Multipoint covalent immobilization of lipase on chitosan hybrid hydrogels: influence of the polyelectrolyte complex type and chemical modification on the catalytic properties of the biocatalysts

This work aimed at the production of stabilized derivatives of Thermomyces lanuginosus lipase (TLL) by multipoint covalent immobilization of the enzyme on chitosan-based matrices. The resulting biocatalysts were tested for synthesis of biodiesel by ethanolysis of palm oil. Different hydrogels were prepared: chitosan alone and in polyelectrolyte complexes (PEC) with kappa-carrageenan, gelatin, alginate, and polyvinyl alcohol (PVA). The obtained supports were chemically modified with 2,4,6-trinitrobenzene sulfonic acid (TNBS) to increase support hydrophobicity, followed by activation with different agents such as glycidol (GLY), epichlorohydrin (EPI), and glutaraldehyde (GLU). The chitosan-alginate hydrogel, chemically modified with TNBS, provided derivatives with higher apparent hydrolytic activity (HA(app)) and thermal stability, being up to 45-fold more stable than soluble lipase. The maximum load of immobilized enzyme was 17.5 mg g(-1) of gel for GLU, 7.76 mg g(-1) of gel for GLY, and 7.65 mg g(-1) of gel for EPI derivatives, the latter presenting the maximum apparent hydrolytic activity (364.8 IU g(-1) of gel). The three derivatives catalyzed conversion of palm oil to biodiesel, but chitosan-alginate-TNBS activated via GLY and EPI led to higher recovered activities of the enzyme. Thus, this is a more attractive option for both hydrolysis and transesterification of vegetable oils using immobilized TLL, although industrial application of this biocatalyst still demands further improvements in its half-life to make the enzymatic process economically attractive.

Biogas production and valorization by means of a two-step biological process

The scope of this research work was to investigate biogas production and purification by a two-step bench-scale biological system, consisting of fed-batch pulse-feeding anaerobic digestion of mixed sludge, followed by methane enrichment of biogas by the use of the cyanobacterium Arthrospira platensis. The composition of biogas was nearly constant, and methane and carbon dioxide percentages ranged between 70.5-76.0% and 13.2-19.5%, respectively. Biogas yield reached a maximum value (about 0.4 m(biogas)(3)/kgCOD(i)) at 50 days-retention time and then gradually decreased with a decrease in the retention time. Biogas CO(2) was then used as a carbon source for A. platensis cultivation either under batch or fed-batch conditions. The mean cell productivity of fed-batch cultivation was about 15% higher than that observed during the last batch phase (0.035 +/- 0.006 g(DM)/L/d), likely due to the occurrence of some shading effect under batch growth conditions. The data of carbon dioxide removal from biogas revealed the existence of a linear relationship between the rates of A. platensis growth and carbon dioxide removal from biogas and allowed calculating carbon utilization efficiency for biomass production of almost 95%. (C) 2009 Elsevier Ltd. All rights reserved.

Two-step liquid-phase microextraction and high-performance liquid chromatography for the simultaneous analysis of the enantiomers of mefloquine and its main metabolite carboxymefloquine in plasma

A method for the simultaneous analysis of the enantiomers of mefloquine (MQ) and its main metabolite carboxymefloquine (CMQ) in plasma is described for the first time. The assay involves two-step liquid-phase micro-extraction (LPME) and enantioselective high-performance liquid chromatography. In the first LPME step, the enantiomers of MQ were extracted from an alkalinized sample through a thin layer of di-n-hexyl ether immobilized in the pores of the hollow fiber and into 0.01 M perchloric acid as acceptor solution. In the second LPME step, the same sample was acidified to enable the extraction of CMQ using the same organic solvent and 0.05 M sodium hydroxide as acceptor phase. The analytes were resolved on a Chirobiotic T column in the polar-organic mode of elution and detected at 285 nm. The recovery rates from 1 mL of plasma were in the range 35-38%. The method presented limits of quantification of 50 ng/mL for all analytes and was linear up to 1,500 and 3,000 ng/mL for the enantiomers of MQ and CMQ, respectively. The plasmatic concentrations of (+)-(RS)-MQ were higher than those of (-)-(SR)-MQ after oral administration of the racemic drug to rats.

Development of novel bioanodes for ethanol biofuel cell using PAMAM dendrimers as matrix for enzyme immobilization

This paper describes the preparation and application of a novel bioanode for use in ethanol/O(2) biofuel cells based upon immobilization of alcohol dehydrogenase (ADH) and polyamidoamine (PAMAM) dendrimers onto carbon cloth platforms. The power density measurements indicated a direct relationship between the amount of anchored ADH and the anode power values, which increased upon enzyme loading. The power density values ranged from 0.04 to 0.28 mW cm(-2), and the highest power density was achieved with the bioanode prepared with 28 U of ADH, which provided a power density of 0.28 mW cm(-2) at 0.3 V. The latter power output values were the maximum observed, even for higher enzyme concentrations. Stability of the bioanodes was quite satisfactory, since there was no appreciable reduction of enzymatic activity during the measurements. The method of bioanode preparation described here has proven to be very effective. The PAMAM dendrimer represents a friendly environment for the immobilization of enzymes, and it is stable and capable of generating high power density compared to other immobilization methods. (C) 2010 Elsevier B.V. All rights reserved.

Molecular View of the Interaction between iota-Carrageenan and a Phospholipid Film and Its Role in Enzyme Immobilization

Proteins incorporated into phospholipid Langmuir-Blodgett (LB) films are a good model system for biomembranes and enzyme immobilization studies. The specific fluidity of biomembranes, an important requisite for enzymatic activity, is naturally controlled by varying phospholipid compositions. In a model system, instead, LB film fluidity may be varied by covering the top layer with different substances able to interact simultaneously with the phospholipid and the protein to be immobilized. In this study, we immobilized a carbohydrate rich Neurospora crassa alkaline phosphatase (NCAP) in monolayers of the sodium salt of dihexadecylphosphoric acid (DHP), a synthetic phospholipid that provides very condensed Langmuir films. The binding of NCAP to DHP Langmuir-Blodgett (LB) films was mediated by the anionic polysaccharide iota-carrageenan (iota-car). Combining results from surface isotherms and the quartz crystal microbalance technique, we concluded that the polysaccharide was essential to promote the interaction between DHP and NCAP and also to increase the fluidity of the film. An estimate of DHP:iota-car ratio within the film also revealed that the polysaccharide binds to DHP LB film in an extended conformation. Furthermore, the investigation of the polysaccharide conformation at molecular level, using sum-frequency vibrational spectroscopy (SFG), indicated a preferential conformation of the carrageenan molecules with the sulfate groups oriented toward the phospholipid monolayer, and both the hydroxyl and ether groups interacting preferentially with the protein. These results demonstrate how interfacial electric fields can reorient and induce conformational changes in macromolecules, which may significantly affect intermolecular interactions at interfaces. This detailed knowledge of the interaction mechanism between the enzyme and the LB film is relevant to design strategies for enzyme immobilization when orientation and fluidity properties of the film provided by the matrix are important to improve enzymatic activity.

Two-step tuberculin skin test and booster phenomenon prevalence among Brazilian medical students

SETTING: Five medical schools in three cities in Rio de Janeiro State, Brazil, with different tuberculosis (TB) incidence rates. OBJECTIVE: To evaluate the prevalence of the booster phenomenon and its associated factors in a voting universally BCG-vaccinated TB-exposed population. DESIGN: A two-step tuberculin skin test (TST) was performed among undergraduate medical students. Boosting was defined as an induration >= 10 mm in the second TST (TST2), with an increase of at least 6 mm over the first TST (TST1). The association of boosting with independent variables was evaluated using multivariate analysis. RESULTS: Of the 764 participants (mean age 21.9 +/- 2.7 years), 672 (87.9%) had a BCG scar. The overall booster SUMMARY phenomenon prevalence was 8.4% (95%CI 6.5-10.6). Boosting was associated with TST1 reactions of 1-9 mm (aOR 2.5, 95%CI 1.04-5.9) and with BCG vaccination, mostly after infancy, i.e., after age two years (aOR 9.1, 95%,CI 1.2-70.7). CONCLUSION: The prevalence of the booster phenomenon was high. A two-step TST in young BCG-vaccinated populations, especially in those with TST1 reactions of 1-9 mm, can avoid misdiagnosis as a false conversion and potentially reduce unnecessary treatment for latent TB infection.

Durability of enamel bonding using two-step self-etch systems on ground and unground enamel

This study examined the early and long-term microtensile bond strengths (mu TBS) and interfacial enamel gap formation (IGW) of two-step selfetch systems to unground and ground enamel. Resin composite (Filtek Z250) buildups were bonded to proximal enamel surfaces (unground, bur-cut or SiC-treated enamel) of third molars after the application of four self-etch adhesives: a mild (Clearfil SE Bond [SE]), two moderate (Optibond Solo Plus Self-Etch Primer [SO] and AdheSE [AD]) and a strong adhesive (Tyrian Self Priming Etchant + One Step Plus [TY]) and two etch-and-rinse adhesive systems (Single Bond [SB] and Scotchbond Multi-Purpose Plus [SBMP]). Ten tooth halves were assigned for each adhesive. After storage in water (24 hours/37 degrees C), the bonded specimens were sectioned into beams (0.9 mm(2)) and subjected to mu TBS (0.5 mm/minute) or interfacial gap width measurement (stereomicroscope at 400x) either immediately (IM) or after 12 months (12M) of water storage. The data were analyzed by three-way repeated measures ANOVA and Tukey`s test (alpha=0.05). No gap formation was observed in any experimental condition. The mu TBS in the Si-C paper and diamond bur groups were similar and greater than the unground group only for the moderate self-etch systems (SO and AD). No reductions in bond strength values were observed after 12 months of water storage, regardless of the adhesive evaluated.

A Randomized Clinical Evaluation of a One- and Two-step Self-etch Adhesive Over 24 Months

Objective: To evaluate the performance of All Bond SE used in a one- or two-step protocol in a 24-month randomized clinical study. Methods: Thirty-three patients with two similarly sized non-carious cervical lesions participated in this study. A total of 66 restorations were placed, half using the one-step All Bond SE protocol (SE-1) and the other half using the two-step All Bond SE protocol (SE-2). The restorations were evaluated at baseline and after 6, 12 and 24 months following the modified USPHS criteria and analyzed by the McNemar`s test and Fisher`s exact test (alpha=0.05). Results: After 24 months, six SE-1 and four SE-2 restorations were rated as Bravo in marginal discoloration The retention rates for SE-1 and SE-2 were 84.8% and 90.9%, respectively, after 24 months. Compared to baseline, the retention rate for SE-1 was statistically lower. Conclusions: All Bond SE used in the one- or two-step protocol resulted in high retention rates after 24 months.

Enzyme immobilization on Ag nanoparticles/polyaniline nanocomposites

We show a simple strategy to obtain all efficient enzymatic broelectrochemical device, in which urease was immobilized oil electroactive nanostructured membranes (ENMs) made with polyaniline and silver nanoparticles (AgNP) stabilized in polyvinyl alcohol (PAni/PVA-AgNP). Fabrication of the modified electrodes comprised the chemical deposition of polyaniline followed by drop-coating of PVA-AgNP and urease, resulting in a final ITO/PAni/PVA-AgNP/urease electrode Configuration. For comparison. the electrochemical performance of ITO/PAni/urease electrodes (without Ag nanoparticles) was also studied. The performance of the modified electrodes toward Urea hydrolysis was investigated via amperometric measurements, revealing a fast increase in cathodic current with a well-defined peak upon addition of urea to the electrolytic solution. The cathodic currents for the ITO/PAni/PVA-AgNP urease electrodes were significantly higher than for the ITO/PAni/urease electrodes. The friendly environment provided by the ITO/PAni/PVA-AgNP electrode to the immobilized enzyme promoted efficient catalytic conversion of urea into ammonium and bicarbonate tons. Using the Michaelis-Menten kinetics equation, a K(M)(aPP) of 2.7 mmol L(-1) was obtained. indicating that the electrode architecture employed may be advantageous for fabrication of enzymatic devices with improved biocatalytic properties. Crown Copyright (C) 2009 Published by Elsevier B.V. All rights reserved.

Urea amperometric biosensors based on a multifunctional bipolymeric layer: Comparing enzyme immobilization methods

This paper outlines the results obtained with biosensors designed for urea amperometric detection. The incorporation of urease into a bipolymeric substrate consisting of poly(pyrrole) and poly(5-amino-1-naphthol) was performed through four different approaches: direct adsorption, entrapment in cellulose acetate layer. cross-linking with glutaraldehyde, and also covalent attachment to the polymeric matrix. Poly(pyrrole) acts as amperometric transducer in these biosensors, while poly(5-amino-1-naphthol) drastically reduces the interference signal of agents such as ascorbic and uric acids. The biosensors containing urease covalently attached to the substrate provided interesting results in terms of sensitivity towards urea (0.50 mu A cm(-2) mmol(-1) L), lifetime (20 days) and short response times, due to the enzyme immobilization method used. All biosensors analyzed showed also a wide linear concentration range (up to 100 mmol L(-1)) and low detection limits (0.22-0.58 mmol L(-1)). (C) 2009 Elsevier B.V. All rights reserved.

The effect of agitation speed, enzyme loading and substrate concentration on enzymatic hydrolysis of cellulose from brewer`s spent grain

Brewer`s spent grain components (cellulose, hemicellulose and lignin) were fractionated in a two-step chemical pretreatment process using dilute sulfuric acid and sodium hydroxide solutions. The cellulose pulp produced was hydrolyzed with a cellulolytic complex, Celluclast 1.5 L, at 45 degrees C to convert the cellulose into glucose. Several conditions were examined: agitation speed (100, 150 and 200 rpm), enzyme loading (5, 25 and 45 FPU/g substrate), and substrate concentration (2, 5 and 8% w/v), according to a 2(3) full factorial design aiming to maximize the glucose yield. The obtained results were interpreted by analysis of variance and response surface methodology. The optimal conditions for enzymatic hydrolysis of brewer`s spent grain were identified as 100 rpm, 45 FPU/g and 2% w/v substrate. Under these conditions, a glucose yield of 93.1% and a cellulose conversion (into glucose and cellobiose) of 99.4% was achieved. The easiness of glucose release from BSG makes this substrate a raw material with great potential to be used in bioconversion processes.

Morphological and mechanical properties of hybrid matrices of polysiloxane-polyvinyl alcohol prepared by sol-gel technique and their potential for immobilizing enzyme

Hybrid matrices of polysiloxane-polyvinyl alcohol (POS-PVA) were prepared by sol-gel technique using different concentrations of the organic component (polyvinyl alcohol, PVA) in the synthesis medium. The goal was to prepare carriers for immobilizing enzyme by taking into consideration properties as hardness, mean pore diameter, specific surface area and pore size distribution. The matrices were activated with sodium metaperiodate to render functional groups for binding the lipase from Candida rugosa, used here as a study model. Results showed that low proportion of PVA gave POS-PVA with low surface area and pore volume, although with higher hardness. The chemical activation decreased the pore volume and increased the pore size with a decrease on the surface area of about 60-75%. The matrices for enzyme immobilization were chosen considering the best combination of high surface area and hardness. Thus, the POS-PVA prepared with 5.56 x 10(-5) M of PVA with a surface area of 123 m(2)/g and hardness of 71 HV (50 gf 30 s) was shown to be suitable to immobilize the lipase, with an immobilization yield of about 40%. (c) 2008 Elsevier B.V. All rights reserved.

Pseudomonas fluorescens lipase immobilization on polysiloxane-polyvinyl alcohol composite chemically modified with epichlorohydrin

The technique based on sol-gel approach was used to generate silica matrices derivatives by hydrolysis of silane compounds. The present work evaluates a hybrid matrix obtained with tetraethoxysilane (TEOS) and polyvinyl alcohol (PVA) on the immobilization yield of lipase from Pseudomonas fluorescens. The resulting polysiloxane-polyvinyl alcohol (POS-PVA) matrix combines the property of PVA as a suitable polymer to retain proteins with an excellent optical, thermal and chemical stability of the host silicon oxide matrix. Aiming to render adequate functional groups to the covalent binding with the enzyme the POS-PVA matrix was chemically modified using epichlorohydrin. The results were compared with immobilized derivative on POS-PVA activated with glutaraldehyde. Immobilization yield based on the recovered lipase activity depended on the activating agent and the highest efficiency (32%) was attained when lipase was immobilized on POS-PVA activated with epichlorohydrin, which, probably, provided more linkage points for the covalent bind of the enzyme on the support. This was confirmed by determining the morphological properties using different techniques as X-ray diffraction and scanning electron microscopy (SEM). Comparative studies were carried out to attain optimal activities for free lipase and immobilized systems. For this purpose, a central composite experimental design with different combinations of pH and temperature was performed. Enzymatic hydrolysis with the immobilized enzyme in the framework of the Michaelis-Menten mechanism was also reported. Under optimum conditions, the immobilized derivative on POS-PVA activated with epichlorohydrin showed to have more affinity for the substrate in the hydrolysis of olive oil, with a Michaelis-Menten constant value (K-m) of 293 mM, compared to the value of 401 mM obtained for the immobilized lipase on support activated with glutaraldehyde. Data generated by DSC showed that both immobilized derivatives have similar thermal stabilities. (C) 2007 Elsevier B.V. All rights reserved.

FtsH2 and FtsH5: two homologous subunits use different integration mechanisms leading to the same thylakoid multimeric complex

P>The Arabidopsis thylakoid FtsH protease complex is composed of FtsH1/FtsH5 (type A) and FtsH2/FtsH8 (type B) subunits. Type A and type B subunits display a high degree of sequence identity throughout their mature domains, but no similarity in their amino-terminal targeting peptide regions. In chloroplast import assays, FtsH2 and FtsH5 were imported and subsequently integrated into thylakoids by a two-step processing mechanism that resulted in an amino-proximal lumenal domain, a single transmembrane anchor, and a carboxyl proximal stromal domain. FtsH2 integration into washed thylakoids was entirely dependent on the proton gradient, whereas FtsH5 integration was dependent on NTPs, suggesting their integration by Tat and Sec pathways, respectively. This finding was corroborated by in organello competition and by antibody inhibition experiments. A series of constructs were made in order to understand the molecular basis for different integration pathways. The amino proximal domains through the transmembrane anchors were sufficient for proper integration as demonstrated with carboxyl-truncated versions of FtsH2 and FtsH5. The mature FtsH2 protein was found to be incompatible with the Sec machinery as determined with targeting peptide-swapping experiments. Incompatibility does not appear to be determined by any specific element in the FtsH2 domain as no single domain was incompatible with Sec transport. This suggests an incompatible structure that requires the intact FtsH2. That the highly homologous type A and type B subunits of the same multimeric complex use different integration pathways is a striking example of the notion that membrane insertion pathways have evolved to accommodate structural features of their respective substrates.

Stabilization of an amylase from Neurospora crassa by immobilization on highly activated supports

The amylase from Neurospora crassa is an interesting enzyme, having higher stability than amylase from Aspergillus oryzea under a broad range of pH values. Moreover, the N. crassa enzyme may be immobilized on different supports with good retention of enzyme activity. The best stabilizations were achieved using Eupergit C 250 L or glyoxyl agarose, with which the enzyme remained fully active at 60C for 24 h while the soluble enzyme remained about 17%. The glyoxyl agarose immobilized enzyme had high thermostability, high optimal temperature (65C) and broad pH/activity profile, suggesting that this enzyme has potential for food and industrial applications for starch modification.