The covalent immobilization of microsomal uridine diphospho-glucuronosyltransferase (UDPGT): initial synthesis and characterization of an UDPGT immobilized enzyme reactor for the on-line study of glucuronidation

The microsomal fraction of rat liver containing uridine diphospho-glucuronosyltransferase (UDPGT; EC 2.4.1.17) has been covalently immobilized on a high performance chromatographic support. In this study Nucleosil Si-500 silica was converted into diol-bonded silica and subsequently converted into an aldehyde form through oxidation with sodium periodate. The microsomal fraction was immobilized via Schiff base formation followed by reduction with sodium cyanoborohydride. The resulting immobilized enzyme reactor (IMER) was placed in a multi-dimensional chromatographic system which utilized a mixed mode (C18 and anion exchange) column to trap the parent compound and glucuronide and a C18 column to separate the substrate and product. The IMER system was used for the online glucuronidation of 4-methylumbelliferone (4Me7OHC) and acetaminophen (APAP). The Michaelis-Menten kinetic parameters (Km and Vmax) associated with the formation of 4Me7OHC and APAP glucuronides demonstrated that the immobilization had not significantly affected the enzymatic activity of the UDPGT relative to the non-immobilized enzyme. The IMER retained enzymatic activity for more than 6 weeks. The results of this study demonstrate an easy and convenient way to identify compounds which may be glucuronidated and to synthesize and characterize the resulting products.     

Immobilization of matrix metalloproteinase 8 (MMP-8) for online drug screening

Matrix metalloproteinase 8 (MMP-8) has been reported to have a key role in several pathologic conditions, like heart diseases, osteoarthritis, multiple sclerosis, and various other inflammatory conditions. Therefore, there is a great interest regarding the development of MMP-8 selective inhibitors. In the recent years, immobilized enzyme reactors (IMERs) proved to be an efficient alternative to solution-based assays. Besides the recycling of the enzyme, IMER approach allows a simple way to determine affinity data and thus the ranking of inhibiting potency of the compounds under study, especially when coupled to MS. In this study, the immobilization of MMP-8 was investigated in terms of type of support, kinetic parameters, storage and pH stability. Epoxy activated silica resulted the best matrix for the preparation of an immobilized enzyme reactor (IMER) containing human MMP-8. The IMER was successfully used for the online screening of known MMP-8 inhibitors in zonal chromatography and inhibition experiments.     

Biosensor system for continuous monitoring of organophosphate aerosols

An enzyme-based monitoring system provides the basis for continuous sampling of organophosphate contamination in air. The enzymes butyrylcholinesterase (BuChE) and organophosphate hydrolase (OPH) are stabilized by encapsulation in biomimetic silica nanoparticles, entrained within a packed bed column. The resulting immobilized enzyme reactors (IMERs) were integrated with an impinger-based aerosol sampling system for collection of chemical contaminants in air. The sampling system was operated continuously and organophosphate detection was performed in real-time by single wavelength analysis of enzyme hydrolysis products. The resulting sensor system detects organophosphates based on either enzyme inhibition (of BuChE) or substrate hydrolysis (by OPH). The detection limits of the IMERs for specific organophosphates are presented and discussed. The system proved suitable for detection of a range of organophosphates including paraoxon, demeton-S and malathion.     

Immobilized horse liver alcohol dehydrogenase as an on‐line high‐performance liquid chromatographic enzyme reactor for stereoselective synthesis

Horse liver alcohol dehydrogenase (HLADH) has been non‐covalently immobilized on an immobilized artificial membrane (IAM) high‐performance liquid chromatography (HPLC) stationary phase. The resulting IAM‐HLADH retained the reductive activity of native HLADH as well as the enzyme's enantioselectivity and enantiospecificity. HLADH was also immobilized in an IAM HPLC stationary phase prepacked in a 13 × 4.1 mm ID column to create an immobilized enzyme reactor (HLADH‐IMER). The reactor was connected through a switching valve to a column containing a chiral stationary phase (CSP) based upon p‐methylphenylcarbamate derivatized cellulose (Chiralcel OJR‐CSP). The results from the combined HLADH‐IMER/CSP and chromatographic system demonstrate that the enzyme retained its activity and stereoselectivity after immobilization in the column and that the substrate and products from the enzymatic reduction could be transferred to a second column for analytical or preparative separation. The combined HLADH‐IMER/CSP system is a prototype for the preparative on‐line use of cofactor‐dependent enzymes in large‐scale chiral syntheses. Chirality 11:39–45, 1999. © 1999 Wiley‐Liss, Inc.     

Application of immobilized enzyme reactor in on-line high performance liquid chromatography: a review

This review summarizes all the research efforts in the last decade (1994-2003) that have been spent to the various application of immobilized enzyme reactor (IMER) in on-line high performance liquid chromatography (HPLC). All immobilization procedures including supports, kind of assembly into chromatographic system and methods are described. The effect of immobilization on enzymatic properties and stability of biocatalysts is considered. A brief survey of the main applications of IMER both as pre-column, post-column or column in the chemical, pharmaceutical, clinical and commodities fields is also reported.     

On-line chromatographic screening of matrix metalloproteinase inhibitors using immobilized MMP-9 enzyme reactor

Matrix metalloproteinase 9 (MMP-9) plays an important role in cancer invasion and metastasis and has been an attractive target for therapeutic intervention of cancer metastasis. However, considering the high cost and intricacy associated with the expression, isolation and purification of the recombinant enzyme for the screening of drug candidates, alternative methods that explore the recycling of enzymes become desirable. In this study, a new immobilized enzyme reactor (IMER) containing human recombinant MMP-9 enzyme was developed and characterized for the on-line screening of MMP-9 inhibitors. The MMP-9 IMER containing active unit of the enzyme (U = 0.08 μmol/min) on the disk was inserted into a HPLC system connected to a UV–vis detector for on-line chromatographic screening. The resulting conjugated enzyme was shown to be an active and stable catalyst for the hydrolysis of MMP-9 chromogenic thiopeptide substrate Ac-PLG-[2-mercapto-4-methyl-pentanoyl]-LG-OC₂H₅. The kinetics profile of the enzyme in IMER and free solution were determined and compared. Selected reversible MMP inhibitors, N-isobutyl-N-(4-methoxyphenylsulfonyl)-glycyl hydroxamic acid (NNGH), doxycycline and minocycline were further characterized using the MMP-9 IMER and free enzyme solution assays. Our system demonstrated applicability as a rapid and cost-effective screening tool for inhibitors of the MMP-9 enzyme.     

Efficient proteolysis using a regenerable metal-ion chelate immobilized enzyme reactor supported on organic-inorganic hybrid silica monolith

A metal‐ion chelate immobilized enzyme reactor (IMER) supported on organic–inorganic hybrid silica monolith was developed for rapid digestion of proteins. The monolithic support was in situ prepared in a fused silica capillary via the polycondensation between tetraethoxysilane hydrolytic sol and iminodiacetic acid conjugated glycidoxypropyltrimethoxysilane. After activated by Cu²⁺, trypsin was immobilized onto the monolithic support via metal chelation. Proteolytic capability of such an IMER was evaluated by the digestion of myoglobin and BSA, and the digests were further analyzed by microflow reversed‐phase liquid chromatography with ESI‐MS/MS. Similar sequence coverages of myoglobin and BSA were obtained by IMER, in comparison to those obtained by in‐solution digestion (91 versus 92% for 200 ng myoglobin, and 26 versus 26% for 200 ng BSA). However, the digestion time was shortened from 12 h to 50 s. When the enzymatic activity was decreased after seven runs, the IMER could be easily regenerated by removing Cu²⁺ via EDTA followed by trypsin immobilization with fresh Cu²⁺ introduced, yielding the equal sequence coverage (26% for 200 ng BSA). For ～5 μg rat liver extract, even more proteins were identified with the immobilized trypsin digestion within 150 s in comparison to the in‐solution digestion for 24 h (541 versus 483), demonstrating that the IMER could be a promising tool for efficient and high‐throughput proteome profiling.     

On-line deconjugation of glucuronides using an immobilized enzyme reactor based upon β-glucuronidase

An immobilized enzyme reactor based upon β-glucuronidase (BG–IMER) has been developed for the on-line deconjugation of substrates. The activity of the BG–IMER and its applicability to on-line deconjugation was investigated. The BG–IMER was coupled to a reversed-phase column (C₈ or C₁₈) and the latter column was used to separate substrates and products eluted from the β-glucuronidase reactor. The activity of the BG–IMER was followed by measurement of percent deconjugation and the parameters investigated were: substrate concentration, pH (4 to 6), temperature (r.t., 37°C), enzyme–substrate contact time using flow-rates of 0.1 to 1.0 ml/min (15–1.5 min). The glucuronides used in the evaluation of the BG–IMER were: 4-methylumbelliferyl-β- -glucuronide, p-acetaminophen-β- -glucuronide, 3′-azido-3′-deoxythymidine-β- -glucuronide, phenyl-β- -glucuronide, chloramphenicol-β- -glucuronide, estradiol-17-β- -glucuronide and morphine-β- -glucuronide. The development of on-line HPLC deconjugation of glucuronide substrates using the BG–IMER will facilitate the identification of metabolites and quantification of aglycones in metabolic and pharmacokinetic studies.     

Determination of NG,NG-dimethyl-L-arginine in rat plasma and dimethylarginine dimethylaminohydrolase activity in rat kidney using a monolithic silica column

A fast, simple and sensitive column-switching high-performance liquid chromatography (HPLC)-fluorescence detection method was developed on a monolithic silica column for the determination of N(G),N(G)-dimethyl-L-arginine (ADMA), which is an endogenous nitric oxide synthase inhibitor. After fluorescence derivatization of plasma samples or homogenized tissues with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), the samples were injected into the HPLC system. The NBD-derivatized ADMA was trapped on a cation-exchange column and separated within 15 min on a monolithic silica column. The detection limit for ADMA was 36 nM (250 fmol per injection) when the signal-to-noise ratio was 3. A good linearity for calibration curve for ADMA was observed within the range of 140 nM (1.0 pmol per injection) - 140 microM (1.0 nmol per injection) using N(G)-monomethyl-L-arginine (L-NMMA) as an internal standard. The proposed method was used for the quantitative determination of ADMA in rat plasma. The concentrations of ADMA in rat plasma were 0.82+/-0.05 microM (n=4). Furthermore, the method developed was applied to determine dimethylarginine dimethylaminohydrolase (DDAH) enzyme activity in rat kidney, which was assayed by measuring the amount of ADMA metabolized by the enzyme.     

A preparative method for purification of riboflavin 5'-monophosphate.

A preparative column chromatography method was developed for preparation of pure riboflavin 5′-monophosphate. A crude preparation of riboflavin phosphate(s) was chromatographed on DEAE-cellulose to provide a mixture of riboflavin 4′- and 5′-monophosphates. The 5′-isomer was isolated by chromatography on a column of silica gel with an ethanol:1 m triethylammonium bicarbonate, pH 7.5 (85:15) solvent system.     

Immobilization of Candida antarctica Lipase B on fumed silica

Enzymes are usually immobilized on solid supports or solubilized when they are to be used in organic solvents with poor enzyme solubility. We have reported previously on a novel immobilization method for subtilisin Carlsberg on fumed silica with results that reached some of the best previously reported catalytic activities in hexane for this enzyme. Here we extend our method to Candida antarctica Lipase B (CALB) as an attractive target due to many potential applications of this enzyme in solvents. Our CALB/fumed silica preparations approached the catalytic activity of commercial Novozym 435 for a model esterification in hexane at 90 wt.% fumed silica (relative to the mass of the preparation). An intriguing observation was that the catalytic activity at first increases as more fumed silica was made available to the enzyme but then decreased precipitously when fumed silica exceeded 90 wt.%. This was not the case for s. Carlsberg where the catalytic activity leveled off at high relative amounts of fumed silica. We determined adsorption kinetics, performed variations of the pre-immobilization aqueous pH, determined the stability, and applied fluorescence microscopy to the preparations. A comparison with recent concepts by Gross et al. may point towards a rationale for an optimum intermediate surface coverage for some enzymes on solid supports.     

Acyl-coenzyme A: cholesterol acyltransferase assay: silica gel column separation of reaction products

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) assays are usually performed by incubation of the enzyme with a labeled substrate followed by thin-layer chromatography separation and subsequent quantification of cholesteryl esters (CE) formed. Herein, a method is described for rapid separation of CE from other lipids, by elution from a silica gel column with a solvent mixture of petroleum ether/diethyl ether (98:2, v/v). Silica gel column chromatography is reliable and more rapid and safer than TLC. The best results were obtained when the reaction was stopped by Dole extraction followed by CE separation on a silica gel column. Assays for ACAT from rat intestinal microsomes showed that the specific activity values obtained using this method were reproducible and in good agreement with those obtained by conventional TLC method.     

Rapid synthesis of long-chain deoxyribooligonucleotides by the N-methylimidazolide phosphotriester method.

A modified phosphotriester method has been employed for the efficient chemical synthesis of long-chain deoxyribooligonucleotides. During the course of this work, a general and rapid procedure was developed for the preparation of 24-62-mers in solution. Preparative reversed phase column chromatography on silanized silica gel was used to purify triester intermediates starting from 10-mers. The rapid synthesis of 32-mer and 42-mer on glass and silica gel supports using suitably protected 2-8-mer blocks as coupling units has been also accomplished. In particular, a convenient procedure for the solid-phase synthesis of oligonucleotide blocks bearing 3'-terminal phosphodiester groups is described.     

Perfusion chromatography separation of the tomato fruit-specific pectin methylesterase from a semipurified commercial enzyme preparation

A rapid and simple method was developed, using perfusion chromatography media, to separate the fruit-specific pectin methylesterase (PME) isoform from the depolymerizing enzyme polygalacturonase (PG) and other contaminating pectinases present in a commercial tomato enzyme preparation. Pectinase activities were adsorbed onto a Poros HS (a strong cation exchanger) column in 20 M HEPES buffer at pH 7.5. The fruit-specific PME was eluted from the column with 80 mM NaCl, followed by a step to 300 mM NaCl to elute PG activity. Rechromatography of the PME activity peak with a linear gradient further resolved two PME isoenzymes and removed residual traces of PG activity. The PG activity peak was further treated with lectin affinity chromatography to provide purified PG enzyme, which was separated from a salt-dependent PME (tentatively identified as a "ubiquitous-type" isoform), and a pectin acetylesterase. The later enzyme has not been reported previously in tomato. This method provides monocomponent enzymes that will be useful for studying enzyme mechanisms and for modifying pectin structure and functional properties.     

Purification of Adenosine Deaminase from Chicken-Egg Yolk by Affinity Column Chromatography

Adenosine deaminase (adenosine aminohydrolase; E.C. 3.5.4.4) has been purified 4686-fold from egg yolk. The procedure developed was used to isolate the enzyme from eight chicken eggs. An easily prepared affinity column employing purine riboside was used as the final step in the purification. The method developed permits the rapid isolation and a high recovery of the protein. The specific activity of the enzyme preparation obtained is 81.4 mU/mg.     

Purification of adenosine deaminase from chicken-egg yolk by affinity column chromatography

Adenosine deaminase (adenosine aminohydrolase; E.C. 3.5.4.4) has been purified 4686-fold from egg yolk. The procedure developed was used to isolate the enzyme from eight chicken eggs. An easily prepared affinity column employing purine riboside was used as the final step in the purification. The method developed permits the rapid isolation and a high recovery of the protein. The specific activity of the enzyme preparation obtained is 81.4 mU/mg.     

Determination of inhibitors’ potency (IC50) by a direct high-performance liquid chromatographic method on an immobilised acetylcholinesterase column

An immobilised acetylcholinesterase (AChE) stationary phase was prepared by using an in situ AChE immobilisation procedure. A stainless steel column packed with epoxide silica was connected to the HPLC system and the enzyme solution at pH 5.8 was recycled through the column at a flow-rate of 0.5 ml/min for 24 h. The activity of the immobilised AChE was determined by injecting the substrate acetylthiocholine, using as mobile phase 0.1 M phosphate buffer (pH 7.4) containing Ellman’s reagent [5,5′-dithio-bis(2-nitrobenzoic acid)] and measuring the area of the obtained peak with UV detection at 412 nm. The effect of AChE inhibitors tacrine, edrophonium and donepezil were evaluated by the simultaneous injection of each inhibitor with the substrate. The resulting decrease in the AChE activity, as expressed by the decrease of the peak area detected at 412 nm, was related to the concentration and potency of the solutes. The obtained IC₅₀ values were compared with those derived by the conventional spectrophotometric method. This immobilized enzyme reactor, included in a chromatographic system, can be used for the rapid screening for new inhibitors allowing for the on-line determination of a compound’s inhibitory potency. The advantages over the conventional methods are the increased enzyme stability and system automation which allows a large number of compounds to be analysed continuously.     

Butyrylcholinesterase in the visual ganglia of the squid todarodes sagittatus I. (cephalopoda). isolation,molecular forms,interaction with substrates and inhibitors

1. Soluble butyrylcholinesterase (BuChE) was isolated from the visual ganglia of the squid Todarodes sagittatus L. Gel-chromatography on Sephadex G-200 columns resulted in its separation into three molecular forms.2. The major component with a molecular mass of 180kDa was used for kinetic study.3. The substrate analysis revealed squid enzyme to be BuChE of unusual type.4. Unlike typical BuChE (EC 3.1.1.8), squid enzyme splits acetyl-β-methylcholine (AMCh) with a relatively high rate, alongside with common BuChE substrates—butyrylcholine (BCh), propionylcholine (PCh), acetylcholine (ACh), butyrylthiocholine (BTCh) and acetylthiocholine (ATCh), the enzymic hydrolysis being suppressed by excess of all these substrates.5. Among them, the highest values of k_{cat and}k_cat/K_m were found for BCh and BTCh. Maximal activity of the enzyme was noticed at low BCh and BTCh concentrations (1–2 mM).6. Tetraalkylammonium ions exhibit a mixed type of inhibition and suppress the substrate inhibition of squid BuChE.7. Among organophosphorus inhibitors (OPI), the methylthiophosphonates are most potent for squid BuChE, and for some phosphates, selective OPI of typical BuChE, are potent as well.8. By the pattern of selectivity to OPI, squid enzyme differs from both typical BuChE of horse serum and acetylcholinesterase (EC 3.1.1.7) from bovine erythrocytes.9. Some details of the active center structure of squid BuChE compared to that of typical enzymes are discussed.     

Plant aspartate transcarbamylase: an affinity chromatographic method for the purification of the enzyme from germinated seedings.

A simple and rapid affinity chromatographic method for the isolation of aspartate transcarbamylase from germinated seedlings of mung bean (Phaseolus aureus) was developed. A partially purified preparation of the enzyme was chromatographed on an affinity column containing aspartate linked to CNBr-activated Sepharose 4B. Aspartate transcarbamylase was specifically eluted from the column with 10 mm aspartate or 0.5 m KCl. The enzyme migrated as a single sharp band during disc electrophoresis at pH 8.6 on polyacrylamide gels. Electrophoresis of the sodium dodecyl sulfate-treated enzyme showed two distinct protein bands, suggesting that the mung bean aspartate transcarbamylase was made up of nonidentical subunits. Like the enzyme purified by conventional procedures, this enzyme preparation also exhibited positive homotropic interactions with carbamyl phosphate and negative heterotropic interactions with UMP. This method was extended to the purification of aspartate transcarbamylase from Lathyrus sativus, Eleucine coracona, and Trigonella foenum graecum.