Biomimetic-dye affinity adsorbents for enzyme purification: application to the one-step purification of Candida boidinii formate dehydrogenase

Formate dehydrogenase (FDH, EC 1.2.1.2) was purified from Candida boidinii cells in a single step by biomimetic-dye affinity chromatography. For this purpose, seven' biomimetic analogues of the monochlorotriazine dye, Cibacron(R) Blue 3GA (CB3GA), and parent dichloro-triazine dye, Vilmafix((R)) Blue A-R (VBAR), bearing a car-boxylated structure as their terminal biomimetic moiety, were immobilized on crosslinked agarose gel, Ultrogel((R)) A6R. The corresponding new biomimetic-dye adsorbents, along with nonbiomimetic adsorbents bearing CB3GA and VBAR, were evaluated for their ability to purify FDH from extracts obtained after press-disintegration of C. boidinii cells. Optimal conditions for maximizing specific activity of FDH in starting extracts (1.8 U/mg) were realized when cell growth was performed on 4% methanol, and press disintegration proceeded in four consecutive passages before the homogenate was left to stand for 1 h (4 degrees C). When compared to nonbiomimetic adsorbents, biomimetic adsorbents exhibited higher purifying ability. Furthermore, one immobilized biomimetic dye, bearing as its terminal biomimetic moiety mercap-topyruvic acid linked on the chlorotriazine ring (BM6), displayed the highest purifying ability. Adsorption equilibrium data which were obtained for the BM6 adsorbent in a batch system corresponded well to the Langmuir isotherm and, in addition, breakthrough curves were taken for protein and FDH adsorption in a fixed bed of BM6 adsorbent. The dissociation constant ( K(D)) of the complex between immobilized BM6 and FDH was found to equal 0.05 muM. Adsorbent BM6 was employed in the purification of FDH from a 18-L culture of C. boidinii in a single step (60% overall yield of FDH). The purified FDH afforded a single-band on sodium dodecyl sulphate poly-acrylamide gel electrophoresis, and a specific activity of 7,0 U/mg (30 degrees C). (c) 1995 John Wiley & Sons, Inc.     

Simultaneous purification of L-malate dehydrogenase and L-lactate dehydrogenase from bovine heart by biomimetic-dye affinity chromatography

Two commercially important enzymes, L-lactate dehydrogenase (LDH) and L-malate dehydrogenase (MDH) were purified simultaneously from bovine heart, on an agarose affinity adsorbent. This adsorbent bears a dye-ligand composed of an anthraquinone chlorotriazine chromophore linked to a biomimetic terminal 4-aminophenyloxanylic acid moiety. The purification protocol exploited the biomimetic affinity adsorbent, in combination with a cross-linked agarose DEAE anion-exchanger. The procedure comprised a preliminary anion-exchange first step, for the separation of the three enzyme activities, mMDH, cMDH and LDH. In the second step, that of affinity chromatography, the unbound mMDH obtained from the first step, was purified by specific elution with NAD⁺/sulphite (22.5-fold purification, 55% step-yield). The procedure afforded mMDH preparation of specific activity approx. 1,300?u/mg (25?°C) at 45% overall yield, free of cytoplasmic MDH, glutamic-oxaloacetic transaminase (GOT) and fumarase. The LDH activity, which, bound to the anion-exchanger during the first step, was recovered from the adsorbent in 200?mM KCl, and finally purified by biomimetic-dye affinity chromatography (NAD⁺/sulphite elution) and a second ion-exchange chromatography step (elution with 200?mM KCl). The LDH preparation exhibited specific activity approx. 500?u/mg at 25?°C (content of impurities: pyruvate kinase and GOT were not detected; MDH, 0.01%).     

Design,synthesis and application of benzyl‐sulfonate biomimetic affinity adsorbents for monoclonal antibody purification from transgenic corn

The human anti‐human immunodeficiency virus (HIV) antibody 2G12 (mAb 2G12) is one of the most broadly neutralizing antibodies against HIV that recognizes a unique epitope on the surface glycoprotein gp120. In the present work, a limited affinity‐ligand library was synthesized and evaluated for its ability to bind and purify recombinant mAb 2G12 expressed in transgenic corn. The affinity ligands were structural fragments of polysulfonate triazine dye Cibacron Blue 3GA (CB3GA) and represent novel lead scaffolds for designing synthetic affinity ligands. Solid phase chemistry was used to synthesize variants of CB3GA lead ligand. One immobilized ligand, bearing 4‐aminobenzyl sulfonic acid (4ABS) linked on two chlorine atoms of the triazine ring (4ABS‐Trz‐4ABS), displayed high affinity for mAb 2G12. Absorption equilibrium, 3D molecular modelling and molecular dynamics simulation studies were carried out to provide a detailed picture of the 4ABS‐Trz‐4ABS interaction with mAb 2G12. This biomimetic affinity ligand was exploited for the development of a facile two‐step purification protocol for mAb 2G12. In the first step of the procedure, mAb 2G12 was purified on an S‐Sepharose FF cation exchanger, and in the second step, mAb 2G12 was purified using affinity chromatography on 4ABS‐Trz‐4ABS affinity adsorbent. Analysis of the antibody preparation by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and enzyme‐linked immunosorbent assay showed that the mAb 2G12 was fully active and of sufficient purity suitable for analytical applications. Copyright © 2013 John Wiley & Sons, Ltd.     

Interaction of L-glutamate oxidase with triazine dyes: selection of ligands for affinity chromatography

Glutamate oxidase (GOX, EC 1.4.3.11) from Streptomyces catalyses the oxidation of L-glutamate to alpha-ketoglutarate. Its kinetic constants for L-glutamate were measured equal to 2 mM for Km and 85.8 s(-1) for kcat. BLAST search and amino acid sequence alignments revealed low homology to other L-amino acid oxidases (18-38%). Threading methodology, homology modeling and CASTp analysis resulted in certain conclusions concerning the structure of catalytic alpha-subunit and led to the prediction of a binding pocket that provides favorable conditions of accommodating negatively charged aromatic ligands, such as sulphonated triazine dyes. Eleven commercial textile dyes and four biomimetic dyes or minodyes, bearing a ketocarboxylated-structure as their terminal biomimetic moiety, immobilized on cross-linked agarose gel. The resulted mini-library of affinity adsorbents was screened for binding and eluting L-glutamate oxidase activity. All but Cibacron Blue 3GA (CB3GA) affinity adsorbents were able to bind GOX at pH 5.6. One immobilized minodye-ligand, bearing as its terminal biomimetic moiety p-aminobenzyloxanylic acid (BM1), displayed the higher affinity for GOX. Kinetic inhibition studies showed that BM1 inhibits GOX in a non-competitive manner with a Ki of 10.5 microM, indicating that the dye-enzyme interaction does not involve the substrate-binding site. Adsorption equilibrium data, obtained from a batch system with BM1 adsorbent, corresponded well to the Freundlich isotherm with a rate constant k of 2.7 mg(1/2)ml(1/2)/g and Freundlich isotherm exponent n of 1. The interaction of GOX with the BM1 adsorbent was further studied with regards to adsorption and elution conditions. The results obtained were exploited in the development of a facile purification protocol for GOX, which led to 335-fold purification in a single step with high enzyme recovery (95%). The present purification procedure is the most efficient reported so far for L-glutamate oxidase.     

Large-scale purification of bovine brain lactate dehydrogenase by affinity chromatography on immobilized colchicine

Lactate dehydrogenase (LDH) [EC 1.1.1.27] in a crude extract (40-80% ammonium sulfate fraction) of bovine brain was adsorbed on an immobilized colchicine column and specifically eluted by addition of 1 mM NADH. The purity and subunit composition of the pooled LDH were estimated by two-dimensional gel electrophoresis. With an increase of NaCl concentration from 0 to 2.0 M, ligand saturation of LDH on immobilized colchicine increased from 6.8 to 14%, whereas that on immobilized Cibacron blue F3GA decreased from 2.1 to 0%. In the presence of high NaCl concentration, immobilized colchicine enabled both large- and small-scale purification of LDH by affinity chromatography and resulted in a yield of 117 mg from 1 kg of bovine brain in the presence of 2.5 M NaCl or higher recoveries of 54-96% from various tissues of one rat in the presence of 1.0 M NaCl. These results indicate that immobilized colchicine is an excellent adsorbent for the isolation and purification of LDH by affinity chromatography and has a high LDH-adsorbing capacity dependent upon a high NaCl concentration. Kinetic studies revealed that colchicine apparently competed with cofactor NAD for the active site of LDH and the Ki values of colchicine decreased with an increase of NaCl concentration. The chemical specificity of the colchicine-binding site of LDH was studied by the use of colchicine analogues and it is concluded that both the tropolone moiety (C-ring) and the amido bond in a side chain of colchicine structure are essential to the colchicine-LDH interaction.     

Galactosyl-mimodye ligands for Pseudomonas fluorescens beta-galactose dehydrogenase.

Protein molecular modelling and ligand docking were employed for the design of anthraquinone galactosyl-biomimetic dye ligands (galactosyl-mimodyes) for the target enzyme galactose dehydrogenase (GaDH). Using appropriate modelling methodology, a GaDH model was build based on a glucose-fructose oxidoreductase (GFO) protein template. Subsequent computational analysis predicted chimaeric mimodye-ligands comprising a NAD-pseudomimetic moiety (anthraquinone diaminobenzosulfonic acid) and a galactosyl-mimetic moiety (2-amino-2-deoxygalactose or shikimic acid) bearing an aliphatic 'linker' molecule. In addition, the designed mimodye ligands had an appropriate in length and chemical nature 'spacer' molecule via which they can be attached onto a chromatographic support without steric clashes upon interaction with GaDH. Following their synthesis, purification and analysis, the ligands were immobilized to agarose. The respective affinity adsorbents, compared to other conventional adsorbents, were shown to be superior affinity chromatography materials for the target enzyme, Pseudomonas fluorescensbeta-galactose dehydrogenase. In addition, these mimodye affinity adsorbents displayed good selectivity, binding low amounts of enzymes other than GaDH. Further immobilized dye-ligands, comprising different linker and/or spacer molecules, or not having a biomimetic moiety, had inferior chromatographic behavior. Therefore, these new mimodyes suggested by computational analysis, are candidates for application in affinity labeling and structural studies as well as for purification of galactose dehydrogenase.     

Biphasic association of T7 RNA polymerase and a nucleotide analogue, cibacron blue as a model to understand the role of initiating nucleotide in the mechanism of enzyme action

T7 RNA polymerase (T7 RNAP) is an enzyme that utilizes ribonucleotides to synthesize the nascent RNA chain in a template-dependent manner. Here we have studied the interaction of T7 RNAP with cibacron blue, an anthraquinone monochlorotriazine dye, its effect on the function of the enzyme and the probable mode of binding of the dye. We have used difference absorption spectroscopy and isothermal titration calorimetry to show that the dye binds T7 RNAP in a biphasic manner. The first phase of the binding is characterized by inactivation of the enzyme. The second binding site overlaps with the common substrate-binding site of the enzyme. We have carried out docking experiment to map the binding site of the dye in the promoter bound protein. Competitive displacement of the dye from the high affinity site by labeled GTP and isothermal titration calorimetry of high affinity GTP bound enzyme with the dye suggests a strong correlation between the high affinity dye binding and the high affinity GTP binding in T7 RNAP reported earlier from our laboratory.     

Probing the nucleotide binding sites in T7 RNA polymerase using cibacron blue

T7 RNA polymerase (T7 RNAP) is an enzyme that utilizes ribonucleotides to synthesize the nascent RNA chain in a template-dependent manner. In this work we have studied the interaction of T7 RNAP with cibacron blue, an anthraquinone monochlorotriazine dye, and its effect on the function of the enzyme. T7 RNAP binds to the dye in a bi-phasic manner. The first phase of the binding is characterized by a high affinity (Kd in the nanomolar range) and reversible inactivation of the enzyme. The second binding site is the common substrate binding site. The association of the dye with T7 RNAP is a good model to understand the physiological significance of a high affinity binding of the initiating nucleotide, GTP, earlier reported from our laboratory. The results will be discussed to understand the role of the high affinity GTP binding.     

Inhibition of NAD(P)H:(quinone-acceptor) oxidoreductase by cibacron blue and related anthraquinone dyes: a structure-activity study.

Cibacron Blue, a widely used ligand for affinity chromatography, is a potent inhibitor of NAD(P)H:(quinone-acceptor) oxidoreductase (EC 1.6.99.2) (quinone reductase). This property has been exploited to purify quinone reductase, to identify its nucleotide-binding site, and to obtain diffraction-grade crystals of this enzyme [Prochaska, H. J. (1988) Arch. Biochem. Biophys. 267, 529-538; Ysern, X., & Prochaska, H. J. (1989) J. Biol. Chem. 264, 7765-7767]. To define the structural region(s) of the dye responsible for its inhibitory potency, Cibacron Blue was synthesized and the dye, its synthetic intermediates, and some analogues of these intermediates were crystallized as novel trialkylamine or choline salts. These compounds were characterized by proton NMR and mass spectrometry, and their inhibitory potencies were measured. Only two of the four ring systems of the Cibacron Blue molecule are required for potent inhibition. Acid Blue 25 [1-amino-4-(phenylamino)anthraquinone-2-sulfonic acid] is an inhibitor (Ki = 22 nM) almost as potent as Cibacron Blue (Ki = 6.2 nM). However, removal of any of the three substituents on the anthraquinone ring of Acid Blue 25 markedly reduced inhibitory potency. These results are consistent with the proposal that Cibacron Blue is primarily a mimic for the ADP fragment of mono- and dinucleotides. The difference absorption spectrum of the Acid Blue 25-quinone reductase complex was very different from that of the complex with Cibacron Blue. In contrast to other compounds tested, Procion Blue M-3GS, the electrophilic dichlorotriazine precursor of Cibacron Blue, was an irreversible inhibitor of quinone reductase (KD = 16 nM, k3 = 0.03 min-1), and the inactivation was blocked by Cibacron Blue, a monochlorotriazine.     

Characterization of specific interactions of coenzymes, regulatory nucleotides and cibacron blue with nucleotide binding domains of enzymes by analytical affinity chromatography

The dissociation constant for the complex of rhodanese and Cibacron Blue, determined by analytical affinity chromatography using rhodanese immobilized on controlled-pore glass (CPG) beads (200 nm pore diameter) and aminohexyl-Cibacron Blue, was 44 microM which agreed well with the kinetic inhibition constant, suggesting that the dye binds at or near the active site of this enzyme. Formation of a binary complex of the dye and lactate dehydrogenase (LDH) was also characterized by direct chromatography of LDH on CPG/immobilized Cibacron Blue (KD = 0.29 microM). The binary complex formed between LDH and NADH was characterized by analytical affinity chromatography using both CPG/immobilized LDH and immobilized Cibacron Blue. Since the dye competes with NADH in binding to the active site of LDH, competitive elution chromatography using the immobilized dye allows determination of the dissociation constant of the soluble LDH.NADH complex. Agreement between the dissociation constants determined by direct chromatography of NADH on immobilized LDH (KD = 1.4 microM) and that determined for the soluble complex (KD = 2.4 microM) indicates that immobilization of LDH did not affect the interaction. Formation of various binary, ternary and quaternary complexes of bovine liver glutamate dehydrogenase (GDH) with glutamate, NADPH, NADH, and ADP was also investigated using immobilized GDH. This approach allows characterization of the enzyme/ligand interactions without the complicating effect of enzyme self-association. The affinity for NADPH is considerably greater in the ternary complex (including glutamate) as compared to the binary complex (0.38 microM vs 22 microM); however, occupancy of the regulatory site by ADP greatly reduces the affinity in both complexes (6.4 microM and 43 microM, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Design and study of peptide-ligand affinity chromatography adsorbents: application to the case of trypsin purification from bovine pancreas

The purification of trypsin from bovine pancreas was employed in a case study concerning the design and optimization of peptide-ligand adsorbents for affinity chromatography. Four purpose-designed tripeptide-ligands were chemically synthesized (>95% pure), exhibiting an Arg residue as their C-terminal (site P(1)) for trypsin bio-recognition, a Pro or Ala in site P(2), and a Thr or Val in site P(3). Each tripeptide-ligand was immobilized via its N-terminal amino group on Ultrogel A6R agarose gel, which was previously activated with low concentrations of cyanuric chloride (10.5 to 42.5 mumol/g gel). Well over 90% of the peptide used was immobilized. Three different concentrations were investigated for every immobilized tripeptide-ligand, 3.5, 7.0, and 14 mumol/g gel. The K(D) values of immobilized tripeptide-trypsin complexes were determined as well as the purifying performance and the trypsin-binding capacity of the affinity adsorbents. The K(D) values determined were in good agreement with the trypsin purification performance of the respective affinity adsorbents. The tripeptide sequence H-TPR-OH displayed the highest affinity for trypsin (K(D) 8.7 muM), whereas the sequence H-TAR-OH displayed the lowest (K(D) 38 muM). Dipeptide-ligands have failed to bind trypsin. When the ligand H-TPR-OH was immobilized via its N-terminal on agarose, at a concentration of 14 mumol/g gel, it produced the most effective affinity chromatography adsorbent. This adsorbent exhibited high trypsin-binding capacity (approximately 310,000 BAEE units/mL of adsorbent); furthermore, it purified trypsin from pancreatic crude extract to a specific activity of 15,200 BAEE units/mg (tenfold purification), and 82% yield. (c) 1997 John Wiley & Sons, Inc.     

Complications encountered using cibacron blue F3G-A as a ligand for affinity precipitation of lactate dehydrogenase

The use of the affinity interaction between Cibacron Blue F3G-A (CB) and NADH-dependent enzymes to selectively precipitate these enzymes has been examined. An attempt was made to form crosslinked precipitates of lactate dehydrogenase (LDH) using bis- and poly-CB conjugates. When precipitation was not observed, an examination of the interaction between the enzyme and the conjugated CB was made. Quasielastic light scattering indicated only a slight radius increase, the greatest being from 50 to 130 A, when a CB-dextran conjugate was added to a solution of LDH, and no increase when bis-CB made with a 1, 6-diaminohexane spacer was added to a similar solution. The results of enzyme inhibition studies showed that conjugated CB bound at the NAD(+) site of LDH. Spectral measurements of the conjugated CB below 5 muM were similar to those reported for a stacking interaction that occurs in solutions with CB concentrations above 5 muM We conclude that the conjugated CB is binding to the LDH, but that a competing dye stacking interaction prevents extensive cross-linking of the LDH, and thus inhibits precipitation.     

Combinatorial de novo design and application of a biomimetic affinity ligand for the purification of human anti‐HIV mAb 4E10 from transgenic tobacco

Monoclonal anti‐HIV antibody 4E10 (mAb 4E10) is one of the most broadly neutralizing antibodies against HIV, directed against a specific epitope on envelope protein gp41. In the present study, a combinatorial de novo design approach was used for the development of a biomimetic ligand for the affinity purification of mAb 4E10 from tobacco transgenic extract in a single chromatographic step. The biomimetic ligand (4E10lig) was based on a L ‐Phe/β‐Ala bi‐substituted 1,3,5‐triazine (Trz) scaffold (β‐Ala‐Trz‐L ‐Phe, 4E10lig) which potentially mimics the more pronounced electrostatic and hydrophobic interactions of mAb 4E10‐binding sequence determined by screening of a random peptide library. This library was comprised of Escherichia coli cells harboring a plasmid (pFlitrx) engineered to express a fusion protein containing random dodecapeptides that were inserted into the active loop of thioredoxin, which itself was inserted into the dispensable region of the flagellin gene. Adsorption equilibrium studies with this biomimetic ligand and mAb 4E10 determined a dissociation constant (K_D) of 0.41 ± 0.05 µM. Molecular modeling studies of the biomimetic ligand revealed that it can potentially occupy the same binding site as the natural binding core peptide epitope. The biomimetic affinity adsorbent was exploited in the development of a facile mAb 4E10 purification protocol, affording mAb 4E10 of high purity (approximately 95%) with good overall yield (60–80%). Analysis of the antibody preparation by SDS‐PAGE, enzyme‐linked immunosorbent assays (ELISA), and western blot showed that the mAb 4E10 was fully active and free of degraded variants, polyphenols, and alkaloids. Copyright © 2009 John Wiley & Sons, Ltd.     

Novel biomimetic affinity ligands for human tissue plasminogen activator

Dyes-based biomimetic affinity chromatography has been used to purify therapeutically useful proteins. In order to design novel biomimetic affinity ligands for purification of tissue-type plasminogen activator (t-PA), small molecular fragments were achieved to fit in S3/4 binding site of t-PA by structure-based ligand design method (InsightII/Ludi). Three biomimetic affinity ligands A, B, and C were then designed, synthesized, and proved to bind the target protein (t-PA), exceeding the binding capacity of the commercial p-amino benzamidine affinity matrix. The designed affinity matrix A showed high efficiency to purify sc-tpa from the crude samples with 18-fold of purification.     

Affinity labeling of annexin VI with a triazine dye, Cibacron blue 3GA. Probable interaction of the dye with C-terminal nucleotide-binding site within the annexin molecule.

Annexin VI (AnxVI) from porcine liver, a member of the annexin family of Ca(2+)- and membrane-binding proteins, has been shown to bind ATP in vitro with a K(d) in the low micromolar concentration range. However, this protein does not contain within its primary structure any ATP-binding consensus motifs found in other nucleotide-binding proteins. In addition, binding of ATP to AnxVI resulted in modulation of AnxVI function, which was accompanied by changes in AnxVI affinity to Ca2+ in the presence of ATP. Using limited proteolytic digestion, purification of protein fragments by affinity chromatography on ATP-agarose, and direct sequencing, the ATP-binding site of AnxVI was located in a C-terminal half of the AnxVI molecule. To further study AnxVI-nucleotide interaction we have employed a functional nucleotide analog, Cibacron blue 3GA (CB3GA), a triazine dye which is commonly used to purify multiple ATP-binding proteins and has been described to modulate their activities. We have observed that AnxVI binds to CB3GA immobilized on agarose in a Ca(2+)-dependent manner. Binding is reversed by EGTA and by ATP and, to a lower extent, by other adenine nucleotides. CB3GA binds to AnxVI also in solution, evoking reversible aggregation of protein molecules, which resembles self-association of AnxVI molecules either in solution or on a membrane surface. Our observations support earlier findings that AnxVI is an ATP-binding protein.     

Affinity extraction of BSA with reversed micellar system composed of unbound Cibacron Blue

Affinity Cibacron Blue 3GA (CB) dye in aqueous phase was directly transferred to the reversed micelles due to electrostatic interaction between anionic CB and cationic cetyltrimethylammonium bromide (CTAB). The bovine serum albumin (BSA) transfer to the reverse micelles increases significantly in a wide range of pH by the addition of a small amount of CB ( approximately 1.0-7.0% of the total surfactant concentration) to the aqueous phase. For pH < pI, the selectivity can be significantly improved with the presence of affinity CB because no BSA was extracted in the absence of CB. For backward extraction of BSA from the micellar phase with stripping aqueous solution, the addition of 2-propanol to the aqueous phase can recover almost all BSA (98.5%) extracted into the reverse micelles.     

Affinity adsorption of lysozyme on a macroligand prepared with Cibacron Blue 3GA attached to yeast cells

The objective of this study was the development of affinity adsorbent particles with the appropriate characteristics to be applied in protein purification using the affinity ultrafiltration method. To prepare affinity macroligands Cibacron Blue 3GA, as a ligand molecule, was immobilized by covalent bonding onto yeast cell walls, the support material or matrix. The maximum attachment of the ligand to the matrix was 212 μmol/g (ligand dry weight/yeast dry weight). Lysozyme was selected as the protein model for the adsorption studies. Its adsorption onto the matrix without ligand and matrix with attached ligand were investigated batch-wise. The adsorption equilibrium isotherms appeared to follow a typical Langmuir isotherm. The maximum adsorption capacity (q(m)) of the Cell-Cibacron macroligand for lysozyme was 110 mg/ml of wet macroligand. The adsorbent was also employed for the separation of lysozyme from hen egg white. High purity lysozyme was obtained.     

One-step purification of Taq DNA polymerase using nucleotide-mimetic affinity chromatography

The thermostable Thermus aquaticus DNA polymerase (Taq Pol) has been the key factor in transforming the initial PCR method into one with huge impact in molecular biology and biotechnology. Therefore, the development of effective affinity adsorbents for the purification of Taq Pol, as well as other DNA polymerases, attracts the attention of the enzyme manufacturers and the research laboratories. In this report we describe a simple protocol for the purification of Taq Pol from E. coli lysates, leading to enzymes of high specific activity and purity. The protocol is based on a single affinity chromatography step, featuring an immobilized ligand selected from a structure-biased combinatorial library of dNTP-mimetic synthetic ligands. The ligand library was screened for its ability to bind and purify Taq Pol from E. coli lysates. One immobilized ligand (mABSGu) of the general formula X-Trz-Y, bearing 9-aminoethylguanine (AEGu) and aniline-2-sulfonic acid (mABS) linked on the triazine scaffold (Trz), displayed the highest purifying ability. Adsorption equilibrium studies with this affinity ligand and Taq Pol determined a dissociation constant (KD) of 0.12 mM for the respective complex, whereas ATP prevented the formation of the mABSGu-Taq Pol complex. The mABSGu affinity adsorbent was exploited in the development of a facile Taq Pol purification protocol, affording homogeneous enzyme (>99% purity, approximately 61 500 U/mg) in a single chromatography step. Quality control tests showed that Taq Pol purified on the mABSGu affinity adsorbent is free of nucleic acids and contaminating nuclease activities.     

Decolorization of the anthraquinone dye Cibacron Blue 3G-A with immobilized Coprinus cinereus in fluidized bed bioreactor

Coprinus cinereus, which was able to decolorize the anthraquinone dye Cibacron Blue 3G-A (CB) enzymatically, was used as a biocatalyst for the decolorization of synthetic solutions containing this reactive dye. Coprinus cinereus was immobilized in both calcium alginate and polyacrylamide gels, and was used for the decolorization of CB from synthetic water by using a fluidized bed bioreactor. The highest specific decolorization rate was obtained when Coprinus cinereus was entrapped in calcium alginate beads, and was of about 3.84 mg g(-1) h(-1) with a 50% conversion time (t1/2) of about 2.60 h. Moreover, immobilized fungal biomass in calcium alginate continuously decolorized CB even after 7 repeated experiments without significant loss of activity, while polyacrylamide-immobilized fungal biomass retained only 67% of its original activity. The effects of some physicochemical parameters such as temperature, pH and dye concentration on decolorization performance of isolated fungal strain were also investigated.     

Adsorption of papain with Cibacron Blue F3GA carrying chitosan-coated nylon affinity membranes

Covalent coupling of chitosan (CS) to activated nylon membrane was performed after the reaction of the microporous nylon membrane with formaldehyde. Non-specific adsorption on the CS-coated nylon membrane decreased greatly, compared with plain nylon membrane. The dye Cibacron Blue F3GA (CB F3GA) as a ligand was then covalently immobilized on the CS-coated membranes. Physical properties of the composite membrane and its applications in affinity membrane chromatography were examined. The contents of CS and CB F3GA-attached membranes were 89.6 mg/g nylon membrane and 146.1 micromol/g nylon membrane, respectively. These CB F3GA-attached composite membranes were used in the papain adsorption studies. Higher papain adsorption capacity, up to 235.3mg/g affinity membrane, was obtained. The adsorption isotherm fitted the Freundlich model well. Significant amount of the adsorbed papain (about 94.3%) was eluted by 1.0M NaSCN at pH 9.0. Experiments on regeneration and dynamic adsorption were also performed. It appears that CB F3GA-CS nylon membranes can be applied for papain separation without causing any denaturation.