Maximum Product Concentration Research Articles

Investigation of the Molecular Origins of Protein-arginine Methyltransferase I (PRMT1) Product Specificity Reveals a Role for Two Conserved Methionine Residues

Protein-arginine methyltransferases aid in the regulation of many biological processes by methylating specific arginyl groups within targeted proteins. The varied nature of the response to methylation is due in part to the diverse product specificity displayed by the protein-arginine methyltransferases. In addition to site location within a protein, biological response is also determined by the degree (mono-/dimethylation) and type of arginine dimethylation (asymmetric/symmetric). Here, we have identified two strictly conserved methionine residues in the PRMT1 active site that are not only important for activity but also control substrate specificity. Mutation of Met-155 or Met-48 results in a loss in activity and a change in distribution of mono- and dimethylated products. The altered substrate specificity of M155A and M48L mutants is also evidenced by automethylation. Investigation into the mechanistic basis of altered substrate recognition led us to consider each methyl transfer step separately. Single turnover experiments reveal that the rate of transfer of the second methyl group is much slower than transfer of the first methyl group in M48L, especially for arginine residues located in the center of the peptide substrate where turnover of the monomethylated species is negligible. Thus, altered product specificity in M48L originates from the differential effect of the mutation on the two rates. Characterization of the two active-site methionines provides the first insight into how the PRMT1 active site is engineered to control product specificity.

Ethanol Production from Molasses with Immobilized Cells Technique in Packed Bed Bioreactor by Extractive

The aim of this research was to study the effect of total sugar concentration, Ca-Alginate and K Carrageenan density in immobilized cells on packed-bed and batch bioreactor performance for ethanol production. In addition, this research was also aimed to study the effect of solvent flow rate on packed column performance expressed as % extraction recovery. This experiment was carried out with total sugar concentration of 10, 14, 18 % (v/v) and immobilized cells Ca-Alginate and K-Carrageenan at a density of 2% (w/v) with amyl alcohol as solvent. Based on the result of this study, it is concluded that the total sugar concentration influenced the concentration, yield and productivity of ethanol. The result of this study for immobilized Ca-Alginate cells showed that the maximum ethanol product concentration, yield and productivity were 7.28% g/l, 57.48 g/l, 38.22%, 71.85 g/l.hr, respectively. Meanwhile, immobilized K-Carrageenan cells showed the maximum ethanol product concentration, yield and productivity as 7.22% /56.99 g/l, 26.73%, and 68.40 g/l.hr, respectively. In extraction process, increasing amyl alcohol solvent flow rate will increase mass transfer and hence increase ethanol recovery.

Enzymatic ketone reductions with co-factor recycling: Improved reactions with ionic liquid co-solvents

Ionic liquids have been applied to two pharmaceutically relevant ketone reductions mediated by isolated enzymes. Alcohol dehydrogenase isolated from Rhodococcus erythropolis (ADH RE) was found to mediate the asymmetric reductions of 4′-Br-2,2,2-trifluoroacetophenone to ( R)-4′-Br-2,2,2-trifluoroacetophenyl alcohol and 6-Br-β-tetralone to its corresponding alcohol ( S)-6-Br-β-tetralol. Both of these reactions employed a second enzyme, glucose dehydrogenase 103 (GDH 103) to recycle the co-factor NAD through the oxidation of glucose to gluconic acid. In the case of 4′-Br-2,2,2-trifluoroacetophenone the traditional organic co-solvent approaches were limited to a maximum product concentration of 10 g L −1 due to substrate deactivation of the biocatalyst. Employing 10% (v/v) [BMP][NTf2], a water immiscible ionic liquid, facilitated conversion of 50 g L −1 ketone to the chiral alcohol in less than 24 h. The initial rate of reaction was improved more than four times in the presence of 10% (v/v) ionic liquid compared to no co-solvent and the product could be readily isolated in 85% (w/w) overall yield with an ee of 99%. In the case of 6-Br-β-tetralone the reaction was found to proceed more favorably with both miscible and immiscible ionic liquid co-solvents compared to a number of organic solvents. Again, the product could be readily isolated in 88% (w/w) overall yield with an ee of >99%. For both bioconversions the stability of both the ADH RE and the GDH 103 co-enzyme was found to be enhanced by the presence of certain ionic liquids compared to both organic solvents and aqueous buffer. In the case of the immiscible ionic liquid [BMP][NTf2] present at a volume fraction of 10%, the measured enzyme half lives were 266 and >300 h, respectively. These promising results were obtained after screening only a limited range (11) of representative, commercially available ionic liquids. Critical factors in the screening of ionic liquids for bioconversion applications appear to be the solubility of the substrate, biocatalyst stability in the presence of the ionic liquid co-solvent and mass transfer rates from the ionic liquid into the aqueous phase.

Effect of Conditioned Medium Factors on Productivity and Cell Physiology in Trichoplusia ni Insect Cell Cultures

The influence of conditioned medium (CM) on cell physiology and recombinant protein production in Trichoplusia ni insect cells (T. ni, BTI-Tn-5B1-4) has been investigated. Cell cycle analysis showed that a high proportion of the cell population (80-90%) was in G1 during the whole culture, indicating that the S and G2/M phases are short relative to the G1 phase. Directly after inoculation, a rapid decrease of the S-phase population occurred, which could be observed as a lag-phase. The following increase in the number of cells in S occurred after 7 h of culture for cells in fresh medium, whereas for cells with the addition of CM it occurred at an earlier time point (5 h) and these cells had therefore a shorter lag-phase. The initial changes in the S-phase population were also affected by the inoculum cell density, as higher seeding cell densities resulted in a more rapid increase in the S-phase population after inoculation. These changes in cell cycle distribution were reflected in the cell size, and the CM-cells were smaller than the cells in fresh medium. Recombinant protein production in T. ni cells was improved by the addition of CM. The specific productivity was increased by 30% compared to cells in fresh medium. This beneficial effect was seen between 20 and 72 h of culture. In contrast, the highest specific productivity was obtained already at 7 h for the cells in fresh medium and then decreased rapidly. The total product concentration was around 30% higher in the culture with CM compared to the culture in fresh medium, and the maximum product concentration was obtained on day 2 compared to day 3 for the cells in fresh medium. Our results indicate that the positive effect on productivity by CM is related to its growth-promoting effect, suggesting that the proliferation potential of the culture determines the productivity.

Model of bioconversion of cholesterol in cloud point system

Bioconversion of cholesterol to androst-1,4-diene-3,17-dione (ADD) and androst-4-ene-3,17-dione (4-AD) in cloud point system (CPS) was studied. A mathematical model was presented based on the mass transfer rate and microbial growth rate. The calculations for cell concentration, substrate concentration, and product concentration as functions of time were made. Sensitive analysis of model was made. Some parameters, such as substrate concentration, mass transfer coefficient, and maximum product inhibition concentration, indicated that the maximum product concentration is the most significant parameter in batch process. The prediction was validated by adjusting substrate concentration, stirring speed, and surfactant concentration. It showed the batch process of cholesterol bioconversion in CPS was controlled by inhibition of product. Adjusting surfactant concentration can diminish inhibition of product and increase final product concentration. The results also indicated that CPS can increase the substrate concentration of aqueous phase in batch process.

The catalytic potency of β-glucosidase from Pyrococcus furiosus in the direct glucosylation reaction

Enzymes from extremophiles operate at conditions that are different from their ‘normal’ counterparts, and are therefore a useful extension of the enzyme toolbox. In this paper, the direct glucosylation reaction mediated by a hyperthermophilic β-glucosidase from Pyrocuccus furiosus was investigated. Hexanol was successfully coupled to glucose with this enzyme. A preliminary study was conducted to improve the product yield. A maximum product concentration of 12.9 g.l −1 was attainable by increasing the glucose concentration to the maximum solubility of 2000 g.(kg buffer solution) −1 at the reaction temperature. The highest glucose based yield of 2.64% was achieved with a glucose concentration of 900 g.(kg buffer solution) −1 at a reaction temperature of 65°C and a pH of 6.0. Performing the reaction at higher pH and temperature led to lower product concentrations. This was caused by deactivation of the enzyme accompanied by browning of the reaction mixture. A pH of 4.4 did have a negative effect on both the storage and the operational stability of the enzyme.

Analysis of two-stage continuous operation of Escherichia coli containing bacteriophage λ vector

Bacteriophage λ containing a cloned-gene is stably maintained in Escherichia coli in the lysogenic state while it is replicated and it overproduces a recombinant protein product in the lytic state. The host cell is eventually lysed in the lytic state. The kinetics of cell lysis and production induction were studied and are reported in this article through model equations. In two-stage continuous operation, the first tank is maintained in the lysogenic state for cell growth and cloned-gene stability while the second tank is in the lytic state for the overproduction of cloned-gene product. Individual cells in the second tank have different extent of the induction for product formation, since each has a different residence time. The different residence time for individual cells was taken into account using a population model. The numerical results show good agreement with the experimental data for the prediction of dilution rate in the second tank which gives the maximum product concentration.

Supersaturation generation at the feed point in reaction crystallization of a molecular compound

The maximum product concentration of a molecular compound at the feed point in reaction crystallization is analysed by modelling. A model describing unsteady mass transfer and chemical reaction in strained, semi-infinite, fluid films is combined with models describing productivity constraints and reactant concentration changes during the course of a process. The maximum product concentration is taken as a first estimate of the maximum supersaturation and is used as a basis to discuss the product crystal size that may be produced. The result suggests that at certain conditions we may find smaller product crystals at decreased reactant concentrations. The maximum concentration varies during the course of a single-feed semibatch process and the highest value is not necessarily found in the beginning. The variation with time, depends on the choice of the reactant to feed, and the results provide an explanation to why the product crystal mean size may depend on the choice of feed reactant. Guidelines are proposed for how to decide on optimum reactant solution concentrations and for how to select the reactant to feed in a semibatch process. In addition it is suggested that larger crystals may be produced if the feed reactant concentration is low early in the process and is allowed to gradually increase with time in a controlled way. The term “programmed feed concentration” is introduced.

Product concentration profile in strained reacting fluid films

The product concentration profile that develops as a result of an instantaneous bimolecular reaction when contacting semi-infinite strained reactant solution films, is modelled and analysed. Analytical solutions to reactant and product concentrations are presented for which initial reactant concentrations, reactant diffusivities, and product diffusivity can be independently and arbitrarily chosen. The validity of the model for finite geometry is thoroughly discussed. It is shown that the product concentration profile may differ significantly depending on diffusivities and reactant concentrations, even for cases when almost the same amount of product is produced. The maximum product concentration is constant in time but varies with diffusivities and reactant concentrations in a quite complicated fashion. Under certain conditions the maximum product concentration may even decrease when initial reactant concentrations or reactant diffusivities are increased. Dimensionless concentration profiles and the maximum concentration are invariant with mixing intensity. The influence of mixing is fully incorporated into the length and time scales. The actual real time to form a certain amount of product is inversely proportional to the square root of the specific local energy dissipation rate. The implications of model results on fast complex reactions are discussed.

Thermodynamically Controlled Synthesis Of Cefamandole

The direct enzymatic synthesis of the antibiotic cefamandole is presented as an alternative for the current processes based on either chemical synthesis or kinetic enzymatic synthesis. The influence of pH and temperature on the apparent equilibrium constant was measured; from these data, the maximum cefamandole concentration was predicted. In spite of a beneficial apparent equilibrium constant at pH 3.75, less product was formed as compared to the concentration at the optimum pH (4.25). This effect is caused by the low solubility of one of the substrates at pH 3.75.The maximum product concentration (at the optimum pH of 4.25) was found to be 22 mM which is a significant concentration. The thermodynamically controlled synthesis may prove to be an alternative for the currently used processes if it can be combined with an effective in-situ cefamandole removal step.

Effect of surfactants on the production of ergot-alkaloids by immobilized mycelia of Claviceps paspali

In a semicontinuous process immobilized Claviceps paspali mycelia produced alkaloids over a period of 60 days (six reincubations). By addition of the surfactant Pluronik, a polyethoxypolypropoxy polymer, a considerable increase in alkaloid biosynthesis occurred. The maximum product concentration achieved was 8.35gl-1, and the overall productivity was 5.80 mgl-1 h-1, which is half the productivity of the batch process. Maximum process productivity for a single reincubation (12.3 mg l-1 h-1) was almost equal to the batch process productivity.

Construction and use of recombinant Escherichia coli strains for the synthesis of toluene cis-glycol

The toluene dioxygenase genes derived from Pseudomonas putida NCIMB 11767 were subcloned from a previously constructed recombinant plasmid, pIG, using pUC18 as the cloning vector and E. coli TG2 as the host strain. The resulting strain, E. coli TG2 (p1/1), produced toluene cis-glycol when grown in LB broth or minimal medium in the presence of toluene. Restriction mapping and partial DNA sequencing provided evidence for the presence of ORFs with extensive homology to parts of the tod operon from P. putida F1. The clones exhibited some residual toluene cis-glycol dehydrogenase activity which resulted in the formation of small amounts of 3-methylcatechol. Expression of the dioxygenase was induced by toluene, but was not directed by the lac promoter within the cloning vector. The clones were assessed for toluene cis-glycol production in pH-controlled batch cultures, and the maximum product concentration obtained was 1.02 g l −1. Product formation was dependent upon the presence of glucose in the culture medium. Although the substrate was toxic, the biotransformation was apparently limited by the supply of toluene. The results suggest that it should be possible to improve toluene cis-glycol production by recombinants substantially by improving both the strain and fermentation process.

Experimental evaluation of designs for the simulated countercurrent moving bed separator

AbstractSimulated countercurrent chromatography is a continuous flow method for separation of binary mixtures or for separation of multicomponent mixtures into two fractions. Countercurrent flow is simulated by moving the feed point past several fixed adsorbent beds at a rate between the single column breakthrough time of each component. The number of columns and the location of the inlet and outlet port were varied to investigate product purity and productivity. Concentration profiles of the effluent product streams were measured. The maximum product concentration exceeds the feed concentration during part of the feed‐switching cycle and drops to zero during part of the cycle. Three configurations were tested with the total number of columns varying between three and eight. For the test separation chosen, gaseous propylene and dimethyl ether on Chromosorb 101, high purity (> 99%) product streams were obtained with an optimal four‐column configuration using three desorbent streams.

Microencapsulation of hybridomas by cellulose sulfate-polydimethyldiallylammonium chloride procedure

Two hybridoma cell lines of human origin and two of murine origin were encapsulated using the cellulose sulfate-polydimethyldiallylammonium chloride procedure. All lines could be cultivated in capsules, but the time of growth and product synthesis were limited. The membranes formed were impermeable to albumin and transferrin over a period of 240 minutes. Therefore, the stop of cell proliferation is assumed to be caused by the lack of at least these two proteins. The cell densities inside the capsules reached a maximum of 4.1 × 106/ml (human line) and 3.9 × 107/ml (murine line). The maximum product concentration in the capsules measured by IgG- and IgM-ELISA was 0.17 mg/ml (human line) and 7 mg/ml (murine line). The calculated corresponding retention was 93% (human IgM) and 52% (murine IgG). The characterization of the product showed that the immunoglobulin was not intact and might have been destroyed during harvest. Using the same cell lines the method was compared with the alginate-PLL procedure originally developed by LIM and SUN [1].

Production of sorbitol and gluconic acid by permeabilized cells of Zymomonas mobilis

Zymomonas mobilis is able to convert glucose and fructose to gluconic acid and sorbitol. The enzyme, glucose-fructose oxidoreductase, catalysing the intermolecular oxidation-reduction of glucose and fructose to gluconolactone and sorbitol, was formed in high amounts [1.4 units (U)·mg-1] when Z. mobilis was grown in chemostats with glucose as the only carbon source under non-carbon-limiting conditions. The activity of a gluconolactone-hydrolysing lactonase was constant at 0.2 U·mg-1. Using glucose-grown cells for the conversion of equimolar fructose and glucose mixtures up to 60% (w/v), a maximum product concentration of only 240 g·1-1 of sorbitol was found. The gluconic acid accumulated was further metabolized to ethanol. After permeabilizing the cells using cationic detergents, maximum sorbitol and gluconic acid concentrations of 295 g·1-1 each were reached; no ethanol production occurred. In a continuous process with κ-carrageenan-immobilized and polyethylenimin-hardened, permeabilized cells no significant decrease in the conversion yield was observed after 75 days. The specific production rates for a high yield conversion ( > 98%) in a continuous two-stage process were 0.19 g·g-1·h-1 for sorbitol and 0.21 g·g-1·h-1 for gluconic acid, respectively. For the sugar conversion of cetyltrimethylammonium bromide-treated κ-carrageenan-immobilized cells a V max of 1.7 g·g-1·h-1 for sorbitol production and a K m of 77.2 g·1-1 were determined

Effect of transcription promoters on the optimal production of secreted protein in fed-batch reactors.

Production of heterologous proteins by yeast secretion imposes additional factors that need to be considered, which do not appear with production by direct expression. These include additional intracellular polypeptide processing dynamics through the secretory organelles and the protein concentration in the culture medium, which is the usual final destination of the product. Optimal control theory is applied to optimize fed-batch production of secreted protein. We maximize an objective function that includes both total production rate and product concentration. A mutant invertase is chosen as the model heterologous secretory protein. Optimal control control strategies have been obtained for the use of two different promoters for the gene transcription, a dere-pressible SUC2 promoter and a strong glycolytic GPD promoter. With the use of the strong GPD promoter, achieving maximum production occurs on the singular arc of maximum specific growth rate. As the object switches to maximum product concentration, operation occurs for longer periods of time at a slow glucose singular arc condition. The optimal control for maximizing protein production with the weak SUC2 promoter requires transitions between high and low glucose concentrations associated with multiple distinct singular arc conditions. For maximum product concentration, the high concentration branches of the singular arc supporting maximum growth rate and maximum secretion rate disappear. Operation stays essentially on the low glucose concentration branch of the singular arc, which maximizes the protein production rate and minimizes the dilution of the broth product concentration.

Fumaric acid production from xylose by immobilized Rhizopus arrhizus cells

The production of fumaric acid by immobilized Rhizopus arrhizus TKK 204-1-1a mycelium was optimized in batch fermentations using statistical experimental design and empiric modelling. The maximum fumaric acid concentration was obtained at a xylose concentration of about 6% and a carbon:nitrogen ratio of about 160. In repeated batch fermentations with immobilized cells the highest volumetric productivity of fumaric acid reached was 87 mg/l per hour when the initial xylose concentration was 10%, the C:N ratio 160 and the residence time 1.75 days. The maximum product concentration was 16.4 g/l when the initial xylose concentration was 10%, the C:N ratio 160 and the residence time 10.25 days. The maximum yield from initial xylose (6.47%) was 23.7% with a product concentration of 15.3 g/l and volumetric productivity of 71 mg/l per hour at a residence time of 9 days and a C:N ratio of 188.3. Immobilization could increase the fumaric acid concentration to a level 3.4 times higher than that produced by free cells.

PRODUCT FORMATION PATTERNS AND THE PERFORMANCE IMPROVEMENT FOR MULTISTAGE CONTINUOUS STIRRED TANK FERMENTORS

The relationship between the product formation patterns and the performance improvement was studied for the multistage chemostat system. The performance evaluation was made based on the noninferior sets defined for the productivity and the product concentration. It is shown that when the strong product inhibition or the substrate inhibition occurs, the significant improvement can be attained by increasing the number of stages. It is also shown that the maximum product concentration attainable can be increased by increasing the number of stages for the case where either the cell maintenance coefficient or the product decomposition rate is nonzero. If the productivity is the only performance criterion, the performance improvement cannot be attained by increasing the number of stages for the growth-associated type of product formation, while it can be expected for the non-growth-associated type.

Effect of oxygen supply and dilution rate on the production of 2,3-butanediol in continuous bioreactor by Klebsiella pneumoniae

Kinetics of 2,3-butanediol production by Klebsiella pneumoniae (NRRL B199) from glucose have been studied in a continuous bioreactor. The effect of oxygen supply rate and dilution rate on the product output rate and yield of 2,3-butanediol were investigated. For a feed glucose concentration of 100 g l −1, the optimum oxygen transfer rate is between 25.0–35.0 mmol l −1 h −1. Under these conditions, maximum product concentration obtained was 35 g l −1 at a dilution rate of 0.1 h −1 and the maximum product output rate obtained was 4.25 g l −1 h −1. The product yield based on the substrate utilized approached the theoretical value (50%) at low values of oxygen transfer rate but decreased with increasing oxygen transfer rate.

A solvent screening criterion for multicomponent extractive fermentation

A solvent screening criterion was developed based on the maximum product concentration attainable for the assessment of batch and semicontinuous multicomponent extractive fermentations. The theorem and corollary developed in this paper have proven useful in screening out oleyl alcohol to be the best solvent for butanol extractive fermentation.