Forgot your password?
New user?
New Window Opens on the Secret Life of Microbes: Scientists Develop First Microbial Profiles of Ecosystems
CAS announces the release of SciFinder Scholar for Mac OS X
Press Releases
Press Releases
| Check out our New Publishers' Select for Free Articles |
Biochemical Engineering Journal (v.54, #1)
Equilibrium adsorption of poly(His)-tagged proteins on immobilized metal affinity chromatographic adsorbents by Yi-Hsing Yang; Tusng-Ta Wu; Shing-Yi Suen; Sung-Chyr Lin (pp. 1-9).
► Selective adsorption of recombinant proteins with the adsorbent is demonstrated. ► Adsorption of proteins is best described by the Langmuir–Freundlich model. ► Adsorption of denatured proteins can be described by the Langmuir model. ► Adsorption is mediated predominantly by affinity-like interations. ► Affinity of proteins is proportional to the number of accessible poly(His) tags.A systematic analysis on the adsorption of homo-oligomeric, poly(His)-tagged recombinant proteins on a hydroxyapatite-based immobilized metal affinity chromatography adsorbent was performed. Under non-denaturing conditions, the Langmuir–Freundlich isotherm model was found ideal for predicting the adsorption behavior of the model proteins. The dissociation constants, as low as 10−9M, decreased with the number of poly(His) tags, suggesting the presence of multi-point attachment. The maximum adsorption capacities, ranging from 79.1nmol/g for the 88kDa epimerase to 42.8nmol/g for the 320kDa racemase, were inversely proportional to the contact surface areas of the proteins. Under denaturing conditions, the Langmuir isotherm model fitted well with the experimental data. The maximum adsorption capacities for and the dissociation constants of the three model proteins were essentially identical, as the subunits of the model proteins were of similar dimensions and behaved similarly in the absence of complex tertiary or quaternary structure. Phosphate buffer at a concentration of 500mM, pH 8.0 was found to be effective for the elution of the model proteins with a recovery yield more than twofold higher than that obtained with 500mM imidazole. The results suggest the hydroxyapatite-based adsorbent is a promising alternative for large scale applications.
Keywords: Hydroxyapatite; Adsorption isotherm; Immobilized metal affinity chromatography; Langmuir; Langmuir–Freundlich
Electricity generation in continuous flow microbial fuel cells (MFCs) with manganese dioxide (MnO2) cathodes by Xiang Li; Boxun Hu; Steven Suib; Yu Lei; Baikun Li (pp. 10-15).
► Manganese dioxide featured with cryptomelane-type octohedral molecular sieve (OMS-2) structure was used as microbial fuel cells (MFCs) cathode catalysts. ► The novel cost-effective OMS-2 catalysts were examined in continuous flow MFCs.A novel cost-effective cathode catalyst, manganese dioxide (MnO2) featured with a cryptomelane-type octahedral molecular sieve (OMS-2) structure, was examined in continuous flow microbial fuel cells (MFCs). Power generation and organic substrate removal efficiency of two metal ions doped OMS-2 cathodes (cobalt (Co)-OMS-2 and copper (Cu)-OMS-2) were compared with platinum (Pt) cathodes under different hydraulic retention times (HRTs) and chemical oxygen demands (CODs). The 600-h continuous flow tests showed that Cu-OMS-2 MFCs and Co-OMS-2 MFCs achieved the stable power generation of 200±8mV and 190±5mV, and were 50–60mV higher than that of Pt MFCs. The COD removal efficiencies of Cu-OMS-2 MFCs and Co-OMS-2 MFCs were 83–87%, which were 15–19% higher than that of Pt MFCs. The power generation and COD removal efficiency increased with longer HRTs. The Cu-OMS-2 exhibited the highest power density (201mW/m2) at the COD of 1000mg/L. However, Co-OMS-2 cathodes had the better performance than Cu-OMs-2 at high COD concentrations of 2000–4000mg/L, with the power density of 897mW/m2 and COD removal efficiency of 46%. The continuous flow MFC tests demonstrated that the fast reaction rate of OMS-2 cathodes enhanced power generation and COD removal efficiency, and had a great potential to be applied in real-world wastewater treatment processes.
Keywords: Microbial fuel cells; Continuous flow; Manganese dioxides; Octahedral molecular sieves; Operation condition effect
Monitoring lentiviral vector production kinetics using online permittivity measurements by Sven Ansorge; Stéphane Lanthier; Julia Transfiguracion; Olivier Henry; Amine Kamen (pp. 16-25).
► In situ real-time permittivity measurements were assessed to document the production of lentiviral vectors (LVs). ► Characteristic process phases associated with LV production kinetics were identified. ► Cellular membrane properties and cell size caused the permittivity variations. ► The release of LVs in the culture supernatant can be monitored in real time by permittivity measurements.Lentiviral vectors (LVs) are effective delivery vehicles that have been successfully used in gene and cell therapy. LVs are most commonly produced via the transient transfection of several plasmid constructs in adherent cell cultures. Recently, we described an efficient and scalable LV production in serum-free suspension cultures. To further facilitate the translation of LV-based interventions to the clinic, the robustness of the production process needs to be ensured to ultimately achieve a specified quality and quantity of LV production lots. However, routine processes are largely empirical, and strategies to monitor LV production kinetics in real-time have not yet been described.In this work, in situ real-time permittivity measurements were assessed to document the production of LVs. Characteristic process phases that were closely associated with LV production kinetics were identified. The permittivity signal evolution was interpreted by exploiting various independent online and offline monitoring measurements. Cellular membrane properties and, to a lesser extent, cell size were the main factors contributing to the permittivity variations. It is concluded that the permittivity-related parameters can be used for the detection of viral release, allowing real-time assessment of process performance. The technology should thus greatly facilitate process development and optimization.
Keywords: Lentiviral vector production; HEK293; Perfusion culture; Online monitoring; Permittivity measurements; Dielectric spectroscopy; Process Analytical Technology (PAT)
Production and characterization of polyhydroxyalkanoates by recombinant Methylobacterium extorquens: Combining desirable thermal properties with functionality by Philipp Höfer; Patrick Vermette; Denis Groleau (pp. 26-33).
► Recombinant Methylobacterium extorquens produces modifiable and processable polyhydroxyalkanoates. ► The biopolyester properties suggest applications as medical biocomposite materials. ► Methanol (substrate) monitoring offers novel process control strategies. ► 5-Hexenoic acid (co-substrate) impacts strongly on the metabolism of M. extorquens.Recombinant Methylobacterium extorquens ATCC 55366 harboring phaC2 from Pseudomonas fluorescens GK13 ( M. ex-phaC2) was capable of producing blends of polyhydroxyalkanoates. By co-feeding methanol and 5-hexenoic acid (C6), functionalized PHAs (4≤chain length≤6) containing C–C double bonds were created. Bioreactor studies revealed negative impacts of 5-hexenoic acid on biomass and PHA production by reducing overall yields. In contrast, there was a positive relationship between 5-hexenoic acid supply and abundance of polymeric functional groups, i.e., molar portions of monomeric units bearing double bonds increased with increased 5-hexenoic acid supply. Correlation of C6 consumption to methanol addition resulted in a model that allowed for on-line estimation of toxic co-substrate concentration. The functionalized PHAs were ductile and showed signs of side chain crosslinking, resulting in reduced degrees of crystallinity. Incorporation of 3-hydroxyhex-5-enoate and 3-hydroxyhexanoate into the polymeric chains produced desirable thermal properties with enhanced thermal stability and reduced melting temperatures (139–168°C). Thermal degradation and melting temperatures obtained suggest a comfortable range for melt-processing of these polymers and allow for autoclaving as convenient sterilization process. Consequently, functionalized PHAs produced in this study are candidates for medical applications as part of biocomposite materials. The use of methanol as main substrate for cultivation of recombinant M. extorquens offers the possibility to reduce production costs and develop new process control strategies.
Keywords: Abbreviations; 3HA; 3-hydroxyalkanoate; 3HB; 3-hydroxybutyrate; 3HHx; 3-hydroxyhexanoate; 3HHx; 3-hydroxyhex-5-enonate; 3HO; 3-hydroxyoctanoate; 3HO; 3-hydroxyoct-7-enoate; 3HV; 3-hydroxyvalerate; C6; 5-hexenoic acid; C8; 7-octenoic acid; CCP; cumulative CO; 2; production; CMA; cumulative methanol addition; LCL; long-chain-length; MCL; medium-chain-length; PHB; poly(3-hydroxybutyrate); PHBV; poly(3-hydroxybutyrate-; co; -3-hydroxyvalerate); PHBHx; poly(3-hydroxybutyrate-; co; -3-hydroxyhexanoate); PHOU; poly(3-hydroxyoctanoate-; co; -3-hydroxyundec-10-enoate); SCL; short-chain-length; T; d; thermal decomposition temperature; T; g; glass transition temperature; T; m; melting temperature; V; w; working volume; X; c; degree of crystallinityFunctionalized polyhydroxyalkanoates; Methylobacterium extorquens; Methanol; Alkenoic acid; Automatic substrate feeding; Thermo-mechanical properties
Effect of gamma irradiation on activities and physicochemical characteristics of sewage sludge by Libing Chu; Jianlong Wang; Bo Wang (pp. 34-39).
► Gamma irradiation could disintegrate sludge flocs and release proteins, polysaccharides and extracellular enzymes into the bulk solution. ► The maximum oxygen uptake rate decreased by 58%, and 99% of the culturable bacteria were inactivated at a dose of 25kGy. ► The tested protease, superoxide dismutase and catalase showed slight inactivation during irradiation treatment. ► Following gamma irradiation, nitrate in the supernatant decreased rapidly to nearly zero. Ammonium was the major inorganic nitrogen product.In this paper, the response mechanism of sewage sludge exposed to60Co gamma irradiation at a dose of 0–25kGy was investigated. Results showed that gamma irradiation could disintegrate sludge flocs and release proteins, polysaccharides and extracellular enzymes into the bulk solution. The maximum oxygen uptake rate decreased by 58%, and 99% of the culturable bacteria were inactivated at 25kGy. However, the tested protease, superoxide dismutase and catalase showed slight inactivation during irradiation treatment. The efficiency of sludge solubilization reached around 6.5% with a dose of 25kGy. Nitrate in the supernatant decreased rapidly to nearly zero after gamma irradiation which may have been due to the conversion of nitrate into N2 or N2O gases through reductive reactions by hydrated electrons. Ammonium was the main inorganic nitrogen product.
Keywords: Gamma irradiation; Sludge activity; Sludge reduction; Sludge solubilization; Sewage sludge; Wastewater treatment
Kinetic study of enantioselective hydrolysis of ( R, S)-ketoprofen ethyl ester using immobilized T. laibacchii lipase by Yuan-Yuan Zhang; Jun-Hong Liu (pp. 40-46).
► An inherent kinetic model based on irreversible ping-pong bi–bi mechanism was developed. ► Immobilized T. laibacchii lipase was used to catalyzed enantioselective hydrolysis of ester. ► The kinetic experiments were carried out in a stirred tank reactor at various temperatures. ► The simulated values of the model fitted the experimental values well. ► High enantiomeric ratio indicates that this biotransformation is a promising example.Immobilized Trichosporon laibacchii lipase was used to catalyzed enantioselective hydrolysis of ( S)-ketoprofen from ( R, S)-ketoprofen ethyl ester in an aqueous system containing Tween-80 as the emulsifier. An inherent kinetic model based on irreversible ping-pong bi–bi mechanism with competitive inhibition of both enantiomer products was developed. The diffusion limitations and enzyme deactivation were proved to be negligible. The solution of ordinary differential equations “ode45” was successfully combined with Matlab optimization function “fmincon” to estimate kinetic parameters. The kinetic experiments were carried out in a 100mL stirred tank reactor at various temperatures (30, 35, 40 and 45°C) at 200rpm. The simulated values of the model fitted the experimental values well and the kinetic constant has a good fitness with Arrhenius equation. Thus, the kinetic model proposed here is feasible and can be used for mechanism interpretation of the hydrolytic resolution of racemic ketoprofen ester and the reactor scale-up design. High enantiomeric ratio ( E of 87) indicates that this biotransformation is a promising example for the industrial production of ( S)-ketoprofen.
Keywords: Immobilized enzymes; Lipase; Trichosporon laibacchii; Kinetics; Ketoprofen ethyl ester; Enantioselective hydrolysis
Optimization of production of an oxidant and detergent-stable alkaline β-keratinase from Brevibacillus sp. strain AS-S10-II: Application of enzyme in laundry detergent formulations and in leather industry by Sudhir K. Rai; Ashis K. Mukherjee (pp. 47-56).
► This is first report on statistical optimization of β-keratinase enzyme production and purification from Brevibacillus sp. AS-S10-II strain. ► An alkaline β-keratinase (Brevicarnase) with molecular mass of 83.2kDa displayed optimum activity at 45°C and pH 12.5. ► Brevicarnase demonstrated application oriented properties such as excellent thermostability, compatibility with commercial laundry detergents at a concentration of 0.1% (v/v) and dehairing activity suggesting its inclusion in commercial laundry detergent formulations and in leather- industry as eco-friendly dehairing agent.Present study is the first report on production and purification of β-keratinase enzyme from a bacterium belongs to the genus Brevibacillus. The response surface optimized alkaline β-keratinase production by this strain was achieved as 923.0×103Ul−1 post 48h of incubation. An alkaline β-keratinase (Brevicarnase) having molecular mass of 83.2kDa purified from this strain showed optimum activity at 45°C and pH 12.5, respectively. The Km and Vmax values of β-keratinase towards keratin were determined as 0.3mgml−1 and 4.5μmolmin−1mg−1, respectively. The Brevicarnase demonstrated appreciable thermo-stability and stability in the presence of anionic and non-ionic surfactants, oxidizing and bleaching agents, EDTA, and compatibility with the tested commercial laundry detergents at a concentration of 0.1%. The purified β-keratinase did not show collagen-degrading activity however, demonstrated dehairing property when tested on goat skin. These properties reinforce the feasibility of inclusion of Brevicarnase in laundry detergent formulations and in leather-industry.
Keywords: Brevibacillus; sp.; Detergent-stable β-keratinase; Submerged fermentation; Protease enzyme; Microbial; Shake-flask
Sensing capability of molecularly imprinted self-assembled monolayer by Min Jae Shin; Won Hi Hong (pp. 57-61).
Display Omitted► Fabrication of a molecularly imprinted self-assembled monolayer (SAM). ► Cholesterol, cholic acid, and deoxycholic acid were used as the template molecules. ► These imprinted monolayers were capable of discriminating the substrate. ► The stability of the imprinted monolayers was estimated.A molecularly imprinted self-assembled monolayer (SAM) was fabricated on a gold plate by forming a monolayer with both 1-hexadecanethiol and the template molecule, and removing the template molecules by solvent extraction. Cholesterol, cholic acid, and deoxycholic acid were used as the template molecules. Cyclic voltammograms were obtained using these imprinted gold plates as a working electrode, with Ag/AgCl reference electrode and Pt counter electrode. Potassium ferricyanide was used as a background material for oxidation and reduction. These imprinted monolayers were capable of discriminating the substrate that had been imprinted. The stability of the imprinted monolayers was estimated precisely and the thickness change of the monolayer was estimated using quartz crystal microbalance (QCM). During repeated detection, 1-hexadecanethiol molecules on the gold plate were tightly adhered to the gold surface. However, the sensing ability was reduced with repeated detection, suggesting that these phenomena were due to the movement of hexadecanethiol molecules on the gold plate.
Keywords: Imprinting; Self-assembled monolayer; Cholesterol; Cyclic voltammogram; Quartz crystal microbalance; Sensor
Production of structured triacylglycerols rich in palmitic acid at sn-2 position and oleic acid at sn-1,3 positions as human milk fat substitutes by enzymatic acidolysis by Luis Esteban; María J. Jiménez; Estrella Hita; Pedro A. González; Lorena Martín; Alfonso Robles (pp. 62-69).
► Structured triacylglycerols (STAGs) of structure oleic acid-palmitic acid-oleic acid (OPO) were obtained by a process with two lipase-catalyzed reactions. ► In presence and absence of solvent STAGs were obtained with 67% OA at sn-1,3 positions and 57-68% PA at sn-2 position. ► These STAGs were purified by two procedures differing in whether or not solvent was used and STAGs of approximately 99% purity and recovery yields of over 96% were obtained. ► The STAGs synthesized may be useful for preparing human milk fat substitutes (HMFS) because present PA contents at sn-2 position and OA at sn-1,3 positions higher than human milk TAGs.This paper studies the synthesis of structured triacylglycerols (STAGs) of type oleic acid–palmitic acid–oleic acid (OA–PA–OA or OPO) by acidolysis of PA enriched triacylglycerols (TAGs) (79.2% PA, 74.5% PA at sn-2 position) and several OA-rich free fatty acid (FFA) fractions. The PA located at sn-1,3 positions was replaced by OA, maintaining the PA at sn-2 position; for this purpose several sn-1,3 specific lipases (lipase DF from Rhizopus oryzae, Palatase 20000L from Mucor miehei, Lipozyme RM IM from Rhizomucor miehei, Lipozyme TL IM from Thermomyces lanuginosus and lipase QLC from Alcaligenes sp.) were tested in experiments carried out in presence of solvent at 37°C. Lipase DF immobilized on Accurel MP1000 was selected because this lipase gave a high incorporation of OA at the extreme positions of TAGs (50.4%) in short reaction times (1h) or IOT (intensity of treatment, lipase amount×reaction time/TAG amount) of only 0.4glipase×h/g TAG, maintaining a high content of PA at sn-2 position (68.6%). Using this lipase, the influence of FFA/TAG molar ratio and purity of OA-rich FFAs were studied. With a 6:1 FFA/TAG molar ratio and 90% OA it was possible to obtain STAGs with 67.2% OA at sn-1,3 positions and 67.8% PA at sn-2 position (57.2% of total PA was located at sn-2 position). Next, the synthesis of OPO STAGs was studied in solvent-free media at 50°C. The best results were also attained with a 6:1 FFA/TAG molar ratio, 90% OA and using an IOT of 1.9glipase×h/g TAG. In these conditions it was possible to obtain STAGs with identical composition to the previously obtained with solvent. These STAGs were purified by two procedures which differed in that solvent was or was not used to separate STAGs and FFAs. Both with and without hexane, STAGs were obtained with approximately 99% purity and over 96% recovery yields.
Keywords: OPO; Structured triacylglycerols; Palmitic acid; Oleic acid; Acidolysis; sn; -1,3 specific lipases; Lipase DF from; Rhizopus oryzae; TAG purification