Development of recombinant, immobilised beta-1,4-mannosyltransferase for use as an efficient tool in the chemoenzymatic synthesis of N-linked oligosaccharides.

The preparation of the conserved core structure of asparagine-linked oligosaccharides found in eukaryotic glycoproteins is an important step towards the synthesis of homogeneous neoglycoproteins. So far, however, the convenient generation of the Manbeta4GlcNAcbeta4GlcNAc (Gn2M) core trisaccharide has proved to be a major obstacle because of the inherent difficulties associated with the synthesis of beta-mannosides. Here we report the overproduction in Escherichia coli of full-length and transmembrane-deleted yeast beta-1, 4-mannosyltransferases as novel N-terminal fusions bearing a decahistidinyl sequence and the minimal human Myc epitope. The recombinant enzymes were highly active and were amenable to immobilisation by nickel(II) chelation and to immunodetection with an anti-Myc monoclonal antibody. The immobilised, transmembrane-deleted enzyme exhibited an apparent Km of 14 microM for the synthetic acceptor substrate analogue, phytanyl-pyrophosphoryl-alpha-N,N'-diacetylchitobioside (PPGn2), under saturating donor conditions. This figure is comparable to those previously reported for native and recombinant yeast beta-1, 4-mannosyltransferases with, respectively, the natural dolichyl-linked acceptor and PPGn2. The validity of the reaction product was confirmed by chromatographic and spectroscopic analysis.     

Features and functions of covalently linked proteins in fungal cell walls

The cell walls of many ascomycetous yeasts consist of an internal network of stress-bearing polysaccharides, which serve as a scaffold for a dense external layer of glycoproteins. GPI-modified proteins are the most abundant cell wall proteins and often display a common organization. Their C-terminus can link them covalently to the polysaccharide network, they possess an internal serine- and threonine-rich spacer domain, and the N-terminal region contains a functional domain. Other proteins bind to the polysaccharide network through a mild-alkali-sensitive linkage. Many cell wall proteins are carbohydrate/glycan-modifying enzymes; adhesion proteins are prominent; proteins involved in iron uptake are present, and also specialized proteins that probably help the fungus to survive in its natural environment. The protein composition of the cell wall depends on environmental conditions and developmental stage. We present evidence that the cell wall of mycelial species of the Ascomycotina is similarly organized and contains glycoproteins with comparable functions.     

Detoxification of hexavalent chromate by Amphibacillus sp. KSUCr3 cells immobilised in silica-coated magnetic alginate beads

Recently isolated Cr(VI)-reducing Amphibacillus KSUCr3 whole cells were immobilised in magnetic gels. Magnetic magnetite (Fe₃O₄) nanoparticles were synthesised with an average particle size of 47 nm and 80 electromagnetic unit (emu)/g saturation magnetisation. Whole cells were immobilised by entrapment in agar, agarose, alginate, or gelatin in the presence or absence of Fe₃O₄ nanoparticles for the preparation of both magnetic and nonmagnetic immobilised cells. Of the gels tested, alginate was selected as the best immobilisation matrix, and following optimisation of the entrapment process, the immobilisation yield reached 92.5%. In addition to the ease of separation and reuse of the magnetic cell-containing alginate beads using an external magnet, the magnetically immobilised cells showed approximately 16% higher Cr(VI) reduction activity compared with nonmagnetic immobilised cells. To improve their physical and mechanical properties, the magnetic alginate beads were successfully coated with a dense silica layer using sol-gel chemistry and Ca(OH)₂, an alkaline catalyst for tetraethyl orthosilicate, to avoid leaching of Ca²⁺ ions. Amphibacillus KSUCr3 cells immobilised in silica-coated magnetic alginate beads showed approximately 1.4- to 3.9-fold enhancement of thermal stability compared with free cells. Furthermore, after seven batch cycles, the Cr(VI) reduction activity of free cells decreased to 48%, whereas immobilised cells still retained 81.1% of their original activity. In addition, the Cr(VI)-reduction rate of immobilised cells was higher relative to free cells, especially at higher Cr(VI) concentrations. These results supported the development of a novel, efficient biocatalysts for Cr(VI) detoxification using a combination of whole cell immobilisation, sol-gel chemistry, and nanotechnology.     

Activated zeolite—suitable carriers for microorganisms in anaerobic digestion processes?

Plant cell wall structures represent a barrier in the biodegradation process to produce biogas for combustion and energy production. Consequently, approaches concerning a more efficient de-polymerisation of cellulose and hemicellulose to monomeric sugars are required. Here, we show that natural activated zeolites (i.e. trace metal activated zeolites) represent eminently suitable mineral microhabitats and potential carriers for immobilisation of microorganisms responsible for anaerobic hydrolysis of biopolymers stabilising related bacterial and methanogenic communities. A strategy for comprehensive analysis of immobilised anaerobic populations was developed that includes the visualisation of biofilm formation via scanning electron microscopy and confocal laser scanning microscopy, community and fingerprint analysis as well as enzyme activity and identification analyses. Using SDS polyacrylamide gel electrophoresis, hydrolytical active protein bands were traced by congo red staining. Liquid chromatography/mass spectroscopy revealed cellulolytical endo- and exoglucanase (exocellobiohydrolase) as well as hemicellulolytical xylanase/mannase after proteolytic digestion. Relations to hydrolytic/fermentative zeolite colonisers were obtained by using single-strand conformation polymorphism analysis (SSCP) based on amplification of bacterial and archaeal 16S rRNA fragments. Thereby, dominant colonisers were affiliated to the genera Clostridium, Pseudomonas and Methanoculleus. The specific immobilisation on natural zeolites with functional microbes already colonising naturally during the fermentation offers a strategy to systematically supply the biogas formation process responsive to population dynamics and process requirements.     

Amperometric enzyme biosensors based on optimised electron-transfer pathways and non-manual immobilisation procedures

Development of reagentless biosensors implies the tight and functional immobilisation of biological recognition elements on transducer surfaces. Specifically, in the case of amperometric enzyme electrodes, electron-transfer pathways between the immobilised redox protein and the electrode surface have to be established allowing a fast electron transfer concomitantly avoiding free-diffusing redox species. Based on the specific nature of different redox proteins and non-manual immobilisation procedures possible biosensor designs are discussed, namely biosensors based on (i) direct electron transfer between redox proteins and electrodes modified with self-assembled monolayers; (ii) anisotropic orientation of redox proteins at monolayer-modified electrodes; (iii) electron-transfer cascades via redox hydrogels; and (iv) electron-transfer via conducting polymers.     

Immobilization and characterization of carbonic anhydrase purified from E. coli MO1 and its influence on CO2 sequestration

The present investigation entails the immobilisation and characterisation of Escherichia coli MO1-derived carbonic anhydrase (CA) and its influence on the transformation of CO₂ to CaCO₃. CA was purified from MO1 using a combination of Sephadex G-75 and DEAE cellulose column chromatography, resulting in 4.64-fold purification. The purified CA was immobilised in chitosan-alginate polyelectrolyte complex (C-A PEC) with an immobilisation potential of 94.5 %. Both the immobilised and free forms of the enzyme were most active and stable at pH 8.2 and at 37 °C. The K _m and V _max of the immobilised enzyme were found to be 19.12 mM and 416.66 μmol min^?1 mg^?1, respectively; whereas, the K _m and V _max of free enzyme were 18.26 mM and 434.78 μmol min^?1 mg^?1, respectively. The presence of metal ions such as Cu²⁺, Fe²⁺, and Mg²⁺ stimulated the enzyme activity. Immobilised CA showed higher storage stability and maintained its catalytic efficiency after repeated operational cycles. Furthermore, both forms of the enzyme were tested for targeted application of the carbonation reaction to convert CO₂ to CaCO₃. The amounts of CaCO₃ precipitated over free and immobilised CA were 267 and 253 mg/mg of enzyme, respectively. The results of this study show that immobilised CA in chitosan-alginate beads can be useful for CO₂ sequestration by the biomimetic route.     

Cell Wall Antigens of Pneumocystis carinii Trophozoites and Cysts: Purification and Carbohydrate Analysis of these Glycoproteins

We have purified and biochemically analyzed individual cell wall glycoproteins of Pneumocystis carinii. Our results show that corresponding core glycoproteins constitute the cell wall antigens in both trophozoites and cysts, and glycosylation of these glycoproteins does not appear to be significantly altered during development. Cysts and trophozoites in rat-derived organism preparations were separated from each other by counterflow centrifugal elutriation, then treated with Zymolyase to obtain the cell wall fractions. Gel electrophoresis patterns of these fractions from both life-cycle stages were qualitatively similar. Ten major antigenic glycoproteins in these fractions were purified by preparative continuous elution gel electrophoresis. All ten glycoproteins from cysts and trophozoites contained mannose, glucose, galactose. and N-acetylglucosamine, and some contained traces of fucose. The glycoproteins of cysts had more mannose than their trophozoite counterparts. The trophozoite glycoproteins differed from those of the cyst by the presence of xylose. To examine the species-specificity of glycoprotein glycosylation, preparations of human-derived P. carinii (comprised of mixed life-cycle stages) were also examined and found to contain the same sugars as those found in rat-derived organisms. Most of the purified rat-derived glycoproteins bound Concanavalin A, which was abolished by treatment with N-glycanase. This suggested that the majority of the oligosaccharides were N-linked to the proteins, but attempts to identify carbohydrate linkage sites by amino acid sequencing were hampered by apparent modifications of residues. The peptides derived by cyanogen bromide cleavage revealed distinct size patterns for each glycoprotein, suggesting that they were distinct proteins. Most of the glycoproteins reacted with monoclonal antibodies which recognize a highly conserved epitope on rat P. carinii. Four of the individually purified glycoprotein preparations elicited in vitro cellular immune responses, implicating their involvement in the recognition of P. carinii by host T cells. The identification and characterization of P. carinii cell wall proteins will be helpful in analyzing the relationship of the organism to its mammalian host. Supplementary key words. Biochemical analysis, developmental stages, opportunistic pathogen, structure.     

Domain conservation in several volvocalean cell wall proteins

Based on our previous work demonstrating that (SerPro)_x epitopes are common to extensin-like cell wall proteins in Chlamydomonas reinhardtii, we looked for similar proteins in the distantly related species C. eugametos. Using a polyclonal antiserum against a (SerPro)₁₀ oligopeptide, we found distinct sets of stage-specific polypeptides immunoprecipitated from in vitro translations of C. eugametos RNA. Screening of a C. eugametos cDNA expression library with the antiserum led to the isolation of a cDNA (WP6) encoding a (SerPro)_x-rich multidomain wall protein. Analysis of a similarly selected cDNA (VSP-3) from a C. reinhardtii cDNA expression library revealed that it also coded for a (SerPro)_x-rich multidomain wall protein. The C-terminal rod domains of VSP-3 and WP6 are highly homologous, while the N-terminal domains are dissimilar; however, the N-terminal domain of VSP-3 is homologous to the globular domain of a cell wall protein from Volvox carteri. Exon shuffling might be responsible for this example of domain conservation over 350 million years of volvocalean cell wall protein evolution.     

Kinetics of invertase immobilised on poly(phe-lys) coated polystyrene beads

Summary Surface of polystyrene beads was modified by poly(phe-lys) for invertase immobilisation. The optimum immobilisation conditions of invertase were; 0.01% (w/v) poly(phe-lys), 2% (v/v) glutaraldehyde at 25 °C and pH 4.5. The kinetics of sucrose hydrolysis by free and immobilised invertase in a batch reactor at pH 4.5 and 55 °C gave K_m and V_max values for sucrose with free and immobilised invertase of 81, 114 mM and 10.1, 9.2 mol glucose/min.mg enzyme, respectively. The deactivation rate constants of free and immobilised invertase were 0.0347 and 0.0098 min^–1, respectively.     

Rapid changes in oxidative metabolism as a consequence of elicitor treatment of suspension-cultured cells of French bean (Phaseolus vulgaris L.)

Stressed plant cells often show increased oxygen uptake which can manifest itself in the transient production of active oxygen species, the oxidative burst. There is a lack of information on the redox status of cells during the early stages of biotic stress. In this paper we measure oxygen uptake and the levels of redox intermediates NAD/NADH and ATP and show the transient induction of the marker enzyme for redox stress, alcohol dehydrogenase. Rapid changes in the redox potential of elicitor-treated suspension cultures of French bean cells indicate that, paradoxically, during the period of maximum oxygen uptake the levels of ATP and the NADH/NAD ratio fall in a way that indicates the occurrence of stress in oxidative metabolism. This period coincides with the maximum production of active oxygen species particularly H₂O₂. The cells recover and start producing ATP immediately upon the cessation of H₂O₂ production. This indicates that the increased O₂ uptake is primarily incorporated into active O₂ species. A second consequence of these changes is probably a transient compromising of the respiratory status of the cells as indicated in expression of alcohol dehydrogenase. Elicitor-induced bean ADH was purified to homogeneity and the M_r 40 000 polypeptide was subjected to amino acid sequencing. 15% of the whole protein was sequenced from three peptides and was found to have nearly 100% sequence similarity to the amino acid sequence for pea ADH1 (PSADH1). The cDNA coding for the pea enzyme was used to demonstrate the transient induction of ADH mRNA in elicitor-treated bean cells. Enzyme activity levels also increased transiently subsequently. Increased oxygen uptake has previously been thought to be associated with provision of energy for the changes in biosynthesis that occur rapidly after perception of the stress signal. However the present work shows that this rapid increase in oxygen uptake as a consequence of elicitor action is not wholly associated with respiration.     

Factors affecting the immobilisation of plant cells on reticulated polyurethane foam particles

Summary Factors affecting the immobilisation and subsequent growth of plant cells in reticulated polyurethane foam particles have been studied using three plant species. Polyurethane foam from a number of commercial sources has been screened and a foam having a low phytotoxicity and good retention of plant cells selected for use. Particles (8×8×8 mm) of the material were seeded with plant cells from suspension culture and cells grown immobilised in particles until they occupied >80% of the available volume. For all species, foams containing small pores (60–80 ppi) were most effective in immobilising and retaining cells. For efficient use of the inoculum, high partial volumes of foam particles are required; with partial volumes above 40%, over 80% of the inoculum is taken up by the particles. While the initial immobilisation process presumably involves weak interactions between cells and the support material, factors such as inoculum size and the length of the loading period have been found to affect the immobilisation of cells and their subsequent growth within the matrix. A preliminary study of the requirements for the maintenance of viability of immobilised cultures at high cell densities has been made.     

Purification and characterization of the major whey proteins from the milks of the bottlenose dolphin (Tursiops truncatus), the Florida manatee (Trichechus manatus latirostris), and the beagle (Canis familiaris)

The major whey proteins of the milks of the dolphin, manatee, and beagle were purified by gel filtration and ion exchange chromatography and characterized and identified by molecular weight determination, amino acid analysis, N-terminal sequencing, and activity measurements. The major whey protein components from all three species were found to be monomeric beta-lactoglobulins. These proteins were all active in binding retinol. Dolphin milk contained two beta-lactoglobulins (designated 1 and 2) which showed a slight difference in molecular weight and considerably divergent N-terminal sequences, whereas the other milks only contained a single form of beta-lactoglobulin. alpha-Lactalbumins were purified from dolphin and dog milks and were active in promoting lactose synthesis by bovine galactosyltransferase. The dolphin protein had an N-terminal sequence more similar to ruminant alpha-lactalbumins than to those known from other species. Although alpha-lactalbumin activity has been detected in manatee milk at low levels, the corresponding protein was not isolated. In addition, dog milk was found to contain high levels of lysozyme (greater than 1.0 mg/ml), which were identified by activity and sequencing. The functional and evolutionary implications of these results are discussed.     

Characterization and oxidative in vitro cross-linking of an extensin-like protein and a proline-rich protein purified from chickpea cell walls

Two cell wall proteins from chickpea, known to be rapidly insolubilised by an elicitor-stimulated oxidative burst in-vivo, were purified from suspension cells. N-terminal protein sequencing revealed them as a proline-rich protein and an extensin-like protein. Oxidative cross-linking could be modelled in an in vitro system utilising horseradish peroxidase, H2O2 and the substrate proteins.     

Inhibition of Xanthine Oxidase by Flavonoids

Various dietary flavonoids were evaluated in vitro for their inhibitory effect on xanthine oxidase, which has been implicated in oxidative injury to tissue by ischemia-reperfusion. Xanthine oxidase activity was determined by directly measuring uric acid formation by HPLC. The structure-activity relationship revealed that the planar flavones and flavonols with a 7-hydroxyl group such as chrysin, luteolin, kaempferol, quercetin, myricetin, and isorhamnetin inhibited xanthine oxidase activity at low concentrations (IC₅₀ values from 0.40 to 5.02 μM) in a mixed-type mode, while the nonplanar flavonoids, isoflavones and anthocyanidins were less inhibitory. These results suggest that certain flavonoids might suppress in vivo the formation of active oxygen species and urate by xanthine oxidase.     

Rates of degradation of glycoproteins from normal and regenerating rat livers: A study using double isotopes

The average decay rates (half-lives) of mixed glycoproteins were measured using double isotopes of fucose and glucosamine and compared to those of mixed overall proteins measured with leucine and NaH¹⁴CO₃ in whole homogenates and plasma membranes from normal and regenerating rat livers. A large reutilization of leucine was observed under both normal and regenerating conditions. Fucose seems to be recycling most predominantly in regenerating liver, whereas glucosamine was found to be very little if not at all reutilized under both conditions. Comparison of the results obtained with NaH¹⁴CO₃ and glucosamine demonstrated that glycoproteins from normal liver homogenate are degraded at a faster rate than mixed proteins. Contrary to that of mixed proteins, the half-life of glycoproteins remains unchanged during liver regeneration, and the use of glucosamine revealed that the degradation of plasma membrane glycoproteins is identical to that found in whole homogenate under both normal and regenerating conditions. Finally, the relative degradation rates of fractionated plasma membrane proteins and glycoproteins were evaluated under the same conditions. During liver regeneration some readjustments are observed in the relative degradation rates of individual species which suggest that the synthesis and degradation of the various surface membrane glycoproteins proceed at rates that are controlled independently.     

Properties of free and immobilised lipase from Burkholderia cepacia in organic media

The purified lipase from Burkholderia cepacia was immobilised on a porous polypropylene support and its biocatalytic properties were compared with those of the free enzyme in organic media. For both lipase preparations, the rate of p-nitrophenyl ester hydrolysis in n-heptane was not restricted by mass transfer limitations. The immobilisation changed neither the temperature at which the reaction rate was maximal, nor the activation energy of the reaction. The enzyme stability was slightly decreased (1.3-fold) upon immobilisation. Moreover, the immobilised enzyme displayed fewer variations of activity with fatty acid chain length. Interestingly, for all the different p-nitrophenyl esters used, the immobilised enzyme was more active (from 5.8- to 18.9-fold) than the free enzyme. Therefore, it would be very useful to use B. cepacia lipase immobilised onto porous polypropylene for applications in organic media, as it displayed high activities on a larger range of substrates. Received: 8 February 1999 / Received revision: 19 March 1999 / Accepted: 20 March 1999     

Isolation and characterization of a thioredoxin-dependent peroxidase from Chlamydomonas reinhardtii.

All living organisms contain redox systems involving thioredoxins (Trx), proteins featuring an extremely conserved and reactive active site that perform thiol-disulfide interchanges with disulfide bridges of target proteins. In photosynthetic organisms, numerous isoforms of Trx coexist, as revealed by sequencing of Arabidopsis genome. The specific functions of many of them are still unknown. In an attempt to find new molecular targets of Trx in Chlamydomonas reinhardtii, an affinity column carrying a cytosolic Trx h mutated at the less reactive cysteine of its active site was used to trap Chlamydomonas proteins that form mixed disulfides with Trx. The major protein bound to the column was identified by amino-acid sequencing and mass spectrometry as a thioredoxin-dependent 2Cys peroxidase. Isolation and sequencing of its gene revealed that this peroxidase is most likely a chloroplast protein with a high homology to plant 2Cys peroxiredoxins. It is shown that the Chlamydomonas peroxiredoxin (Ch-Prx1) is active with various thioredoxin isoforms, functions as an antioxidant toward reactive oxygen species (ROS), and protects DNA against ROS-induced degradation. Expression of the peroxidase gene in Chlamydomonas was found to be regulated by light, oxygen concentration, and redox state. The data suggest a role for the Chlamydomonas Prx in ROS detoxification in the chloroplast.     

Studies on chemiluminescence in the enzymatic and autoxidative transformation of 3,4-dihydroxyphenylalanine into eumelanins

The catechol oxidase-catalysed and autoxidative transformation of 3,4-dihydroxyphenylalanine (DOPA) to eumelanin have been studied by oxygen consumption, energy transfer, absorption and fluorescence spectroscopy. Formation of transient dopachrome (λ_max = 480 nm) and dopalutin (λ_ex = 423 nm, λ_em = 491 nm) have been found in the enzymatic and autoxidative reaction. In the enzymatic reaction, neither a photon emission with quantum yield Φ > 10^?13 nor energy transfer to triplet and singlet energy acceptors (sensitizers such as anthracene derivatives, xanthene dyes and chlorophyll-a) in water and micellar solutions have been found. The autoxidative reaction is chemiluminescent (Φ = 10^?9), the emission occurring in the 400-600 nm range. The excitation energy is not transferred to sensitizers. The effect of various enzymes and traps of active oxygen species as well as the spectral distribution of chemiluminescence indicate that there is no emission from oxygen dimoles. Carbonates and active species of oxygen are shown to participate in the chemiexcitation reaction.     

Immobilisation of Pseudomonas gessardii acidic lipase derived from beef tallow onto mesoporous activated carbon and its application on hydrolysis of olive oil

The study was carried out to immobilise the acidic lipase derived from Pseudomonas gessardii onto mesoporous activated carbon (MAC₄₀₀) for the application of hydrolysis of olive oil. MAC₄₀₀ was prepared from rice husk by the two stages process. P. gessardii was isolated from the beef tallow acclimatised soil. The acidic lipase (ALIP) was produced from a slaughterhouse waste, namely beef tallow as a substrate and immobilised onto MAC₄₀₀. The maximum immobilisation capacity of the MAC₄₀₀ was 3570 U/g at optimum immobilisation conditions; time (180 min), pH (5.0) and temperature (30 °C). The immobilised lipase had better thermal stability and reusability than the free lipase. The immobilisation of ALIP onto MAC₄₀₀ (MAC₄₀₀–ALIP) followed pseudo-first-order rate kinetics with rate constant 0.012/min. The Michaelis–Menten constant of MAC₄₀₀–ALIP was lower than that of the ALIP, which confirmed the higher affinity between enzyme and substrate. The immobilization of acidic lipase onto MAC₄₀₀ was confirmed by Fourier transform-infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and X-ray diffraction (XRD) pattern analysis. Reusability study of MAC₄₀₀–ALIP on olive oil hydrolysis showed 82% of hydrolysis up to 13 runs and 50% of hydrolysis up to 35 cycles of reuse. This work concludes that the acidic lipase immobilised mesoporous activated carbon matrix can be considered as a potential biocatalyst for the hydrolysis of olive oil. Thus, the enzyme immobilised matrix has potential industrial applications.”     

Adsorption and immobilisation of human insulin on graphene monoxide,silicon carbide and boron nitride nanosheets investigated by molecular dynamics simulation

The adsorption and immobilisation of human insulin onto the bio-compatible nanosheets including graphene monoxide, silicon carbide and boron nitride nanosheets were studied by molecular dynamics simulation at the temperature of 310 K. After equilibration, heating and 100 ns production molecular dynamic runs, it was found that the insulin was adsorbed and immobilised onto the considered surfaces in a native folded state. The structural parameters, including root-mean-square deviation and fluctuation, surface accessible solvent area, radius of gyration (R_g) and the distance between the centre of the mass of immobilised protein and the surface of the considered nanosheets, were measured, analysed and discussed. The energetics of the studied systems such as the interaction energy between protein and nanosheet was also measured and addressed. The discussions were centred on the structural and energetic parameters of the protein and nanosheets, including charge density, hydrophobicity, hydrophilicity and residue polarity. The results also showed that the active site of C-termini of chain B played an important role in the adsorption process and this could be helpful in the protection of insulin in its smart delivery and release applications.