l-DOPA production by immobilized tyrosinase

The production of l-DOPA using l-tyrosine as substrate, the enzyme tyrosinase (EC 1.14.18.1) as biocatalyst, and l-ascorbate as reducing agent for the o-quinones produced by the enzymatic oxidation of the substrates was studied. Tyrosinase immobilization was investigated on different supports and chemical agents: chitin flakes activated with hexamethylenediamine and glutaraldehyde as crosslinking agent, chitosan gel beads, chitosan gel beads in the presence of glutaraldehyde, chitosan gel beads in the presence of polyvinyl pyrrolidone, and chitosan flakes using glutaraldehyde as crosslinking agent. The last support was considered the best using as performance indexes the following set of immobilization parameters: efficiency (90.52%), yield (11.65%), retention (12.87%), and instability factor (0.00). The conditions of immobilization on chitosan flakes were optimized using a two-level full factorial experimental design. The independent variables were enzyme-support contact time (t), glutaraldehyde concentration (G), and the amount of enzyme units initially offered (U _C). The response variable was the total units of enzymatic activity shown by the immobilized enzyme (U _IMO). The optimal conditions were t=24 h, G=2% (v/v), and U _C=163.7 U. Under these conditions the total units of enzymatic activity shown by the immobilized enzyme (U _IMO) was 23.3 U and the rate of l-DOPA production rate was 53.97 mg/(L·h).     

DNA interaction with synthetic polymers in solution

DNA complexes with cationic polymers (polyvinylamine (PVA), polyallylamine (PAA), polydimethylaminoethylmethacrylate (PDMAEM), poly-(N,N,N-trimethylammonio)ethyl methacrylate chloride (PTMAEM), poly-l-lysine (PLL)) were investigated. It was shown that volume and persistent length of DNA do not change essentially at low cationic polymer concentration in a solution. DNA packaging in 0.005 M NaCl was observed at charge ratio N/P ≈ 1. Secondary DNA structure in complexes was not disrupted, and DNA was protected from protonation. The comparison between DNA packaging in complexes with polycations and DNA condensation induced by trivalent ions was made.     

On-line study of polyelectrolyte network formation by interfacial reaction

A modified synthetic boundary experiment of analytical ultracentrifugation has been employed to examine, on-line, polyelectrolyte complex formation at flat interfaces yielding highly swollen membranes/networks. Systematic experiments with sodium alginate as a polyanion and chitosan and poly(l-lysine) as polycations identified the influence of concentration, pH, molar mass, and polycation type on the membrane characteristics and the formation process. The membranes have been evaluated by five characteristics defined herein: total thickness, compactness, heterogeneity, symmetry, and growth. The results confirm the sensitivity of the method suited to elaborate general relationships for polyelectrolyte membrane design.     

A new method for microcapsule characterization

Numerous microcapsule systems have been developed for a wide range of applications, including the sustained release of drugs, cell transplantation for therapy, cell immobilization, and other biotechnological applications. Despite the fact that microcapsule membrane is a dominant factor governing overall microcapsule performance, its characterization is challenging. We report a new method for characterizing microcapsule membranes, using the most common alginate-poly-l-lysine-alginate (APA) microcapsule as an example. Our data demonstrate that genipin, a naturally derived reagent extracted from gardenia fruits, interacts with poly-l-lysine (PLL) and generates fluorescence. This fluorescence allows clear visualization and easy analysis of the PLL membrane in the APA microcapsules using confocal laser scanning microscopy. The results also show that PLL binding correlates to the reaction variables during PLL coating such as PLL concentration and coating time. In addition, five other different microcapsule formulations consisting of PLL and/or chitosan membranes were examined, and the results imply that this method can be extended to characterize a variety of microcapsule membranes. These findings suggest that genipin can serve as a fluorogenic marker for rapid characterization of microcapsule membranes, a superior method that would have important implications for microcapsule research and potential in many other applications.     

Purification and characterization of an extracellular α-l-arabinofuranosidase from Fusarium oxysporum

An α-l-arabinofuranosidase from Fusarium oxysporum F3 was purified to homogeneity by a two-step ion exchange intercalated by a gel filtration chromatography. The enzyme had a molecular mass of 66 kDa and was optimally active at pH 6.0 and 60°C. It hydrolyzed aryl α-l-arabinofuranosides and cleaved arabinosyl side chains from arabinoxylan and arabinan. There was a marked synergistic effect between the α-l-arabinofuranosidase and an endo-(1 →4)-β-d-xylanase produced by F. oxysporum in the extensive hydrolysis of arabinoxylan.     

Electrochemical reduction of nitroprusside on a glassy carbon electrode modified by poly-l-lysine films

The present work describes the preparation and characterization of polyelectrolyte coatings of poly-l-lysine (PLL) to modify a glassy carbon electrode and its application to the pre-accumulation of nitroprusside (NP). The effects of the coating on the electrochemical reduction of NP were investigated. The performance of the modified electrode indicates that the drug can be immobilized by electrostatic interaction and the voltammetric signal monitored at all pH values in the range of 2–12. The strong interaction between NP and PLL stabilizes the complex on the electrode surface and minimizes the chemical reaction of lost CN⁻ ions as a subsequent reaction of electron transfer, which could improve the action mechanism of NP.     

Complexes of titanium(IV) chloride with N-(3,5-R,R′-salicylidene)-2(3,4)-[bis(5-methyl-2-furyl)methyl]aniline, a novel type of phenoxyimine catalysts for olefin polymerization

New representatives of chelate-type titanium(IV) salicylideneaniline complexes with bis(5-methyl-2-furyl)methyl substituents in the aniline fragment are synthesized. In the presence of poly(methylalumoxane), these complexes catalyze ethylene and propylene polymerization. The effect of the position of substituents in the ligands on the activities of the catalysts is studied. High-molecular-weight linear polyethylene (M _w ≈ 172200–300000, M _w/M _n ≈ 2–3) and high-molecular-weight atactic elastic polypropylene (M _w ≈ 1000000, M _w/M _n ≥ 7.0) are obtained. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 10, pp. 1730–1737, October, 2006.     

Quantitative characterization of membrane formation process of alginate–chitosan microcapsules by GPC

The semi-permeable membrane of alginate–chitosan (AC) microcapsules has been proven important to control the microcapsule stability and selective substance diffusion rate. Therefore, a novel and operable methodology based on gel permeation chromatography (GPC) was established for quantitative characterization of the membrane formation process, so as to provide guidance on performance improvement of AC microcapsules in biomedical applications. Not only the molecular weight (M_w) and its distribution of chitosan can be obtained by GPC, but also the area integral of molecular weight peaks can be linearly correlated to chitosan concentration in certain range. The dynamic membrane formation process was then obtained by quantitatively analyzing reaction amount of chitosan with time, which showed that for chitosan molecules with wide M_w distribution, only parts of molecules bind with alginate to form microcapsule membrane. Moreover, the contribution of chitosan molecules participating in the membrane formation process was also different. These new findings, therefore, are helpful for adjusting and controlling the membrane formation process and properties of microcapsule membrane.     

Metabolic engineering of Saccharomyces cerevisiae for efficient production of pure l ?(+)?lactic acid

We developed a metabolically engineered Saccharomyces cerevisiae, which produces optically pure l-lactic acid efficiently using cane juice-based medium. In this recombinant, the coding region of pyruvate decarboxylase (PDC)1 was completely deleted, and six copies of the bovine l-lactate dehydrogenase (l-LDH) genes were introduced on the genome under the control of the PDC1 promoter. To confirm optically pure lactate production in lowcost medium, cane juice-based medium was used in fermentation with neutralizing conditions. l-lactate production reached 122 g/L, with 61% of sugar being transformed into l-lactate finally. The optical purity of this l-lactate, that affects the physical characteristics of poly-l-lactic acid, was extremely high, 99.9% or over. These two authors contributed equally to this work.     

Enhancement of plant growth stimulation activity of irradiated alginate by fractionation

Alginate with the weight-average molecular weight (M_w) approximately 900 kDa and ratio of M (mannuronate)/G (guluronate) about 1.3 was irradiated by gamma Co-60 in aqueous solution at doses up to 200 kGy. The irradiation dose was shown to be a function for reducing M_w and molecular weight distribution of irradiated alginates. The distribution of oligomer fractions in irradiated products was also investigated by separation using ultrafiltration membranes. The irradiated alginate with M_w approximately 14.2 kDa was found to have a positive influence for growing of barley and soybean. The irradiated oligoalginate fraction with M_w ranging from 1 to 3 kDa displayed the strongest effect on the growth and development of the mentioned plants at low concentration (20 ppm). It is suggested that oligoalginate with M_w in the range 1–3 kDa is a trigger for the growth and development of plants.     

Inexpensive isolation of beta-D-glucopyranosidase from alpha-L-arabinofuranosidase,alpha-L-rhamnopyranosidase,and o-acetylesterase

β-d-Glucopyranosidase (βG, EC 3.2.1.21) has been isolated from some collateral activities, α-l-arabinofuranosidase (Ara, EC3.2.1.55), α-l-rhamnopyranosidase (Rha, EC 3.2.1.40), and o-acetylesterase (Est, EC 3.1.1.53), using a commercial enzyme preparation and a simple method economically sustainable for the food industry. The procedure comprises precipitation of extraneous substances by adding ethanol and CaCl₂, ultrafiltration, and adsorption, first on bentonite and then on chitosan. The results obtained were the complete isolation of βG from the above-mentioned activities, a drastic reduction in extraneous compounds, such as brown substances and polysaccharides, and a slight increase in purification.     

Intrinsic viscosity–molecular weight relationship for chitosan

The intrinsic viscosity–molecular weight relationship for chitosan was determined in 0.25 M acetic acid/0.25M sodium acetate. Chitosan samples with a degree of acetylation (DA) between 20 and 26% were prepared from shrimp‐shell chitosan by acid hydrolysis (HCl) and oxidative fragmentation (NaNO₂). Absolute molecular weights were measured by light scattering and membrane osmometry. Size exclusion chromatography (SEC) was used to determine average molecular weights (M_n, M_v, and M_w) and polydispersity. The following Mark–Houwink–Sakurada equation (MHS) is proposed for chitosan of M_w in the range of 35–2220 kDa: The value of the MHS exponent a suggests that chitosan behaves as a flexible chain in this solvent. Examination of MHS constants obtained in this work and those available in the literature with other solvents indicates that a and K are inversely related and that they are influenced by DA, and pH and ionic strength of the solvent. © 2000 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 38: 2591–2598, 2000     

Quartz crystal microbalance immunosensor for the detection of antibodies to double-stranded DNA

We report the development of a novel quartz crystal microbalance immunosensor with the simultaneous measurement of resonance frequency and motional resistance for the detection of antibodies to double-stranded DNA (dsDNA). The immobilization of poly(l-lysine) and subsequent complexation with DNA resulted in formation of a sensitive dsDNA-containing nanofilm on the surface of a gold electrode. Atomic force microscopy has been applied for the characterization of a poly(l-lysine)–DNA film. After the blocking with bovine serum albumin, the immunosensor in flow-injection mode was used to detect the antibodies to dsDNA in purified protein solutions of antibodies to dsDNA and to single-stranded DNA, monoclonal human immunoglobulin G, DNase I and in blood serum of patients with bronchial asthma and systemic lupus erythematosus. Experimental results indicate high sensitivity and selectivity of the immunosensor. In memoriam Prof. Victor G. Vinter     

Synthesis and in vitro Property Study of Polyaspartamides

Cationic polyaspartamides including poly-α,β-[N＇-（2-aminoethyl）-L-aspartamide] （PAEA）, poly-α,β-[N＇-（4- aminobutyl）-L-aspartamide] （PABA）, poly-α,β-[N＇-（6-aminohexyl）-L-aspartamide] （PAHA）, poly-α,β-[N＇-（5-amino- 3-azapentyl）-L-aspartamide] （PAAPA） and poly-α,β-[N＇-（8-amino-3,6-diazaoctyl）-L-aspartamide] （PADAOA） were synthesized from polysuccinimide. Their properties were evaluated by ^1H NMR, IR, GPC, fluorescence measurement and in vitro cytotoxicity assays. The molecular weights per primary amine charge group of PAEA（1） （Mn= 2229）, PAAPA and PADAOA are 212, 279, and 226. Polyaspartamides including PAEA（1）, PAAPA, PADAOA and low molecular weight PAHA are markedly less toxic than poly（ethyleneimine） and poly（L-lysine）, however, PABA and higher molecular weight PAHA are slightly less toxic than poly（L-lysine）. Cell cytotoxicity of PAHA was seen to decrease with increasing molecular weight of PAHA, due to water solubility reduction. The negatively charged plasmid DNA has been found to be completely neutralized and complexed by the cationic polyaspartamides at an N/P ratio of 5 ： 1 to 10 ： 1, forming self-assembled polyplexes via ionic interactions. These polyaspartamide/DNA complexes possess stable zeta potentials and mean particle diameters of about 180 nm for PAEA （1）/DNA and PAAPA/DNA complexes and 280 nm for PADAOA/DNA complexes.     

Improved postharvest quality in patagonian squash (Cucurbita moschata) coated with radiation depolymerized chitosan

Different molecular weight chitosans were evaluated on the decay of coated Anquito squashes (Cucurbita moschata) as well as the maintenance of the fruit quality along five storage months. The original chitosan (Mw=391 kDa, 83% DD), was depolymerized by gamma radiation. Apart from chain scission, other chemical changes were not detected by FTIR or UV–vis analyses. The molecular weight characterization of chitosans was done by size exclusion chromatography with dual light scattering and concentration detection (SEC-MALLS-RI). The coating effectiveness was evaluated on the following parameters: fungal decay incidence, weight loss, firmness, total reducing sugar, soluble solid, flesh color, carotene content, pH and titratable acidity. No sign of fungal decay was observed in squashes coated with 122 and 56 kDa chitosans, which were also the most effective treatments in reducing the weight loss. The chitosan with Mw=122 kDa was also the best treatment considering firmness, internal aspect, sugar and carotene content. Then, radiation degraded chitosan was better in C. moschata preservation than the original chitosan.     

A Novel Approach for Testing the Hixon‐Crowel Model For In Vitro Release of Vitamin B2 From Chitosan Coated Calcium Alginate Beads

The dynamic release of a model drug (vitamin B₂) from chitosan coated calcium alginate beads has been studied in the media of varying pH and the Hixon‐Crowel model has been applied to the experimental data, using a novel ‘curve area measurement’ (CAM) approach. The two release profiles, namely experimental and ideal, were found to be in close agreement except for the initial phase of the release process.     

Chiral ligand-exchange chromatography on an RP HPLC column coated with a new chiral selector derived froml-spinacine

Summary A commercial reversed-phase (RP) C₁₈ HPLC column has been dynamically coated with the chiral selectorN ^τ-n-decyl-l-spinacine and then loaded with copper(II) ions. Several racemic mixtures of underivatized amino acids and oligopeptides were resolved on the column by chiral ligand-exchange chromatography. The most important experimental conditions affecting column efficiency, retention, and selectivity (temperature and mobile phase flow rate and composition) were extensively investigated.     

l-Lactat-Durchflu?elektrode mit immobilisierter Lactat-Oxidase

Zusammenfassung Eine leicht zu handhabende l-Lactat-Durchflußelektrode mit immobilisierter Lactat-Oxidase wird beschrieben. Sie ist Bestandteil eines dreikanaligen O₂-sensitiv-enzymatischen Meßsystems für l-Lactat, Pyruvat und -d-Glucose. Der l-Lactat-Sensor weist eine schnelle Einstellzeit, geringe Streuung um die errechnete Regressionsgerade und einen für die klinisch-chemische Analytik adäquaten linearen Anzeigebereich auf. Meßkurven werden demonstriert. Auf den Chemismus anderer üblicher elektrochemisch-enzymatischer Meßverfahren von l-Lactat wird hingewiesen und auf die klinische Bedeutung der l-Lactat-Bestimmung Bezug genommen.

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l-lactate flow-through electrode with immobilized lactate oxidase

Summary An l-lactate flow-through electrode with immobilized lactate oxidase is described that is easy to handle. It is part of a three-channel O₂-sensitive enzymatical measuring system for l-lactate, pyruvate and -d glucose. The l-lactate sensor features a short response time, little variation about the regression line computed and a linear indicating range adequate for clinical-chemical analysis. Measurement diagrams are demonstrated. The chemism of other conventional electrochemical-enzymatical measuring methods for l-lactate is mentioned, and reference is made to the clinical importance of l-lactate determination.

     

Sulfation of Hydroxyethyl Cellulose by N(SO3Na)3 and the Anticoagulant Activity of Sulfated Hydroxyethyl Cellulose

Hydroxyethyl cellulose sulfate (HECS) was synthesized by sulfation of hydroxyethyl cellulose with N(SO₃Na)₃, which was manufactured by reaction of sodium bisulfite and sodium nitrite. Barium sulfate nephelometry and light scattering method were used to determine degrees of substitution (DSs) and molecular weights (M_ws), respectively. Sulfate products with DS values from 0.26 to 1.88 and M_w in the range of 12.1–54.8 kDa were obtained at temperatures from 30°C to 80°C. Furthermore, the anticoagulant activity of HECS with different DSs, concentrations, and M_ws was studied. Clotting assays revealed that the introduction of sulfate groups into hydroxyethyl cellulose could improve its anticoagulant activity.     

Temperature- and pressure-responsive properties of <Emphasis Type="SmallCaps">l</Emphasis>- and<Emphasis Type="SmallCaps"> dl</Emphasis>-forms of poly(<Emphasis Type="Italic">N</Emphasis>-(1-hydroxymethyl)propylmethacrylamide) in aqueous solutions

The structural transition of the l- and dl forms of poly(N-(1- hydroxymethyl)propylmethacrylamide (PHMPMA) in aqueous solution was studied by measuring the pressure dependence of the apparent scattering intensity, differential scanning calorimetry (DSC), and circular dichroism (CD). The thermodynamic implications of the results are discussed in relation to the chiral structure of the side chain, and differences in the thermal and barometric transitions. T-P diagrams of the transition showed characteristic ellipsoid features. Antagonism of the temperature and pressure effects was observed only for P(dl-HMPMA). For P(l-HMPMA), the transition temperature (T _tr) decreased with increasing pressure, and the highest T _tr was observed at atmospheric pressure (0.1 MPa). For both polymers, the highest P _trs were observed at the lowest temperatures. The l polymer showed a specific negative peak in its CD spectrum at around 220 nm in the lower temperature region and the temperature dependence was reproduced by a single-step transition, with the midpoint corresponding to the T _tr obtained from the scattering measurements. Coupled with the results from the DSC, the different behavior between the P(l-HMPMA) and P(dl-HMPMA) could be explained in terms of the chain states before and after the transition. The cooperative factors derived from the DSC measurement revealed that about 4 to 5 polymers of the present size were necessary to perform a thermal transition for P(l-HMPMA), and that P(dl-HMPMA) underwent its transition as an almost single molecular event.This revised version was published online in June 2005 with correction to the article category.