Methanol biosynthesis by covalently immobilized cells of Methylosinus trichosporium: batch and continuous studies

The DEAE-cellulose linked cells of Methylosinus trichosporium displaying high specific methane mono-oxygenase activity (66 mumol methane oxidized/h mg cells) were used for methanol biosynthesis from biogas derived methane in a batch and a continuous cell reactor. The optimum cell-to-carrier ratio was determined to be 0.5 g cells/g dry weight cellulose. Batch experiments indicated that 100 mM phosphate ion concentration was necessary to inhibit further oxidation of methanol; excess oxygen supply favored methanol accumulation with an increase in methane conversion efficiency to 27%. A pulse of 40 mM sodium formate at the end of 6 h resulted in restoration of methanol accumulation by regenerating NADH(2) required for the sustained activity of methane mono-oxygenase. Maximum methanol level of 50 mumol/mg cells was obtained in the batch reactor. In a standard 50-mL ultrafiltration continuous reactor, the covalently linked cells produced methanol at a continuous rate of 100 mumol/h for the first 10 h, after which the methanol accumulation rate fell low due to the depletion of NADH(2). The methanol accumulation could be stimulated by supplying sodium formate (40 mM) in either 20 or 100 mM phosphate buffer. Maximum methanol accumulation rate of 267 mumol/h was obtained when 20 mM formate was supplied in the feed stream containing 100 mM phosphate ions, and this level of biosynthesis was maintained for over 72 h. The stoichiometric balance made at various points of formate addition indicated that the molar amount of methanol generated at steady state is dependent on the equimolar addition of sodium formate to the feed. The half-life t(1/2) and thermal denaturation rate constant K(d) were computed to be 108 h and 6.42 x 10(-3) h(-1), respectively.     

Morphological studies of oligodeoxyribonucleotides probes covalently immobilized at polystyrene modified surfaces

The immobilization of short ss-DNA (18- and 36-mer) and their hybridization were studied at gold and glassy carbon substrates modified with low molecular weight (approximately 12, 18 and 24 kg/mol) polystyrene thin films. Amino-modified DNA was attached to the surface by reaction with succinimide ester groups bound to the polystyrenes. A ferrocene modified DNA target was used to confirm the probe-target hybridization. Atomic force microscopy studies showed significant morphological changes after probe immobilization and hybridization compared to the featureless structure of the polystyrene film. Single-stranded DNA samples had a globular morphology with an average density of 3.8 and 2.2 (x 10(11)) globules/cm2 for the 18- and 36-mer, respectively. The formation of a porous structure with a 2.0 and 1.0 (x10(11)) average pore density corresponding to the 18- and 36-mer was observed after hybridization. A surface composition analysis was done by X-ray photoelectron spectroscopy to confirm and support the images interpretation. Ferrocene oxidation (+323 mV/18-mer, +367 mV/36-mer, versus Ag/AgCl) proved the presence of ds-DNA at the modified surfaces.     

Distribution of TMV movement protein in single living protoplasts immobilized in agarose

Recent studies of the tobacco mosaic virus (TMV) P30 movement protein (MP) fused with green fluorescent protein (GFP) during TMV infection described the involvement of elements of the cytoskeleton and components of the endoplasmic reticulum (ER) in the intracellular trafficking of MP:GFP from the sites of synthesis in the cytoplasm to plasmodesmata. To examine in real-time the pattern of synthesis, accumulation and degradation of MP:GFP, we developed a method to immobilize protoplasts in agarose such that they are maintained alive for extended periods of time. The pattern of MP:GFP accumulation in single living protoplasts visualized by confocal laser scanning microscopy (CLSM) was parallel to that previously described in a population of protoplasts harvested at different times post-infection. Additionally, a network of weakly fluorescent filaments, which are apparently different from microtubules, was observed to surround the nucleus and these filaments were associated with fluorescent bodies (previously identified as ER-derived structures). Later in infection, the fluorescent bodies increased in size and coalesced to form larger structures that accumulated near the periphery of the cells while highly fluorescent non-cortical filaments were observed distributed in the cytoplasm. The putative involvement of these filaments in targeting the fluorescent bodies to the periphery of the cell is discussed. Studies of single, embedded protoplasts make it possible to observe changes in amount and subcellular localization of viral and other proteins.     

Determination of protein covalently bound to agarose supports using bicinchoninic acid

A method using bicinchoninic acid (BCA) for the direct determination of protein covalently bound to agarose is described. The method involves the preparation of a standard curve using solubilized protein, the determination of the slurry concentration of the gel sample, the colorimetric assay of the gel slurry using BCA in a serum separator device, and the calculation of the amount of protein bound to the gel using the standard curve of the solubilized protein. The use of the BCA method for direct estimation of immobilized protein agrees well with the "balance" method which depends upon the measurement of protein depletion and yields highly reproducible results with a variety of coupling chemistries commonly used with agarose beads. The use of commercially available serum filters provides for a fast, less than 1 h, and convenient analytical format.     

Preparation of immobilized alpha-amylase covalently attached to granular polyacrylonitrile

In this report, alpha-Amylase originating from Bacillus subtilis (liquefying type) was immobilized on partially imidoesterized polyacrylonitrile (PAN) by covalent bonding. For the preparation of immobilized alpha-amylase, which has a high activity and high stability to repeated use, the optimum conditions for the preparation reaction were investigated. The optimum conditions for the preparation reaction were quantified on the basis of the enzymatic activity, the preservation of the activity during repeated use in batch process and the protein content on the support. Further-more, enzymatic properties of immobilized alpha-amylase prepared at optimum conditions were compared with the native enzyme. The optimum temperature and reaction time for the imidoes-terification reaction were 30 degrees c and 6 h, respectively, whereas those of the amidinatin reaction were 30-40 degrees C and more than 3 h, respectively; the optimum pH range was 9-10. Immobilized alpha-amylase prepared at the optimum conditions was very stable against the repeated use and had more than 90% of relative to activity of the first use after the tenth procedure. The initial reaction rate of immobilized alpha-amylase was lower than native alpha-amylase, but same amount of reducing sugars were produced after the reaction passed for more than 90 min. The immobilized alpha-amylase was less stabel at the high temperature and the more basic media. However, after long incubation time, immobilized alpha-amylase was more stable than the native enzyme in exposure to heat and a storng base.     

Synthesis of covalently immobilized phospholipid analogues

Cyclic 1-O-acyl-2-O-alkyl-glycero-3-phosphotriesters and 1-O-acyl-2-O-alkyl-glycero-3-bromoethylphosphate with a free acyl moiety in position 1 of the glycerol backbone were synthesized. These phospholipid intermediates were covalently bound to AH-Sepharose via the carbodiimide method. After immobilization the corresponding phosphatidylethanolamine analogues were obtained by acid hydrolysis of the cyclic phosphotriesters and by direct amination of the bromoethylphosphate. Thus, in a short, stepwise synthesis including minimum use of protecting groups, a variety of immobilized phospholipid analogues are available as affinity adsorbents for the purification of enzymes related to phospholipid metabolism.     

Permeabilization of covalently immobilized saccharomyces cerevisiae

The effect of solvents, ionic detergent, and drying on the passive membrane permeability of free and immobilized yeast cells was studied. The immobilization by covalent linkage was shown to affect the membrane composition as well as the individual susceptibility of cells to a permeabilization procedure.     

A simple method for quantitating ligands covalently bound to agarose beads

Agarose derivatives, as used for affinity chromatography, can be dissolved by warming in aqueous media at suitable pH values. Dilute solutions so formed are stable and transparent in regions of the ultraviolet and visible range, depending on the method of solubilization. Covalently bound ligands which possess chromophoric groups, or functional groups which can be converted to chromophores, can be quantitated by direct spectral analysis provided a solubilizing medium is chosen which results in minimal interference by absorbing decomposition products of the matrix.     

Interaction of thyroid peroxidase with concanavalin A covalently coupled to agarose.

We have investigated the interaction between concanavalin A-agarose (Con A-agarose) and thyroid peroxidase, an integral membrane protein found in the 105,000 X g, 1-h particulate fraction of thyroid tissue. An intact form of porcine thyroid peroxidase was obtained by solubilization with the nonionic detergent Triton X-100 and two fragmented, hydrophilic forms of the enzyme were prepared by trypsin treatment of the membrane. The three types of thyroid peroxidase bind to Con A-agarose and can be eluted with alpha-methyl-D-mannoside. The alpha-methyl-D-mannoside eluate of the most purified thyroid peroxidase preparation has been analyzed by polyacrylamide gel electrophoresis. Peroxidase activity corresponds with a glycoprotein band. The binding of thyroid peroxidase to Con A-agarose can be inhibited by sugars in the following order: alpha-methyl-D-mannoside greater than D-mannose greater than alpha-methyl-D-glucoside greater than D-glucose greater than D-galactose. This order of specificity is typical of Con A-sugar interactions. Furthermore, inactivation of the carbohydrate binding site of Con A by demetallization greatly reduces the extent of thyroid peroxidase binding. Reactivation of the carbohydrate binding site by the addition of Ca2+ and Mn2+ to demetallized Con A-agarose restores thyroid peroxidase binding. These and other experiments suggest that htyroid peroxidase is, like several other peroxidases, a glycoprotein. In addition, the interaction between thyroid peroxidase and Con A-agarose may provide a new purification tool for thyroid peroxidase.     

Stability of nicotinamide adenine dinucleotide immobilized to cyanogen bromide activated agarose

The stability of NAD(H) immobilized to a crosslinked agarose support (Sepharose(R)-4B) was examined in buffer solutions at a pH of 7.0 and 8.5. Specifically, this study investigated particle attrition and ligand leakage rates from a cyanogen bromide activated agarose support. Particle attrition did not occur under the experimental conditions. Ligand leakage rates were found to be first order in immobilized ligand concentration with two labile populations of ligand. The two-population model is consistent with the cyanogen bromide coupling chemistry, which results in both an isourea and imidocarbonate ligand linkage. The rate of ligand leakage was found to occur over a time scale of days, with first order rate constants ranging from 0.007 to 0.15 d(-1), depending on solution pH. (c) 1997 John Wiley & Sons, Inc.     

Selective adsorption of proteins to their ligands covalently immobilized onto microfibers

Following ozone oxidation of polyester microfibers of 3.5 mum average diameter and 0.83 m(2)/g specific area, the fiber surface was subjected to graft polymerization of acrylic acid and subsequently immobilized with serologically active proteins including Staphylococcus aureus protein A, a specific antigen, and a specific antibody. The immobilization reaction was mediated by a watersoluble carbodiimide, which allowed formation of a co-valent linkage between the ligand proteins and the grafted poly(acrylic acid)chains. The yields of the immobilized ligand proteins were of the order of 1 mg/g fiber. Their binding affinity and capacity to respective specific proteins were studied in vitro from a buffered solution and serum. It was found that the specific proteins were selectively adsorbed with dissociation constants as low as 1x 10(-6) M, suggesting the adsorption to take place through highly specific protein-protein interaction. An addition of serum albumin did not significantly affect the specific binding, regardless of the ligand proteins. The binding capacity ranged from 1 x 10(-13) to 1x 10(-11) mol/cm(2) primarily depending on the surface density of the immobilized ligands and the number of their binding sites per molecule. (c) 1995 John Wiley & Sons Inc.     

Experimental and theoretical microdialysis studies of in situ metabolism

Microdialysis sampling was performed to monitor localized metabolism in vivo and in vitro. A mathematical model that accounts for analyte mass transport during microdialysis sampling was used to predict metabolite concentrations in the microdialysis probe during localized metabolism experiments. The model predicts that metabolite concentrations obtained in the microdialysis probe are a function of different experimental parameters including membrane length, perfusion fluid flow rate, and sample diffusive and kinetic properties. Different microdialysis experimental parameters including membrane length and perfusion fluid flow rate were varied to affect substrate extraction efficiency (E(d)), or loss to the sample matrix, in vivo and in vitro. Local hepatic metabolism was studied in vivo in male Sprague-Dawley rats by infusing acetaminophen through the microdialysis probe. Acetaminophen sulfate concentrations increased linearly with respect to acetaminophen E(d) in contrast to modeling predictions. Xanthine oxidase was used as an in vitro model of localized metabolism. In vitro experimental results partially matched modeling predictions for 10-mm probes. These results suggest that monitoring local metabolism using microdialysis sampling is feasible. It is important to consider system parameters such as dialysis flow rate, membrane length, and sample properties because these factors will affect analyte concentrations obtained during local metabolism experiments.     

Refolding of firefly luciferase immobilized on agarose beads

The renaturation yield of the denatured firefly luciferase decreased strongly with increasing protein concentration in a renaturation buffer, because of aggregation. In this study, firefly luciferase was immobilized on agarose beads at a high concentration. Although the protein concentration was extremely high (about 100-fold) compared to that of soluble luciferase, the renaturation yield was comparable with that for the soluble one. Thus, immobilization was shown to be effective for avoiding aggregation of firefly luciferase. It was also shown that the optimum buffer conditions for renaturation of the immobilized luciferase were the same as those for the renaturation in solution. Also, it was indicated that electrostatic interactions between a protein and the matrix have a negative effect on renaturation of the immobilized luciferase since the renaturation yield decreased at acidic pH only for the immobilized luciferase. These novel observations are described in detail in this paper.     

Reactivation of covalently immobilized lipase from Thermomyces lanuginosus

Lipase from Thermomyces lanuginosus (TLL) immobilized on cyanogen bromide agarose (CNBr) may be fully inactivated when incubated in saturated solutions of guanidine. When this inactivated enzyme is re-incubated in aqueous medium, 20% of the activity may be recovered for several cycles. However, if the activity was determined in the presence of a detergent (CTAB, an activator of this enzyme), 100% of the initial activity in the presence of detergent was recovered. The enzyme was also inactivated in the presence of organic solvents and at high temperatures. Inactivations were more rapid when the activity was determined in absence of detergent. In both cases, some activity could be recovered just by incubation under mild conditions, and this increase was higher if the activity measurements were performed in the presence of CTAB. These results suggested that the opening of the lipase could be a critical step in the inactivation or reactivation of immobilized TLL. In inactivations in the presence of solvents, 100% of activity could be recovered during several cycles, while in thermal inactivations, the recovered activity decreased in each inactivation–reactivation cycle. The incubation of the enzyme inactivated by temperature in guanidine improved the results, but still 100% could not be achieved during several cycles even measured in the presence of CTAB.Thus, the simple incubation of the partially or fully inactivated enzyme under mild conditions permitted to recover some activity (enhancing the half life of the biocatalysts), even in thermal inactivations.     

Effects of microwave irradiation on triglyceride transesterification: Experimental and theoretical studies

An improved reaction rate of biodiesel fuel formation was observed under microwave irradiation. Such irradiation is effective for not only heating during the reaction but also preheating. The advantages observed in this study suggest that a continuous process, that is, the introduction of reactant preheated by microwave irradiation into reactor will be feasible. The reasons for the improved reaction rate are explained through the use of optimized structures, dipole moments, IR spectra and activation energies obtained using molecular orbital calculations. Planar triolein formed by microwave irradiation has higher reactivity, a lower dipole moment, a lower activation energy and stronger vibration around the carboxyl carbon, and is more reactive than triolein with a higher dipole moment.     

Inhibition of Escherichia coli growth and respiration by polymyxin B covalently attached to agarose beads.

Polymyxin B was attached to agarose beads by stable covalent bonds and the antimicrobial activity of the immobilized peptide was examined. Polymyxin-agarose inhibited the growth of Escherichia coli and Pseudomonas aeruginosa, but not Bacillus subtilis. In addition, the respiration of E. coli, E. coli spheroplasts, and B. subtilis protoplasts was inhibited by immobilized polymyxin, whereas the respiration of B. subtilis was unaffected by polymyxin-agarose. The activity of polymyxin-agarose was not due to the release of free peptide from the derivative. These data indicate that polymyxin can inhibit the growth and respiration of gram-negative bacteria by interacting with the outer surface of these cells. It is proposed that perturbation of outer membrane structure by polymyxin-agarose indirectly affected the selective permeability of the inner membrane and inhibited respiration. The results of this study emphasize the importance of outer membrane structural integrity for the normal functions of gram-negative bacteria.     

Experimental and theoretical studies of mechanical unfolding of different proteins

Mechanical properties of proteins are important for a wide range of biological processes including cell adhesion, muscle contraction, and protein translocation across biological membranes. It is necessary to reveal how proteins achieve their required mechanical stability under natural conditions in order to understand the biological processes and also to use the knowledge for constructing new biomaterials for medical and industrial purposes. In this connection, it is important to know how a protein will behave in response to various impacts. Theoretical and experimental works on mechanical unfolding of globular proteins will be considered in detail in this review.