Enhanced production and organic solvent stability of a protease fromBrevibacillus laterosporus strain PAP04

A bacterial strain (PAP04) isolated from cattle farm soil was shown to produce an extracellular, solvent-stable protease. Sequence analysis using 16S rRNA showed that this strain was highly homologous (99%) to Brevibacillus laterosporus. Growth conditions that optimize protease production in this strain were determined as maltose (carbon source), skim milk (nitrogen source), pH 7.0, 40°C temperature, and 48 h incubation. Overall, conditions were optimized to yield a 5.91-fold higher production of protease compared to standard conditions. Furthermore, the stability of the enzyme in organic solvents was assessed by incubation for 2 weeks in solutions containing 50% concentration of various organic solvents. The enzyme retained activity in all tested solvents except ethanol; however, the protease activity was stimulated in benzene (74%) followed by acetone (63%) and chloroform (54.8%). In addition, the plate assay and zymography results also confirmed the stability of the PAP04 protease in various organic solvents. The organic solvent stability of this protease at high (50%) concentrations of solvents makes it an alternative catalyst for peptide synthesis in non-aqueous media.

SEPARATION OF SATURED AND UNSATURATED FATTY ACIDS FROM PALM FATTY ACIDS DISTILLATES IN CONTINUOUS MULTISTAGE COUNTERCURRENT COLUMNS WITH SUPERCRITICAL CARBON DIOXIDE AS SOLVENT: A PROCESS DESIGN METHODOLOGY

In this work the separation of multicomponent mixtures in counter-current columns with supercritical carbon dioxide has been investigated using a process design methodology. First the separation task must be defined, then phase equilibria experiments are carried out, and the data obtained are correlated with thermodynamic models or empirical functions. Mutual solubilities, Ki-values, and separation factors aij are determined. Based on this data possible operating conditions for further extraction experiments can be determined. Separation analysis using graphical methods are performed to optimize the process parameters. Hydrodynamic experiments are carried out to determine the flow capacity diagram. Extraction experiments in laboratory scale are planned and carried out in order to determine HETP values, to validate the simulation results, and to provide new materials for additional phase equilibria experiments, needed to determine the dependence of separation factors on concetration. Numerical simulation of the separation process and auxiliary systems is carried out to optimize the number of stages, solvent-to-feed ratio, product purity, yield, and energy consumption. Scale-up and cost analysis close the process design. The separation of palmitic acid and (oleic+linoleic) acids from PFAD-Palm Fatty Acids Distillates was used as a case study.

Continuous Counter-current Solvent Extraction of Magnesium and Calcium from Lithium-rich Brine

Solvent extraction of calcium and magnesium impurities from a lithium-rich brine (Ca ~ 2,000 ppm, Mg ~ 50 ppm, Li ~ 30,000 ppm) was investigated using a continuous counter-current solvent extraction mixer-settler set-up. The literature review includes a general review about resources, demands and production methods of Li followed by basics of solvent extraction. Experimental section includes batch experiments for investigation of pH isotherms of three extractants; D2EHPA, Versatic 10 and LIX 984 with concentrations of 0.52, 0.53 and 0.50 M in kerosene respectively. Based on pH isotherms LIX 984 showed no affinity for solvent extraction of Mg and Ca at pH ≤ 8 while D2EHPA and Versatic 10 were effective in extraction of Ca and Mg. Based on constructed pH isotherms, loading isotherms of D2EHPA (at pH 3.5 and 3.9) and Versatic 10 (at pH 7 and 8) were further investigated. Furthermore based on McCabe-Thiele method, two extraction stages and one stripping stage (using HCl acid with concentration of 2 M for Versatic 10 and 3 M for D2EHPA) was practiced in continuous runs. Merits of Versatic 10 in comparison to D2EHPA are higher selectivity for Ca and Mg, faster phase disengagement, no detrimental change in viscosity due to shear amount of metal extraction and lower acidity in stripping. On the other hand D2EHPA has less aqueous solubility and is capable of removing Mg and Ca simultaneously even at higher Ca loading (A/O in continuous runs > 1). In general, shorter residence time (~ 2 min), lower temperature (~23 °C), lower pH values (6.5-7.0 for Versatic 10 and 3.5-3.7 for D2EHPA) and a moderately low A/O value (< 1:1) would cause removal of 100% of Ca and nearly 100% of Mg while keeping Li loss less than 4%, much lower than the conventional precipitation in which 20% of Li is lost.

The role of organic solvent amount in the lipase-catalyzed biodiesel production

This research note addresses the role of organic solvent amount in the production of fatty acid ethyl esters from soybean oil. N-hexane was chosen as solvent and two commercial immobilized lipases as catalysts, Novozym 435 and Lipozyme IM. The reactions were conducted in 6 hours, varying the solvent to oil ratio from zero to 50 (v/wt) and adopting adopting for Novozym 435: 65 ºC, enzyme concentration (E, wt%) = 5, oil to ethanol molar ratio (R) = 1:10, water addition (H, wt%) = 0, and for Lipozyme IM: 35 ºC, E = 5 wt%, R = 1:3, H = 10 wt%. For Lipozyme IM, an increase in solvent amount is shown to lead to an enhancement of reaction conversion, while a negligible effect was found for Novozym 435. When using 30 mL of solvent the reaction conversions were 88% for Lipozyme IM and 15% for Novozym 435.

Ultrafiltration performance of PVDF, PES, and cellulose membranes for the treatment of coconut water (Cocos nucifera L.)

Ultrafiltration (UF) inhibits the enzymatic activity which is responsible for color changes of coconut water without the need for heat treatment. In the present study, UF performance in terms of the permeate flux and enzymatic retention of the coconut water was evaluated at laboratory unit (LU) and pilot unit (PU). The membranes studied were polyethersulfone 150 kDa (UP150), polyvinylidene fluoride 150 kDa (UV150) and cellulose 30 kDa (UC030). The UP150 membrane showed the best permeate flux. The UC030 membrane showed the lowest flux, but it resulted in 100% enzymatic retention, while the other membranes showed enzymatic retentions between 71 and 85%. The application of the UC030 in the pilot unit (PU) resulted in a flux value higher than that obtained in the LU due to the tangential velocity effect. The UC030 membrane has proved adequate for industrial applications.

Resistant starch in cassava products

Found in different foods, starch is the most important source of carbohydrates in the diet. Some factors present in starchy foods influence the rate at which the starch is hydrolyzed and absorbed in vivo. Due the importance of cassava products in Brazilian diet, the objective of this study was to analyze total starch, resistant starch, and digestible starch contents in commercial cassava products. Thirty three commercial cassava products from different brands, classifications, and origin were analyzed. The method used for determination of resistant starch consisted of an enzymatic process to calculate the final content of resistant starch considering the concentration of glucose released and analyzed. The results showed significant differences between the products. Among the flours and seasoned flours analyzed, the highest levels of resistant starch were observed in the flour from Bahia state (2.21%) and the seasoned flour from Paraná state (1.93%). Starch, tapioca, and sago showed levels of resistant starch ranging from 0.56 to 1.1%. The cassava products analyzed can be considered good sources of resistant starch; which make them beneficial products to the gastrointestinal tract.

Steroid-resistant idiopathic nephrotic syndrome in children: long-term follow-up and risk factors for end-stage renal disease

INTRODUTION: Steroid resistant idiopathic nephrotic syndrome (SRINS) in children is one of the leading causes of progression to chronic kidney disease stage V (CKD V)/end stage renal disease (ESRD). OBJECTIVE: The aim of this retrospective study is to evaluate the efficacy of immunosuppressive drugs (IS) and to identify risk factors for progression to ESRD in this population. METHODS: Clinical and biochemical variables at presentation, early or late steroid resistance, histological pattern and response to cyclosporine A (CsA) and cyclophosfamide (CP) were reviewed in 136 children with SRINS. The analyzed outcome was the progression to ESRD. Univariate as well as multivariate Cox-regression analysis were performed. RESULTS: Median age at onset was 5.54 years (0.67-17.22) and median follow up time was 6.1 years (0.25-30.83). Early steroid-resistance was observed in 114 patients and late resistance in 22. Resistance to CP and CsA was 62.9% and 35% respectively. At last follow-up 57 patients reached ESRD. The renal survival rate was 71.5%, 58.4%, 55.3%, 35.6% and 28.5% at 5, 10, 15, 20 and 25 years respectively. Univariate analysis demonstrated that older age at onset, early steroid-resistance, hematuria, hypertension, focal segmental glomerulosclerosis (FSGS), and resistance to IS were risk factors for ESRD. The Cox proportional-hazards regression identified CsAresistance and FSGS as the only predictors for ESRD. CONCLUSION: Our findings showed that CsA-resistance and FSGS were risk factors for ESRD.

Immunosupressive therapy in children with steroid-resistant nephrotic syndrome: single center experience

INTODUCTION: Nephrotic syndrome is one of the most frequent glomerular diseases among children, and steroid therapy remains as the treatment choice. In spite of this, 10 to 15% of the patients are steroidresistant, and the best therapy for such cases has never been defined. Mycophenolate acid (MA) is one of the treatments used in such situations. OBJECTIVE: To describe the clinical behavior of children diagnosed with steroid-resistant nephrotic syndrome (SRNS) and to assess the therapeutic response to MA. METHODS: This was a retrospective and descriptive study. RESULTS: 26 clinical records of patients with SRNS; 70% male and 30% female. All patients underwent kidney biopsies, which showed a predominance of focal segmental glomerulosclerosis (FSGS). The immunosuppresive drugs used were: Mycophenolate mofetil (MMF) 100%, Cyclosporine 69.2%, Cyclophosphamide 23.1%, and Rituximab 23%. One month after treatment initiation with MMF 61.5% achieved remission. The median of relapses per year for the patients was 3 (p25: 2.75 - p75: 4). This median became 1 (p25: 1 - p75: 3.25) after using this medication (p = 0.08). Furthermore, prior to the start of the MMF treatment, the median of the steroid dose was 1 (p25: 0.5- p75: 1.62) mg/k/day. After using MMF, this median became 0.07 (p25: 0 - p75: 0.55) mg/k/day (p < 0.001), in 8 patients prednisolone was stopped. CONCLUSION: In our experience, treatment with MMF showed positive results such as decrease in the frequency of relapses, less proteinuria, and reduction in the dose of steroids administered without deterioration of glomerular filtration rates. However, more studies are needed to assess efficacy, safety, and optimal dosage.

The structural effects of divalent cations and insulin on phospholipid model membranes /

It is well accepted that structural studies with model membranes are of considerable value in understanding the structure of biological membranes. Many studies with models of pure phospholipids have been done; but the effects of divalent cations and protein on these models would make these studies more applicable to intact membrane. The present study, performed with above view, is a structural analysis of divalent io~cardio1ipin complexes using the technique of x-ray diffraction. Cardiolipin, precipitated from dilute solution by divalent ionscalcium, magnesium and barium, contains little water and the structure formed is similar to the structure of pure cardiolipin with low water content. The calcium-cardiolipin complex forms a pure hexagonal type II phase that exists from 40 to 400 C. The molar ratio of calcium and cardiolipin in the complex is 1 : 1. Cardiolipin, precipitated with magnesium and barium forms two co-existing phases, lamellar and hexagonal, the relative quantity of the two phases being dependent on temperature. The hexagonal phase type II consisting of water filled channels formed by adding calcium to cardiolipin may have a remarkable permeability property in intact membrane. Pure cardiolipin and insulin at pH 3.0 and 4.0 precipitate but form no organised structure. Lecithin/cardiolipin and insulin precipitated at pH 3.0 give a pure lamellar phase. As the lecithin/cardiolipin molar ratio changes from 93/7 to SO/50, (a) the repeat distance of the lamellar changes from 72.8 X to 68.2 A; (b) the amount of protein bound increases in such a way that cardiolipin/insulin molar ratio in the complex reaches a maximum constant value at lecithin/cardiolipin molar ratio 70/30. A structural model based on these data shows that the molecular arrangement of lipid and protein is a lipid bilayer coated with protein molecules. The lipid-protein interaction is chiefly electrostatic and little, if any, hydrophobic bonding occurs in this particular system. So, the proposed model is essentially the same as Davson-Daniellifs model of biological membrane.

Effect of the position of the double-bond in monounsaturated phospholipids on the dynamics of model membranes

Therllloelynalllics of lllodel 11lel1ll)rane systeills containing 1110nollnsaturatecl I)lloSI)holil) ids is strongly infllienced l)y the I)osition of the C==C dOlll)le })ond in tIle acyl chain. The telllI)eratllres of both chain-nlelting (TM) and La -+ HI! (TH) I)hase traIlsitions are lowered by IIp to 20°C when C==C is Inoved froln positions 6 or 11 to I)osition 9 in an 18-carl)on chain. This work is an attellll)t to ellicidate the uIlderlying Illoleclilar Illechanisllls reSI)Onsi])le for tllese draillatic tllerillodynaillic changes. Mixtllres of di-18: 1 l)hoSI)hatidylethanolanline with C==C at l)ositioIlS 6, 9, 11 were llsed, witll a sI1lall aI1lOlint of I)erdellterated tetradecanol, known to })e a gooel rel)Orter of the cllain Illoleclilar order. SI)ectral second 11I0I1lents were llsed to Illonitor tIle La -+ HII I)hase transition, which was fOllnd to ])e ])road (2-6°C), with a slight llysteresis on heatiIlg/cooling. The orientational order I)rofiles were nleasllred 1lSiIlg 2H Illiclear Illagnetic resonance and changes in these order I)rofiles between La aIld HII I)hases silow l)oth a local increase in order in the vicinity of the C==C bonds and an o\Terall decrease ill the average orientational order of the chain as a whole. These Sll])tle changes recluire })oth high-fidelity SI)ectrosCol)y and a careflll data analysis that takes into aCCOllnt the effects due to l)artiall1lagnetically-indllced orientational ordering of the l)ilayers. In tIle COIltext of SOllle recently rel)Orted cross-relaxation 11leaSlirenlents in Silllilar l)llOSI)llolil)iels, 0111' reslilts sllggest that large-anll)litllde conforlllational changes in the interior of tIle I110del 111eI11])ranes I)lay a 1110re significant role than I)reviollsly thOllght.

Effects of sample solvent composition and injection volume on chromatographic peak profiles of methyl w- benzimidazolecarbamate and 3-butyl1-2,4-dioxo[1,2-a]-s- triazinobenzimidazole in RP-HPLC

The effects of sample solvent composition and the injection volume, on the chromatographic peak profiles of two carbamate derivatives, methyl 2-benzimidazolecarbamate (MBC) and 3-butyl-2,4-dioxo[1,2-a]-s-triazinobenzimidazole (STB), were studied using reverse phase high performance liquid chromatograph. The study examined the effects of acetonitrile percentage in the sample solvent from 5 to 50%, effects of methanol percentage from 5 to 50%, effects of pH increase from 4.42 to 9.10, and effect of increasing buffer concentration from ° to 0.12M. The effects were studied at constant and increasing injection mass and at four injection volumes of 10, 50, 100 and 200 uL. The study demonstrated that the amount and the type of the organic solvents, the pH, and the buffer strength of the sample solution can have a pronounced effect on the peak heights, peak widths, and retention times of compounds analysed. MBC, which is capable of intramolecular hydrogen bonding and has no tendency to ionize, showed a predictable increase .in band broadening and a decrease in retention times at higher eluting strengths of the sample solvent. STB, which has a tendency to ionize or to strongly interact with the sample solvent, was influenced in various ways by the changes in ths sample solvent composition. The sample solvent effects became more pronounced as the injection volume increased and as the percentage of organic solvent in the sample solution became greater. The peak height increases for STB at increasing buffer concentrations became much more pronounced at higher analyte concentrations. It was shown that the widely accepted procedure of dissolving samples in the mobile phase does not yield the most efficient chromatograms. For that reason samples should be dissolved in the solutions with higher aqueous content than that of the mobile phase whenever possible. The results strongly recommend that all the samples and standards, regardless whether the standards are external or internal, be analysed at a constant sample composition and a constant injection volume.

Localization of the mechanism of pH-dependent non- photochemical fluorescence quenching in thylakoid membranes

Higher plants have evolved a well-conserved set of photoprotective mechanisms, collectively designated Non-Photochemical Quenching of chlorophyll fluorescence (qN), to deal with the inhibitory absorption of excess light energy by the photosystems. Their main contribution originates from safe thermal deactivation of excited states promoted by a highly-energized thylakoid membrane, detected via lumen acidification. The precise origins of this energy- or LlpH-dependent quenching (qE), arising from either decreased energy transfer efficiency in PSII antennae (~ Young & Frank, 1996; Gilmore & Yamamoto, 1992; Ruban et aI., 1992), from alternative electron transfer pathways in PSII reaction centres (~ Schreiber & Neubauer, 1990; Thompson &Brudvig, 1988; Klimov et aI., 1977), or from both (Wagner et aI., 1996; Walters & Horton, 1993), are a source of considerable controversy. In this study, the origins of qE were investigated in spinach thylakoids using a combination of fluorescence spectroscopic techniques: Pulse Amplitude Modulated (PAM) fluorimetry, pump-probe fluorimetry for the measurement of PSII absorption crosssections, and picosecond fluorescence decay curves fit to a kinetic model for PSII. Quenching by qE (,..,600/0 of maximal fluorescence, Fm) was light-induced in circulating samples and the resulting pH gradient maintained during a dark delay by the lumenacidifying capabilities of thylakoid membrane H+ ATPases. Results for qE were compared to those for the addition of a known antenna quencher, 5-hydroxy-1,4naphthoquinone (5-0H-NQ), titrated to achieve the same degree of Fm quenching as for qE. Quenching of the minimal fluorescence yield, F0' was clear (8 to 130/0) during formation of qE, indicative of classical antenna quenching (Butler, 1984), although the degree was significantly less than that achieved by addition of 5-0H-NQ. Although qE induction resulted in an overall increase in absorption cross-section, unlike the decrease expected for antenna quenchers like the quinone, a larger increase in crosssection was observed when qE induction was attempted in thylakoids with collapsed pH gradients (uncoupled by nigericin), in the absence of xanthophyll cycle operation (inhibited by DTT), or in the absence of quenching (LlpH not maintained in the dark due to omission of ATP). Fluorescence decay curves exhibited a similar disparity between qE-quenched and 5-0H-NQ-quenched thylakoids, although both sets showed accelerated kinetics in the fastest decay components at both F0 and Fm. In addition, the kinetics of dark-adapted thylakoids were nearly identical to those in qEquenched samples at F0' both accelerated in comparison with thylakoids in which the redox poise of the Oxygen-Evolving Complex was randomized by exposure to low levels of background light (which allowed appropriate comparison with F0 yields from quenched samples). When modelled with the Reversible Radical Pair model for PSII (Schatz et aI., 1988), quinone quenching could be sufficiently described by increasing only the rate constant for decay in the antenna (as in Vasil'ev et aI., 1998), whereas modelling of data from qE-quenched thylakoids required changes in both the antenna rate constant and in rate constants for the reaction centre. The clear differences between qE and 5-0H-NQ quenching demonstrated that qE could not have its origins in the antenna alone, but is rather accompanied by reaction centre quenching. Defined mechanisms of reaction centre quenching are discussed, also in relation to the observed post-quenching depression in Fm associated with photoinhibition.