzymes are approximate, whereas at pH 8 and over the difference betweenk _in values exceeds four orders of magnitude. The activity dependence of the immobilized enzyme on pH is shifted two units to the alkaline region. This shift depends on the ionic strength of the solution. This dependence is best reflected in the high gel concentrations. A mechanism of enzymes stabilization in the concentrated HEMA gel is discussed.     

Liposome-based flow injection enzyme immunoassay for theophylline

Preliminary results are presented on the development of a sensitive, quantitative immunoassay based on a regenerable, flow injection analysis system incorporating a double-amplification approach. The double amplification is achieved by means of liposome-encapsulated peroxidase enzyme molecules which are released subsequent to a competitive immunological reaction with analyte molecules for immobilized antibodies. The released peroxidase enzymatically cleaves, from an organofluorine substrate, fluoride ions which are then potentiometrically measured. The entire process is carried out in a flow injection analysis system. The competition between the analyte molecules (theophylline) and theophylline-derivatized liposomes for immobilized antibody sites in flow-through immunoreactor column results in unbound liposomes being carried downstream where they are ruptured in the presence of hydrogen peroxide andp-fluorophenol. The peroxidase molecules released react enzymatically to produce fluoride ions which are measured with an ion-selective electrode. The immunoreactor column is then regenerated with a chaotropic agent and the next sample or calibration solution is injected. By means of column regeneration and calibration, accurate quantitation can be achieved; a feature missing from conventional batch-type immunoassays. By means of this liposome/enzyme double-amplification approach, theophylline was determined over a range of concentrations from 0.2 to 4000 ng/ml. The detection limit of 200 pg/ml corresponds to about 100 femtomole of theophylline measured in the 100 l sample injected.Dedicated to Professor W. Simon on the occasion of his 60th birthday     

Capillary electrophoresis-integrated immobilized enzyme microreactor with graphene oxide as support: Immobilization of negatively charged L-lactate dehydrogenase via hydrophobic interactions

We report the first application of hydrophobic interaction between graphene oxide (GO) and negatively charged enzymes to fabricate CE-integrated immobilized enzyme microreactors (IMERs) by a simple and reliable immobilization procedure based on layer by layer assembly. L -lactate dehydrogenase (L -LDH), which is negatively charged during the enzymatic reaction, is selected as the model enzyme. Various spectroscopic techniques, including SEM, FTIR, and UV-vis are used to characterize the fabricated CE-IMERs, demonstrating the successful immobilization of enzymes on the negatively charged GO layer in the capillary surface. The IMER exhibits excellent repeatability with RSDs of inter-day and batch-to-batch less than 3.49 and 6.37%, respectively, and the activity of immobilized enzymes remains about 90% after five-day usage. The measured K_m values of pyruvate and NADH of the immobilized L -LDH are in good agreement with those obtained by free enzymes. The results demonstrate that the hydrophobic interactions and/or π-π stacking is significant between the GO backbone and the aromatic residues of L -LDH and favorable to fabrication of CE-integrated IMERs. Finally, the method is successfully applied to the determination of pyruvate in beer samples.     

Surface characterization and direct electrochemistry of redox copper centers of bilirubin oxidase from fungi Myrothecium verrucaria

The key characteristics of multicopper oxidases are redox potentials of Type 1, Type 2 and Type 3 copper centers of enzymes. However, there is still a challenge to obtain a value of the redox "signature" of the enzymes. In this study, the electrochemical behavior of T1 and T2/T3 redox copper centers of bilirubin oxidase (BOD) from the fungi Myrothecium verrucaria was studied based on direct bioelectrocatalysis. Two distinct redox peaks corresponding to reduction and oxidation of T1 and T2/T3 redox centers of enzymes have been clearly detected in anaerobic conditions. The bioelectrocatalytic activity of the enzyme was studied in the presence of oxygen and redox mediators. The electron-transfer rate constant for BOD immobilized on carbon electrode (CE) is 1.5 s(-1). The mechanism of enzyme inactivation by ABTS has been proposed. The physical architecture of BOD layers immobilized on the electrode surface, including elemental and chemical composition, relative thickness and assembly of layers was investigated by Angle Resolved X-ray photoelectron spectroscopy. Unique peaks of BOD at 288.5 eV and of CE at 284.6 eV were used in a substrate over layer model for estimation of the thickness of the of BOD film on the carbon electrode surface.     

Immobilized citrate synthase

Kinetic properties of pig heart citrate synthase immobilized on Sepharose were determined. Compared to the free enzyme the K_m values for both acetyl-CoA and oxalacetate were increased. The kinetic pattern of the Lineweaver-Burk plots of both substrates for the immobilized enzyme was that of lines intersecting on thex axis. This is the same as that obtained for the free enzyme and indicates that there is no change in the kinetic mechanism of the reaction. The pH response and the Arrhenius plot of both the immobilized and free enzyme were the same. The enzymes show a break in their Arrhenius plots. The immobilized enzyme exhibits greater heat stability than does the free enzyme.     

Enhanced Properties and Lactose Hydrolysis Efficiencies of Food-Grade β-Galactosidases Immobilized on Various Supports: a Comparative Approach

In this study, a fungal and two yeast β-galactosidases were immobilized using alginate and chitosan. The biochemical parameters and lactose hydrolysis abilities of immobilized enzymes were analyzed. The pH optima of immobilized fungal β-galactosidases shifted to more acidic pH compared to free enzyme. Remarkably, the optimal temperature of chitosan-entrapped yeast enzyme, Maxilact, increased to 60 °C, which is significantly higher than that of the free Maxilact (40 °C) and other immobilized forms. Chitosan-immobilized A. oryzae β-galactosidase showed improved lactose hydrolysis (95.7%) from milk, compared to the free enzyme (82.7%) in 12 h. Chitosan-immobilized Maxilact was the most efficient in lactose removal from milk (100% lactose hydrolysis in 2 h). The immobilized lactases displayed excellent reusability, and chitosan-immobilized Maxilact hydrolyzed >?95% lactose in milk after five reuses. Compared to free enzymes, the immobilized enzymes are more suitable for cost-effective industrial production of low-lactose milk due to improved thermal activity, lactose hydrolysis efficiencies, and reusability.

     

Enzyme immobilization strategies for the design of robust and efficient biocatalysts

Different strategies for the preparation of efficient and robust immobilized biocatalysts are here reviewed. Different physico-chemical approaches are discussed.i.- The stabilization of enzyme by any kind of immobilization on pre-existing porous supports.ii.- The stabilization of enzymes by multipoint covalent attachment on support surfaces.iii.- Additional stabilization of immobilized-stabilized enzyme by physical or chemical modification with polymers.These three strategies can be easily developed when enzymes are immobilized in pre-existing porous supports. In addition to that, these immobilized-stabilized derivatives are optimal to develop enzyme reaction engineering and reactor engineering. Stabilizations ranging between 1000 and 100,000 folds regarding diluted soluble enzymes are here reported.