Kinetics and bioreactor studies of immobilized invertase on polyurethane rigid adhesive foam

A new support, polyurethane rigid adhesive foam (PRAF), which can be used to cover internal surface of metallic tubes, was used to immobilize invertase for application in an enzymatic bioreactor. The kinetic parameters were: K_m - 46.5 ± 1.9 mM (PRAF-invertase) and 61.2 ± 0.1 mM (free enzyme) and V_max 42.0 ± 4.3 U/mg protein/min (PRAF-invertase) and 445.3 ± 24.0 U/mg protein/min (free invertase). The PRAF-invertase derivative maintained 50.1% of initial activity (69.17 U/g support) for 8 months (4 °C) and was not observed microbial contamination. The bioreactor showed the best production of inverted sugar syrup using up-flow rate (0.48 L/h) with average conversion of 10.64 ± 1.5% h⁻¹ at feeding rate (D) of 104 h⁻¹. The operational inactivation rate constant (k_opi) and half-life were 1.92 × 10⁻⁴ min⁻¹ and 60 h (continue use). The PRAF spray support looks promising as a new alternative to produce immobilized derivatives on reactor surfaces.     

Immobilization of invertase via carbohydrate moiety on chitosan to enhance its thermal stability

A new technique using chitosan as support for covalent coupling of invertase via carbohydrate moiety improved the activity and thermal stability of immobilized invertase. The best preparation of immobilized invertase retained 91% of original specific activity (412 U mg^–1). The half-life at 60°C was increased from 2.3 h (free invertase) to 7.2 h (immobilized invertase). In contrast, the immobilization of invertase via protein moiety on chitosan or using Sepharose as support resulted in less thermostable preparations. Additionally, immobilization of invertase on both supports caused the optimal reaction pH to shift from 4.5 to 2.5 and the substrate (sucrose) concentration for maximum activity to increase from 0.5 M to 1.0 M.     

Immobilization of invertase on a new type of macroporous glycidyl methacrylate

Invertase was immobilized via its carbohydrate moiety. The immobilized enzyme has a specific activity of 5500 IU g^–1, with 45% activity yield on immobilization. In a packed bed reactor, 90% 2.5 M sucrose was converted at a flow rate of 4 bed volumes h^–1. The obtained specific productivity at 40 °C of 3 kg l^–1 h^–1 is the best one so far. Long-term stability was 290 days in 2.5 M sucrose at 40 °C and at a flow rate of 3 bed volumes h^–1.     

Biodegradation of feather keratin with a PEGylated protease of Chryseobacterium gleum

Present study deals with the covalent modification of keratinolytic protease of Chryseobacterium gleum with higher enzyme activity, improved stability, non-immunogenicity and reusability. Protease of C. gleum showing feather degradation ability was modified by covalent attachment to polyethylene glycol. This modification culminated the change in electrophoretic mobility of protease in acrylamide gel. The modified enzyme showed 1.4 times more catalytic activity with better stability than native in aqueous system containing whole feathers as keratin. It showed improved pH, thermal, storage and solvent stability with a broadened range of pH (7–9) and temperature (25–50 °C) than native. The differentiation between modified and native enzyme was authenticated through UV–vis spectroscopy, SEM, XRD, FTIR and DSC. This modification of protease proved to be non-immunogenic in rats. The enzyme extracted after first run could be used for several cycles which clearly demonstrated its reusability in catalytic bioprocess of keratin degradation.     

Invertase embedded-PVC tubing as a flow-through reactor aimed at conversion of sucrose into inverted sugar

A simple flow-through reactor system is prepared by covalent linking of a biomolecule on the inner surface of a polyvinyl chloride (PVC) tube. This is achieved by introducing an active functional group on the surface of an inert PVC tube through 1-fluoro-2-nitro-4-azidobenzene (FNAB), a precursor of highly reactive nitrene, which can insert to any C–H bond. CCl₄ lacking C–H bond is taken as a solvent for loading FNAB solution into the tube. FNAB loaded tube is then allowed to expose to sunlight for 20 min during which azido group of FNAB generates nitrene and attaches itself to PVC tube through insertion reaction. Invertase is immobilized in the activated PVC tube at 50 °C in 45 min. Invertase embedded-PVC tube is used as a flow-through reactor to convert sucrose to invert sugar. The flow-through reactor converted sucrose into invert sugar with 97% yield as shown by HPLC analysis. The reactor is reused for eight cycles with 17% loss of its initial activity. The reactor system is cheap as PVC tube is working both as a carrier of biomolecule and a reaction vessel. This reactor system overcomes the problem of back pressure and can be used for any enzymatic conversion in a flow-through system.     

The thermal stability of the external invertase isoforms from Saccharomyces cerevisiae correlates with the surface charge density

Understanding the effect of surface charge on the stability of proteins is one prerequisite for "tailoring" proteins with increased thermal stability. Here, we investigated the origin of the altered thermal stability observed between the four recently isolated isoforms (EINV1-EINV4) of external invertase. External invertase from yeast Saccharomyces cerevisiae, a homodimeric glycoprotein, represents a widely used model for studying the influence of the glyco component on protein stability. The stability of the four isoforms of invertase decreases from EINV1 to EINV4, which is accompanied by an increase in negative surface charge density. Mass spectrometry analysis revealed that the isoforms share identical protein parts indicating that the differences in stability are the result of post-translational modifications. (31)P NMR analysis revealed that the isoforms contain negatively charged phosphate groups in diester and monoester forms attached to the glycan part. The total amount of phosphate bound to the polymannan component varies between the different isoforms. These results, together with the analysis of the amount of polymannan components, show that negative surface charge density does not entirely depend on the amount of phosphate but rather on its distribution. This suggests that charged groups bound to the glyco-component of a protein can influence the stability of glycoproteins.     

Expression,purification and immobilization of the intracellular invertase INVA,from <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> on crystalline cellulose and Nylon-6

This paper presents two immobilization methods for the intracellular invertase (INVA), from Zymomonas mobilis. In the first method, a chimeric protein containing the invertase INVA, fused through its C-terminus to CBD _Cex from Cellulomonas fimi was expressed in Escherichia coli strain BL21 (DE3). INVA was purified and immobilized on crystalline cellulose (Avicel) by means of affinity, in a single step. No changes were detected in optimal pH and temperature when INVA-CBD was immobilized on Avicel, where values of 5.5 and 30 °C, respectively, were registered. The kinetic parameters of the INVA-CBD fusion protein were determined in both its free form and when immobilized on Avicel. K _m and V _max were affected with immobilization, since both showed an increase of up to threefold. Additionally, we found that subsequent to immobilization, the INVA-CBD fusion protein was 39% more susceptible to substrate inhibition than INVA-CBD in its free form. The second method of immobilization was achieved by the expression of a 6xHis-tagged invertase purified on Ni-NTA resin, which was then immobilized on Nylon-6 by covalent binding. An optimal pH of 5.5 and a temperature of 30 °C were maintained, subsequent to immobilization on Nylon-6 as well as with immobilization on crystalline cellulose. The kinetic parameters relating to V _max increased up to 5.7-fold, following immobilization, whereas K _m increased up to 1.7-fold. The two methods were compared showing that when invertase was immobilized on Nylon-6, its activity was 1.9 times that when immobilized on cellulose for substrate concentrations ranging from 30 to 390 mM of sucrose.     

Solubilisation of a cell wall bound invertase in Aspergillus nidulans

Aspergillus nidulans produces an extracellular invertase when incubated in the presence of sucrose and about half of the activity produced was found to be associated with the mycelium. Sixty percent of this mycelial invertase could be solubilised by simple mechanical disruption. Among the agents tested for solubilisation of invertase, proteinase K and dithiothreitol were the most effective.     

Polyelectrolyte complex formation mediated immobilization of chitosan-invertase neoglycoconjugate on pectin-coated chitin

Saccharomyces cerevisiae invertase, chemically modified with chitosan, was immobilized on pectin-coated chitin support via polyelectrolyte complex formation. The yield of immobilized enzyme protein was determined as 85% and the immobilized biocatalyst retained 97% of the initial chitosan-invertase activity. The optimum temperature for invertase was increased by 10 °C and its thermostability was enhanced by about 10 °C after immobilization. The immobilized enzyme was stable against incubation in high ionic strength solutions and was 4-fold more resistant to thermal treatment at 65 °C than the native counterpart. The biocatalyst prepared retained 96 and 95% of the original catalytic activity after ten cycles of reuse and 74 h of continuous operational regime in a packed bed reactor, respectively.     

Multiple steady states in a continuous stirred tank reactor: an experimental case study for hydrolysis of sucrose by invertase

The purpose of this work was to validate experimentally that multiple steady states may be achieved in a continuous stirred tank reactor (CSTR) during hydrolysis of sucrose by invertase. Experiments were done with four initial sucrose concentrations (0.1, 0.175, 0.584 and 1 M) to study their effect on residual sucrose and reaction rate at steady state. Two different steady states (S=0.7 M, r=9×10⁻⁴ mol/l min and S=0.135 M, r=1.54×10⁻³ mol/l min) were found depending on initial concentration of sucrose in the reactor. Two stable steady states were possible in a CSTR using invertase for the hydrolysis of sucrose. A third possible steady state can be derived theoretically, but it should be a metastable condition because any small disturbance in the system will result in transitory states stabilizing at sugar concentrations of either 0.135 or 0.7 M.     

Properties of a glucose oxidase covalently immobilized on amorphous AlPO4 support

Glucose oxidase (GOD) was covalently immobilized on amorphous AlPO₄ as well as on an AlPO₄/clay mineral Sepiolite system. Immobilization of the enzyme was carried out through the -amino group of lysine residues through an aromatic Schiff's-base. Activation of the support was obtained after reaction of appropriate molecules with support surface –OH groups. The enzymatic activities of native, and different immobilized GOD systems and filtrates, were followed by the amount of liberated -gluconic acid obtained in the enzymatic β- -glucose oxidation with the aid of an automatic titrator. The kinetic properties of native and immobilized GOD were obtained for glucose concentrations in the range of physiological conditions and at different working conditions such as reaction temperature, reaction pH, and enzyme concentration.

The binding percentage of enzymes was in the 50–80% range, with residual and specific activities in the 65–80% and 90–150% ranges, respectively. No change in the pH optimum and only slight changes in the V_max and K_M kinetic parameters with respect to native GOD were observed, so that not only was little deactivation of enzyme obtained throughout the immobilization process but also that the stability of the covalently bound enzyme in the two supports appeared to have increased with respect to the soluble enzyme. GOD immobilization also increased its efficiency and operational stability in repeated uses on increasing the amount of immobilized enzyme.     

Structural investigations of a GYF domain covalently linked to a proline-rich peptide

Protein structure determination of low affinity complexes of interacting macromolecules is often hampered by a lack of observable NOEs between the binding partners. Covalent linkage offers a way to shift the equilibrium of the interaction partners to the bound state. Here we show that a single-chain protein containing the GYF domain of CD2BP2 and the target peptide SHRPPPPGHRV from CD2 allows for the intramolecular association of the binding partners. We obtained NOEs between the GYF domain and the peptide that could define the principal orientation of the peptide in the complex. In conjunction with general recognition rules for proline-rich sequence recognition these NOEs allowed the accurate modeling of the protein-peptide complex.     

Immobilization of catalases from Bacillus SF on alumina for the treatment of textile bleaching effluents

A catalase preparation from a newly isolated Bacillus sp. was covalently immobilized on silanized alumina using glutaraldehyde as crosslinking agent. The effect of the coupling time of the enzyme-support reaction was determined in terms of protein recovery and immobilization yield and a certain balance point was found after which the activity recovery decreased. The activity profile of the immobilized catalase at high pH and temperature was investigated. The immobilized enzyme showed higher stabilities (214 h at pH 11, 30°C) at alkaline pH than the free enzyme (10 h at pH 11, 30°C). The immobilized catalase was inhibited by anionic stabilizers or surfactants added to the hydrogen peroxide substrate solution.     

Solution structure of oligonucleotides covalently linked to a psoralen derivative.

Psoralen (pso) was attached via its C-5 position to the 5'-phosphate group of an oligodeoxynucleotide d(TAAGCCG) by a hexamethylene linker (m6). Complex formation between pso-m6-d(TAAGCCG) and the complementary strands d(CGGCTTA)[7-7mer] or d(CGGCTTAT)[7-8mer] was investigated by nuclear magnetic resonance in aqueous solution. Structural informations derived from DQF-COSY and NOESY maps, revealed that the mini double helix adopts a B-form conformation and that the deoxyriboses preferentially adopt a C2'-endo conformation. The nOe connectivities observed between the protons of the bases or the sugars in each duplex, and the protons of the psoralen and the hexamethylene chain, led us to propose a model involving an equilibrium between two conformations due to different locations of the psoralen. Upon UV-irradiation, the psoralen moiety cross-linked the two DNA strands at the level of 5'TpA3' sequences. NMR studies of the single major photo-cross-linked duplex pso-m6-d(TAAGCCG) and d(CGGCTTA) were performed. The stereochemistry of the diadduct is indeed cis-syn at both cyclobutane rings. In addition, the effects of this diadduct on the helical structure are analyzed in detail.     

Immobilization of the recombinant invertase INVB from <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> on Nylon-6

The recombinant invertase INVB (re-INVB) from Zymomonas mobilis was immobilized on microbeads of Nylon-6, by means of covalent bonding. The enzyme was strongly and successfully bound to the support. The activity of the free and immobilized enzyme was determined, using 10% (w/v) sucrose, at a temperature ranging between 15 and 60 °C and a pH ranging between 3.5 and 7. The optimal pH and temperature for the immobilized enzyme were 5.5 and 25 °C, respectively. Immobilization of re-INVB on Nylon-6 showed no significant change in the optimal pH, but a difference in the optimal temperature was evident, as that for the free enzyme was shown to be 40 °C. The values for kinetic parameters were determined as: 984 and 98 mM for of immobilized and free re-INVB, respectively. values for immobilized and free enzymes were 6.1 × 10² and 1.2 × 10⁴ s⁻¹, respectively, and immobilized re-INVB showed of 158.73 μmol h min⁻¹ mg⁻¹. Immobilization of re-INVB on Nylon-6 enhanced the thermostability of the enzyme by 50% at 30 °C and 70% at 40 °C, when compared to the free enzyme. The immobilization system reported here may have future biotechnological applications, owing to the simplicity of the immobilization technique, the strong binding of re-INVB to the support and the effective thermostability of the enzyme.     

Versatile method employing basic techniques of genetic engineering to study the ability of low-molecular-weight compounds to bind covalently with DNA in cell-free systems

Numerous antitumor and carcinogenic compounds and free radicals are able to modify DNA by forming covalent bonds, mainly with nucleophilic centers in nucleobases. Such a binding is usually of utmost importance for the biological outcome. The level of DNA adducts formed by a given agent is in most cases extremely low; hence their detection is very difficult. Here we propose a simple approach, exploiting techniques widely used in genetic engineering, to demonstrate and characterize the covalent modification of a DNA fragment by any low-molecular-weight compound of interest in a cell-free system. The specifically designed, several-hundred-base-pairs-long double-stranded deoxyoligonucleotide (PCR amplified)--subject to modification--includes two restriction sites: one containing only GC base pairs recognized by restriction endonuclease MspI and the other including only AT base pairs recognized by restriction endonuclease Tru1I. The covalent modification of the restriction sites abolishes their recognition and thus cleavage by the endonucleases applied. The formation of DNA adducts is induced by incubating the oligonucleotide with increasing concentrations of a studied compound, in the appropriate activating system if required. Then, the modified oligonucleotide is submitted to digestion by the above-mentioned restriction endonucleases and the DNA fragments are separated by polyacrylamide gel electrophoresis. The inhibition of cleavage indicates the occurrence of covalent modification of the restriction site(s) while simultaneously pointing at the kind of base pairs involved in DNA adduct formation. The validation of the method was performed for two DNA binding antitumor compounds, cisplatin and CC-1065, which form adducts preferentially with guanine and adenine, respectively.