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Free Enzyme (free + enzyme)
Selected AbstractsKINETIC BEHAVIOR OF SOYBEAN LIPOXYGENASE: A COMPARATIVE STUDY OF THE FREE ENZYME AND THE ENZYME IMMOBILIZED IN AN ALGINATE SILICA SOL-GEL MATRIX,JOURNAL OF FOOD BIOCHEMISTRY, Issue 1 2000AN-FEI HSU Lipoxygenase (LOX) is an enzyme that regioselectively introduces a hydroperoxide into polyunsaturated fatty acids (PUFA). We recently reported a procedure that immobilizes soybean LOX within an alginate sol-gel matrix. In this study, the kinetic profile of free LOX was compared with that of the sol-gel immobilized LOX. The temperature dependent activity profile of free LOX was optimal at 25C whereas immobilized LOX had optimal activity over the temperature range of 25,35C. Enzyme activity, measured in aqueous buffer, for both the free and immobilized LOX preparations had Km values of 2.5 and 1.40 mmoles/L, respectively, and Vmax values of 0.056 and 0.02 ,mol/min, respectively. The relative rates of oxidation of linoleic acid and acylgfycerols containing linoleoyl residues catalyzed by free and immobilized LOX also were determined The results showed that both free and immobilized LOX favor linoleic acid as a substrate. Relative substrate preference for free LOX was linoleic acid >1-monolinolein > 1,3-dilinolein >trilinolein, and for immobilized LOX was linoleic acid >l, 3-dilinolein >1-monolinolein >trilinolein. In general, LOX immobilized in alginate silica sol-gel matrix retained the physical and chemical characteristics of free LOX. [source] Chitosan-Glutamate Oxidase Gels: Synthesis, Characterization, and Glutamate DeterminationELECTROANALYSIS, Issue 23 2005Maogen Zhang Abstract The biopolymer chitosan (CHIT) was chemically modified with glutaric dialdehyde (GDI) and used for the covalent immobilization of enzyme glutamate oxidase (GmOx). The relationships between the loaded, retained, and active units of GmOx in the CHIT-GDI-GmOx gels were determined by electrochemical assays. The latter indicated that on average ca. 95% of the GmOx was retained in the CHIT-GDI matrix that was loaded with 0.10,3.0 units of the enzyme. The maximum activity of the GmOx immobilized in the gels corresponded to ca. 5% of the activity of the free enzyme. Platinum electrodes coated with CHIT-GDI-GmOx gels (films) were used as amperometric biosensors for glutamate. Such biosensors displayed good operational and long-term stability (at least 11,h and 100 days, respectively) in conjunction with low detection limit of 0.10,,M glutamate (S/N=3), linear range up to 0.5,mM (R2=0.991), sensitivity of 100 mA M,1 cm,2, and short response time (t90%=2,s). This demonstrated an efficient signal transduction in the Pt/CHIT-GDI-GmOx+glutamate system. The CHIT-GDI-GmOx gels constitute a new biosensing element for the development of glutamate biosensors. [source] Capillary electrophoresis versus differential scanning calorimetry for the analysis of free enzyme versus enzyme-ligand complexes: In the search of the ligand-free status of cholinesterasesELECTROPHORESIS, Issue 2 2006Daniel Rochu Dr. Abstract Cholinesterases (ChEs) are highly efficient biocatalysts whose active site is buried in a deep, narrow gorge. The talent of CE to discover inhibitors in the gorge of highly purified preparations has fairly altered the meaning of a ChE ligand-free status. To attempt at a description of this one, we investigated the stability of Bungarus fasciatus acetylcholinesterase (AChE), alone or complexed with different inhibitors. Determination of midtransition temperature for thermal denaturation, using differential scanning calorimetry (DSC) and CE, provided conflicting results. Discrepancies strongly question the reality of a ligand-free AChE state. DSC allowed estimation of the stability of AChE-ligands complexes, and to rank the stabilizing effect of different inhibitors. CE acted as a detector of hidden ligands, provided that they were charged, reversibly bound, and thus dissociable upon action of electric fields. Then, CE allowed quantification of the stability of ligand-free AChE. CE and DSC providing each fractional and nonredundant information, cautious attention must be paid for actual estimation of the conformational stability of ChEs. Because inhibitors used in purification of ChEs by affinity chromatography are charged, CE remains a leading method to estimate enzyme stability and detect the presence of bound hidden ligands. [source] Synthesis and Characterization of Thrombin Conjugated ,-Fe2O3 Magnetic Nanoparticles for HemostasisADVANCED ENGINEERING MATERIALS, Issue 12 2009Ofra Ziv Abstract Thrombin is the final protease produced in the clotting pathways. Thrombin has been used in the clinic more than six decades for topical hemostasis and wound management. In human plasma the half-life of thrombin is shorter than 15 seconds due to close control by inhibitors. In order to stabilize thrombin, this enzyme was conjugated covalently and physically to ,-Fe2O3 magnetic nanoparticles. The physical conjugation was accomplished through adsorption of thrombin to BSA coating on the nanoparticles. The coagulant activity of the covalently bound thrombin was significantly lower than that of the physically adsorbed thrombin. Leakage of the physically bound thrombin into PBS containing 4% HSA was negligible. The physical conjugation of thrombin onto the nanoparticles stabilized the thrombin against its major inhibitor antithrombin III and improved its storage stability. At optimal CaCl2 concentration, the clotting time by the bound thrombin is shorter than that of the free enzyme. This novel conjugated thrombin may be an efficient candidate for topical hemostasis and wound healing. [source] Kinetics of inhibition of acetylcholinesterase in the presence of acetonitrileFEBS JOURNAL, Issue 8 2009Markus Pietsch The hydrolysis of acetylthiocholine by acetylcholinesterase from Electrophorus electricus was investigated in the presence of the inhibitors tacrine, gallamine and compound 1. The interaction of the enzyme with the substrate and the inhibitors was characterized by the parameters KI, ,,, b or ,, Km and Vmax, which were determined directly and simultaneously from nonlinear Michaelis,Menten plots. Tacrine was shown to act as a mixed-type inhibitor with a strong noncompetitive component (,, , 1) and to completely block deacylation of the acyl-enzyme. In contrast, acetylcholinesterase inhibition by gallamine followed the ,steric blockade hypothesis', i.e. only substrate association to as well as substrate/product dissociation from the active site were reduced in the presence of the inhibitor. The relative efficiency of the acetylcholinesterase,gallamine complex for the catalysis of substrate conversion was determined to be 1.7,25% of that of the free enzyme. Substrate hydrolysis and the inhibition of acetylcholinesterase were also investigated in the presence of 6% acetonitrile, and a competitive pseudo-inhibition was observed for acetonitrile (KI = 0.25 m). The interaction of acetylcholinesterase with acetonitrile and tacrine or gallamine resulted in a seven- to 10-fold increase in the KI values, whereas the principal mode of inhibition was not affected by the organic solvent. The determination of the inhibitory parameters of compound 1 in the presence of acetonitrile revealed that the substance acts as a hyperbolic mixed-type inhibitor of acetylcholinesterase. The complex formed by the enzyme and the inhibitor still catalysed product formation with 8.7,9.6% relative efficiency. [source] The phosphate site of trehalose phosphorylase from Schizophyllum commune probed by site-directed mutagenesis and chemical rescue studiesFEBS JOURNAL, Issue 5 2008Christiane Goedl Schizophyllum commune,,,-trehalose phosphorylase utilizes a glycosyltransferase-like catalytic mechanism to convert its disaccharide substrate into ,- d -glucose 1-phosphate and ,- d -glucose. Recruitment of phosphate by the free enzyme induces ,,,-trehalose binding recognition and promotes the catalytic steps. Like the structurally related glycogen phosphorylase and other retaining glycosyltransferases of fold family GT-B, the trehalose phosphorylase contains an Arg507-XXXX-Lys512 consensus motif (where X is any amino acid) comprising key residues of its putative phosphate-binding sub-site. Loss of wild-type catalytic efficiency for reaction with phosphate (kcat/Km = 21 000 m,1·s,1) was dramatic (,107 -fold) in purified Arg507,Ala (R507A) and Lys512,Ala (K512A) enzymes, reflecting a corresponding change of comparable magnitude in kcat (Arg507) and Km (Lys512). External amine and guanidine derivatives selectively enhanced the activity of the K512A mutant and the R507A mutant respectively. Analysis of the pH dependence of chemical rescue of the K512A mutant by propargylamine suggested that unprotonated amine in combination with H2PO4,, the protonic form of phosphate presumably utilized in enzymatic catalysis, caused restoration of activity. Transition state-like inhibition of the wild-type enzyme A by vanadate in combination with ,,,-trehalose (Ki = 0.4 ,m) was completely disrupted in the R507A mutant but only weakened in the K512A mutant (Ki = 300 ,m). Phosphate (50 mm) enhanced the basal hydrolase activity of the K512A mutant toward ,,,-trehalose by 60% but caused its total suppression in wild-type and R507A enzymes. The results portray differential roles for the side chains of Lys512 and Arg507 in trehalose phosphorylase catalysis, reactant state binding of phosphate and selective stabilization of the transition state respectively. [source] Kinetic mechanism for p38 MAP kinase ,FEBS JOURNAL, Issue 18 2005A partial rapid-equilibrium random-order ternary-complex mechanism for the phosphorylation of a protein substrate p38 Mitogen-activated protein kinase alpha (p38 MAPK,) is a member of the MAPK family. It is activated by cellular stresses and has a number of cellular substrates whose coordinated regulation mediates inflammatory responses. In addition, it is a useful anti-inflammatory drug target that has a high specificity for Ser-Pro or Thr-Pro motifs in proteins and contains a number of transcription factors as well as protein kinases in its catalog of known substrates. Fundamental to signal transduction research is the understanding of the kinetic mechanisms of protein kinases and other protein modifying enzymes. To achieve this end, because peptides often make only a subset of the full range of interactions made by proteins, protein substrates must be utilized to fully elucidate kinetic mechanisms. We show using an untagged highly active form of p38 MAPK,, expressed and purified from Escherichia coli[Szafranska AE, Luo X & Dalby KN (2005) Anal Biochem336, 1,10) that at pH 7.5, 10 mm Mg2+ and 27 °C p38 MAPK, phosphorylates ATF2,115 through a partial rapid-equilibrium random-order ternary-complex mechanism. This mechanism is supported by a combination of steady-state substrate and inhibition kinetics, as well as microcalorimetry and published structural studies. The steady-state kinetic experiments suggest that magnesium adenosine triphosphate (MgATP), adenylyl (,,,-methylene) diphosphonic acid (MgAMP-PCP) and magnesium adenosine diphosphate (MgADP) bind p38 MAPK, with dissociation constants of KA = 360 µm, KI = 240 µm, and KI > 2000 µm, respectively. Calorimetry experiments suggest that MgAMP-PCP and MgADP bind the p38 MAPK,,ATF2,115 binary complex slightly more tightly than they do the free enzyme, with a dissociation constant of Kd , 70 µm. Interestingly, MgAMP-PCP exhibits a mixed inhibition pattern with respect to ATF2,115, whereas MgADP exhibits an uncompetitive-like pattern. This discrepancy occurs because MgADP, unlike MgAMP-PCP, binds the free enzyme weakly. Intriguingly, no inhibition by 2 mm adenine or 2 mm MgAMP was detected, suggesting that the presence of a ,-phosphate is essential for significant binding of an ATP analog to the enzyme. Surprisingly, we found that inhibition by the well-known p38 MAPK, inhibitor SB 203580 does not follow classical linear inhibition kinetics at concentrations >,100 nm, as previously suggested, demonstrating that caution must be used when interpreting kinetic experiments using this inhibitor. [source] Cross-Linked Enzyme Aggregates of Chloroperoxidase: Synthesis, Optimization and CharacterizationADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 13 2009Daniel Abstract In this study we have optimized the conditions to precipitate and cross-link the enzyme chloroperoxidase (EC,1.11.1.10) from Caldariomyces fumago (CPO) using 1,2-dimethoxyethane as the precipitating agent. The coprecipitation of the enzyme with albumin and pentaethylenehexamine was needed for optimum results, presumably due to the low number of lysines available in CPO. The enzyme was immobilized with an activity recovery of 68%. The cross-linked enzyme aggregate showed higher temperature and pH stability, and better hydrogen peroxide tolerance than the free enzyme. [source] Covalent immobilization of ,-galactosidase on carrageenan coated with chitosanJOURNAL OF APPLIED POLYMER SCIENCE, Issue 1 2009Magdy M.M. Elnashar Abstract ,-Galactosidase was covalently immobilized to carrageenan coated with chitosan for the hydrolysis of lactose. The chitosan-carrageenan polyelectrolyte interaction was found to be dependent on the chitosan pH. At pH 4, the chitosan reached its maximum binding of 28.5% (w/w) where the chitosan surface density was 4.8 mg chitosan/cm2 g of carrageenan gel disks, using Muzzarelli method. Glutaraldehyde was used as a mediator to incorporate new functionality, aldehydic carbonyl group, to the bio-polymers for covalent attachment of ,-galactosidase. The enzyme was covalently immobilized to the biopolymer at a concentration of 2.73 mg protein per g of wet gel. FTIR proved the incorporation of the aldehydic carbonyl group to the carrageenan coated with chitosan at 1720 cm,1. The optimum time for enzyme immobilization was found to be 16 h, after which a plateau was reached. The enzyme loading increased from 2.65 U/g (control gel) to 10.92 U/g gel using the covalent technique. The gel's modification has shown to improve the carrageenan gel thermal stability as well as the immobilized enzyme. For example, the carrageenan gel treated with chitosan showed an outstanding thermal stability at 95°C compared with 35°C for the untreated carrageenan gel. Similarly, the immobilization process shifted the enzyme's optimum temperature from 50°C for the free enzyme towards a wider temperature range 45,55 °C indicating that the enzyme structure is strengthened by immobilization. In brief, the newly developed immobilization method is simple; the carrier is cheap, yet effective and can be used for the immobilization of other enzymes. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009 [source] Chitosan-grafted poly(hydroxyethyl methacrylate- co -glycidyl methacrylate) membranes for reversible enzyme immobilizationJOURNAL OF APPLIED POLYMER SCIENCE, Issue 5 2007M. Yakup Ar Abstract Epoxy group-containing poly(hydroxyethyl methacrylate/glycidyl methacrylate), p(HEMA/GMA), membrane was prepared by UV initiated photopolymerization. The membrane was grafted with chitosan (CH) and some of them were chelated with Fe(III) ions. The CH grafted, p(HEMA/GMA), and Fe(III) ions incorporated p(HEMA/GMA)-CH-Fe(III) membranes were used for glucose oxidase (GOD) immobilization via adsorption. The maximum enzyme immobilization capacity of the p(HEMA/GMA)-CH and p(HEMA/GMA)-CH-Fe(III) membranes were 0.89 and 1.36 mg/mL, respectively. The optimal pH value for the immobilized GOD preparations is found to have shifted 0.5 units to more acidic pH 5.0. Optimum temperature for both immobilized preparations was 10°C higher than that of the free enzyme and was significantly broader at higher temperatures. The apparent Km values were found to be 6.9 and 5.8 mM for the adsorbed GOD on p(HEMA/GMA)-CH and p(HEMA/GMA)-CH-Fe(III) membranes, respectively. In addition, all the membranes surfaces were characterized by contact angle measurements. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 103: 3084,3093, 2007 [source] Improvement of enantioselectivity and stability of Klebsiella oxytoca hydrolase immobilized on Eupergit C 250LJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 11 2008Pei-Yun Wang Abstract BACKGROUND: A simple procedure was employed to covalently immobilize a Klebsiella oxytoca hydrolase (SNSM-87) onto epoxy-activated supports of Eupergit C 250L via multipoint covalent attachment. The resultant biocatalyst was explored for the hydrolytic resolution of a variety of (R,S)-2-hydroxycarboxylic acid ethyl esters. RESULTS: With the hydrolytic resolution of (R,S)-ethyl mandelate in biphasic media as the model system, optimal conditions of 55 °C, pH 6 buffer and isooctane as the organic phase were selected for improving the enzyme stability (activity retained from 10% to 50% at 96 h) and enantioselectivity (VSVR,1 value enhanced from 44 to 319) in comparison to the performance of free enzyme. Moreover, the immobilized enzyme retained its activity and enantioselectivity after eight cycles of hydrolysis at 55 °C. When applying the resolution process to other (R,S)-2-hydroxycarboxylic acid ethyl esters, 2.4- to 4.0-fold enhancements of the enantioselectivity in general were obtainable. CONCLUSIONS: The enantioselectivity enhancement, good reusability and easy recovery after reaction indicate that the immobilized SNSM-87 may have the potential as an industrial biocatalyst for the preparation of optically pure 2-hydroxycarboxylic acids. Copyright © 2008 Society of Chemical Industry [source] COVALENT IMMOBILIZATION OF INVERTASE ON CHEMICALLY ACTIVATED POLY (STYRENE-2-HYDROXYETHYL METHACRYLATE) MICROBEADSJOURNAL OF FOOD BIOCHEMISTRY, Issue 3 2008HAYDAR ALTINOK ABSTRACT A carrier for invertase enzyme was synthesized from styrene (S) and 2- hydroxyethyl methacrylate (HEMA) in the form of microbeads. These poly (styrene-2-hydroxyethyl methacrylate), P(S-HEMA) microbeads were activated by epichlorohydrin (ECH) treatment for covalent immobilization. The free and immobilized invertase were assayed in the hydrolysis of sucrose to glucose, and the obtained results were compared. The optimum pH was 4.5 for free and 5.5 for immobilized invertase. The optimum temperature of invertase shifted from 45C to 55C upon immobilization. For free and immobilized enzymes, kinetic parameters were calculated as 4.1 × 10,3 mol L,1and 9.2 × 10,3 mol L,1for Km, and 6.6 × 10,2 mol L,1 min,1and 4.1 × 10,1 mol L,1 min,1for Vmax, respectively. After 1 month of storage at 4C, free enzyme retained 36% of its initial activity, while for the ECH-activated P(S-HEMA) immobilized enzyme, P(S-HEMA)-E, this value was observed as 67%. In repeated batch use, i.e., 20 times in 3 days, 78% retention of the initial activity was observed for P(S-HEMA)-E system. PRACTICAL APPLICATIONS Immobilization of enzymes are very important for many industrial applications, e.g., food, medicine, pharmacology, etc. Invertase converts sucrose to glucose and fructose, which have wide applications in food industry especially as sweeteners. Glucose,fructose mixture has much lower crystallinity compared to sucrose and therefore used in the production of noncrystallizing jams and creams. They are also used as liquid sweeteners. Immobilization enables repeated use, provides significant reduction in the operation costs, facilitates easy separation and speeds up recovery of enzyme and extends the stability of enzyme by protecting the active material from deactivation. Industrial application of immobilized invertase may decrease the production cost of glucose,fructose mixture because it could be used repeatedly for long periods. Although invertase is not a very expensive enzyme, the technique can also be applied to expensive ones for biotechnological productions. [source] Kinetic behaviour and stability of Escherichia coli ATCC27257 alkaline phosphatase immobilised in soil humatesJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 3 2003María C Pilar Abstract Alkaline phosphatase (EC 3.1.3.1) extracted from Escherichia coli ATCC27257 was immobilised by co-flocculation with soil humates in the presence of Ca2+. The effects of time, temperature, pH and concentration of enzyme and support on immobilisation were studied. Between 58 and 92% of the added phosphatase was strongly bound to the humates, depending on the conditions of immobilisation used. Some characteristics of the humate,phosphatase complexes and of the free enzyme were compared. The enzymatic complexes showed values of Km (2.22,mM) and activation energy (33.4,kJ,mol,1) similar to those of the free enzyme (2.00,mM and 27.6,kJ,mol,1). The pH/activity profiles revealed no change in terms of shape or optimum pH (10.5) upon immobilisation of alkaline phosphatase. However, the immobilised enzyme showed maximal activity in the range of 80,100,°C, while the free enzyme had its highest activity at 60,°C. The thermal stability of alkaline phosphatase was enhanced by complexation to the soil humates. © 2003 Society of Chemical Industry [source] The Properties of Covalently Immobilized Trypsin on Soap-Free P(MMA-EA-AA) Latex ParticlesMACROMOLECULAR BIOSCIENCE, Issue 4 2005Kai Kang Abstract Summary: The covalent immobilization of trypsin onto poly[(methyl methacrylate)- co -(ethyl acrylate)- co -(acrylic acid)] latex particles, produced by a soap-free emulsion polymerization technique, was carried out using the carbodiimide method. The catalytic properties and kinetic parameters, as well as the stability of the immobilized enzyme were compared to those of the free enzyme. Results showed that the optimum temperature and pH for the immobilized trypsin in the hydrolysis of casein were 55,°C and 8.5, both of which were higher than that of the free form. It was found that Km (Michaelis constant) was 45.7 mg,·,ml,1 and Vmax (maximal reaction rate) was 793.0 ,g,·,min,1 for immobilized trypsin, compared to a Km of 30.0 mg,·,ml,1 and a Vmax of 5,467.5 ,g,·,min,1 for free trypsin. The immobilized trypsin exhibited much better thermal and chemical stabilities than its free counterpart and maintained over 63% of its initial activity after reusing ten times. TEM photograph of latex particles after trypsin immobilization. [source] Stability of ,-galactosidase immobilized on composite microspheres of artemisia seed gum and chitosanPOLYMER COMPOSITES, Issue 1 2008Ji Zhang In this work, composite microspheres were prepared by using artemisia seed gum and chitosan as a source. The composite microspheres have activated aldehyde groups by using glutaraldehyde. ,-Galactosidase was covalently bound on these activated microspheres. The properties of the immobilized enzyme were investigated and compared with those of the free enzyme, for which o -nitrophenol ,- D -galactopyranoside (ONPG) was chosen as a substrate. The results showed that the pH and thermal stability of the immobilized ,-galactosidase were higher than those of the soluble one. Apart from these, the Michaelis constant Km was evaluated for the immobilized ,-galactosidase and the soluble enzyme. The immobilized ,-galactosidase exhibited better environmental adaptability and reusability than the soluble one. POLYM. COMPOS., 29:9,14, 2008. © 2007 Society of Plastics Engineers [source] Biocatalytic hydrogels by template polymerizationPOLYMERS FOR ADVANCED TECHNOLOGIES, Issue 5 2008H. El-Sherif Abstract Novel ionizable hydrogels were prepared from poly(acrylic acid) and dimethylaminoethyl methacrylate monomer employing template polymerization technique as an alternative to traditional physical and chemical crosslinking. The mode of interaction, as proved by Fourier Transform Infrared Spectroscopy (FTIR), was multiple H-bonding between the tertiary amino group of the monomer and the carboxylic groups of the polymer. The hydrogels represented suitable matrices for enzyme immobilization. The effect of varying the polymer,monomer molar ratio on the swelling kinetics and parameters was investigated. The dynamic swelling isotherm exhibited a Fickian mode of penetrant sorption and a plateau that increases with the amino group content. A polymer complex of molar ratio (polymer:monomer) 0.5:0.8 had a weight swelling ratio of 10 and 7 at pHs 3 and 8, respectively. The proven pH sensitivity together with the amphoteric character of these hydrogels make them good candidates for another bioapplication such as oral delivery systems of therapeutic peptides and proteins. The structural integrity of the hydrogels was proved by their swelling reversibility. , -Galactosidase, as an acidic model enzyme, was immobilized covalently on the synthesized hydrogels. The maximum enzyme velocity (Vmax) was enhanced to 19,µmol/min/mg, for polycomplex of molar ratio 0.5:0.8, compared with 3.2,µmol/min/mg for the free enzyme. Copyright © 2008 John Wiley & Sons, Ltd. [source] Crystallization and preliminary crystallographic analysis of endonuclease VIII in its uncomplexed formACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2004Gali Golan The Escherichia coli DNA repair enzyme endonuclease VIII (EndoVIII or Nei) excises oxidized pyrimidines from damaged DNA substrates. It overlaps in substrate specificity with endonuclease III and may serve as a back-up for this enzyme in E. coli. The three-dimensional structure of Nei covalently complexed with DNA has been recently determined, revealing the critical amino-acid residues required for DNA binding and catalytic activity. Based on this information, several site-specific mutants of the enzyme have been tested for activity against various substrates. Although the crystal structure of the DNA-bound enzyme has been fully determined, the important structure of the free enzyme has not previously been analyzed. In this report, the crystallization and preliminary crystallographic characterization of DNA-free Nei are described. Four different crystal habits are reported for wild-type Nei and two of its catalytic mutants. Despite being crystallized under different conditions, all habits belong to the same crystal form, with the same space group (I222) and a similar crystallographic unit cell (average parameters a = 57.7, b = 80.2, c = 169.7,Å). Two of these crystal habits, I and IV, appear to be suitable for full crystallographic analysis. Crystal habit I was obtained by vapour diffusion using PEG 8000, glycerol and calcium acetate. Crystal habit IV was obtained by a similar method using PEG 400 and magnesium chloride. Both crystals are mechanically strong and stable in the X-ray beam once frozen under cold nitrogen gas. A full diffraction data set has recently been collected from a wild-type Nei crystal of habit I (2.6,Å resolution, 85.2% completeness, Rmerge = 9.8%). Additional diffraction data were collected from an Nei-R252A crystal of habit IV (2.05,Å resolution, 99.9% completeness, Rmerge = 6.0%) and an Nei-E2A crystal of habit IV (2.25,Å resolution, 91.7% completeness, Rmerge = 6.2%). These diffraction data were collected at 95,100 K using a synchrotron X-ray source and a CCD area detector. All three data sets are currently being used to obtain crystallographic phasing via molecular-replacement techniques. [source] Crystallization and preliminary X-ray analysis of 4-pyridoxolactonase from Mesorhizobium lotiACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2009Sayoko Matsuda 4-Pyridoxolactonase from Mesorhizobium loti MAFF303099 has been overexpressed in Escherichia coli. The recombinant enzyme was purified and was crystallized by the sitting-drop vapour-diffusion method using PEG 4000 and ammonium sulfate as precipitants. Crystals of the free enzyme (form I) and of the 5-pyridoxolactone-bound enzyme (form II) grew under these conditions. Crystals of form I diffracted to 2.0,Å resolution and belonged to the monoclinic space group C2, with unit-cell parameters a = 77.93, b = 38.88, c = 81.60,Å, , = 117.33°. Crystals of form II diffracted to 1.9,Å resolution and belonged to the monoclinic space group C2, with unit-cell parameters a = 86.24, b = 39.35, c = 82.68,Å, , = 118.02°. The calculated VM values suggested that the asymmetric unit contains one molecule in both crystal forms. [source] Complete amino-acid sequence, crystallization and preliminary X-ray diffraction studies of leucurolysin-a, a nonhaemorrhagic metalloproteinase from Bothrops leucurus snake venomACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2009Rodrigo Novaes Ferreira Leucurolysin-a (leuc-a) is a class P-I snake-venom metalloproteinase isolated from the venom of the South American snake Bothrops leucurus (white-tailed jararaca). The mature protein is composed of 202 amino-acid residues in a single polypeptide chain. It contains a blocked N-terminus and is not glycosylated. In vitro studies revealed that leuc-a dissolves clots made either from purified fibrinogen or from whole blood. Unlike some other venom fibrinolytic metalloproteinases, leuc-a has no haemorrhagic activity. Leuc-a was sequenced and was crystallized using the hanging-drop vapour-diffusion technique. Crystals were obtained using PEG 6000 or PEG 1500. Diffraction data to 1.80 and 1.60,Å resolution were collected from two crystals (free enzyme and the endogenous ligand,protein complex, respectively). They both belonged to space group P212121, with very similar unit-cell parameters (a = 44.0, b = 56.2, c = 76.3,Å for the free-enzyme crystal). [source] Crystallization and preliminary X-ray analysis of d -2-hydroxyacid dehydrogenase from Haloferax mediterraneiACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2009J. Domenech d -2-Hydroxyacid dehydrogenase (D2-HDH) from Haloferax mediterranei has been overexpressed in Escherichia coli, solubilized in 8,M urea and refolded by rapid dilution. The protein was purified and crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate or PEG 3350 as precipitant. Two crystal forms representing the free enzyme and the nonproductive ternary complex with ,-ketohexanoic acid and NAD+ grew under these conditions. Crystals of form I diffracted to beyond 3.0,Å resolution and belonged to the monoclinic space group P21, with unit-cell parameters a = 66.0, b = 119.6, c = 86.2,Å, , = 96.3°. Crystals of form II diffracted to beyond 2.0,Å resolution and belonged to the triclinic space group P1, with unit-cell parameters a = 66.5, b = 75.2, c = 77.6,Å, , = 109.1, , = 107.5, , = 95.9°. The calculated values for VM and analysis of the self-rotation and self-Patterson functions suggest that the asymmetric unit in both crystal forms contains two dimers related by pseudo-translational symmetry. [source] Enzyme immobilization via silaffin-mediated autoencapsulation in a biosilica supportBIOTECHNOLOGY PROGRESS, Issue 2 2009Wesley D. Marner II Abstract Enzymes and other biomolecules are often immobilized in a matrix to improve their stability or to improve their ability to be reused. Performing a polycondensation reaction in the presence of a biomolecule of interest relies on random entrapment events during polymerization and may not ensure efficient, homogeneous, or complete biomolecule encapsulation. To overcome these limitations, we have developed a method of incorporating autosilification activity into proteins without affecting enzymatic functionality. The unmodified R5 silaffin peptide from Cylindrotheca fusiformis is capable of initiating silica polycondensation in vitro at ambient temperatures and pressures in aqueous solution. In this study, translational fusion proteins between R5 and various functional proteins (phosphodiesterase, organophosphate hydrolase, and green fluorescent protein) were produced in Escherichia coli. Each of the fusion proteins initiated silica polycondensation, and enzymatic activity (or fluorescence) was retained in the resulting silica spheres. Under certain circumstances, the enzymatically-active biosilica displayed improved stability relative to free enzyme at elevated temperatures. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Immobilization of a thermostable ,-amylase by covalent binding to an alginate matrix increases high temperature usabilityBIOTECHNOLOGY PROGRESS, Issue 2 2009Boon L. Tee Abstract Thermostable ,-amylase was covalently bound to calcium alginate matrix to be used for starch hydrolysis at liquefaction temperature of 95°C. 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride (EDAC) was used as crosslinker. EDAC reacts with the carboxylate groups on the calcium alginate matrix and the amine groups of the enzyme. Ethylenediamine tetraacetic acid (EDTA) treatment was applied to increase the number of available carboxylate groups on the calcium alginate matrix for EDAC binding. After the immobilization was completed, the beads were treated with 0.1 M calcium chloride solution to reinstate the bead mechanical strength. Enzyme loading efficiency, activity, and reusability of the immobilized ,-amylase were investigated. Covalently bound thermostable ,-amylase to calcium alginate produced a total of 53 g of starch degradation/mg of bound protein after seven consecutive starch hydrolysis cycles of 10 min each at 95°C in a stirred batch reactor. The free and covalently bound ,-amylase had maximum activity at pH 5.5 and 6.0, respectively. The Michaelis-Menten constant (Km) of the immobilized enzyme (0.98 mg/mL) was 2.5 times greater than that of the free enzyme (0.40 mg/mL). The maximum reaction rate (Vmax) of immobilized and free enzyme were determined to be 10.4-mg starch degraded/mL min mg bound protein and 25.7-mg starch degraded/mL min mg protein, respectively. The high cumulative activity and seven successive reuses obtained at liquefaction temperature make the covalently bound thermostable ,-amylase to calcium alginate matrix, a promising candidate for use in industrial starch hydrolysis process. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Enhancing the Reusability of Endoglucanase-Gold Nanoparticle Bioconjugates by Tethering to Polyurethane MicrospheresBIOTECHNOLOGY PROGRESS, Issue 6 2004Sumant Phadtare The synthesis of polyurethane microsphere-gold nanoparticle "core-shell" structures and their use in the immobilization of the enzyme endoglucanase are described. Assembly of gold nanoparticles on the surface of polymer microspheres occurs through interaction of the nitrogens in the polymer with the nanoparticles, thereby precluding the need for modifying the polymer microspheres to enable such nanoparticle binding. Endoglucanse could thereafter be bound to the gold nanoparticles decorating the polyurethane microspheres, leading to a highly stable biocatalyst with excellent reuse characteristics. The immobilized enzyme retains its biocatalytic activity and exhibits improved thermal stability relative to free enzyme in solution. The high surface area of the host gold nanoparticles renders the immobilized enzyme "quasi free", while at the same time retaining advantages of immobilization such as ease of reuse, enhanced temporal and thermal stability, etc. [source] Invertase-Lipid Biocomposite Films: Preparation, Characterization, and Enzymatic ActivityBIOTECHNOLOGY PROGRESS, Issue 1 2004Sumant Phadtare The formation of biocomposite films of the industrially important enzyme invertase and fatty lipids under enzyme-friendly conditions is described. The approach involves a simple beaker-based diffusion protocol wherein invertase diffuses into the cationic lipid octadecylamine during immersion of the lipid film in the enzyme solution. Entrapment of invertase in the octadecylamine film is highly pH-dependent, underlining the role of attractive electrostatic interactions between the enzyme and the lipid in the biocomposite film formation. The kinetics of formation of the enzyme-lipid biocomposites has been studied by quartz crystal microgravimetry (QCM) measurements. The stability of the enzyme in the lipid matrix was confirmed by fluorescence spectroscopy and biocatalytic activity measurements. The biocatalytic activity of the invertase-lipid biocomposite films was comparable to that of the free enzyme in solution and showed marginally higher temperature stability. Particularly exciting was the excellent reuse characteristics of the biocomposite films, indicating potential industrial application of these films. [source] A Feasible Enzymatic Process for d -Tagatose Production by an Immobilized Thermostable l -Arabinose Isomerase in a Packed-Bed BioreactorBIOTECHNOLOGY PROGRESS, Issue 2 2003Hye-Jung Kim To develop a feasible enzymatic process for d -tagatose production, a thermostable l -arabinose isomerase, Gali152, was immobilized in alginate, and the galactose isomerization reaction conditions were optimized. The pH and temperature for the maximal galactose isomerization reaction were pH 8.0 and 65 °C in the immobilized enzyme system and pH 7.5 and 60 °C in the free enzyme system. The presence of manganese ion enhanced galactose isomerization to tagatose in both the free and immobilized enzyme systems. The immobilized enzyme was more stable than the free enzyme at the same pH and temperature. Under stable conditions of pH 8.0 and 60 °C, the immobilized enzyme produced 58 g/L of tagatose from 100 g/L galactose in 90 h by batch reaction, whereas the free enzyme produced 37 g/L tagatose due to its lower stability. A packed-bed bioreactor with immobilized Gali152 in alginate beads produced 50 g/L tagatose from 100 g/L galactose in 168 h, with a productivity of 13.3 (g of tagatose)/(L-reactor·h) in continuous mode. The bioreactor produced 230 g/L tagatose from 500 g/L galactose in continuous recycling mode, with a productivity of 9.6 g/(L·h) and a conversion yield of 46%. [source] High Production of D -Tagatose, a Potential Sugar Substitute, Using Immobilized L -Arabinose IsomeraseBIOTECHNOLOGY PROGRESS, Issue 1 2001Pil Kim An L -arabinose isomerase of Escherichia coli was immobilized using covalent binding to agarose to produce D -tagatose, a bulking sweetener that can be economically used as a sugar substitute. The immobilized L -arabinose isomerase stably produced an average of 7.5 g-tagatose/L·day for 7 days with a productivity exceeding that of the free enzyme (0.47 vs 0.30 mg/U·day). Using a scaled-up immobilized enzyme system, 99.9 g-tagatose/L was produced from galactose with 20% equilibrium in 48 h. The process was repeated two more times with production of 104.1 and 103.5 g-tagatose/L. D -Tagatose production using an immobilized L -arabinose isomerase has a high potential for commercial application. [source] Polysaccharide hydrolysis in aggregates and free enzyme activity in aggregate-free seawater from the north-eastern Gulf of MexicoENVIRONMENTAL MICROBIOLOGY, Issue 2 2008Kai Ziervogel Summary Marine snow aggregates represent hotspots of carbon remineralization in the ocean. Various aspects of bacterial dynamics have been investigated on marine snow. To date, extracellular enzymatic activities in aggregates have been measured using small substrate proxies that do not adequately reflect the complexity of biomacromolecules such as polysaccharides, proteins and lipids. To address this issue, we used six structurally distinct, fluorescently labelled polysaccharides to measure enzymatic hydrolysis on aggregates formed with a roller table and in aggregate-free (ambient) seawater from two near-coast sites, north-eastern Gulf of Mexico. A single polysaccharide was incubated in aggregates and ambient seawater. Changes in polysaccharide molecular weight were monitored over time to measure the course of enzymatic hydrolysis. All six polysaccharides were hydrolysed in aggregates, indicating a broad range of enzyme activities in aggregate-associated bacteria. Four substrates were also hydrolysed in ambient waters. Epifluorescence microscopy revealed that nearly all of the bacteria present in original waters were incorporated into aggregates. Therefore hydrolytic activities in ambient waters were presumably due to enzymes spatially disconnected from cells and aggregates. Our results show substantial enzymatic activity in cell/aggregate-free seawater, suggesting a significant role of free enzymes in hydrolytic activity in waters from the north-eastern Gulf of Mexico. [source] Coimmobilization of malic enzyme and alanine dehydrogenase on organic,inorganic hybrid gel fibers and the production of L -alanine from malic acid using the fibers with coenzyme regenerationJOURNAL OF APPLIED POLYMER SCIENCE, Issue 5 2010Koji Nakane Abstract Malic enzyme (EC 1.1.1.39) and alanine dehydrogenase (EC 1.4.1.1) were entrap-immobilized on hybrid gel fibers of cellulose acetate (CA) and zirconium (Zr) alkoxide by air-gap wet spinning. The production of L -alanine from malic acid with coenzyme regeneration was examined with the enzymes immobilized on the fibers. The productivity of L -alanine of the immobilized enzymes decreased to approximately one-fifth of that of free enzymes, but the CA,Zr-fiber-immobilized enzymes retained a high level of productivity after repeated use. Reduced form of nicotinamide adenine dinucleotide (NADH) recycling also occurred effectively for the enzymes immobilized on the fiber. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010 [source] Catalytic behaviors of enzymes attached to nanoparticles: the effect of particle mobilityBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2003Hongfei Jia Abstract Nanoparticles provide an ideal remedy to the usually contradictory issues encountered in the optimization of immobilized enzymes: minimum diffusional limitation, maximum surface area per unit mass, and high effective enzyme loading. In addition to the promising performance features, the unique solution behaviors of the nanoparticles also point to a transitional region between the heterogeneous (with immobilized enzymes) and homogeneous (with soluble free enzymes) catalysis. The particle mobility, which is related to particle size and solution viscosity through Stokes-Einstein equation, may impact the reaction kinetics according to the collision theory. The mobility-activity relationship was examined through experimental studies and theoretical modeling in the present work. Polystyrene particles with diameters ranging from 110,1000 nm were prepared. A model enzyme, ,-chymotrypsin, was covalently attached to the nanoparticles up to 6.6 wt%. The collision theory model was found feasible in correlating the catalytic activities of particles to particle size and solution viscosity. Changes in the size of particles and the viscosity of reaction media, which all affect the mobility of the enzyme catalyst, evidently altered the intrinsic activity of the particle-attached enzyme. Compared to KM, kcat appeared to be less sensitive to particle size and viscosity. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng84: 406,414, 2003. [source] Effect of surfactants on separate hydrolysis fermentation and simultaneous saccharification fermentation of pretreated lodgepole pineBIOTECHNOLOGY PROGRESS, Issue 4 2009Maobing Tu Abstract The effects of surfactants addition on enzymatic hydrolysis and subsequent fermentation of steam exploded lodgepole pine (SELP) and ethanol pretreated lodgepole pine (EPLP) were investigated in this study. Supplementing Tween 80 during cellulase hydrolysis of SELP resulted in a 32% increase in the cellulose-to-glucose yield. However, little improvement was obtained from hydrolyzing EPLP in the presence of the same amount of surfactant. The positive effect of surfactants on SELP hydrolysis led to an increase in final ethanol yield after the fermentation. It was found that the addition of surfactant led to a substantial increase in the amount of free enzymes in the 48 h hydrolysates derived from both substrates. The effect of surfactant addition on final ethanol yield of simultaneous saccharification and fermentation (SSF) was also investigated by using SELP in the presence of additional furfural and hydroxymethylfurfural (HMF). The results showed that the surfactants slightly increased the conversion rates of furfural and HMF during SSF process by Saccharomyces cerevisiae. The presence of furfural and HMF at the experimental concentrations did not affect the final ethanol concentration either. The strategy of applying surfactants in cellulase recycling to reduce enzyme cost is presented. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] | ||||||||||||||||||||||||||||