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Biochemical Engineering Journal (v.38, #1)
Statistical optimization of immunoaffinity purification of hepatitis B surface antigen using response surface methodology by Jorge Sánchez-Romeu; José Manuel País-Chanfrau; Yanelys Pestana-Vila; Ivette López-Larraburo; Yamile Masso-Rodríguez; Marbelis Linares-Domínguez; Gabriel Márquez-Perera (pp. 1-8).
Response surface methodology (RSM) was successfully applied to find an elution condition enhancing the performance of immunoaffinity (IAF) purification of hepatitis B surface antigen (HBsAg), conducted in batch chromatography. The immunosorbent efficiency measured as HBsAg eluted per milliliter of immunosorbent was selected as the response. Two numerical independent variables were studied: concentration of potassium thiocyanate (KSCN) and ethanol, and a categorical variable formed by two levels of sodium deoxycholate under defined ionic strength (DCS/NaCl). A three-level factorial design of RSM was developed and gets a reduced cubic model which accurately fits the data ( R2≈0.97). RSM predicted an optimal region where the response could allow maximum values. At the optimal condition 1.7M KSCN, 30% (v/v) ethanol, and 0% (w/v) DCS and 1M NaCl a response of 582±11μg/ml (559±52 by the model), wherein the measured response does not differs statistically with the maximum experimental value. At optimized condition, a recovery of HBsAg of ∼94% of the adsorbed antigen was obtained, representing a ∼69% more HBsAg than the previously reported. Due to less accumulation of HBsAg this optimum could increase the immunosorbent life in a multiple cycle's operation.
Keywords: Abbreviations; ANOVA; analysis of variance; API; active pharmaceutical ingredient; CI; confidence interval; CDR; complementary determining region; CV; coefficient of variations; DCS; sodium deoxycholate; ELISA; enzyme-linked immunosorbent assay; EtOH; ethanol; HBsAg; hepatitis B surface antigen; mAb; monoclonal anti-body; RC; reduced cubic; RSM; response surface methodology; SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresisAffinity; Chromatography; Hepatitis B surface antigen; Monoclonal antibodies; Optimization; Response surfaces methodology
Electrochemical immunoassay for α-1-fetoprotein based on CdS nanoparticles and Thionine bilayer films modified glass carbon electrode by Xiangmin Miao; Ruo Yuan; Yaqin Chai; Yintao Shi; Yanru Yuan (pp. 9-15).
A novel and effective electrochemical immunosensor for the rapid determination of α-1-fetoprotein (AFP), based on electrostatic interaction of CdS nanoparticles (nano-CdS) and thionine (Thi) on poly-congo red (PCR) modified glass carbon electrode (GCE), alternately was developed. The formation of the {nano-CdS/Thi+}2 bilayer films showed a good electrochemical activity, and a couple of stable and well-defined Thi redox peaks were obtained in pH 7.0 phosphate buffer solution (PBS). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were adopted to confirm the successful stepwise layer-by-layer (LBL) assembly procedure of the immunosensor. Horseradish peroxidase (HRP) was employed to block sites against non-specific binding instead of bovine serum albumin (BSA) and amplify the response currents. Tests showed that the reduction current of the immunosensor was proportional to AFP concentration in the range of 0.30–250.00ng/mL with a detection limit of 0.12ng/mL. Moreover, the prepared immunosensor exhibited high sensitivity and good stability, would be valuable for clinical immunoassay of AFP.
Keywords: Immunosensor; CdS nanoparticles; α-1-fetoprotein; Thionine; Congo red; Horseradish peroxidase
Synthesis of xylitoyl fatty acid monoesters by immobilized lipase in subcritical acetone by Norihisa Iwamoto; Motohiro Shima; Shuji Adachi (pp. 16-21).
The thermostability of immobilized lipase from Candida antarctica in subcritical acetone, which was in the liquid state under pressurized conditions, was examined in the temperature range from 60 to 90°C. The inactivation process of the enzyme did not obey first-order kinetics and the enzyme activity leveled off after a prolonged incubation. The level depended on the temperature, and the remaining activity at 80°C was 15.4% after 1.5-h incubation. A reactor system consisting of columns packed with xylitol and the immobilized lipase, which were connected in series, was constructed and operated at 80°C to continuously produce the xylitoyl fatty acid esters. Xylitoyl laurate, myristate and palmitate were synthesized at the constant conversion of ca. 70%, and the productivities of 512, 519 and 551g/day/L-reactor, respectively, were realized.
Keywords: Condensation; Lipase; Surfactant; Subcritical acetone; Xylitol
Mathematical modeling of glycerol fermentation by Klebsiella pneumoniae: Concerning enzyme-catalytic reductive pathway and transport of glycerol and 1,3-propanediol across cell membrane by Ya Qin Sun; Wen Tao Qi; Hu Teng; Zhi Long Xiu; An Ping Zeng (pp. 22-32).
Glycerol can be converted to 1,3-propanediol by Klebsiella pneumoniae under anaerobic conditions. The main metabolic pathways of glycerol fermentation include oxidative and reductive pathway. In this study, a novel mathematical model is set up to describe the continuous and batch fermentations of glycerol by K. pneumoniae, in which the enzyme-catalytic kinetics on the reductive pathway, the transport of glycerol and diffusion of 1,3-propanediol across cell membrane, and the inhibition of 3-hydroxypropionaldehyde (3-HPA) to glycerol dehydratase (GDHt) and 1,3-PD oxydoreductase (PDOR) are all taken into consideration. Comparisons between simulated and experimental results indicate that the model can be used to describe the continuous fermentation under steady states reasonably. Moreover, the intracellular concentrations of glycerol, 1,3-propanediol, and 3-HPA could also be predicted for continuous and batch cultivations. 3-HPA can be dramatically accumulated during the fermentation process to an uppermost value of 275.41mmolL−1 under the continuous culture of dilute rate of 0.1h−1 when extracellular residual glycerol concentration is changed from 0.07 to 1495.09mmolL−1. Furthermore, the inhibition of 3-HPA to PDOR is stronger than that to GDHt. This work is helpful for deeply understanding metabolic and genetic regulation of dha regulon of glycerol metabolism.
Keywords: Glycerol; 1,3-Propanediol; Klebsiella pneumoniae; Mathematical model; Enzyme-catalytic kinetics; Transport across cell membrane
Isolation, purification and characterization of superoxide dismutase from garlic by Ning He; Qingbiao Li; Daohua Sun; Xueping Ling (pp. 33-38).
A simple and cost-effective way to purify superoxide dismutase (SOD) from garlic was proposed. The stepwise isolation and purification procedure consists of phosphate buffer and chloroform–ethanol extraction, acetone precipitation followed by ion exchange chromatography on DEAE column. The specific enzyme activity was 4124U/mg protein with the recovery rate of 53.3% and concentration factor of 192, respectively. SDS-PAGE analysis showed high consistency of garlic SOD with cattle blood SOD. The enzyme activity of garlic SOD was greatly inhibited by cyanide and hydrogen peroxide while it could be well maintained at pH values ranging from 4 to 11. Only slight decrease of enzyme activity was identified at temperatures lower than 50°C or by treatment with ultrasonic wave for 30min. The ultraviolet absorbance of garlic SOD was found to be 269nm.
Keywords: Superoxide dismutase; Garlic; Purification; Chromatography
Recovery and separation of surfactin from pretreated fermentation broths by physical and chemical extraction by Huei-Li Chen; Ruey-Shin Juang (pp. 39-46).
The recovery of surfactin from fermentation broths with the culture of Bacillus subtilis ATCC 21332 by physical and chemical extraction was studied, in which the broths were pretreated by acid precipitation and, if necessary, the precipitate was further dissolved in NaOH solution. The physical solid–liquid and liquid–liquid extractions were performed with different organic solvents (ethyl acetate, n-hexane) and at different times of extraction run. It was shown that better extraction was obtained with ethyl acetate than n-hexane. The extraction could be improved by increasing the times of extraction run under a given volume of the organic solution. For chemical liquid–liquid extraction of surfactin with Aliquat 336 (5–200mM) in n-hexane, the efficiency was improved. The amounts of inorganic salt in the strip solution were also optimized. Surfactin recovered after physical or chemical extraction was finally characterized by mass and NMR spectroscopies. The results of chemical extraction showed that surfactin would readily bind with the quaternary ammonium cations of Aliquat 336.
Keywords: Surfactin; Recovery; Fermentation broths; Extraction; Ethyl acetate; n; -Hexane; Aliquat 336
Chromium binding capacity of Lyngbya putealis exopolysaccharides by Bala Kiran; Anubha Kaushik (pp. 47-54).
This paper presents the chromium adsorptive potential of polysaccharide produced by a novel cyanobacterium Lyngbya putealis HH-15. Batch mode experiments were performed to determine the adsorption equilibrium and the equilibrium data was applied to different two-parameter models (Langmuir, Freundlich, Temkin, Dubinin–Radushkevich, Flory–Huggins, and Brunauer, Emmer & Teller (BET) model). The highest coefficient of determination (0.9925) for Langmuir and BET models indicates best fitness of these models in explaining the sorption as a multilayer process. Effect of different variables like initial metal ion concentration (10–100mg/L), pH (2–6) and temperature (25–45°C) on chromium adsorption of exopolysaccharides (EPS) were also examined, using Box–Behnken design model. Very high value of regression coefficient between the variables and the response ( R2=0.9982) indicates excellent evaluation of experimental data by second-order polynomial regression model. The response surface method indicated that 30–40mg/L initial chromium concentration, pH 2 and 45°C temperature were optimal for biosorption of chromium by the cyanobacterial exopolysaccharides. Adsorption capacity of EPS increased from 45 to 157mg/g of EPS as initial Cr(VI) concentration increased from 10 to 30mg/L. Surface adsorption of the metal at surface of EPS was confirmed through scanning electron microscopy.
Keywords: Exopolysaccharides; Chromium; Lyngbya putealis; Response surface methodology; Adsorption isotherms
Improvement of propagation frequency with two-step direct somatic embryogenesis from carrot hypocotyls by Miki Mizukami; Toshiya Takeda; Hideho Satonaka; Hiroshi Matsuoka (pp. 55-60).
Mass production of the regenerated plantlets from carrot hypocotyls via two-step direct somatic embryogenesis (DSE) was investigated. Somatic embryos were induced in the induction culture with medium containing 2,4-dichlorophenocxyacetic acid (2,4-D) prior to the development culture in this method. The optimum condition for the regeneration efficiency was found to be 2 weeks of induction culture using hypocotyls shorter than 3cm. The distribution profile of the regeneration efficiency corresponded to that of the normal distribution. The distribution range was observed to depend on the length of the hypocotyls. Calcium addition increased the regeneration efficiency but did not affect the distribution range. We also found that this procedure was applicable to the mass production of regenerated plants using a bioreactor.
Keywords: Direct somatic embryo; Carrot; Plant cell culture; Tissue cell culture; Plant cell bioreactor; Airlift bioreactor
Biosorption of direct azo dye from aqueous phase onto Spirogyra sp. I02: Evaluation of kinetics and mechanistic aspects by S. Venkata Mohan; S.V. Ramanaiah; P.N. Sarma (pp. 61-69).
Sorption of azo dye from aqueous phase was evaluated using non-viable algal Spirogyra I02 as biosorbent. Agitated non-flow batch sorption studies revealed the capacity of algal biosorbent to adsorb the azo dye. Influence of dye concentration, effect of pH, biosorbent dosage, temperature effect and sorption–desorption were studied to evaluate the algal-dye sorption mechanism. Sorption interaction of dye on to algal species obeyed the pseudo second-order rate equation. Experimental data showed good fit with the Langmuir's adsorption isotherm model. Dye sorption was found to be dependent on the aqueous phase pH and the uptake was observed to be higher at lower pH. Maximum dye sorption was observed at 30°C temperature. Sorption–desorption of dye into organic and/or inorganic solutions were observed and this indicated the combined effect of chemical and ion-exchange sorption phenomena.
Keywords: Biosorption; Algal; Spirogyra; Kinetics; pH; Intraparticle diffusion model; Pseudo first-order; Pseudo second-order; Biosorbent; Isotherm
Measurement of kinetic parameters in a submerged aerobic membrane bioreactor fed on acetate and operated without biomass discharge by M. Heran; C. Wisniewski; J. Orantes; A. Grasmick (pp. 70-77).
The objective of this work was to study the biological performance of a submerged membrane bioreactor working with complete sludge retention that is susceptible to induce high biomass concentration and consequently low F/M ratio. A synthetic organic influent was used and the biological behavior of the system was studied for three different volumetric organic loads. In spite of continuous increase in the VSS concentration, a steady state in terms of removal efficiency and respiratory activity was achieved. High organic matter removal efficiency and no decline of the membrane performance were observed. Cycles of biomass synthesis and biomass loss by predation, lysis and decay could be considered so that a reduction of the biomass production seemed to be possible while keeping high treatment efficiency. Most of the cells were assumed to stay in a physiological state in which cell division was not favored, and the degraded substrate was essentially used to ensure maintenance requirements and storage products synthesis.
Keywords: Wastewater treatment; Immersed membrane bioreactors; Biokinetics; Bioconversion yield; Oxygen uptake rate; Sludge production
Reaction kinetics of bacteria disinfection employing hydrogen peroxide by Marisol D. Labas; Cristina S. Zalazar; Rodolfo J. Brandi; Alberto E. Cassano (pp. 78-87).
The inactivation reaction of Escherichia coli bacteria employing hydrogen peroxide at 20°C and pH=7 was studied in a well-mixed batch reactor. The proposed objective, as far as the extent of inactivation is concerned, was obtained for H2O2 concentrations above 100ppm (1ppm=2.94×10−5mmolcm−3) but, compared with other disinfection technologies, for too long reaction times. Below 40ppm of the oxidant concentration inactivation was practically ineffective. Results were analyzed employing Modified forms of the Series-Event and Multitarget mechanistic models. At concentrations above 100ppm the induction time in the semi-logarithmic plot of bacteria concentration versus time was reduced. With both modified models it was found that the reaction order with respect to the hydrogen peroxide concentration was different than one. Both mathematical descriptions provide a good representation of the experimental results in an ample range of the disinfectant concentrations and confirm a methodology that renders the starting point of a reaction kinetic expression useful for further studies regarding the optimization of the operating conditions (pH and temperature, for example), including also combination with other advanced oxidation technologies. An interpretation of the data in terms of a Weibull-like model is also included.
Keywords: Water disinfection; Kinetic parameters; Hydrogen peroxide; Modelling; Waste-water treatment; Escherichia coli
Cost-effective xylanase production from free and immobilized Bacillus pumilus strain MK001 and its application in saccharification of Prosopis juliflora by Mukesh Kapoor; Lavanya M. Nair; Ramesh Chander Kuhad (pp. 88-97).
Cultural conditions for xylanase production by an alkalophilic Bacillus pumilus strain MK001 were optimized under submerged fermentation. An initial medium pH 9.0, agitation 200rpm, inoculum size 1.25% (v/v) and inoculum age 2h were found to be optimal for maximum enzyme production. The bacterium secretes high levels of xylanase on agricultural residues (wheat bran 1220.0IUml−1; wheat straw 900.0IUml−1) as well as on synthetic xylans (birch wood xylan 1190IUml−1; oat spelt xylan 1150.0IUml−1). Among different inorganic/organic nitrogen sources tested, a combination of yeast extract and peptone (0.66% N2 equivalent) produced maximum (1288.0IUml−1) xylanase titers. Among various amino acids, vitamins and surfactants,dl-β-phenylalanine, niacin, and polyethylene glycol (PEG)-3330 maximally enhanced xylanase production by 136.0% (2880.0IUml−1), 79.0% (2190.0IUml−1), and 107% (2536.0IUml−1) respectively. Batch fermentation in 5-l laboratory fermentor allowed reduction (8.0h) in incubation period for optimal (2886.0IUml−1) xylanase production. Whole-cell immobilization of B. pumilus strain MK001 on inert supports (polyurethane foam, PUF and scotch brite, SB) was found to be superior in maximizing xylanase production (up to 4000.0IUml−1) as compared to natural supports (cotton and silk) (up to 3854.0IUml−1). Saccharification of chemically pretreated Prosopis juliflora by enzymatic cocktail (xylanase, cellulase and cellobiase) resulted in release of reducing sugars (15.5gg−1; saccharification efficiency of 20.0%). Fermentation of saccharified hydrolysates by Pichia stiptis resulted in an ethanol yield of 0.36gg−1.
Keywords: Bacillus pumilus; Ethanol; Submerged fermentation; Wheat bran; Xylanase
Enhancement of methane fermentation in the presence of Ni2+ chelators by Hu Qing-Hao; Li Xiu-Fen; Liu He; Du Guo-Cheng; Chen Jian (pp. 98-104).
The enhancement of methane fermentation in the presence of Ni2+ chelators, citric acid (CA), nitrilotriacetic acid (NTA), and ethylene diamine tetraacetic acid (EDAT) respectively, was investigated in this study. The results showed that the addition of chelating agents promoted methane fermentation of acetate. At a sodium acetate concentration of 7g/L, temperature of 35°C, and Ni2+ concentration of 20μM, methane production was enhanced 34.1%, 49.2%, and 38.6%, by the addition of 10μM CA, NTA and EDTA, respectively. The formation of soluble complexes between chelating agents and Ni2+ favored the dissolution of Ni2+ from their sulfides, and the bioavailability of Ni2+ was dramatically increased. By the addition of 10μM NTA, the concentration of dissolved Ni2+ was greatly increased from 58 to 1020μg/L. Accompanying with that, the concentrations of F430 and coenzyme M of the biomass increased 37% and by four-fold, respectively.
Keywords: Anaerobic; Chelators; Bioavailability; Methane; Coenzyme; Nickel
Sugarcane bagasse hemicellulose hydrolysate for ethanol production by acid recovery process by Ke-Ke Cheng; Bai-Yan Cai; Jian-An Zhang; Hong-Zhi Ling; Yu-Jie Zhou; Jing-Ping Ge; Jing-Ming Xu (pp. 105-109).
In order to increase the reducing sugar concentration in the sugarcane bagasse hemicellulose acid hydrolysate and recover the acid, the acid hydrolysis was carried out in an acid recycle process and detoxification of hydrolysate was performed by electrodialysis. Two cycles of acidic treatments increased the reducing sugar concentration from 28 to 63.5gl−1 and sulphuric acid consumption decreased to 0.056gg−1 bagasse. After treatment by electrodialysis, 90% of acetic acid in hydrolysate was removed and the recovery ratio of sulphuric acid was 88%. The pretreated hydrolysates, supplemented with nutrient materials, were fermented to ethanol using Pachysolen tannophilus DW06. A batch culture with pretreated hydrolysate as substrate was developed giving 19gethanoll−1 with a yield of 0.34gg−1 sugar and productivity of 0.57gl−1h−1.
Keywords: Acid recovery; Ethanol; Fermentation; Hemicellulose; Hydrolysate; Sugarcane bagasse
Kinetic stabilities of soybean and horseradish peroxidases by J. K. Amisha Kamal; Digambar V. Behere (pp. 110-114).
Peroxidases have attractive biocatalytic properties and are used in biosensing and immunoassays. Among various peroxidases, isoenzyme C of horseradish peroxidase (HRP-C) is the most studied, and is also the most commercially used due to its high structural stability. Soybean peroxidase (SBP) and horseradish peroxidase share strikingly similar three-dimensional structures with ∼60% sequence homology. We reported previously, that the conformational and thermal stabilities of SBP are substantially higher than HRP-C. In the present study, we show that the kinetic stability of SBP is much higher than HRP-C as obtained by measuring their unfolding rates at various guanidine hydrochloride (GdnHCl) concentrations. In contrast, the heme-free forms of SBP and HRP-C showed similar kinetic stabilities. We conclude that the higher structural stability of SBP compared to HRP-C stems from the heme binding to the apo protein. Commercial interest of these results is twofold. A cheaper, abundant, better active, and more stable SBP could replace HRP-C. The stability and hence the biocatalytic property of a peroxidase can be improved by suitably engineering the heme active-site that enhances the heme-apo-protein interaction.
Keywords: Abbreviations; SBP; soybean peroxidase; HRP-C; horseradish peroxidase isoenzyme C; GdnHCl; Guanidine hydrochloride (enclosing square brackets indicate concentration); ΔG(H; 2; O); equilibrium free energy of unfolding in water; k; U; (H; 2; O); unfolding rate constant in water; t; 1/2; (H; 2; O); half-life of unfolding in water; ΔG*(H; 2; O); activation free energy of unfolding in waterSoybean peroxidase; Horseradish peroxidase; Homologous proteins; Unfolding rate; Kinetic stability; Heme binding