Hydrolysis of crambe oil by enzymatic catalysis: An evaluation of the operational conditions

Abstract

In this work, the enzymatic hydrolysis of the crambe oil by using a commercial immobilized lipase Lipozyme RM IM was evaluated. The effect of the operational conditions, such as temperature, water/oil molar ratio, enzyme/substrate mass ratio and stirring speed were assessed based on the experimental designs. The experiments were performed in a closed and batch system with controlled temperature and stirring speed. In addition, the kinetics of the process was studied in the best operational conditions, wherein the experimental data were obtained and described by a mathematical model. The influence of the operational conditions was assessed based on the measured values of the free fatty acids (FFA) produced by the enzymatic hydrolysis. In 4?h of reaction, a yield of 42.6% was observed and the most significant operational conditions were the enzyme/substrate mass ratio and stirring speed. By the kinetic investigation, an initial reaction rate of 3.5?×?10⁴?mol?mL^?1?h^?1 and a maximum yield of 74% were observed after 40?h of reaction (in the equilibrium condition). The mathematical model was not only able to adequately describe the experimental data of FFA concentrations profiles but also showed predictive capacity to independents assays in different operational conditions. Therefore, based on the simulation analysis of the enzymatic hydrolysis of the crambe oil, the model can be useful for process optimization and phenomenological studies.     

Stimulus amplification, efficacy, and the operational model. Part II--ternary complex occupancy mechanisms.

To understand how deviations from simple binary occupancy affect measures of efficacy, the generalized stimulus function developed in Part I was used to examine the actions of drugs in systems where occupation of the receptor was modeled using a two-state, ternary complex, or combination of mechanisms.Amplification of drug responses can occur during formation of an active agonist-receptor complex, during generation of the initial stimulus, and during signal transduction. Expressions were derived to characterize the separate contributions of these three phases. Ideally, comparison of relative intrinsic efficacy measures differences in the ability of the agonists to convert active complex into an active stimulus. In practice, differences in the ability of the drugs to form the stimulus-generating complex may also contribute to the efficacy ratio and must be taken into consideration. Failure to adequately account for differences in occupancy can result in overestimation of the efficacy ratio. The magnitude of the difference between true and experimental measures of intrinsic efficacy may be affected by G protein concentration, by the affinity between the G protein and receptor, and (in some models) by the receptor activation constant. Provided that the dissociation constant between the G protein and receptor is of the same order of magnitude as, or lower than the receptor concentration, however, experimental estimates should provide reasonably accurate estimates of the true efficacy ratio.In agreement with previously published experimental data, total G protein level was found capable of influencing agonist maximal response, Emax, and EC50 values in all four ternary complex models. The magnitude of the changes in Emax and EC50 appear to be dependent upon the efficacy of the agonist as well as characteristics of the post-receptor stimulus sequence.Additionally, the concentration-response relations for all four ternary complex models could be reduced to a modified operational format in which the apparent dissociation constant Kapp replaced the true KA, and an apparent operational efficacy, tauapp, replaced tau. tauapp can be estimated experimentally from measurements of the Kapp and EC50, while the operational maximum, Em, may be found from the calculated tauapp and the measured Emax of the response curve. These findings support the use of direct operational model-fitting in a variety of systems, regardless of the mechanisms underlying occupancy.Values of Kapp calculated using the exact formula for [ARG] displayed an anomalous rise or discontinuity where the concentrations of total G protein equaled that of the receptor protein. This discontinuity is not observed in the estimates based on approximations to [ARG], and may explain practical difficulties in evaluating the dissociation constant under these conditions.     

Production of hemicellulosic sugars and glucose from residual corrugated cardboard

Corrugated cardboard samples were subjected to two-step saccharification. A first prehydrolysis stage was carried out to solubilise the hemicellulosic fraction as hemicellulosic sugars, and the solid phase from prehydrolysis was used as a substrate for the enzymic hydrolysis of cellulose. The prehydrolysis step was carried out for 0–180 min in media containing 1–3 wt.% of H₂SO₄ and the fraction of solid recovered after treatments and the compositions of solid and liquid phases from treatments were measured. The susceptibility of prehydrolysed solids towards the enzymic hydrolysis was assessed in further experiments. Under selected prehydrolysis conditions (3% H₂SO₄, 180 min), 78.2% of initial hemicelluloses was saccharified, leading to liquors containing up to 10 g hemicellulosic sugars/l and 9.2 g glucose/l. The corresponding solid phase, enriched in cellulose, showed good susceptibility towards enzymatic hydrolysis, leading to solutions containing up to 17.9 g glucose/l (conversion yield=63.6%) and a glucose/total sugar ratio of 0.93 g/g. Mathematical models assessing the effects of the operational conditions on both the prehydrolysis stage and the susceptibility of substrates towards enzymic hydrolysis have been developed.     

On the infusion of a therapeutic agent into a solid tumor modeled as a poroelastic medium

The direct infusion of an agent into a solid tumor, modeled as a spherical poroelastic material with anisotropic dependence of the tumor hydraulic conductivity upon the tissue deformation, is treated both by solving the coupled fluid/elastic equations, and by expressing the solution as an asymptotic expansion in terms of a small parameter, ratio between the driving pressure force in the fluid system, and the elastic properties of the solid. Results at order one match almost perfectly the solutions of the full system over a large range of infusion pressures. Comparison with experimental results is acceptable after the hydraulic conductivity of the medium is properly calibrated. Given the uncertain estimates of some model constants, the order zero solution of the expansion, for which fluid and porous matrix are decoupled, yields acceptable values and trends for all the physical fields of interest, rendering the coupled analysis (in the limit of small displacements) of little use. When the deformation of the tissue becomes large nonlinear elasticity theory must be resorted to.     

次氯酸钠离析法观测甘蓝叶片脉序的处理条件优化筛选

次氯酸钠（NaClO）离析法主要用于植物叶片表皮的观测,在研究过程中发现该法也可用于叶片脉序的观测。以甘蓝（Brassica oleracea var.capitata）为实验材料,采用二次正交设计方法对水煮时间、NaOH浓度、NaOH处理时间、NaClO浓度、NaClO处理温度和处理时间等各种处理条件进行优化筛选,以期得到适合于甘蓝叶片脉序观测的最佳处理条件组合。实验结果表明,新鲜甘蓝叶片水煮3分钟,10%的NaOH溶液60°C水浴处理2.5小时,3%的NaClO溶液40°C水浴离析2小时,叶片脉序的观测效果最佳。     

Properties of membrane stationary states. 2. The isobaric isothermal ensemble

A multi-state membrane transport system serves as the basic model for a comparison of equilibrium and stationary state distributions of ions within an ensemble of membrane channels. Transition probabilities for intramembrane transitions are developed for consistency with an equilibrium Donnan potential and the magnitude of the flux in the stationary state can be controlled with either the bath concentrations or an externally applied potential. The stationary state distribution and operational thermodynamic parameters for the stationary state are expressed as functions of the ratio (j^net/p) where j^net is the observable net membrane flux and p is a characteristic decay constant for the intramolecular transitions. Both parameters are experimentally accessible so that their time independent ratio serves as an experimental measure of the membrane configuration. When operational thermodynamic parameters are expressed as functions of the ratio (j/p) for systems which obey the condition of detailed balance, the free energy varies quadratically with the ratio while the enthalpy, expressed as an average membrane potential, varies linearly with the ratio. As the system is driven from its equilibrium configuration by the net membrane flux the free energy increases relative to the enthalpy of the membrane. The free energy is localized in the membrane region at the expense of dissipation in the bathing solutions.     

Carboxymethylcellulose from totally chlorine-free-bleached milox pulps

High purity cellulose pulp was obtained from Eucalyptus globulus wood by using an environmentally friendly delignification technique (Milox pulping) and subsequent bleaching by totally chlorine free technology. The pulp obtained under optimised experimental conditions was used for the manufacture of carboxymethylcellulose in a heterogeneous medium. By means of an experimental design, the effects of selected operational variables on the composition and chemical properties of reaction products from the carboxymethylation reaction were assessed for optimisation purposes. The distribution of the different carboxymethylglucose mole fractions (un-, mono-, di- and tri-substituted) was determined and compared with theoretical predictions. The maximum average degree of substitution (1.26) was determined at a NaOH/cellulose mole ratio of 4.8 and at a MCA/cellulose mole ratio of 2.0.     

次氯酸钠离析法观测甘蓝叶片脉序的处理条件优化筛选

次氯酸钠(NaClO)离析法主要用于植物叶片表皮的观测, 在研究过程中发现该法也可用于叶片脉序的观测。以甘蓝(Brassica oleracea var. capitata)为实验材料, 采用二次正交设计方法对水煮时间、NaOH浓度、NaOH处理时间、NaClO浓度、NaClO处理温度和处理时间等各种处理条件进行优化筛选, 以期得到适合于甘蓝叶片脉序观测的最佳处理条件组合。实验结果表明, 新鲜甘蓝叶片水煮3分钟, 10%的NaOH溶液60°C水浴处理2.5小时, 3%的NaClO溶液40°C水浴离析2小时,叶片脉序的观测效果最佳。     

Production and recovery of an alkaline exo-polygalacturonase from Bacillus subtilis RCK under solid-state fermentation using statistical approach

The empirical models developed through two independent RSM (RSM-I, 2(3); RSM-II, 2(5)) in terms of effective operational factors of inoculum age, inoculum volume, wheat bran-to-moisture ratio (RSM-I) and contact time, extraction temperature, agitation, fermented bran-to-solvent ratio and SDS (RSM-II) were found adequate to describe the optimization of exo-polygalacturonase from Bacillus subtilis RCK under solid-state fermentation (SSF) conditions. Through the analysis of RSM-I, wheat bran-to-moisture ratio and inoculum volume were found to be the most significant factors and an increment in both had a positive effect in enhancing enzyme yield, while in RSM-II all the factors significantly affected enzyme recovery except fermented bran-to-solvent ratio, which had the least impact within the ranges investigated in enhancing enzyme recovery. Based on contour plots and variance analysis, optimum operational conditions for maximum exo-polygalacturonase yield were achieved when 1.5% (v/w) of 24h old (OD(600 nm) approximately 2.7+/-0.2) B. subtilis RCK cells were inoculated on moistened wheat bran (1:7 solid substrate-to-moisture ratio) and enzyme was harvested by addition of solvent (1:6 fermented bran-to-solvent ratio) under shaking conditions (200 rpm) in presence of SDS (0.25% w/v) for 15 min at 35 degrees C. An over all 3.4 fold (1.7-fold RSM-I; 2.0 fold RSM-II) increase in enzyme production was attained because of optimization by RSM.     

Electron and hole transfer from DNA base radicals to oxidized products of guanine in DNA

An investigation of electron and hole transfer to oxidized guanine bases in DNA is reported. Guanine in DNA was preferentially oxidized by Br(2)(*-) at 298 K to 8-oxo-7,8-dihydro-guanine (8-oxo-G) and higher oxidation products. HPLC-EC analysis of irradiated DNA shows that the formation of 8-oxo-G could not be increased above the ratio of one 8-oxo-G to 127 +/- 6 bp regardless of dose. 8-oxo-G can be produced only at low levels because it is further oxidized to other species. These oxidation products of guanine have been extensively investigated and identified by others. Our electron spin resonance studies suggest that at 77 K 8-oxo-G is a trap for radiation-produced holes, but certain further oxidation products of 8-oxo-G (G(ox)) are found to be efficient electron traps. Gamma irradiation of oxidized DNA samples in frozen (D(2)O) aqueous ices and glassy 7 M LiBr solutions resulted in radicals formed by electron attachment to the G(ox) sites that were monitored by electron spin resonance spectroscopy (ESR) at 77 K. These ESR spectra suggest that those oxidation products of 8-oxo-G containing alpha-diketo groups account for the electron traps (G(ox)) in oxidized DNA with oxaluric acid a likely major trap. Electron transfer from DNA anion radicals to G(ox) was followed by monitoring of their ESR signals with time at 77 K. Using typical values for the tunneling constant beta estimates of the relative amount of G(ox) to base pairs were obtained. Radicals formed by UV photolysis of oxidized DNA in 8 M NaClO(4) glassy aqueous solutions were also investigated. The 8-oxo-G cation accounts for less than 10% of all the radicals observed after either gamma irradiation of oxidized DNA in frozen (D(2)O) aqueous solution or UV photolysis of oxidized DNA in 8 M NaClO(4) glassy aqueous solutions. We estimate hole transfer distances of about 7 +/- 1 bp at 1 min from G(*+) to 8-oxo-G.     

均匀设计法优化高丽参多糖的提取

利用均匀设计法优化了高丽参多糖的提取过程.主要考察料液质量比、浸泡时间、提取温度和提取时间四个因素对提取率的影响.通过数学模型得到最优提取状况和3D曲面图.结果表明,提取温度对高丽参多糖得率影响最大,在料液质量比1:35、浸泡时间135 min、水提取温度81℃条件下提取156 min,高丽参多糖最大理论提取率为49.51%.最优化模型下实际多糖得率49.29%与理论得率相符.     

A high sensitivity amperometric biosensor using laccase as biorecognition element

An amperometric flow biosensor, using laccase from Rigidoporus lignosus as bioelement was developed. The laccase was kinetically characterized towards various phenolics both in solution and immobilized to a hydrophilic matrix by carbodiimide chemistry. A bioreactor connected to an amperometric flow cell by a FIA system was filled with the immobilized enzyme and the operational conditions of this biosensor were optimized as regards pH. Under the adopted experimental conditions, the immobilized enzyme oxidizes all the substrate molecules avoiding the need of cumbersome calibration procedures. The biosensor sensitivity, which was found to be 100 nA/microM for some of the tested substrates, resulted to be constant for more than 100 working days. This biosensor permits the detection of phenolics in aqueous solutions at concentrations in the nanomolar range and was successfully used to detect phenolics in wastewaters from olive oil mill without sample preparation.     

柑橘皮中果胶的提取工艺条件研究

目的:研究柑橘皮中果胶的提取工艺条件,提高果胶资源的开发及利用度,为农产品深加工提供理论基础。方法:以柑橘皮为原料,采用盐酸提取、真空浓缩、乙醇沉淀的方法,以果胶提取率和吸光度为考察指标,在单因素试验基础上,进行多因素的L16(45)正交试验设计,研究了浸提时间、浸提温度、溶液pH值、乙醇用量和料液比等重要影响因素,对柑橘皮果胶提取率的影响,获得果胶提取最优化组合,建立最佳的果胶提取工艺参数。结果:最佳的工艺条件为提取温度80℃,料液比1:15,提取液pH1.5,浸提时间2h,乙醇用量80%。在此条件下,果胶提取率可达到11.82%。结论:本实验所得的果胶制备最佳工艺参数组合,能获得较理想的橘皮中果胶的提取率。果胶产品各项检测指标均达到或超过国家标准。     

用SDS．NaClO从重组大肠杆菌中分离聚-β-羟基丁酸酯

聚β羟基丁酸酯（ＰＨＢ）是微生物合成的一种以颗粒状态存在于细胞中的高分子聚合物,由于它具有生物可降解性、生物相容性等特性,在医学上具有独特而广阔的应用前景。从微生物细胞中分离ＰＨＢ的方法有溶剂萃取法［１］、化学试剂法［２４］和酶法［５］。目前工...     

A neutron diffraction study of the influence of ions on phospholipid membrane interactions.

Neutron diffraction is used to examine the effects of Ca2+ and ClO4- ions on interactions and some structural features of dipalmitoylphosphatidylcholine membranes in both solid and fluid lamellar phases. The results are described within the framework of Derjaguin-Landau-Verwey-Overbeek (DLVO) theory with reference to electrostatic, van der Waals, and hydration components of disjoining pressure. The Hamaker constants are evaluated under equilibrium conditions. Addition of 100 mM CaCl2 to the aqueous phase substantially increases the lamellar repeat spacing (d), which is interpreted in terms of adsorption of Ca2+ ions to bilayers followed by electrostatic repulsion between membranes. The rise of NaClO4 concentration in the presence of 100 mM CaCl2 leads to gradual decrease in d, evidently resulted from the diminution of Ca(2+)-induced positive surface potential by both electrostatic screening and binding of ClO4- ions. In the absence of CaCl2, elevation of NaClO4 concentration to 100-300 mM drastically enhances the repeat spacing and then dramatically decreases d at about 1 M NaClO4. Estimation of the hydration coefficients showed that the pronounced decrease of the repeat spacing at high NaClO4 concentrations was resulted mainly from the (partial) disruption of the structure of intermembrane bound water by chaotropic ClO4- ions and subsequent decrease in hydration repulsive pressure. Moreover, in the case of solid membranes (20 degrees C) high concentrations of ClO4- induced formation of interdigitated phase paralleled with marked reduction in bilayer thickness and corresponding increase in the effective cross-sectional area per lipid molecule.     

Eucalyptus globulus wood fractionation by autohydrolysis and organosolv delignification

This work provides an assessment on the fractionation of Eucalyptus globulus wood by sequential stages of autohydrolysis (to cause the solubilization of hemicelluloses) and organosolv pulping (to dissolve lignin, leaving solids enriched in cellulose). With this approach, valuable products (hemicellulose-derived saccharides, sulphur-free lignin fragments and cellulosic substrates with low contents of residual hemicelluloses) are obtained in separate streams, according to the biomass refinery approach. Autohydrolysis was carried out under optimized operational conditions, and organosolv pulping was performed using uncatalyzed ethanol-water solutions. The effects of the most influential operational variables (autohydrolysis severity, delignification temperature and ethanol concentration in the organosolv stage) on solid yield, solid composition, cellulose susceptibility and recovery of the various fractions was assessed using statistical methods, which enabled the identification of the most favourable operational conditions.     

Modeling of polyphenols extraction from pomegranate by-product using rotatable central composite design of experiments

There is a growing request to find an effective method of polyphenols extraction from agro-industry by-product as pomegranate. In this study, response surface methodology (RSM) was used to explore the effect of three factors on ultrasonic assisted extraction (UAE) of total polyphenols (TP), total flavonoids (TF) and condensed tannins (CT) from pomegranate peels. The optimal conditions were determined for each phenolic compound using regression model equations and 3-D plots. The high TP, TF and CT content were obtained with, respectively, liquid/solid ratio of 20.00, 9.77, 9.77, extraction time of 36.38, 41.82, 30.39 min and 36.00, 83.64, 59.26% of ethanol percentage. In fact, liquid/solid ratio of 20, extraction time of 30.94 min and 59.26% of ethanol gives the highest contents of TP, TF and CT simultaneously. The experimental values using optimal conditions agreed with the predicted values. The pomegranate peels extract obtained under optimum conditions have an effective antioxidant activity as determined by ABTS and DPPH assays.     

B to Z transitions of the short DNA hairpins formed from the oligomer sequences: d[(CG)3X4(CG)3] (X = A, T, G, C).

Circular Dichroism (CD) spectra were collected as a function of sodium perchlorate concentration [NaClO4] for the set of DNA hairpins formed from the oligomer sequences d[(CG)3X4(CG)3] where X = A, T, G or C. Over the range in salt concentration from 0 to 4.0 M NaClO4, the CD spectra invert in a manner characteristic of the B to Z transition. A factor analysis routine is described and employed to determine the least number of basis spectra required to fit the measured spectra of each hairpin over the entire salt range examined. In every case, linear combinations of only two sub-spectra fit the experimental spectra of the hairpins with greater than 98% accuracy, indicating the spectrally monitored structural transitions are two-state. From the relative weights of the individual sub-spectra, B-Z transition curves are constructed. The transitions are analyzed in terms of a simple two-state equilibrium model which yields an evaluation of the transition free-energy, delta GB-Z, as a function of NaClO4 concentration. At 1.0 M NaClO4 and 21 degrees C, delta GB-Z = 5.4, 4.9, 3.6 and 2.3 kcal/mole for the G4, T4, A4 and C4 loop hairpins, respectively.     

Beta-glucan derived from zymosan acts as an adjuvant for collagen-induced arthritis

Collagen-induced arthritis (CIA) is an experimental model of rheumatoid arthritis (RA) and has helped researchers to analyze the pathogenesis of inflammatory joint disease. In classical CIA, Freund's complete adjuvant (FCA), which contains heat-killed Mycobacterium tuberculosis, is used as an adjuvant. In our previous study, we reported that particles of beta-glucan, OX-CA, derived from Candida albicans, acted as a proper adjuvant in the CIA model. In this study, to establish pure beta-glucan as an adjuvant for CIA, we tested a commercially available preparation of Zymosan A (ZYM) and modified its products. beta-Glucan fractions of ZYM were prepared by oxidation with various concentrations of NaClO. The oxidized ZYM (OX-ZYM) was mainly composed of beta-glucan. In this study, we examined its effect as an adjuvant for CIA. DBA/1 mice injected with CII and OX-CA developed arthritis 7-10 days after receiving booster injections; the OX-ZYM fractions induced arthritis with the same time course. 0.01% OX-ZYM (oxidized with a 0.01% NaClO solution) caused arthritis faster than 0.1% OX-ZYM or 0.5% OX-ZYM. In conclusion, beta-glucan derived from ZYM by brief oxidation with NaClO is a suitable adjuvant for a CIA model with anti-CII antibody production.     

Production of xylitol from raw wood hydrolysates by Debaryomyces hansenii NRRL Y-7426

Xylose solutions were obtained from the hemicellulosic fraction of Eucalyptus globulus by means of sulphuric acid treatments performed under selected operational conditions. The hydrolysates were neutralized, supplemented with nutrients, sterilized and utilised as fermentation media for xylitol production using the yeast Debaryomyces hansenii NRRL Y-7426. Preliminary experiments allowed the adaptation of the strain to the culture media. Further fermentation trials were performed with adapted cells according to an incomplete, factorial design of experiments. Empirical models describing the bioconversion were derived from the experimental results. The effects of three operational variables (nutrient concentration, initial pH and shaking speed) on both xylose consumption and xylitol production rates were described by significant mathematical equations. Additional aspects in relation to the process were also discussed. The authors are grateful to Xunta de Galicia and “Orember” for their financial support to this work; as well as to Ms. Rocío Rodríguez Fontán for her excellent technical assistance.