Refolding of recombinant N-acetyl-d-glucosamine 2-epimerase by a fed-batch process

N-Acetyl-d-glucosamine 2-epimerase is one of the key enzymes for the enzymatic synthesis of N-acetylneuraminic acid, a sialic acid and a critical precursor for the synthesis of some antiviral agents. Overexpression of the recombinant epimerase in Escherichia coli led to the formation of protein inclusion bodies. Refolding of guanidine HCl-solubilized protein by direct dilution resulted in the formation of soluble oligomers, mediated probably by hydrophobic interactions. The extent of aggregation of protein subunits into inactive oligomers could be efficiently reduced by employing fed-batch refolding process, in which the solubilized proteins were added continuously at a pre-determined rate. The yields of soluble proteins decreased with the feeding rates. The addition of glutathione into refolding buffer at certain stage of the refolding process could enhance the yield of soluble proteins more than two-fold, possibly by resolving the inadvertently formed disulfide bridges among the protein subunits that contain 10 cysteine residues each. Folding aids such as l-arginine and glycerol were found effective in increasing the yield of soluble proteins and the specific activity of the refolded proteins. Under the optimal condition, a specific activity of 0.47 IU/mg was obtained with an activity recovery yield of ca. 30%. The specific activity of the refolded proteins was significantly lower than that of the native protein, 1.23 IU/mg, indicating that more information concerning the 3D structure of native N-acetyl-d-glucosamine 2-epimerase and the role of its cofactor, ATP, for catalytic activity is needed for the development of a more efficient refolding process.     

Tailor-made gold brush nanoelectrode ensembles modified with l-cysteine for the detection of daunorubicine

A new functionalized l-cysteine surface modified 3D gold brush nanoelectrode assembly BNEE (l-cys/BNEEs) was prepared. The BNEEs consisted of gold nanowires 100 nm in diameter and up to 400 nm in length fabricated by template synthesis in track etched polycarbonate membranes. The nanowires were exposed by controlled chemical etching of the membrane and were then modified by coating l-cys on the surface of the exposed gold nanowires. The morphology of the BNEEs was imaged by scanning electron microscopy and the real active area of BNEEs was determined by electrochemical impedance spectroscopy. The redox of daunorubicine (DNR) at the l-cys/BNEEs exhibited absorption-controlled characteristics and higher current activity than that at l-cys surface modified 2D disk NEEs (l-cys/DNEEs). The square wave voltammetry technique was employed to detect DNR. The detection limit was 1.0 × 10⁻⁸ M (s/n = 3). The linear detection concentration range of DNR was from 2.5 × 10⁻⁸ to 4.0 × 10⁻⁷ M.     

Studies on the oxidation reaction of l-cysteine in a confined matrix of layered double hydroxides

The amino acid l-cysteine (l-Cys) was intercalated into a MgAl layered double hydroxide (LDH), and its oxidation reaction by hexacyanoferrate (III) (Fe(CN)₆³⁻) in the confined region between sheets of LDH has been studied in detail. Based on the measurement results of XRD, Raman and FT-IR, it was found that the interlayer l-Cys was oxidized to cystine by Fe(CN)₆³⁻. Furthermore, the kinetics of this reaction was investigated in batch mode. The influences of initial Fe(CN)₆³⁻concentration, l-Cys-LDH quantity and reaction temperature on the interlayer oxidation reaction have been studied, respectively. The reaction follows a diffusion-controlled mechanism represented by Crank-Ginstling and Brounshtein kinetic model with the apparent activation energy of 29.93 kJ/mol. Therefore, this layered material may have prospective application as a novel “molecular reactor” for confined chemical reactions.     

Electrochemical reduction of Zn(II) ions on l-cysteine coated gold electrodes

Zn(II) ions have been selectively bound to the l-cysteine coated gold electrode in the form of a four-coordinated complex. Voltammograms of the Zn complex on the l-cysteine coated gold electrode showed a cathodic wave at ca. 0.05 V in the pH 7.54 phosphate buffered saline. The charge transfer coefficient and rate constant for the reduction of this Zn complex were 0.65 and 0.003 s⁻¹, respectively. The complexation of Zn(II) ions with l-cysteine on the gold electrode resulted in the maximum surface coverage of the Zn complex of 0.35 nmol cm⁻² and the Gibbs energy change of −27.6 kJ mol⁻¹. The cathodic peak current, influenced by the types of the end functional groups in thiols, the preconcentration time, and pH values of the supporting electrolyte, was linear with the concentration of Zn(II) ions in the range of 5.0 nM to 5 μM with a detection limit of 2.1 nM. The proposed voltammetric method was utilized successfully to detect the concentration of Zn(II) ions in hairs.     

pH affected migrational transport of ferrocene derivative of l-cysteine in aqueous solutions: Voltammetric studies

The mass transport of biologically-active l-cysteine derivatized with the ferrocenyl group (FcCH₂Cys) was investigated voltammetrically at a microelectrode in aqueous solutions under the conditions of varying content of supporting electrolyte and at different pH values. By varying the pH conditions one could obtain samples containing differently ionized forms of the l-cysteine derivative, from a monovalent cation via a neutral form (zwitterion) to a monovalent anion. Due to the acid-base equilibrium the obtained solutions were, in fact, mixtures composed predominantly of either anionic or cationic, or neutral species of FcCH₂Cys, respectively. Under the conditions of low ionic support the mass transport of these forms is differently affected by the migrational contribution. The results obtained experimentally were in good agreement with the theoretical predictions. In the calculations it was assumed that the FcCH₂Cys forms, coexisting in the solution, contribute independently to the steady-state transport-limited current. It was also assumed that the diffusion coefficients of the FcCH₂Cys forms were equal. This was validated by the voltammetric measurements at the supporting electrolyte excess (purely diffusional conditions). The diffusion coefficients of different forms of FcCH₂Cys are very similar and the average diffusion coefficient is (5.35 ± 0.05) × 10⁻¹⁰ m²/s.The studies clearly show that the variation in the conditions of pH or/and concentration of electrolyte can change the transport rate of l-cysteine even by several tens percent.     

Enhanced production of l(+)-lactic acid by floc-form culture of Rhizopus oryzae

A process to optimize l-lactic acid production from glucose by Rhizopus oryzae, based on sustaining floc morphology throughout the fermentation process, is herein performed. During the fermentation, supplementary ammonium sulfate was added intermittently to maintain the ammonia level of the culture medium always higher than 0.1 g/L. With replenish of nitrogen source, mycelia flocs did not aggregate, and the lactic acid production was optimized upon the fermentation being controlled at pH 4.3–4.5 by adding calcium carbonate slurry. In contrast, without supplementary addition of nitrogen source, mycelial clumps formed, resulting in a poor production of lactic acid. In the initial batch fermentation process, the final concentration of lactic acid produced was 109 g/L, with a yield (g lactic acid/g glucose consumed) of 0.87 and a productivity of 2.73 g/L h, using 125 g/L of glucose as substrate. For the first four cycles of repeated-batch fermentation, the average final concentration, the productivity and the yield of lactic acid were 113 g/L, 4.03 g/L h and 0.90, respectively.     

A homogeneous redox catalytic process for the paired synthesis of l-cysteine and l-cysteic acid from l-cystine

Redox catalytic process involved in the paired electrosynthesis of l-cysteine and l-cysteic acid from l-cystine is investigated by cyclic voltammetric technique and also confirmed by preparative electrolysis. The cyclic voltammetric behaviour shows that in the catholyte, in situ deposited tin (Sn) surface acts as a redox catalyst for the electro-reduction of l-cystine to l-cysteine whereas in the anolyte, the electro-generated bromine acts as a homogeneous redox mediator to enhance the electro-oxidation of l-cystine. l-Cysteine hydrochloride monohydrate (l-cysteine) and l-cysteic acid are prepared from l-cystine by preparative electrolysis with high purity and high yield using graphite cathode and DSA anode. At optimum concentration of l-cystine with 1:1 concentration ratio (catholyte:anolyte), the material yield obtained for l-cysteine is above 80% and that for l-cysteic acid is close to 60% in the paired electrosynthesis process in the batch operation. Scope for further experiments in conversion efficiency is also discussed.     

Electrochemical detection of l-cysteine using a boron-doped carbon nanotube-modified electrode

A boron-doped carbon nanotube (BCNT)-modified glassy carbon (GC) electrode was constructed for the detection of l-cysteine (L-CySH). The electrochemical behavior of BCNTs in response to l-cysteine oxidation was investigated. The response current of L-CySH oxidation at the BCNT/GC electrode was obviously higher than that at the bare GC electrode or the CNT/GC electrode. This finding may be ascribed to the excellent electrochemical properties of the BCNT/GC electrode. Moreover, on the basis of this finding, a determination of L-CySH at the BCNT/GC electrode was carried out. The effects of pH, scan rate and interferents on the response of L-CySH oxidation were investigated. Under the optimum experimental conditions, the detection response for L-CySH on the BCNT/GC electrode was fast (within 7 s). It was found to be linear from 7.8 × 10⁻⁷ to 2 × 10⁻⁴ M (r = 0.998), with a high sensitivity of 25.3 ± 1.2 nA mM⁻¹ and a low detection limit of 0.26 ± 0.01 μM. The BCNT/GC electrode exhibited high stability and good resistance against interference by other oxidizable amino acids (tryptophan and tyrosine).     

Mathematical modelling of the solubility of supercritical CO2 in poly(l-lactide) and poly(d,l-lactide-co-glycolide)

Two mathematical models, Sanchez–Lacombe equation of state and the Perturbed-Chain Statistical Associating Fluid Theory were applied for modelling the phase equilibrium for the poly(l-lactide)–CO₂ and poly(d,l-lactide-co-glycolide)–CO₂ systems. Aspen Polymer Plus software was used. The results were compared with previously obtained experimental values for solubility. The solubility of scCO₂ in the two biodegradable polymers was calculated for three different temperatures (308, 313 and 323 K) in the pressure range (10–30 MPa). The characteristic parameters for the components and the binary interaction parameters for the models were optimized in order to obtain the best fit between the estimated and the experimental gas solubility data. The results suggest that both SL EOS and PC-SAFT are reliable models in describing the phase equilibrium of the PLLA–CO₂ and PLGA–CO₂ systems at the proposed working conditions.     

Synthesis and characterization of poly(l-glutamic acid)-block-poly(l-phenylalanine)

Poly(γ-benzyl l-glutamate)-block-poly(l-phenylalanine) was prepared via the ring opening polymerization of γ-benzyl l-glutamate N-carboxyanhydride and l-phenylalanine N-carboxyanhydride using n-butylamine·HCl as an initiator for the living polymerization. Polymerization was confirmed by ¹H-nuclear magnetic resonance spectroscopy and matrix assisted laser desorption ionization time of flight mass spectroscopy. After deprotection, the vesicular nanostructure of poly(l-glutamic acid)-block-poly(l-phenylalanine) particles was examined by transmission electron microscopy and dynamic light scattering. The pH-dependent properties of the nanoparticles were evaluated by means of ζ-potential and transmittance measurements. The results showed that the block copolypeptide could be prepared using simple techniques. Moreover, the easily prepared PGA-PPA block copolypeptide showed pH-dependent properties due to changes in the PGA ionization state as a function of pH; this characteristic could potentially be exploited for drug delivery applications.     

Drowning-out crystallization of l-proline: Effect of anti-solvent composition and processing parameters on crystal size and shape

Crystallization of l-proline was investigated using a drowning-out method. Due to the high water and alcohol solubility of l-proline, the recovery of an l-proline product by precipitation using the drowning-out method required suitable anti-solvents selected from immiscibility and solubility studies. Through past experience, acetone and NMP were carefully chosen for analogy and solubility tests in pure anti-solvent. Although phase separation occurred in acetone, ultrasound was used to mix the two immiscible phases and generate fine emulsion droplets. l-proline crystals were obtained using NMP with a general drowning-out method. A spherical agglomerate of l-proline was also obtained using acetone by emulsion solvent diffusion methods. The ultrasound power controlled the agglomerate sizes, and the agglomerate surface transformed from amorphous to crystalline as the residence time increased. Alternatively, when NMP was used as an anti-solvent, l-proline monocrystals with needle type morphology were produced.     

Feasibility study of using montmorillonite for stability enhancement of l-ascorbic acid

This study tended to construct new l-ascorbic acid (LAA) composites in low toxicity and high stability for feasible application. LAA is chemically very unstable, since it is easily oxidized into biologically inactive compounds naturally. Our finding showed that introduction of montmorillonite (MMT) could significantly attenuate its toxicity and to sustain the stability of LAA with economic feasibility for practical uses. In addition, as phosphoric acid was biologically compatible, it was used for the pretreatment of MMT to obtain a promising stabilization of LAA. Toxicity assessment also showed that MMT treated with low-concentration acids should be considered as biologically safe according to our assessment. Thus, using acid treated MMT to stabilize LAA in a long-term might be technically feasible for further uses.     

Spherulite growth of l-lactide copolymers: Effects of tacticity and comonomers

The spherulite growth behavior and mechanism of l-lactide copolymers, poly(l-lactide-co-d-lactide) [P(LLA-DLA)], poly(l-lactide-co-glycolide) [P(LLA-GA)], and poly(l-lactide-co-ε-caprolactone) [P(LLA-CL)] have been studied using polarization optical microscopy in comparison with poly(l-lactide) (PLLA) having different molecular weights to elucidate the effects of incorporated comonomer units. The incorporation of comonomer units reduced the radius growth rate of spherulites (G) and increased the induction period of spherulite formation (t_i), irrespective of the kind of comonomer unit. Such effects became remarkable with the content of comonomers. At a crystallization temperature (T_c) of 130 °C, the disturbance effects of comonomers on the spherulite growth decreased in the following order: d-lactide>glycolide>ε-caprolactone, when compared at the same comonomer unit or reciprocal of averaged l-lactyl unit sequence length (l_l). The t_i estimation indicated that the glycolide units have the lowest disturbance effects on the formation of spherulite (crystallite) nuclei. The PLLA having the number-average molecular weight (M_n) exceeding 3.1×10⁴ g mol⁻¹ showed the transition from regime II to regime III at T_c=120 °C, whereas PLLA with the lowest M_n of 9.2×10³ g mol⁻¹ crystallized solely in regime III kinetics and the copolymers excluding P(LLA-DLA) with 3% of d-lactide units crystallized solely according to regime II kinetics. The nucleation and front constant for regime II and III [K_g(II), K_g(III), G₀(II), and G₀(III), respectively] estimated with each (not with a fixed for high-molecular-weight PLLA) decreased with increasing the amount of defects per unit mass of the polymer for crystallization, i.e. with increasing the comonomer content and the density of terminal group through decreasing the molecular weight.     

Thermal degradation of poly(l-lactide): effect of alkali earth metal oxides for selective l,l-lactide formation

To achieve the feed stock recycling of poly(l-lactide) (PLLA) to l,l-lactide, PLLA composites including alkali earth metal oxides, such as calcium oxide (CaO) and magnesium oxide (MgO), were prepared and the effect of such metal oxides on the thermal degradation was investigated from the viewpoint of selective l,l-lactide formation. Metal oxides both lowered the degradation temperature range of PLLA and completely suppressed the production of oligomers other than lactides. CaO markedly lowered the degradation temperature, but caused some racemization of lactide, especially in a temperature range lower than 250 °C. Interestingly, with MgO racemization was avoided even in the lower temperature range. It is considered that the effect of MgO on the racemization is due to the lower basicity of Mg compared to Ca. At temperatures lower than 270 °C, the pyrolysis of PLLA/MgO (5 wt%) composite occurred smoothly causing unzipping depolymerization, resulting in selective l,l-lactide production. A degradation mechanism was discussed based on the results of kinetic analysis. A practical approach for the selective production of l,l-lactide from PLLA is proposed by using the PLLA/MgO composite.     

Experimental and theoretical studies of l-cysteine adsorbed at Ag(1 1 1) electrodes

We have investigated l-cysteine adsorbed on Ag(1 1 1) electrodes under different conditions. We have employed experimental and theoretical approaches to obtain a better understanding of the adsorbed layer. An estimation of the coverage from charge measurements and the second harmonic response shows C_3v symmetry for the interface indicating a (√3 × √3)R°30 overlayer. The theoretical calculations show a variety of different structures with local adsorption energy minima. Particularly, under special initial conditions, zwitterionic structures adsorbed at different sites have been found. This can account for the multiplicity of redox processes observed experimentally below the potential of zero charge. The presence of an external field produces the stabilization of the zwitterion by interaction of the amino/carboxylic groups with the substrate.     

Functionalized micelles from new ABC polyglycidol-poly(ethylene oxide)-poly(d,l-lactide) terpolymers

New ABC type terpolymers of poly(ethoxyethyl glycidyl ether)/poly(ethylene oxide)/poly(d,l-lactide) were obtained by multi-mode anionic polymerization. After successive deprotection of the ethoxyethyl groups from the first block, highly hydroxyl functionalized copolymers of polyglycidol/poly(ethylene oxide)/poly(d,l-lactide) were obtained. These copolymers form elongated ellipsoidal micelles by direct dissolution in water. The micelles consist of a poly(d,l-lactide) core and stabilizing shell of polyglycidol/poly(ethylene oxide). The hydroxyl groups of polyglycidol blocks situated at the micelle surface provide high functionality, which could be engaged in further chemical modification resulting in a potential drug targeting agents. The micellization process of the copolymers in aqueous media was studied by hydrophobic dye solubilization, static and dynamic light scattering, and transmission electron microscopy.     

Controlled crystal nucleation in the melt-crystallization of poly(l-lactide) and poly(l-lactide)/poly(d-lactide) stereocomplex

Among the various inorganic nucleators examined, Talc and an aluminum complex of a phosphoric ester combined with hydrotalcite (NA) were found to be effective for the melt-crystallization of poly(l-lactide) (PLLA) and PLLA/poly(d-lactide) (PDLA) stereo mixture, respectively. NA (1.0 phr (per one hundred resin)) can exclusively nucleate the stereocomplex crystals, while Talc cannot suppress the homo crystallization of PLLA and PDLA in the stereo mixture. Double use of Talc and NA (in 1.0 phr each) is highly effective for enhancing the crystallization temperature of the stereo complex without forming the homo crystals. The stereocomplex crystals nucleated by NA show a significantly lower melting temperature (207 °C) than the single crystal of the stereocomplex (230 °C) in spite of recording a large heat of crystallization ΔH_c (54 J/g). Photomicrographic study suggests that the spherulites with a symmetric morphology are formed in the stereo mixture added with NA while the spherulites do not grow in size in the mixture added with Talc. The exclusive growth of the stereocomplex crystals by the melt-crystallization process will open a processing window for the PLLA/PDLA.     

A study on cytocompatible poly(chitosan-g-l-lactic acid)

A novel cytocompatible graft copolymer of chitosan and l-lactic acid (CL) was prepared by grafting l-lactic acid onto the amino groups in chitosan without a catalyst. The structures of the CL graft copolymers were characterized by FTIR, ¹³C-NMR and X-ray measurements. Degree of substitution and side-chain length were evaluated from salicylaldehyde and elemental analysis. The tensile strength and water uptake of the CL copolymers films were investigated as a function of feed ratio of LA/CS. The influence of pH on the swelling behavior of the copolymer films was determined and interpreted. Fibroblast culture was performed to evaluate cell proliferation on the copolymers films. The results showed that the cell growth rate on the copolymers films is faster than chitosan obviously.     

Ring-opening polymerization of l-lactide catalyzed by a biocompatible calcium complex

A novel calcium complex, [(DAIP)₂Ca]₂ (where DAIP-H = 2-[(2-dimethylamino-ethylimino)methyl]phenol), is prepared in a one flask reaction by condensation of Ca(OMe)₂ with DAIP-H in toluene/THF. Experimental results show that in the presence of various alcohols, [(DAIP)₂Ca]₂ efficiently initiates the ring-opening polymerization of l-lactide in a controlled fashion, yielding polymers with expectative molecular weight and low polydispersity indexes. Furthermore, kinetic studies show a first-order dependency on both [LA] and [BnOH].     

Properties of l-tyrosine based polyphosphates pertinent to potential biomaterial applications

Since their introduction by Kohn and Langer et al. in 1984, l-tyrosine based ‘pseudo’ poly(amino acids) have undergone extensive research in the area of polymeric biomaterials. Starting from l-tyrosine based diphenolic monomers, polyiminocarbonates, polycarbonates and polyarylates have been developed by Kohn and co-workers and are being investigated for potential orthopedic biomaterial applications. Mao et al. have reported development of l-tyrosine based polyphosphates and polyphosphonates in a patent, however, detailed structural and physico-chemical characterization studies on such polymers have not been reported yet. For the purpose of the current paper, using a novel solid phase process for synthesis of l-tyrosine based diphenolic monomers and adapting the polymerization process described by Mao et al., l-tyrosine based polyphosphates were developed and investigated for their pertinent bioengineering properties. The properties investigated consist of chemical solubility, hydrophilicity and hydrolytic degradation. The results of this investigation are crucial to validate further investigation of biomaterial applications of these polymers.