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Biochemical Engineering Journal (v.53, #2)
Mathematical modeling of biomass and α-amylase production kinetics by Bacillus sp. in solid-state fermentation based on solid dry weight variation by M. Hashemi; S.M. Mousavi; S.H. Razavi; S.A. Shojaosadati (pp. 159-164).
Modeling the different phases of the bacterial growth curve and the production of α-amylase by Bacillus sp. KR-8104 in a solid-state fermentation process based on variation in dry weight was the aim of this study. The result showed that the growth of bacterial cells and the production of α-amylase on a wheat bran substrate could be expressed by simple models incorporating the mathematical definition of each phase and the variation in dry substrate weight over the incubation time. Experimental data collected from a series of batch fermentations were used to validate the models proposed. In both cases, the model simulation matched well with the experimental observations, which made it possible to conclude that these models can be successfully employed for the development of growth and α-amylase production in solid-state fermentation processes.
Keywords: Modeling; Biomass; α-Amylase production; Solid-state fermentation; Dry weight
Mathematical modeling of biomass and α-amylase production kinetics by Bacillus sp. in solid-state fermentation based on solid dry weight variation by M. Hashemi; S.M. Mousavi; S.H. Razavi; S.A. Shojaosadati (pp. 159-164).
Modeling the different phases of the bacterial growth curve and the production of α-amylase by Bacillus sp. KR-8104 in a solid-state fermentation process based on variation in dry weight was the aim of this study. The result showed that the growth of bacterial cells and the production of α-amylase on a wheat bran substrate could be expressed by simple models incorporating the mathematical definition of each phase and the variation in dry substrate weight over the incubation time. Experimental data collected from a series of batch fermentations were used to validate the models proposed. In both cases, the model simulation matched well with the experimental observations, which made it possible to conclude that these models can be successfully employed for the development of growth and α-amylase production in solid-state fermentation processes.
Keywords: Modeling; Biomass; α-Amylase production; Solid-state fermentation; Dry weight
Partition of lectin from Canavalia grandiflora Benth in aqueous two-phase systems using factorial design by Camila S. Porto; Tatiana S. Porto; Kyria S. Nascimento; Edson H. Teixeira; Benildo S. Cavada; José L. Lima-Filho; Ana L.F. Porto (pp. 165-171).
▶ The ATPS proved to be efficient as a first step in lectin purification from ConGF seeds.This work explores the possibility of using a polyethylene glycol (PEG)/sodium citrate aqueous two-phase system (ATPS) as a first step in a process for the purification of lectin from Canavalia grandiflora seed. Purification of lectins is a limiting step for its uses in the area of biotechnology. Extraction by an aqueous two-phase system is a powerful technique for separation, concentration, and purification of biomolecules and pharmaceutical products. Four factors (PEG's molar mass, PEG's concentration, pH, citrate concentration) affecting the lectin ConGF partitioning were studied. A two-level factorial design (24) was carried out. Lectin ConGF preferentially partitioned to the top (polyethylene glycol) phase. The statistical analysis showed that the citrate concentration chloride significantly affects the KL (partitioning coefficient for protein) value for lectin partitioning. An ATPS composed of 20% (w/w) PEG 400 and 20% (w/w) citrate, at pH 6, allowed the recovery of lectin ConGF with an 8.67 partition coefficient and 104% yield. Consequently, the system proved to be efficient and can be used as a first step in lectin purification from crude extract of ConGF seeds.
Keywords: ATPS; Lectin; Canavalia grandiflora; Partition; Factorial design; PEG/citrate
Partition of lectin from Canavalia grandiflora Benth in aqueous two-phase systems using factorial design by Camila S. Porto; Tatiana S. Porto; Kyria S. Nascimento; Edson H. Teixeira; Benildo S. Cavada; José L. Lima-Filho; Ana L.F. Porto (pp. 165-171).
▶ The ATPS proved to be efficient as a first step in lectin purification from ConGF seeds.This work explores the possibility of using a polyethylene glycol (PEG)/sodium citrate aqueous two-phase system (ATPS) as a first step in a process for the purification of lectin from Canavalia grandiflora seed. Purification of lectins is a limiting step for its uses in the area of biotechnology. Extraction by an aqueous two-phase system is a powerful technique for separation, concentration, and purification of biomolecules and pharmaceutical products. Four factors (PEG's molar mass, PEG's concentration, pH, citrate concentration) affecting the lectin ConGF partitioning were studied. A two-level factorial design (24) was carried out. Lectin ConGF preferentially partitioned to the top (polyethylene glycol) phase. The statistical analysis showed that the citrate concentration chloride significantly affects the KL (partitioning coefficient for protein) value for lectin partitioning. An ATPS composed of 20% (w/w) PEG 400 and 20% (w/w) citrate, at pH 6, allowed the recovery of lectin ConGF with an 8.67 partition coefficient and 104% yield. Consequently, the system proved to be efficient and can be used as a first step in lectin purification from crude extract of ConGF seeds.
Keywords: ATPS; Lectin; Canavalia grandiflora; Partition; Factorial design; PEG/citrate
Behavior of HepG2/C3A cell cultures in a microfluidic bioreactor by Régis Baudoin; Laurent Griscom; Jean Matthieu Prot; Cécile Legallais; Eric Leclerc (pp. 172-181).
▶ New models avoiding labor intensive methods are under development for drug screening, pharmacological discovery and toxicology assessment. ▶ Microfluidic bioreactors have the advantage of including a whole new set of technologies. ▶ However, there is lack of parametrical studies and systematic analysis of the cell behavior in such new environment. ▶ The research highlight of our study is to give a parametric investigation of the inoculated cell density, the perfusion flow rate on the hepG2/C3a behavior cultivated in a microfluidic bioreactors. ▶ We also compared the parameters with a first type of well-known chemical used in the toxicity benchmark. ▶ The HepG2/C3a cells are also one of the references in the toxicity analysis concerning liver cells. ▶ The main interest of the paper is thus to provide a view of the sensitivity of the cell culture in bioreactor when compared to a flat Petri culture control.An important issue in toxicity studies is the development of pertinent new in vitro tests that will be able to provide an alternative to in vivo testing methods. Current developments in the fields of tissue engineering and microtechnology make it possible to propose the use of microfluidic bioreactors as a tool for enhanced in vitro investigations. However, both the cells’ behavior in complex environments and their response to chemicals need to be better understood, especially for future validation of any new assay. To characterize the sensitivity of this approach, we investigated the behavior of a liver cell model with respect to variations of two cell culture parameters in a microfluidic bioreactor: inoculated cell density (0.35×106, 0.45×106 and 0.65×106 cells/bioreactor) and microfluidic flow rates (0, 10 and 25μL/min). We also investigated an environmental pollutant modeled with three ammonia concentrations (0, 5 and 10mM). Proliferation in the bioreactor was found to be flow rate and inoculated cell density dependent. This led to a mean value of 1.2±0.2×106 cells in the 3D microenvironment of the bioreactor without ammonia loadings after 96h of cultures. Cell metabolism rates, such as glucose and glutamine consumption or CYP1A detoxification, were found to be higher in dynamic conditions than in static conditions. Furthermore, increased ammonium chloride concentration in turn increased glucose and glutamine consumptions and CYP1A activity. Inhibition of 50% of cell proliferation (IC50) during the ammonium chloride analysis was found at 5mM when cell concentrations of 0.35×106 cells/bioreactor were inoculated. In contrast, no effect could be detected at 5mM for larger cell densities of 0.65×106 cells/bioreactor, demonstrating concentration and cell density dependence in the bioreactors. This study highlighted the sensitivity of the HepG2/C3A cells to microfluidic culture conditions and illustrated the potential for larger in vitro toxicity studies using microfluidic bioreactors.
Keywords: PDMS microfluidic bioreactor; HepG2/C3A; Ammonia toxicity
Behavior of HepG2/C3A cell cultures in a microfluidic bioreactor by Régis Baudoin; Laurent Griscom; Jean Matthieu Prot; Cécile Legallais; Eric Leclerc (pp. 172-181).
▶ New models avoiding labor intensive methods are under development for drug screening, pharmacological discovery and toxicology assessment. ▶ Microfluidic bioreactors have the advantage of including a whole new set of technologies. ▶ However, there is lack of parametrical studies and systematic analysis of the cell behavior in such new environment. ▶ The research highlight of our study is to give a parametric investigation of the inoculated cell density, the perfusion flow rate on the hepG2/C3a behavior cultivated in a microfluidic bioreactors. ▶ We also compared the parameters with a first type of well-known chemical used in the toxicity benchmark. ▶ The HepG2/C3a cells are also one of the references in the toxicity analysis concerning liver cells. ▶ The main interest of the paper is thus to provide a view of the sensitivity of the cell culture in bioreactor when compared to a flat Petri culture control.An important issue in toxicity studies is the development of pertinent new in vitro tests that will be able to provide an alternative to in vivo testing methods. Current developments in the fields of tissue engineering and microtechnology make it possible to propose the use of microfluidic bioreactors as a tool for enhanced in vitro investigations. However, both the cells’ behavior in complex environments and their response to chemicals need to be better understood, especially for future validation of any new assay. To characterize the sensitivity of this approach, we investigated the behavior of a liver cell model with respect to variations of two cell culture parameters in a microfluidic bioreactor: inoculated cell density (0.35×106, 0.45×106 and 0.65×106 cells/bioreactor) and microfluidic flow rates (0, 10 and 25μL/min). We also investigated an environmental pollutant modeled with three ammonia concentrations (0, 5 and 10mM). Proliferation in the bioreactor was found to be flow rate and inoculated cell density dependent. This led to a mean value of 1.2±0.2×106 cells in the 3D microenvironment of the bioreactor without ammonia loadings after 96h of cultures. Cell metabolism rates, such as glucose and glutamine consumption or CYP1A detoxification, were found to be higher in dynamic conditions than in static conditions. Furthermore, increased ammonium chloride concentration in turn increased glucose and glutamine consumptions and CYP1A activity. Inhibition of 50% of cell proliferation (IC50) during the ammonium chloride analysis was found at 5mM when cell concentrations of 0.35×106 cells/bioreactor were inoculated. In contrast, no effect could be detected at 5mM for larger cell densities of 0.65×106 cells/bioreactor, demonstrating concentration and cell density dependence in the bioreactors. This study highlighted the sensitivity of the HepG2/C3A cells to microfluidic culture conditions and illustrated the potential for larger in vitro toxicity studies using microfluidic bioreactors.
Keywords: PDMS microfluidic bioreactor; HepG2/C3A; Ammonia toxicity
Kinetic study of 7- O-ethyl ganoderic acid O stability and its importance in the preparative isolation by Jia-Le Wang; Ying-Bo Li; Han-Lin Qin; Jian-Jiang Zhong (pp. 182-186).
Ganoderic acid (GA) is a kind of important bioactive triterpenoid with anti-cancer effect. 7- O-Ethyl ganoderic acid O (7- O-ethyl GA-O) was recently found to be a promising target for mass production of bioactive individual GA due to its high content. However, its apparent degradation was observed during preparation procedures. In this research, to understand and control its degradation process during the preparative isolation, a systematic kinetic investigation on 7- O-ethyl GA-O stability was done not only under different conditions of pH and temperature, but in different solvents as well. 7- O-Ethyl GA-O was proved to possess an optimal stability in neutral and non-polar environment. Its degradation kinetics and thermodynamics could be effectively described by first-order reaction model and Arrhenius equation. The activation energy was estimated to be 38.2±3.6kJ/mol in acidic methanol (0.01mM HCl, pH=5.1), 103.2±2.4kJ/mol in aqueous methanol (20% water), and 105.8±3.3kJ/mol in methanol. Furthermore, the possible mechanism of acid-catalyzed 7- O-ethyl GA-O decomposition in methanol was proposed, consisting of a fast protonation, followed by a committed step of ethanol removal.
Keywords: Ganoderma lucidum; 7-; O; -Ethlyl ganoderic acid O; Stability; Degradation; Kinetics; Preparative isolation
Kinetic study of 7- O-ethyl ganoderic acid O stability and its importance in the preparative isolation by Jia-Le Wang; Ying-Bo Li; Han-Lin Qin; Jian-Jiang Zhong (pp. 182-186).
Ganoderic acid (GA) is a kind of important bioactive triterpenoid with anti-cancer effect. 7- O-Ethyl ganoderic acid O (7- O-ethyl GA-O) was recently found to be a promising target for mass production of bioactive individual GA due to its high content. However, its apparent degradation was observed during preparation procedures. In this research, to understand and control its degradation process during the preparative isolation, a systematic kinetic investigation on 7- O-ethyl GA-O stability was done not only under different conditions of pH and temperature, but in different solvents as well. 7- O-Ethyl GA-O was proved to possess an optimal stability in neutral and non-polar environment. Its degradation kinetics and thermodynamics could be effectively described by first-order reaction model and Arrhenius equation. The activation energy was estimated to be 38.2±3.6kJ/mol in acidic methanol (0.01mM HCl, pH=5.1), 103.2±2.4kJ/mol in aqueous methanol (20% water), and 105.8±3.3kJ/mol in methanol. Furthermore, the possible mechanism of acid-catalyzed 7- O-ethyl GA-O decomposition in methanol was proposed, consisting of a fast protonation, followed by a committed step of ethanol removal.
Keywords: Ganoderma lucidum; 7-; O; -Ethlyl ganoderic acid O; Stability; Degradation; Kinetics; Preparative isolation
Improvement of medium composition for heterotrophic cultivation of green microalgae, Tetraselmis suecica, using response surface methodology by Mojtaba Azma; Mohd Shamzi Mohamed; Rosfarizan Mohamad; Raha Abdul Rahim; Arbakariya B. Ariff (pp. 187-195).
▶ Medium for heterotrophic growth of Tetraselmis suecica cells was optimized using CCD design. ▶ Medium: glucose, 5.8g/L; peptone, 9g/L; yeast extract, 4.5g/L; meat extract, 3g/L. ▶ T. suecica cells, yield and productivity: 28.8g/L, 9.1g cell/g glucose and 0.2g/L/h. ▶ Cells in heterotrophic (28.8g/L) thrice higher than photoautotrophic (8.4g/L).Optimization of medium composition for the improvement of heterotrophic cultivation of green microalgae, Tetraselmis suecica, was performed using response surface methodology (RSM). Heterotrophic cultivation of T. suecica was conducted in total darkness using Walne medium formulated with natural sea water. Initially, the effect of two types of carbon source (glucose and sodium acetate) and various types of nitrogen source (peptone, yeast extract, meat extract, malt extract, urea, sodium nitrate and ammonium nitrate) on growth of T. suecica was studied. The concentration of medium component that was found to significantly influence the heterotrophic growth of T. suecica (glucose, peptone, yeast extract and meat extract) was further optimized using RSM. The medium that consists of 5.78g/L glucose, 9g/L peptone, 4.48g/L yeast extract and 3.01g/L meat extract was found optimal for heterotrophic cultivation of T. suecica. The final cell concentration (28.88g/L) obtained in heterotrophic cultivation using this optimized medium was about 3 and 2 times higher than obtained in photoautotrophic culture (8.40g/L) and non-optimized medium for heterotrophic cultivation (13.81g/L), respectively. In addition, the cell yield based on glucose consumed (9.31g cell/g glucose) was increased by about 3 times as compared to non-optimized medium (3.61g cell/g glucose).
Keywords: Green microalgae; Tetraselmis suecica; Heterotrophic algae culture; Medium optimization; Response surface methodology
Improvement of medium composition for heterotrophic cultivation of green microalgae, Tetraselmis suecica, using response surface methodology by Mojtaba Azma; Mohd Shamzi Mohamed; Rosfarizan Mohamad; Raha Abdul Rahim; Arbakariya B. Ariff (pp. 187-195).
▶ Medium for heterotrophic growth of Tetraselmis suecica cells was optimized using CCD design. ▶ Medium: glucose, 5.8g/L; peptone, 9g/L; yeast extract, 4.5g/L; meat extract, 3g/L. ▶ T. suecica cells, yield and productivity: 28.8g/L, 9.1g cell/g glucose and 0.2g/L/h. ▶ Cells in heterotrophic (28.8g/L) thrice higher than photoautotrophic (8.4g/L).Optimization of medium composition for the improvement of heterotrophic cultivation of green microalgae, Tetraselmis suecica, was performed using response surface methodology (RSM). Heterotrophic cultivation of T. suecica was conducted in total darkness using Walne medium formulated with natural sea water. Initially, the effect of two types of carbon source (glucose and sodium acetate) and various types of nitrogen source (peptone, yeast extract, meat extract, malt extract, urea, sodium nitrate and ammonium nitrate) on growth of T. suecica was studied. The concentration of medium component that was found to significantly influence the heterotrophic growth of T. suecica (glucose, peptone, yeast extract and meat extract) was further optimized using RSM. The medium that consists of 5.78g/L glucose, 9g/L peptone, 4.48g/L yeast extract and 3.01g/L meat extract was found optimal for heterotrophic cultivation of T. suecica. The final cell concentration (28.88g/L) obtained in heterotrophic cultivation using this optimized medium was about 3 and 2 times higher than obtained in photoautotrophic culture (8.40g/L) and non-optimized medium for heterotrophic cultivation (13.81g/L), respectively. In addition, the cell yield based on glucose consumed (9.31g cell/g glucose) was increased by about 3 times as compared to non-optimized medium (3.61g cell/g glucose).
Keywords: Green microalgae; Tetraselmis suecica; Heterotrophic algae culture; Medium optimization; Response surface methodology
Simultaneous syntheses of polyhydroxyalkanoates and rhamnolipids by Pseudomonas aeruginosa IFO3924 at various temperatures and from various fatty acids by Katsutoshi Hori; Rieko Ichinohe; Hajime Unno; Sidik Marsudi (pp. 196-202).
▶ The optimum temperature for PHA synthesis is 30°C. ▶ The optimum temperature for rhamnolipid (RL) synthesis is 28°C. ▶ Codd-3-hydroxyalkanoates (3HAs) can be incorporated into RLs. ▶ PHAs and RLs produced from Ceven-fatty acids contain only Ceven-3HAs. ▶ PHAs and RLs produced from Codd-fatty acids contain Codd- and Ceven-3HAs.Medium-chain-length polyhydroxyalkanoates (mcl-PHAs) and rhamnolipids produced by Pseudomonas aeruginosa share 3-hydroxyalkanoates (3HAs) as common structural units. Previously, we demonstrated their simultaneous production to share the cultivation cost for the intracellular products, PHAs, with the extracellular products, rhamnolipids. Here, we investigated the effects of temperature and carbon length of fatty acid substrates on the simultaneous production by P. aeruginosa IFO3924. The optimum temperatures for PHA and rhamnolipid syntheses were 30°C and 28°C, respectively, suggesting that the product ratio between these two products can be controlled by changing temperature. After the induction periods, both PHAs and rhamnolipids were produced at constant rates by resting cells. The apparent activation energies were determined as 45kJ/mol for PHA synthesis and 40kJ/mol for rhamnolipid synthesis from Arrhenius plots. These values in a similar level suggest that a common reaction such as β-oxidation before the synthesis of 3HA units is the rate-determining step. PHAs and rhamnolipids synthesized from fatty acids of even-number carbon length (Ceven) consisted of only Ceven-3HAs. By contrast, PHAs and rhamnolipids synthesized from fatty acids of odd-number carbon length (Codd) consisted of not only Codd-3HAs but also Ceven-3HAs. This is the first report to show the incorporation of Codd-3HAs into rhamnolipids.
Keywords: Key words; Polyhydroxyalkanoate (PHA); Rhamnolipid; Pseudomonas aeruginosa; Fatty acid; β-Oxidation; Simultaneous syntheses
Simultaneous syntheses of polyhydroxyalkanoates and rhamnolipids by Pseudomonas aeruginosa IFO3924 at various temperatures and from various fatty acids by Katsutoshi Hori; Rieko Ichinohe; Hajime Unno; Sidik Marsudi (pp. 196-202).
▶ The optimum temperature for PHA synthesis is 30°C. ▶ The optimum temperature for rhamnolipid (RL) synthesis is 28°C. ▶ Codd-3-hydroxyalkanoates (3HAs) can be incorporated into RLs. ▶ PHAs and RLs produced from Ceven-fatty acids contain only Ceven-3HAs. ▶ PHAs and RLs produced from Codd-fatty acids contain Codd- and Ceven-3HAs.Medium-chain-length polyhydroxyalkanoates (mcl-PHAs) and rhamnolipids produced by Pseudomonas aeruginosa share 3-hydroxyalkanoates (3HAs) as common structural units. Previously, we demonstrated their simultaneous production to share the cultivation cost for the intracellular products, PHAs, with the extracellular products, rhamnolipids. Here, we investigated the effects of temperature and carbon length of fatty acid substrates on the simultaneous production by P. aeruginosa IFO3924. The optimum temperatures for PHA and rhamnolipid syntheses were 30°C and 28°C, respectively, suggesting that the product ratio between these two products can be controlled by changing temperature. After the induction periods, both PHAs and rhamnolipids were produced at constant rates by resting cells. The apparent activation energies were determined as 45kJ/mol for PHA synthesis and 40kJ/mol for rhamnolipid synthesis from Arrhenius plots. These values in a similar level suggest that a common reaction such as β-oxidation before the synthesis of 3HA units is the rate-determining step. PHAs and rhamnolipids synthesized from fatty acids of even-number carbon length (Ceven) consisted of only Ceven-3HAs. By contrast, PHAs and rhamnolipids synthesized from fatty acids of odd-number carbon length (Codd) consisted of not only Codd-3HAs but also Ceven-3HAs. This is the first report to show the incorporation of Codd-3HAs into rhamnolipids.
Keywords: Key words; Polyhydroxyalkanoate (PHA); Rhamnolipid; Pseudomonas aeruginosa; Fatty acid; β-Oxidation; Simultaneous syntheses
Production and properties of the highly efficient raw starch digesting α-amylase from a Bacillus licheniformis ATCC 9945a by Nataša Božić; Jordi Ruiz; Josep López-Santín; Zoran Vujčić (pp. 203-209).
▶ A highly efficient, thermostable, raw starch digesting α-amylase was produced. ▶ α-Amylase was purified and its enzymological and molecular properties were studied. ▶ Enzyme has a high hydrolytic affinity towards cereal, tuber and root raw starches. ▶ Very low enzyme doses hydrolyze raw starches below gelatinization temperatures.Highly efficient raw starch digesting α-amylase was produced after 24h of batch fermentation of Bacillus licheniformis ATCC 9945a in laboratory bioreactor at 37°C. The enzyme was purified by gel filtration chromatographies with 6-fold increase of specific activity and 38% recovery and showed a molecular mass of 31kDa by SDS-PAGE. The purified enzyme had an optimum pH of 6.5 and optimum temperature of 90°C. The purified α-amylase in the presence of CaCl2 retained 55% of its activity after 6h of incubation at 70°C. Co2+, Ni2+ and Ca2+ slightly stimulated, while Hg2+ completely inhibited α-amylase activity. Hydrolysis rates of raw triticale, wheat, potato, horseradish and corn starches, at 1% concentration were 63, 60, 59, 52 and 37%, respectively, in a period of 4h. The properties of the purified enzyme proved its high efficacy for digesting diverse raw starches below gelatinization temperature and, hence, its potential commercial value to use as an industrial enzyme.
Keywords: α-Amylase; Bacillus licheniformis; Raw starch hydrolysis; Thermostable; Purification
Production and properties of the highly efficient raw starch digesting α-amylase from a Bacillus licheniformis ATCC 9945a by Nataša Božić; Jordi Ruiz; Josep López-Santín; Zoran Vujčić (pp. 203-209).
▶ A highly efficient, thermostable, raw starch digesting α-amylase was produced. ▶ α-Amylase was purified and its enzymological and molecular properties were studied. ▶ Enzyme has a high hydrolytic affinity towards cereal, tuber and root raw starches. ▶ Very low enzyme doses hydrolyze raw starches below gelatinization temperatures.Highly efficient raw starch digesting α-amylase was produced after 24h of batch fermentation of Bacillus licheniformis ATCC 9945a in laboratory bioreactor at 37°C. The enzyme was purified by gel filtration chromatographies with 6-fold increase of specific activity and 38% recovery and showed a molecular mass of 31kDa by SDS-PAGE. The purified enzyme had an optimum pH of 6.5 and optimum temperature of 90°C. The purified α-amylase in the presence of CaCl2 retained 55% of its activity after 6h of incubation at 70°C. Co2+, Ni2+ and Ca2+ slightly stimulated, while Hg2+ completely inhibited α-amylase activity. Hydrolysis rates of raw triticale, wheat, potato, horseradish and corn starches, at 1% concentration were 63, 60, 59, 52 and 37%, respectively, in a period of 4h. The properties of the purified enzyme proved its high efficacy for digesting diverse raw starches below gelatinization temperature and, hence, its potential commercial value to use as an industrial enzyme.
Keywords: α-Amylase; Bacillus licheniformis; Raw starch hydrolysis; Thermostable; Purification
Ion-selective electrodes for determination of fluoxetine in capsules and in biological fluids by E.M. Hussien; F.M. Abdel-Gawad; Y.M. Issa (pp. 210-215).
Fluoxetine (FX) ion selective liquid membrane and coated wire graphite electrodes have been constructed from poly(vinyl chloride) containing FX-tetraphenylborate (FX-TPB) as the sensing element in the presence of DOP as the plasticizing solvent mediator. The two electrodes show nearly Nernestian response over the concentration range 2×10−5–1×10−2mol/L of the drug with slopes of 58.5 and 55.5mV/decade for the liquid membrane and the coated wire graphite electrodes, respectively. The electrodes exhibit good selectivity for the FX with respect to a large number of inorganic cations and organic substances of biological fluids. The method is precise as shown by the mean recoveries range of 100.50–101.04% and 99.69–100.17% with mean relative standard deviations 0.96–1.10% and 0.82–1.03% for liquid membrane and coated wire graphite electrodes, respectively. Fluoxetine is determined successfully in pure solutions and in capsules or in biological fluids using the standard additions and potentiometric titrations methods.
Keywords: Fluoxetine; Ion-selective electrodes; Potentiometric determination; Capsules; Biological fluids
Ion-selective electrodes for determination of fluoxetine in capsules and in biological fluids by E.M. Hussien; F.M. Abdel-Gawad; Y.M. Issa (pp. 210-215).
Fluoxetine (FX) ion selective liquid membrane and coated wire graphite electrodes have been constructed from poly(vinyl chloride) containing FX-tetraphenylborate (FX-TPB) as the sensing element in the presence of DOP as the plasticizing solvent mediator. The two electrodes show nearly Nernestian response over the concentration range 2×10−5–1×10−2mol/L of the drug with slopes of 58.5 and 55.5mV/decade for the liquid membrane and the coated wire graphite electrodes, respectively. The electrodes exhibit good selectivity for the FX with respect to a large number of inorganic cations and organic substances of biological fluids. The method is precise as shown by the mean recoveries range of 100.50–101.04% and 99.69–100.17% with mean relative standard deviations 0.96–1.10% and 0.82–1.03% for liquid membrane and coated wire graphite electrodes, respectively. Fluoxetine is determined successfully in pure solutions and in capsules or in biological fluids using the standard additions and potentiometric titrations methods.
Keywords: Fluoxetine; Ion-selective electrodes; Potentiometric determination; Capsules; Biological fluids
Self-interaction chromatography of proteins on a microfluidic monolith by Cristina Martin; Abraham M. Lenhoff (pp. 216-222).
▶ The osmotic second virial coefficient B22 is a measure of protein interactions. ▶ B22 can be measured efficiently by self-interaction chromatography (SIC). ▶ Microfluidic SIC measurements in 100μm diameter monoliths are demonstrated. ▶ This miniaturized system makes B22 measurement possible with minimal protein consumption.A novel miniaturized system has been developed for measuring protein–protein interactions in solution with high efficiency and speed, and minimal use of protein. A chromatographic monolith synthesized in a capillary is used in the method to make interaction measurements by self-interaction chromatography (SIC) in a manner that, compared to column methods, is more efficient as well as more readily practicable even if only small amounts of protein are available. The microfluidic monolith requires much less protein for both column preparation and the chromatographic measurements than a conventional SIC system, and in addition offers improved mass transfer and hence higher chromatographic efficiency than for previous SIC miniaturization systems. Protein self-interactions for catalase as a model protein, quantified by measurement of second virial coefficients, B22, were determined by SIC and follow trends that are consistent with previously reported values. Different column derivatization conditions were studied in order to optimize the chromatographic behavior of the microfluidic system for SIC measurements. Chromatographic sensitivity can be further increased by using different column synthesis conditions.
Keywords: Protein–protein interactions; Protein solution thermodynamics; Osmotic second virial coefficient; Catalase; Chromatographic monolith
Self-interaction chromatography of proteins on a microfluidic monolith by Cristina Martin; Abraham M. Lenhoff (pp. 216-222).
▶ The osmotic second virial coefficient B22 is a measure of protein interactions. ▶ B22 can be measured efficiently by self-interaction chromatography (SIC). ▶ Microfluidic SIC measurements in 100μm diameter monoliths are demonstrated. ▶ This miniaturized system makes B22 measurement possible with minimal protein consumption.A novel miniaturized system has been developed for measuring protein–protein interactions in solution with high efficiency and speed, and minimal use of protein. A chromatographic monolith synthesized in a capillary is used in the method to make interaction measurements by self-interaction chromatography (SIC) in a manner that, compared to column methods, is more efficient as well as more readily practicable even if only small amounts of protein are available. The microfluidic monolith requires much less protein for both column preparation and the chromatographic measurements than a conventional SIC system, and in addition offers improved mass transfer and hence higher chromatographic efficiency than for previous SIC miniaturization systems. Protein self-interactions for catalase as a model protein, quantified by measurement of second virial coefficients, B22, were determined by SIC and follow trends that are consistent with previously reported values. Different column derivatization conditions were studied in order to optimize the chromatographic behavior of the microfluidic system for SIC measurements. Chromatographic sensitivity can be further increased by using different column synthesis conditions.
Keywords: Protein–protein interactions; Protein solution thermodynamics; Osmotic second virial coefficient; Catalase; Chromatographic monolith
Sandwich-type conductometric immunoassay of alpha-fetoprotein in human serum using carbon nanoparticles as labels by Juan Tang; Jianxin Huang; Biling Su; Huafeng Chen; Dianping Tang (pp. 223-228).
▶ We design a conductrometric immunoassay of alpha-fetoprotein in human serum. ▶ Carbon nanoparticles as labels. ▶ Analysis of real samples and intralaboratory validation. ▶ We compare the analytical performance of the immunosensor with commercially available Electrochemiluminescent.A simple and sensitive conductometric immunosensor for detection of alpha-fetoprotein (AFP) was designed using carbon nanoparticles as labels. The immunosensing probe was fabricated by means of the immobilization of monoclonal anti-AFP primary antibodies on an interdigitated conductometric transducer, while the detection antibodies were prepared using nanocarbon-conjugated horseradish peroxidase-labeled anti-AFP (CNP-HRP- anti-AFP). With a sandwich-type immunoassay format, the conjugated CNP-HRP- anti-AFP on the transducer was increased with the increase of AFP in the sample, and the conductivity of the immunosensor was decreased in the H2O2–KI system. Under optimal conditions, the immunosensor exhibited a wide dynamic range of 0.1–500ng/mL with a detection limit of 50pg/mL AFP at 3 σ. The reproducibility and recovery were <10% and 83.9–112.3%, respectively. Interestingly, 45 clinical serum specimens were assayed using the conductometric immunosensor, and the results were in accordance with those obtained from our Clinical Laboratory using Roche 2010 Electrochemiluminescent Automatic Analyzer.
Keywords: Alpha-fetoprotein; Carbon nanoparticles; Conductivity; Immunosensor; Gold colloids; Sandwich-type immunoassay
Sandwich-type conductometric immunoassay of alpha-fetoprotein in human serum using carbon nanoparticles as labels by Juan Tang; Jianxin Huang; Biling Su; Huafeng Chen; Dianping Tang (pp. 223-228).
▶ We design a conductrometric immunoassay of alpha-fetoprotein in human serum. ▶ Carbon nanoparticles as labels. ▶ Analysis of real samples and intralaboratory validation. ▶ We compare the analytical performance of the immunosensor with commercially available Electrochemiluminescent.A simple and sensitive conductometric immunosensor for detection of alpha-fetoprotein (AFP) was designed using carbon nanoparticles as labels. The immunosensing probe was fabricated by means of the immobilization of monoclonal anti-AFP primary antibodies on an interdigitated conductometric transducer, while the detection antibodies were prepared using nanocarbon-conjugated horseradish peroxidase-labeled anti-AFP (CNP-HRP- anti-AFP). With a sandwich-type immunoassay format, the conjugated CNP-HRP- anti-AFP on the transducer was increased with the increase of AFP in the sample, and the conductivity of the immunosensor was decreased in the H2O2–KI system. Under optimal conditions, the immunosensor exhibited a wide dynamic range of 0.1–500ng/mL with a detection limit of 50pg/mL AFP at 3 σ. The reproducibility and recovery were <10% and 83.9–112.3%, respectively. Interestingly, 45 clinical serum specimens were assayed using the conductometric immunosensor, and the results were in accordance with those obtained from our Clinical Laboratory using Roche 2010 Electrochemiluminescent Automatic Analyzer.
Keywords: Alpha-fetoprotein; Carbon nanoparticles; Conductivity; Immunosensor; Gold colloids; Sandwich-type immunoassay
New strategy to enhance catalytic performance of Escherichia coli whole cell biocatalysts harboring P450cam mutants by Tsuyoshi Mouri; Noriho Kamiya; Masahiro Goto (pp. 229-233).
Cytochrome P450cam mutants (Y96F and F87W-Y96F) can catalyze the oxidation of dichlorobenzene and other aromatic compound that wild type P450cam cannot catalyze. The expression of the single mutant Y96F in an Escherichia coli host resulted in indigo formation (approximately 23mg/l), of which productivity was comparable to the highest reported so far using other P450cam mutant cultures. On the other hand, recombinant E. coli that harbored F87W-Y96F exhibited little activity for indigo production. However, co-expression of E. coli glycerol dehydrogenase (GLD) with F87W-Y96F triggered a marked increase in the productivity of indigo (approximately 20mg/l) without external glycerol addition. These results indicate the potential of introducing GLD-mediated NADH regeneration that utilizes endogenous NAD+ and glycerol in E. coli to enhance markedly the catalytic performance of a low-activity recombinant P450cam system. The present E. coli whole-cell biocatalysts might be applicable to practical microbial indigo production.
Keywords: Cytochrome P450; Cofactor regeneration; Glycerol dehydrogenase; Whole cell biocatalyst; Indigo
New strategy to enhance catalytic performance of Escherichia coli whole cell biocatalysts harboring P450cam mutants by Tsuyoshi Mouri; Noriho Kamiya; Masahiro Goto (pp. 229-233).
Cytochrome P450cam mutants (Y96F and F87W-Y96F) can catalyze the oxidation of dichlorobenzene and other aromatic compound that wild type P450cam cannot catalyze. The expression of the single mutant Y96F in an Escherichia coli host resulted in indigo formation (approximately 23mg/l), of which productivity was comparable to the highest reported so far using other P450cam mutant cultures. On the other hand, recombinant E. coli that harbored F87W-Y96F exhibited little activity for indigo production. However, co-expression of E. coli glycerol dehydrogenase (GLD) with F87W-Y96F triggered a marked increase in the productivity of indigo (approximately 20mg/l) without external glycerol addition. These results indicate the potential of introducing GLD-mediated NADH regeneration that utilizes endogenous NAD+ and glycerol in E. coli to enhance markedly the catalytic performance of a low-activity recombinant P450cam system. The present E. coli whole-cell biocatalysts might be applicable to practical microbial indigo production.
Keywords: Cytochrome P450; Cofactor regeneration; Glycerol dehydrogenase; Whole cell biocatalyst; Indigo
Evaluation of enzyme kinetic parameters using explicit analytic approximations to the solution of the Michaelis–Menten equation by Marko Goličnik (pp. 234-238).
The time-course of product accumulation during an enzyme-catalyzed reaction that conforms to the Michaelis–Menten rate equation is expressed by an explicit closed-form equation in terms of the Lambert W( x) function. Unfortunately, the use of direct solution of the Michaelis–Menten equation is limited, because the W( x) function is not widely available in curve-fitting software. The present commentary suggests an alternative to surmount this difficulty. The Lambert W( x) function can be approximated in terms of the elementary mathematical functions that enable the fitting of particular equations on time-course data of the Michaelis–Menten enzyme reaction by any nonlinear regression computer program. Three different demonstrated approximations of the W( x) with relatively high accuracies are shown here to be appropriate for use when progress curves are analyzed by the direct solution of the integrated Michaelis–Menten equation. The elementary and precise nature of these approximations makes them the most user-friendly open candidates for simple, but operative, kinetic parameter estimations from experimental time-course data.
Keywords: Michaelis–Menten equation; Enzyme-kinetics analysis; Progress curve; Nonlinear regression; Lambert; W; function
Evaluation of enzyme kinetic parameters using explicit analytic approximations to the solution of the Michaelis–Menten equation by Marko Goličnik (pp. 234-238).
The time-course of product accumulation during an enzyme-catalyzed reaction that conforms to the Michaelis–Menten rate equation is expressed by an explicit closed-form equation in terms of the Lambert W( x) function. Unfortunately, the use of direct solution of the Michaelis–Menten equation is limited, because the W( x) function is not widely available in curve-fitting software. The present commentary suggests an alternative to surmount this difficulty. The Lambert W( x) function can be approximated in terms of the elementary mathematical functions that enable the fitting of particular equations on time-course data of the Michaelis–Menten enzyme reaction by any nonlinear regression computer program. Three different demonstrated approximations of the W( x) with relatively high accuracies are shown here to be appropriate for use when progress curves are analyzed by the direct solution of the integrated Michaelis–Menten equation. The elementary and precise nature of these approximations makes them the most user-friendly open candidates for simple, but operative, kinetic parameter estimations from experimental time-course data.
Keywords: Michaelis–Menten equation; Enzyme-kinetics analysis; Progress curve; Nonlinear regression; Lambert; W; function
Comparison of β-1,3-glucanase production by Botryosphaeria rhodina MAMB-05 and Trichoderma harzianum Rifai and its optimization using a statistical mixture-design by Ellen C. Giese; Robert F.H. Dekker; Ieda S. Scarminio; Aneli M. Barbosa; Roberto da Silva (pp. 239-243).
Botryosphaeria rhodina MAMB-05 produced β-1,3-glucanases and botryosphaeran when grown on glucose, while Trichoderma harzianum Rifai only produced the enzyme. A comparison of long-term cultivation (300h) by B. rhodina demonstrated a correlation between the formation of botryosphaeran (48h) and its consumption (after 108h), and de-repression of β-1,3-glucanase synthesis when glucose was depleted from the nutrient medium, whereas for T. harzianum enzyme production commenced during exponential growth. Growth profiles and levels of β-1,3-glucanases produced by both fungi on botryosphaeran also differed, as well as the production of β-1,3-glucanases and β-1,6-glucanases on glucose, lactose, laminarin, botryosphaeran, lasiodiplodan, curdlan, Brewer's yeast powder and lyophilized fungal mycelium, which were dependent upon the carbon source used. A statistical mixture-design used to optimize β-1,3-glucanase production by both fungi evaluated botryosphaeran, glucose and lactose concentrations as variables. For B. rhodina, glucose and lactose promoted enzyme production at the same levels (2.30UmL−1), whereas botryosphaeran added to these substrates exerted a synergic effect favorable for β-glucanase production by T. harzianum (4.25UmL−1).
Keywords: β-1,3-Glucanases; Botryosphaeran; Statistical mixture-design optimization
Comparison of β-1,3-glucanase production by Botryosphaeria rhodina MAMB-05 and Trichoderma harzianum Rifai and its optimization using a statistical mixture-design by Ellen C. Giese; Robert F.H. Dekker; Ieda S. Scarminio; Aneli M. Barbosa; Roberto da Silva (pp. 239-243).
Botryosphaeria rhodina MAMB-05 produced β-1,3-glucanases and botryosphaeran when grown on glucose, while Trichoderma harzianum Rifai only produced the enzyme. A comparison of long-term cultivation (300h) by B. rhodina demonstrated a correlation between the formation of botryosphaeran (48h) and its consumption (after 108h), and de-repression of β-1,3-glucanase synthesis when glucose was depleted from the nutrient medium, whereas for T. harzianum enzyme production commenced during exponential growth. Growth profiles and levels of β-1,3-glucanases produced by both fungi on botryosphaeran also differed, as well as the production of β-1,3-glucanases and β-1,6-glucanases on glucose, lactose, laminarin, botryosphaeran, lasiodiplodan, curdlan, Brewer's yeast powder and lyophilized fungal mycelium, which were dependent upon the carbon source used. A statistical mixture-design used to optimize β-1,3-glucanase production by both fungi evaluated botryosphaeran, glucose and lactose concentrations as variables. For B. rhodina, glucose and lactose promoted enzyme production at the same levels (2.30UmL−1), whereas botryosphaeran added to these substrates exerted a synergic effect favorable for β-glucanase production by T. harzianum (4.25UmL−1).
Keywords: β-1,3-Glucanases; Botryosphaeran; Statistical mixture-design optimization