A dynamic membrane reactor with immobilized α-chymotrypsin for continuous kyotorphin synthesis in organic media

-Chymotrypsin was covalently attached to an -alumina ultrafiltration membrane coated with an inert protein. This derivative was used as catalyst for the continuous kinetically controlled synthesis of kyotorphin in both membrane and packed bed reactors, using aqueous (water/dimethyl sulfoxide, 60:40, v/v) and nearly-dry (hexane/ethanol/water, 57:40:3, by vol.) organic media, respectively. In both media, the synthetic activity and operational stability of the enzyme-membrane derivative was compared with an adsorbed -chymotrypsin Celite derivative, being 2-times and 4-times respectively, higher than the Celite one. The enzyme-membrane derivative showed half-life times higher than 36 days, with a selectivity near to 100%.     

Immunoscintigraphic localization of renal tumours in an extracorporeal perfusion model with a monoclonal antibody against γ-glutamyltransferase

Summary Monoclonal antibody 138H11 against human -glutamyltransferase has been shown to react immunohistochemically with 98% of all tested clear-cell type and chromophilic renal cell carcinomas, but not with renal chromophobic carcinomas, Duct-Bellini carcinomas or oncocytomas. In normal kidney the target epitopes of mAb 138H11 are located in the luminal brush-border membrane of proximal tubule cells, whereas in renal carcinomas the epitopes are found surrounding the whole tumour cells. These results form the basis of the present immunoscintigraphic study designed to evaluate mAb 138H11 in an extracorporeal perfusion model. Immediately after nephrectomy, human tumour-bearing kidneys were perfused with^99mTc-labelled mAb 138H11 in Euro-Collins solution. High specific uptake in 4/4 renal clear cell carcinomas could be demonstrated by planar immunoscintigraphy and single-photon-emission computed tomography, regions of interest investigation and immunohistochemistry. In contrast, a perfused oncocytoma showed up as an unlabelled lesion. The results indicate a possible use for mAb 138H11 in immunoscintigraphy or even therapy, provided high tumour uptake can be confirmed in patients.     

Continuous hydrolysis ofβ-casein in a membrane reactor: Preparation of a bioactive peptide

Summary Continuous Stirred Tank Membrane Reactor was used to investigate the continuous and selective extraction of a bioactive peptide-CN (193–209) from bovine-casein/chymosin hydrolysate. It was shown that the feasibility of the process depends on the nature and the area of ultrafiltration membrane used. With an inorganic (carbon-zirconia) membrane, high retention of the peptide constitutes a limit to the operation. However, when the reactor was equipped with a cellulosic type membrane, satisfactory transmission of the peptide was obtained. As shown by RP-HPLC and mass spectrometry analysis, only-CN (193–209) permeates through the membrane.     

Characterization and optimization of β-galactosidase immobilization process on a mixed-matrix membrane

β-Galactosidase is an important enzyme catalyzing not only the hydrolysis of lactose to the monosaccharides glucose and galactose but also the transgalactosylation reaction to produce galacto-oligosaccharides (GOS). In this study, β-galactosidase was immobilized by adsorption on a mixed-matrix membrane containing zirconium dioxide. The maximum β-galactosidase adsorbed on these membranes was 1.6 g/m², however, maximal activity was achieved at an enzyme concentration of around 0.5 g/m². The tests conducted to investigate the optimal immobilization parameters suggested that higher immobilization can be achieved under extreme parameters (pH and temperature) but the activity was not retained at such extreme operational parameters. The investigations on immobilized enzymes indicated that no real shift occurred in its optimal temperature after immobilization though the activity in case of immobilized enzyme was better retained at lower temperature (5 °C). A shift of 0.5 unit was observed in optimal pH after immobilization (pH 6.5 to 7). Perhaps the most striking results are the kinetic parameters of the immobilized enzyme; while the Michaelis constant (K_m) value increased almost eight times compared to the free enzyme, the maximum enzyme velocity (V_max) remained almost constant.     

Characterization and optimization of β-galactosidase immobilization process on a mixed-matrix membrane

β-Galactosidase is an important enzyme catalyzing not only the hydrolysis of lactose to the monosaccharides glucose and galactose but also the transgalactosylation reaction to produce galacto-oligosaccharides (GOS). In this study, β-galactosidase was immobilized by adsorption on a mixed-matrix membrane containing zirconium dioxide. The maximum β-galactosidase adsorbed on these membranes was 1.6 g/m2, however, maximal activity was achieved at an enzyme concentration of around 0.5 g/m2. The tests conducted to investigate the optimal immobilization parameters suggested that higher immobilization can be achieved under extreme parameters (pH and temperature) but the activity was not retained at such extreme operational parameters. The investigations on immobilized enzymes indicated that no real shift occurred in its optimal temperature after immobilization though the activity in case of immobilized enzyme was better retained at lower temperature (5 °C). A shift of 0.5 unit was observed in optimal pH after immobilization (pH 6.5 to 7). Perhaps the most striking results are the kinetic parameters of the immobilized enzyme; while the Michaelis constant (K(m)) value increased almost eight times compared to the free enzyme, the maximum enzyme velocity (V(max)) remained almost constant.     

Development of an in vitro system for screening the ligands of a membrane glycoprotein CD36

It has well been known that human and rodents exhibit a preference for fats. This suggests the existence of an orosensory system responsible for the detection of dietary fats. A plasma membrane glycoprotein CD36, besides the role in the uptake of long-chain fatty acids (LCFAs) as well as oxidized low-density lipoprotein (OxLDL) in a variety of cells, has been postulated to be a candidate fat taste receptor on the tongue. Therefore, molecules that bind with CD36 to cause intracellular signaling but have fewer calories could be substitutes for dietary fats. In the present study, we developed an in vitro system for the screening of CD36 ligands using Chinese hamster ovary-K1 cells (CHO-K1) stably transfected with human or mouse CD36. When incubated with OxLDL labeled with fluorescence dye, the fluorescence was much higher in the transfected CHO-K1 cells than in non-transfected CHO-K1 cells. Incubation of the transfected cells with OxLDL caused a rapid phosphorylation of extracellular signal regulated kinase, and the degree was significantly higher compared with that in non-transfected CHO-K1 cells. The expression system using CHO-K1 cells could be a convenient tool to screen the novel ligands of CD36.     

Apoplastic and cytosolic expression of full‐size antibodies and antibody fragments in Nicotiana tabacum

We compared the expression of a functional recombinant TMVspecific fullsize antibody (rAb29) in both the apoplast and cytosol of tobacco plants and a single chain antibody fragment (scFv29), derived from rAb29, was expressed in the cytosol. Cloned heavy and light chain cDNAs of fullsize rAb29, which binds to TMV coat protein monomers, were integrated into the plant expression vector pSS. The fullsize rAb29 was expressed in the cytosol and targeted to the apoplast by including the original murine antibody leader sequences. Levels of functional fullsize rAb29 expression were high in the apoplast (up to 8.5g per gram leaf tissue), whereas cytosolic expression was low or at the ELISA detection limit. Sequences of the variable domains of rAb29 light and heavy chain were used to generate the single chain antibody scFv29, which was expressed in the periplasmic space of E.coli and showed the same binding specificity as fullsize rAb29. In addition, scFv29 was functionally expressed in the cytosol of tobacco plants and plant derived scFv29 maintained same binding specificity to TMVcoat protein monomers as rAb29.     

Development of an anti-Aβ monoclonal antibody for in vivo imaging of amyloid angiopathy in Alzheimer's disease

We evaluated the efficacy of murine monoclonal antibodies (MAbs) targeted to the Aβ amyloid of Alzheimer's disease for development of procedures for the in vivo identification of amyloid angiopathy (AA). MAbs to Aβ were prepared and screened for effectiveness in visualizing AA and neuritic plaques in postmortem AD brain sections. They were assessed again after enzymatic cleavage to produce Fab fragments and after labeling with technetium-99m (^99mTc) using a diamide dimercaptide ligand system. Modified and radiolabeled Fab fragments retained activity and specificity toward amyloid-laden blood vessels and neuritic plaques. A highly specific murine MAb, 10H3, was identified and characterized that fulfills criteria necessary for the development of an in vivo diagnostic imaging agent. Toxicity studies in rats showed the MAb to be safe. Biodistribution studies in mice demonstrated desirable properties for use as an imaging agent. Expansion and adaptation of these strategies may provide the methods and materials for the noninvasive analysis of AA in living patients, and permit assessment of the contribution of AA to the clinical and pathological features of AD.     

Is arterial wall-strain stiffening an additional process responsible for atherosclerosis in coronary bifurcations?: an in vivo study based on dynamic CT and MRI

Coronary bifurcations represent specific regions of the arterial tree that are susceptible to atherosclerotic lesions. While the effects of vessel compliance, curvature, pulsatile blood flow, and cardiac motion on coronary endothelial shear stress have been widely explored, the effects of myocardial contraction on arterial wall stress/strain (WS/S) and vessel stiffness distributions remain unclear. Local increase of vessel stiffness resulting from wall-strain stiffening phenomenon (a local process due to the nonlinear mechanical properties of the arterial wall) may be critical in the development of atherosclerotic lesions. Therefore, the aim of this study was to quantify WS/S and stiffness in coronary bifurcations and to investigate correlations with plaque sites. Anatomic coronary geometry and cardiac motion were generated based on both computed tomography and MRI examinations of eight patients with minimal coronary disease. Computational structural analyses using the finite element method were subsequently performed, and spatial luminal arterial wall stretch (LW(Stretch)) and stiffness (LW(Stiff)) distributions in the left main coronary bifurcations were calculated. Our results show that all plaque sites were concomitantly subject to high LW(Stretch) and high LW(Stiff), with mean amplitudes of 34.7 ± 1.6% and 442.4 ± 113.0 kPa, respectively. The mean LW(Stiff) amplitude was found slightly greater at the plaque sites on the left main coronary artery (mean value: 482.2 ± 88.1 kPa) compared with those computed on the left anterior descending and left circumflex coronary arteries (416.3 ± 61.5 and 428.7 ± 181.8 kPa, respectively). These findings suggest that local wall stiffness plays a role in the initiation of atherosclerotic lesions.     

Resurrection of a clinical antibody: template proteogenomic de novo proteomic sequencing and reverse engineering of an anti-lymphotoxin-α antibody

A mouse hybridoma antibody directed against a member of the tumour necrosis factor (TNF)-superfamily, lymphotoxin-alpha (LT-α), was isolated from stored mouse ascites and purified to homogeneity. After more than a decade of storage the genetic material was not available for cloning; however, biochemical assays with the ascites showed this antibody against LT-α (LT-3F12) to be a preclinical candidate for the treatment of several inflammatory pathologies. We have successfully rescued the LT-3F12 antibody by performing MS analysis, primary amino acid sequence determination by template proteogenomics, and synthesis of the corresponding recombinant DNA by reverse engineering. The resurrected antibody was expressed, purified and shown to demonstrate the desired specificity and binding properties in a panel of immuno-biochemical tests. The work described herein demonstrates the powerful combination of high-throughput informatic proteomic de novo sequencing with reverse engineering to reestablish monoclonal antibody-expressing cells from archived protein sample, exemplifying the development of novel therapeutics from cryptic protein sources.     

Structure of an ‘open’ clamp type II topoisomerase-DNA complex provides a mechanism for DNA capture and transport

Type II topoisomerases regulate DNA supercoiling and chromosome segregation. They act as ATP-operated clamps that capture a DNA duplex and pass it through a transient DNA break in a second DNA segment via the sequential opening and closure of ATPase-, G-DNA- and C-gates. Here, we present the first ‘open clamp’ structures of a 3-gate topoisomerase II-DNA complex, the seminal complex engaged in DNA recognition and capture. A high-resolution structure was solved for a (full-length ParE-ParC55)₂ dimer of Streptococcus pneumoniae topoisomerase IV bound to two DNA molecules: a closed DNA gate in a B-A-B form double-helical conformation and a second B-form duplex associated with closed C-gate helices at a novel site neighbouring the catalytically important β-pinwheel DNA-binding domain. The protein N gate is present in an ‘arms-wide-open’ state with the undimerized N-terminal ParE ATPase domains connected to TOPRIM domains via a flexible joint and folded back allowing ready access both for gate and transported DNA segments and cleavage-stabilizing antibacterial drugs. The structure shows the molecular conformations of all three gates at 3.7 Å, the highest resolution achieved for the full complex to date, and illuminates the mechanism of DNA capture and transport by a type II topoisomerase.     

Sequence‐ and activity‐based screening of microbial genomes for novel dehalogenases

Dehalogenases are environmentally important enzymes that detoxify organohalogens by cleaving their carbon-halogen bonds. Many microbial genomes harbour enzyme families containing dehalogenases, but a sequence-based identification of genuine dehalogenases with high confidence is challenging because of the low sequence conservation among these enzymes. Furthermore, these protein families harbour a rich diversity of other enzymes including esterases and phosphatases. Reliable sequence determinants are necessary to harness genome sequencing-efforts for accelerating the discovery of novel dehalogenases with improved or modified activities. In an attempt to extract dehalogenase sequence fingerprints, 103 uncharacterized potential dehalogenase candidates belonging to the α/β hydrolase (ABH) and haloacid dehalogenase-like hydrolase (HAD) superfamilies were screened for dehalogenase, esterase and phosphatase activity. In this first biochemical screen, 1 haloalkane dehalogenase, 1 fluoroacetate dehalogenase and 5 l -2-haloacid dehalogenases were found (success rate 7%), as well as 19 esterases and 31 phosphatases. Using this functional data, we refined the sequence-based dehalogenase selection criteria and applied them to a second functional screen, which identified novel dehalogenase activity in 13 out of only 24 proteins (54%), increasing the success rate eightfold. Four new l -2-haloacid dehalogenases from the HAD superfamily were found to hydrolyse fluoroacetate, an activity never previously ascribed to enzymes in this superfamily.     

Synthesis and degradation of poly-β-hydroxybutyrate in a sequencing batch biofilm reactor

The aim of this work was the study of poly-β-hydroxybutyrate (PHB) formation and degradation in a sequencing batch biofilm reactor (SBBR). The SBBR was operated in cycles comprising three individual phases: mixed fill, aeration and draw. A synthetic substrate solution with acetate and ammonium was used.PHB was formed during the aeration phase immediately after acetate depletion, and was subsequently consumed for biomass growth, owing to the high oxygen concentration in the reactor. It was observed a combination of suspended and biofilm growth in the SBBR with predominance of the fixed form of biomass (506 Cmmol and 2102 Cmmol, respectively). Maximum PHB fraction of suspended biomass (0.13 Cmol/Cmol) was considerably higher than that of biofilm (0.01 Cmol/Cmol). This may possibly be explained by a combination of two factors: lower mass transfer limitation of acetate and higher fraction of heterotrophs in suspended biomass compared to the ones of biofilm.     

Characterization of membrane foulants in a pilot-scale powdered activated carbon–membrane bioreactor for drinking water treatment

The external and internal foulants of a pilot-scale powdered activated carbon–membrane bioreactor (PAC–MBR) used for drinking water treatment were systematically examined by scanning electron microscopy (SEM), three-dimensional excitation emission matrix (EEM) fluorescence spectroscopy, Fourier transform infrared spectroscopy (FTIR) and energy diffusive X-ray (EDX) analysis. The results showed that external fouling, which comprised 31.68% of the total fouling, was caused by the deposition of a large amount of biological PAC on the membrane surface. Bacteria and organic matter comprised only a small fraction of the external foulants. Biologically derived proteins and polysaccharides were the major constituents of the internal foulants. EDX analysis indicated that the external and internal foulants also included inorganic elements such as Mg, Al, Si, Ca, Mn and Fe. During the operation of PAC–MBR, low flux and effective physical cleaning protocols should be adopted; proteins, polysaccharides and inorganic elements in the bioreactor should also be controlled.     

Selective growth of a mutant in continuous culture of Bacillus caldolyticus for production of α-amylase

Summary In a continuous culture of Bacillus caldolyticus strain SP, which requires maltose as an inducer for production of -amylase in batch culture, a predominant mutant strain M1 which produced high amounts of -amylase in the absence of maltose in batch culture, developed. The change of cell population from strain SP to strain M1 in maltose-casitone medium was linear with time in the transient state after the change from batch to continuous culture at a dilution rate of 0.17 h^-1, and was completed in about 11 generations of bacterial growth. The dilution rate effect of continuous culture on -amylase activity was almost the same with both strains SP and M1. The maximum -amylase activity of 380 units/ml was observed at an intermediate dilution rate that was 11.5 times higher than -amylase activity at the end of a batch culture using the same medium. It was deduced that the enhancement of -amylase production in continuous culture was attributed partly to the predominant growth of a mutant strain with higher -amylase productivity.     

Performance of an anaerobic baffled reactor and hybrid constructed wetland treating high-strength wastewater in Nepal—A model for DEWATS

Centralized wastewater treatment systems require sophisticated technologies and skilled manpower for their operation and maintenance (O&M). These systems have huge construction as well as O&M costs. Therefore, a Decentralized Wastewater Treatment System (DEWATS) rather than a centralized system might be especially beneficial in developing countries. A model for DEWATS is developed in Nepal with Anaerobic Baffled Reactor (ABR) and hybrid Constructed Wetland (CW). The DEWATS treats high-strength wastewater from 80 households (400 PE). This paper summarizes the performance of the DEWATS from July 2006 to August 2007 in the removal efficiencies of TSS, BOD₅, COD, NH₄–N, TP and FC. The ABR is very effective in the removal of organic pollutants and could achieve TSS removal up to 91%, BOD₅ up to 78% and COD up to 77%. The average removal efficiencies of the DEWATS is 96% TSS, 90% BOD₅, 90% COD, 70% NH₄–N, 26% TP and 98% FC.     

Influence of alterations in culture condition and changes in perfusion parameters on the retention performance of a 20 μm spinfilter during a perfusion cultivation of a recombinant CHO cell line in pilot scale

Since 1969 much attention has been devoted to the useof spinfilter systems for retention of mammalian cellsin continuous perfusion cultivations. Previousinvestigations dealt with hydrodynamic conditions,fouling processes and upscaling. But hydrodynamicconditions and fouling processes seem to have asecondary importance in spinfilter performance duringauthentic perfusion cultivations. Obviously,alterations in culture condition are more relevantespecially during long-term processes. Therefore, ourpratical approach focussed on the performance qualityof a commercially available 20 m spinfilterduring a perfusion cultivation of a recombinant CHOcell line in pilot scale regarding the followingissues: 1) retention of viable cells in thebioreactor; 2) removal of dead cells and cell debrisfrom the bioreactor; 3) alterations in culturecondition; and 4) changes in perfusion mode.Furthermore, we tested the performance of 20 mspinfilters in 2 and 100 l pilot scale using solidmodel particles instead of cells. Our investigationsshowed that retention of viable cells in pilot scalewas independent of spinfilter rotation velocity andperfusion rate; the retention increased from 75 to 95%corresponding to operation time, enlarging celldiameter and enhanced formation of aggregates in theculture during the perfusion cultivation. By means ofthe Cell Counter and Analyzer System (CASY) anoperation cut off of 13 m was determined forthis spinfilter. Using solid model particles in 2 lscale, optimal retention was achieved at a tip speedof 0.43 m s^-1 (141 rpm) – furtherenhancement of spinfilter rotation velocity up to0.56 m s^-1 (185 rpm) decreased the retentionrapidly. In pilot scale best retention performance wasobtained with tip speeds of 0.37 m s^-1(35 rpm) and 1.26 m s^-1 (120 rpm). Hence,significant retention in pilot scale could already beachieved with low agitation. Therefore, the additionof shear force protectives could be avoided so thatthe purification of the target protein from thesupernatant would be facilitated.     

Inversion of sucrose in a continuous process with β-d-fructofuranosidase (invertase) immobilized on bead DEAHP-cellulose

Inversion of sucrose with β-d-fructofuranosidase (EC 3.2.1.26) immobilized by the ionic bond on bead DEAHP-cellulose has been studied under flow conditions. Under these conditions, the inversion of sucrose is affected by the concentration and flow rate of the substrate and by the reaction temperature. The effect of substrate concentration on the reaction was investigated in the range 19.5–64.2 wt %; the effect of flow rate was examined in the range 0.25–5.57 g solution per min, and the temperature range used was 25–50°C. It was found that the activities of immobilized β-d-fructofuranosidase in stirred and flow reactors were the same. The lower activities of β-d-fructofuranosidase in the case of concentrated solutions, and of immobilized β-d-fructofuranosidase compared with the native enzyme are attributed to more difficult diffusion through the beads of the ion exchanger, especially of the strongly viscous substrate. A long-term investigation of the enzyme activity over a period of three months demonstrated the stability of the β-d-fructofuranosidase immobilized by the ionic bond on bead DEAHP-cellulose; the half-life of the enzyme was 215 days. It was also found that the immobilization of the enzyme on a carrier was more effective under flow conditions, i.e. through an ion exchanger in the column, than under the equilibrium conditions of a stirred reactor.     

Membrane interactions and fibrillization of α-synuclein play an essential role in membrane disruption

We studied α-synuclein (αS) aggregation in giant vesicles, and observed dramatic membrane disintegration, as well as lipid incorporation into micrometer-sized suprafibrillar aggregates. In the presence of dye-filled vesicles, dye leakage and fibrillization happen concurrently. However, growing fibrils do not impair the integrity of phospholipid vesicles that have a low affinity for αS. Seeding αS aggregation accelerates dye leakage, indicating that oligomeric species are not required to explain the observed effect. The evolving picture suggests that fibrils that appear in solution bind membranes and recruit membrane-bound monomers, resulting in lipid extraction, membrane destabilization and the formation of lipid-containing suprafibrillar aggregates.     

Reconstitution and alignment by a magnetic field of a β-barrel membrane protein in bicelles

A protocol is described for the reconstitution of a transmembrane β-barrel protein domain, tOmpA, into lipid bicelles. tOmpA is the largest protein to be reconstituted in bicelles to date. Its insertion does not prevent bicelles from orienting with their plane either parallel or perpendicular to the magnetic field, depending on the absence or presence of paramagnetic ions. In the latter case, tOmpA is shown to align with the axis of the β-barrel parallel to the magnetic field, i.e. perpendicular to the plane of the bilayer, an orientation conforming to that in natural membranes and favourable to structural studies by solid-state NMR. Reconstitution into bicelles may offer an interesting approach for structural studies of membrane proteins in a medium resembling a biological membrane, using either NMR or other biophysical techniques. Our data suggest that alignment in the magnetic field of membrane proteins included into bicelles may be facilitated if the protein is folded as a β-barrel structure.