Immobilization of enzyme on detonation nanodiamond for highly efficient proteolysis

Immobilization of enzyme on detonation nanodiamond (dND, 3-10 nm) and its application for efficient proteolysis have been demonstrated. By evaluation of the Michaelis constant (K_m) and maximum velocity (V_max) of immobilized enzyme, its activity was not impaired significantly by immobilization. And enzyme immobilized on dNDs exhibited much better thermal and chemical stabilities than its free counterpart and maintained high activity even after 10 times reuse. The efficient proteolysis by trypsin immobilized on dNDs (dND-trypsin) is demonstrated with the digestion of myoglobin (or other model protein) in a short time (5 min). Large numbers of identified peptides obtained by dNDs-trypsin enable a higher degree of sequence coverage and more positive identification of proteins than those obtained by in-solution digestion and the commercial immobilized trypsin beads, respectively. Moreover, immobilization of peptide-N-glycosidase F (PNGase F) on dNDs was realized and resulted in faster sequential glycosidase digestion of glycopeptides in less than 10 min.     

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。     

Analytical applications of reactors containing immobilized enzymes

The theory for analytical packed-enzyme reactors is discussed and it is shown that a 100% conversion efficiency gives many advantages. This concept has been applied to methods for substrate determinations of urea, amino acids, and glucose. Enzyme reactors have also been used in the effluent from a Chromatographic column to enhance selectivity and sensitivity for cholesterol. Enzyme reactors for the determination of inhibitors, e.g. mercury ions, should be designed differently. A low conversion efficiency gives high sensitivity and a linear response.     

Recent advances in immobilized enzymatic reactors and their applications in proteome analysis

Immobilized enzymatic reactors recently have drawn much attention because of the striking advantages, such as high substrate turnover rate and ease in coupling with the separation and detection systems. Carrier materials, which have great effects on the development of the immobilized enzymatic reactors, have always being the focus of study. In this paper, the contributions, mainly in the last 5 years, on the enzymatic reactors and their applications in proteome study are reviewed, with some newly developed inorganic and organic carriers for enzyme immobilization described in details. Moreover, the hyphenation of immobilized enzymatic reactors with the separation and identification systems is also summarized. By reviewing these achievements, it could be seen that enzymatic reactors have very bright future, especially in proteome analysis.     

Immobilization of trypsin on silica-coated fiberglass core in microchip for highly efficient proteolysis

In this report, trypsin was immobilized on silica-coated fiberglass core in microchip to form a core-changeable bioreactor for highly efficient proteolysis. To prepare the fiber core, a layer of organic-inorganic hybrid silica coating was prepared on the surface of a piece of glass fiber by a sol-gel method with tetraethoxysilane (TEOS) and 3-aminopropyltriethoxysilane (APTES) as precursors. Subsequently, trypsin was immobilized on the coating with the aid of glutaraldehyde. Prior to use, the enzyme-immobilized fiber was inserted into the channel of a microchip to form an in-channel fiber bioreactor. The novel bioreactor can be regenerated by changing its fiber core. The scanning electron microscopy images of the cross-section of a trypsin-immobilized fiber indicated that a layer of ∼1 μm thick film formed on the glass substrate. The feasibility and performance of the unique bioreactor were demonstrated by the tryptic digestion of bovine serum albumin (BSA) and cytochrome c (Cyt-c) and the digestion time was significantly reduced to less than 10 s. The digests were identified by MALDI-TOF MS with sequence coverages of 45% (BSA) and 77% (Cyt-c) that were comparable to those obtained by 12-h conventional in-solution tryptic digestion. The fiber-based microchip bioreactor provides a promising platform for the high-throughput protein identification.     

Immobilized enzyme reactors in proteomics

Fast, efficient characterization of proteins is becoming one of the hottest topics in the bioanalytical community, especially for large-scale proteomic studies. As an attractive approach, protein digestion by enzymes supported on various matrices (referred to as immobilized enzyme reactors, IMERs) has recently attracted much attention.In this article, we present a critical overview of some highly efficient IMERs and related analytical systems. We give major coverage to applications of IMERs in proteomic analysis, including protein-expression profiling, characterization of proteins with post-translational modifications, and protein quantification. We also comment on promising trends for IMERs in proteomics.     

Hydrophilic monolith based immobilized enzyme reactors in capillary and on microchip for high-throughput proteomic analysis

A novel kind of hydrophilic monolith based immobilized enzyme reactors (IMERs) was prepared both in UV-transparent capillaries and on glass microchips by the photopolymerization of N-acryloxysuccinimide and poly(ethylene glycol)diacrylate, followed by trypsin immobilization. The performance of capillary IMERs for protein digestion was evaluated by the digestion of myoglobin with the residential time from 12s to 71 s. With μRPLC-ESI-MS/MS analysis, the obtained sequence coverages were all over 80%, comparable to that obtained by in-solution digestion for 12 h. The nonspecific absorption of BSA on monolithic support was evaluated, and no obvious protein residue was observed by a fluorescence assay. Moreover, no carry-over of the digests on the capillary IMER was found after the digestion of myoglobin (24 μg) and BSA (9 μg), which further demonstrated the good hydrophilicity of such matrix. In addition, an integrated microchip-based system involving on-line protein digestion by microchip-based IMER, peptides separation by nanoRPLC and identification by ESI-MS/MS was established, by which a mixture of standard proteins and one RPLC fraction of Escherichia coli extract were successfully identified, indicating that the hydrophilic monolith based IMER might provide a promising tool for high-throughput proteomic analysis.     

Investigation of micro-immobilised enzyme reactors containing endoglucanases for efficient hydrolysis of cellodextrins and cellulose derivatives

Cellulases hydrolysing the interior parts of cellulose, also called endoglucanases, were immobilised in micro-immobilised enzyme reactors (μIMER) made of porous silicon with the purpose of investigating the use of such μIMERs for hydrolysis of cellodextrins and soluble cellulose derivatives. The endoglucanases Trichoderma reesei Cel 12A (TrCel 12A) and Bacillus agaradhaerens Cel 5A (BaCel 5A) were covalently coupled to the surface of a silicon microchip through Schiff base formation. For characterisation cellohexaose was used as substrate for the immobilised enzymes. The characteristics of the μIMER were investigated by studying the product formation when varying the concentration, flow-rate, temperature and pH of the substrate solution. Hydrolysis was performed in the μIMER connected on-line to a chromatographic system, where the products were separated and detected using high-performance anion exchange chromatography (HPAEC) coupled to pulsed amperometric detection (PAD). A comparison of the hydrolytic pattern between BaCel 5A and TrCel 12A was carried out and the results show that the two investigated endoglucanases give specific hydrolytic patterns in the products formed that provide important information about the enzymes. The μIMERs are robust and can be employed continuously over a period of at least several days. Moreover, on appropriate storage, no activity loss is seen after 60 days. The ability of the BaCel 5A containing μIMER to perform hydrolysis of derivatised cellulose was also investigated using carboxymethyl cellulose (CMC) as substrate. Separation and detection were carried out using size exclusion chromatography (SEC) with refractive index detection (RI). The results show that the μIMERs are robust and can be employed for on-line hydrolysis of both cellodextrins and derivatised cellulose of high molecular weight.     

A multimembrane enzyme reactor operating in an electric field

Summary A multimembrane enzyme reactor, operating under an electric field, is described. This reactor is based on the concept of isoelectric traps, by which a pair of membranes, of appropriate pI value, are able to trap the enzyme, the buffering ions (which have to be amphoteric), the substrate and, if needed, cofactors. Charged reaction products can thus leave the enzyme chamber and be recovered in other chambers where they are trapped by the same isoelectric mechanism (if amphoteric). The unique advantages of such reactors are absence of enzyme inhibition by a feed-back mechanism and reaction yields approaching 100%. Examples of a urease and a trypsin/casein reactor are given. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996     

Novel monolithic enzymatic microreactor based on single-enzyme nanoparticles for highly efficient proteolysis and its application in multidimensional liquid chromatography

In this work, a novel and facile monolithic enzymatic microreactor was prepared in the fused-silica capillary via a two-step procedure including surface acryloylation and in situ aqueous polymerization/immobilization to encapsulate a single enzyme, and its application to fast protein digestion through a direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF-MS) analysis was demonstrated. At first, vinyl groups on the protein surface were generated by a mild acryloylation with N-acryloxysuccinimide in alkali buffer. Then, acryloylated enzyme was encapsulated into polyacrylates by free-radical copolymerization with acrylamide as the monomer, N,N′-methylenebisacrylamide as the cross-linker, and N,N,N′,N′-tetramethylethylenediamine/ammonium persulfate as the initiator. Finally, polymers were immobilized onto the activated inner wall of capillaries via the reaction of vinyl groups. Capability of the enzyme-immobilized monolithic microreactor was demonstrated by myoglobin and bovine serum albumin as model proteins. The digestion products were characterized using MALDI-TOF-MS with sequence coverage of 94% and 29% observed. This microreactor was also applied to the analysis of fractions through two-dimensional separation of weak anion exchange/reversed-phase liquid chromatography of human liver extract. After a database search, 16 unique peptides corresponding to 3 proteins were identified when two RPLC fractions of human liver extract were digested by the microreactor. This opens a route for its future application in top–down proteomic analysis.     

纳米花型酶-无机杂化固定化酶研究进展

酶是一种绿色高效的生物催化剂,被广泛应用于工业生产中,为了更好地提升游离酶的性能,酶固定化技术应运而生.然而,与游离酶相比,固定化酶活性下降以及传质受限一直是酶固定化技术亟待解决的关键问题.作为一种新型酶固定化技术,纳米花型酶-无机杂化固定化酶因具有高比表面积、高酶活性和高催化效率且制备过程简单、绿色无污染而受到广泛关...     

Flow‐through immobilized enzyme reactors based on monoliths: II. Kinetics study and application

In the last decade, the application of monolithic materials has rapidly expanded to the realization of flow‐through bioconversion processes. Up to these days, different classes of enzymes such as hydrolases, lyases, and oxidoreductases have been immobilized on organic, inorganic, or hybrid monolithic materials to prepare the effective flow‐through enzymes reactors for application in proteomics, biotechnology, pharmaceutics, organic synthesis, and biosensoring. Current review describes the results of kinetic study and specialties of flow‐through immobilized enzyme reactors based on the existing monolithic materials.     

Determination of various alcohols based on a new immobilized enzyme fluorescence capillary analysis

A novel method for the determination of ethanol in tequila based on the immobilized enzyme fluorescence capillary analysis (IE-EFCA) has been proposed. Alcohol dehydrogenase (ADH) was immobilized in inner surface of a capillary and an immobilized enzyme capillary bioreactor (IE-ECBR) was formed. After nicotinamide adenine dinucleotide (NAD⁺) as an oxidizer is mixed with alcohol sample solution, it was sucked into the IE-ECBR. The fluorescence intensity of the mixed solution in the IE-ECBR was detected at λ_ex = 350 nm and λ_em = 459 nm. The experimental conditions are as follows: The reaction time is 20 min; temperature is 40 °C; the concentrations of phosphate buffer solution (pH 7.5) and NAD⁺ are 0.1 mol L⁻¹ and 5 mmol L⁻¹, respectively; immobilization concentration of ADH is 10 U L⁻¹. The determination range of ethanol is 2.0-15.0 g L⁻¹ (F = 10.44C + 6.6002, r > 0.9958); its detection limit is 1.11 g L⁻¹; and relative standard deviation is 1.9%. IE-EFCA method is applicable for the determination of the samples containing alcohol in medicine, industry and environment.     

Flow-injection method for the determination of serine using immobilized enzyme

A flow-injection method for the determination of serine using a mini-column containing immobilized serine dehydratase isolated and purified from rat liver is described. Ammonia produced from the enzymatic reaction is coupled with hypochlorite and phenol in an alkaline medium yields a blue product due to the indophenol anion formation, which is the basis of a spectrophotometric detection at 640 nm. The limit of detection (2×blank noise) is 0.01 mM with a sample throughput of 25 h⁻¹. Calibration graph is linear in the range 0.2–1.0 mM, with relative standard deviation 0.6–1.0%.     

Rapid measurement of fish freshness indices by an amperometric flow-injection system with a 16-way switching valve and immobilized enzyme reactors

A novel flow-injection system is proposed for the rapid measurement of the fish freshness indices K₁ and K₂: K₁=[([HxR+[Hx])×100/([IMP]+[HxR]+[Hx])] and K₂=[[Hx]×100/([HxR]+[Hx])], where [IMP], [HxR] and [Hx] are inosine-5′-monophosphate, inosine and hypoxanthine concentrations, respectively. For the estimation of index K₁, 5′-nucleotidase immobilized reactor and nucleoside phosphorylase (NP)/xanthine oxidase (XO) coimmobilized reactor were incorporated in series in the flow-injection line made up by a 16-way switching valve with two sample loops. For the estimation of index K₂, NP and XO immobilized reactors were also incorporated in the similar flow-line. Two sample portions passed through the flow-line with different residence times so that two peaks were obtained. The first and second peaks obtained in the K₁-determining system corresponded to the total of HxR and Hx and the total of Hx, HxR and IMP, respectively. Similarly, the first and second peaks obtained in the K₂-determining system corresponded to Hx and the total of Hx and HxR, respectively. Therefore, the indices K₁ and K₂ can be estimated by

where i₁ and i₂ present the peak current of the first and second peaks, respectively, and f the ratio of the peak currents of the first and second peaks for a Hx standard solution. A sea bream was selected as a model fish and it was stored at 4 °C after death. A good correlation was found between the index K₁ and the storage time over a period of 400 h, with a correlation coefficient of 0.992, but no correlation between the index K₂ and the storage time. The measurements could be performed at a rate of 22 samples per hour with satisfactory precision (0.6–1.2% R.S.D.), without calibration for each species, accurate weighing of fish meat and any interferences in fish meat.     

Disparities between immobilized enzyme and solution based digestion of transferrin with trypsin

Trypsin digestion is a major component of preparing proteins for peptide based identification and quantification by mass spectral (MS) analysis. Surprisingly proteolysis is the slowest part of the proteomics process by an order of magnitude. Numerous recent efforts to reduce protein digestion to a few minutes have centered on the use of an immobilized enzyme reactor (IMER) to minimize both trypsin autolysis and vastly increase the trypsin to protein ratio. A central question in this approach is whether proteolysis with an IMER produces the same peptide cleavage products as derived from solution based digestion. The studies reported here examined this question with transferrin; a model protein of known resistance to trypsin digestion. Results from these studies confirmed that a trypsin‐IMER can in fact digest transferrin in a few minutes; providing tryptic peptides that subsequent to MS analysis allow sequence identification equivalent to solution digestion. Although many of the peptides obtained from these two trypsin digestion systems were identical, many were not. The greatest difference was that the trypsin‐ IMER produces (i) numerous peptides bearing multiple lysine and/or arginine residues and (ii) identical portions of the protein sequence were found in multiple peptides. Most of these peptides were derived from five regions in transferrin. These results were interpreted to mean that proteolysis in the case of transferrin occurred faster than the rate at which buried lysine and arginine residues were unmasked in the five regions providing peptides that were only partially digested.     

High temporal resolution monitoring of enzyme reaction and inhibition using optically gated vacancy capillary electrophoresis and immobilized enzyme

A novel method for monitoring of enzyme reaction and inhibition with high temporal resolution was developed by using optically gated vacancy capillary electrophoresis (OGVCE) with laser-induced fluorescence (LIF) detection and immobilized enzyme. Trypsin cleavage reaction and inhibition were investigated by the presented OGVCE-LIF assay, using carboxyfluorescein (FAM) end-labeled Angiotensin as the substrate and commercially available immobilized trypsin. The substrate and the product were continuously loaded into the capillary by the electroosmotic flow while the immobilized enzyme remained in the sample vial. Substrate consumption and product formation were monitored simultaneously at 5 s interval during the whole reaction time. The enzymatic reaction rates obtained from the substrate and the product were highly consistent. The enzyme activity and the Michaelis constants of trypsin cleavage reaction, as well as the inhibition constant (for reversible competitive inhibitor) and the inhibition fraction (for irreversible inhibitor), were obtained. It was showed that the reported OGVCE-LIF method can perform fast, accurate, sensitive and reproducible CE enzyme assay with high temporal resolution, thus has great potential in application of the enzyme-substrate systems with fast reaction rate and the fluorescent substrate and products.     

Immobilized‐enzyme reactors integrated with capillary electrophoresis for pharmaceutical research

Enzymes play an essential role in many aspects of pharmaceutical research as drug targets, drug metabolizers, enzyme drugs and more. In this specific field, enzyme assays are required to meet a number of specific requirements, such as low cost, easy automation, and high reliability. The integration of an immobilized‐enzyme reactor to capillary electrophoresis represents a unique approach to fulfilling these criteria by combining the benefits of enzyme immobilization, that is, increased stability and repeated use, as well as the minute sample consumption, short analysis time, and efficient analysis provided by capillary electrophoresis. In this review, we summarize, analyze, and discuss published works where pharmaceutically relevant enzymes were used to prepare capillary electrophoresis‐integrated immobilized‐enzyme reactors in an online manner. The presented assays are divided into three distinct groups based on the drug–enzyme relationship. The first, more extensively studied group employs enzymes that are considered to be therapeutic targets, the second group of assays present tools to assess drug metabolism and the third group assesses enzyme drugs. Furthermore, we examine various methods of enzyme immobilization and their implications for assay properties.