L-glutamic Acid Fermentation Research Articles

Production Of L-Glutamic Acid As A Function Of Urease Activity By Corynebacterium glutamicum X680 Using Agro-Based Raw Materials

Urease catalyzes hydrolysis of urea. The present investigation was undertaken to investigate the urease activity and its optimization during L-glutamic acid fermentation by a biotin auxotroph Corynebacterium glutamicum X680 using urea as the principal nitrogen source. Another interesting part of this study was to investigate the efficiency of this microorganism for utilization of indigenous raw materials (such as hydrolysates of cassava starch, rice bran and wheat bran) as a cheap carbon sources instead of glucose. Among different raw materials, hydrolysate of cassava starch appeared to be the most suitable. However, the production efficiency is significantly less (p<0.01) with cassava starch compared to glucose. When the medium is supplemented with equivalent amount of (10g%) cassava starch hydrolysate, the L-glutamic acid accumulation was reported to be 18.2mg/ml with maximum urease activity (0.18U/mg protein) with pH7 at 300C.Thus, among the different agro-based wastes were examined, cassava starch hydrolysate appeared to be the best alternative of glucose to minimize L- glutamic acid production cost.

Enhanced L-glutamic acid production by immobilized Corynebacterium glutamicum X680 using Response Surface Methodology

L-glutamic acid is a non-essential amino acid largely used as flavor enhancer and food additive. It also has several therapeutic applications. Fermentation has gained superiority over its chemical synthesis as it produced stereo-specific isomer. Corynebacterium glutamicum is mostly used microorganism for Lglutamic acid fermentation. The study was dealing with optimization of L-glutamic acid production by immobilized mutant Corynebacterium glutamicum X680 in calcium alginate beads using response surface methodology as effective statistical tool. Among several parameters studied, pH, inoculums size, incubation time, concentration of sodium alginate, agitation and cell:alginate ration showed the most significant effect. Immobilized cells produced significantly (p<0.01) lower amount of L-glutamic acid (24.3mg/ml) compared to the production by free cells (27.6mg/ml). However, reusability of the beads minimized production cost and hence conferred benefit as far as the market economy is concerned.

Effects of proteases on L-glutamic acid fermentation

ABSTRACT In the process of L-glutamic acid fermentation, there are proteins that cannot be decomposed and utilized by bacteria and that secreted by bacteria at the same time, which cause problems such as increased foam production in the fermentation broth that lowers the dissolved oxygen, which makes the total fermentation efficiency low. Therefore, these proteins can be decomposed by adding proteases in the fermentation broth, and it is found that the best results are obtained by adding 0.5 g/L of trypsin. Proteins can be used by bacteria after being decomposed as well. The final L-glutamic acid production in our research was 177.0 g/L, which is 14.9% more than the control fermentation (154.0 g/L). Similarly, the glucose conversion rate was 68.3%, which is an increase of 4.0% as compared to the control fermentation (65.6%).

MODELING AND OPTIMIZATION OF GLUTAMIC ACID PRODUCTION USING MIXED CULTURE OF Corynebacterium glutamicum NCIM2168 AND Pseudomonas reptilivora NCIM2598

In this study, a hybrid system of response surface methodology followed by genetic algorithm has been adopted to optimize the production medium for L-glutamic acid fermentation with mixed cultures of Corynebacterium glutamicum and Pseudomonas reptilovora. The optimal combination of media components for maximal production of L-glutamic acid was found to be 49.99 g L−1 of glucose, 10 g L−1 of urea, 18.06% (v/v) of salt solution, and 4.99% (v/v) of inoculum size. The experimental glutamic acid yield at optimum condition was 19.69 g L−1, which coincided well to the value predicted by the model (19.61 g L−1). Using this methodology, a nonlinear regression model was developed for the glutamic acid production. The model was validated statistically and the determination coefficient (R 2) was found to be 0.99.

Analysis of l-glutamic acid fermentation by using a dynamic metabolic simulation model of Escherichia coli

BackgroundUnderstanding the process of amino acid fermentation as a comprehensive system is a challenging task. Previously, we developed a literature-based dynamic simulation model, which included transcriptional regulation, transcription, translation, and enzymatic reactions related to glycolysis, the pentose phosphate pathway, the tricarboxylic acid (TCA) cycle, and the anaplerotic pathway of Escherichia coli. During simulation, cell growth was defined such as to reproduce the experimental cell growth profile of fed-batch cultivation in jar fermenters. However, to confirm the biological appropriateness of our model, sensitivity analysis and experimental validation were required.ResultsWe constructed an l-glutamic acid fermentation simulation model by removing sucAB, a gene encoding α-ketoglutarate dehydrogenase. We then performed systematic sensitivity analysis for l-glutamic acid production; the results of this process corresponded with previous experimental data regarding l-glutamic acid fermentation. Furthermore, it allowed us to predicted the possibility that accumulation of 3-phosphoglycerate in the cell would regulate the carbon flux into the TCA cycle and lead to an increase in the yield of l-glutamic acid via fermentation. We validated this hypothesis through a fermentation experiment involving a model l-glutamic acid-production strain, E. coli MG1655 ΔsucA in which the phosphoglycerate kinase gene had been amplified to cause accumulation of 3-phosphoglycerate. The observed increase in l-glutamic acid production verified the biologically meaningful predictive power of our dynamic metabolic simulation model.ConclusionsIn this study, dynamic simulation using a literature-based model was shown to be useful for elucidating the precise mechanisms involved in fermentation processes inside the cell. Further exhaustive sensitivity analysis will facilitate identification of novel factors involved in the metabolic regulation of amino acid fermentation.

Estimation of biochemical variables using quantum- behaved particle swarm optimization (QPSO)-trained radius basis function neural network: A case study of fermentation process of L-glutamic acid

Due to the difficulties in the measurement of biochemical variables in fermentation process, softsensing model based on radius basis function neural network had been established for estimating the variables. To generate a more efficient neural network estimator, we employed the previously proposed quantum-behaved particle swarm optimization (QPSO) algorithm for neural network training. The experiment results of L-glutamic acid fermentation process showed that our established estimator could predict variables such as the concentrations of glucose, biomass and glutamic acid with higher accuracy than the estimator trained by the most widely used orthogonal least squares (OLS). According to its global convergence, QPSO generated a group of more proper network parameters than the most popular OLS. Thus, QPSO-RBF estimator was more favorable to the control and fault diagnosis of the fermentation process, and consequently, it increased the yield of fermentation. Key words: Soft-sensing model, quantum-behaved particle swarm optimization algorithm, neural network.

Modeling the growth of Corynebacterium glutamicum under product inhibition in l-glutamic acid fermentation

In the fermentation of l-glutamic acid by Corynebacterium glutamicum, the growth inhibition by the substrate (glucose) at higher concentrations, and by the product at almost all concentrations seem to occur. In order to identify the range of concentrations for substrate limitation/inhibition, the experiments were conducted separately with different initial glucose concentrations. Proof of growth inhibition by the product was established by analyzing the data obtained from the time course of batch fermentation. Based on the experimental observations, a product inhibition model has been developed by modifying the Monod's kinetic equation for cell growth. This model simulates the growth satisfactorily. The same model is also able to describe the experimental data for growth of C. glutamicum obtained from different investigators.

Metabolic Analysis of Glutamate Production by Corynebacterium glutamicum

The dynamic behavior of the metabolism of Corynebacterium glutamicum during l-glutamic acid fermentation, was evaluated by quantitative analysis of the evolution of intracellular metabolites and key enzyme concentrations. Glutamate production was induced by an increase of the temperature and a final concentration of 80g/l was attained. During the production phase, various other compounds, notably lactate, trehalose, and DHA were secreted to the medium. Intracellular metabolites analysis showed important variations of glycolytic intermediates and NADH, NAD coenzymes levels throughout the production phase. Two phenomena occur during the production phase which potentially provoke a decrease in the glutamate yield: Both the intracellular concentrations of glycolytic intermediates and the NADH/NAD ratio increase significantly during the period in which the overall metabolic rates decline. This correlates with the decrease in glutamate yield due in part to the production of lactate and also to the period of the fermentation in which growth no longer occurred.

しょ糖転化ケインモラセスを原料としたグルタミン酸発酵の工業的実施

しょ糖転化ケインモラセスを原料としたグルタミン酸発酵の工業的実施

Amperometric L-glutamate sensor using a novel L-glutamate oxidase from Streptomyces platensis NTU 3304

Streptomyces platensis NTU 3304, isolated from soil samples, produces extracellular L-glutamate oxidase in liquid culture. Strains of this species have never been reported to be able to produce this enzyme. The purified enzyme was immobilized onto a cellulose triacetate membrane which was held at an oxygen electrode. The sensor was specific to L-glutamate in accordance with the properties of the novel L-glutamate oxidase. The time required for each assay in batch operation was less than 3 min. A linear relationship is observed between the decrease in dissolved oxygen and the concentration of L-glutamate between 20 and 140 mg l −1 (ca. 0.12 and 0.84 mM). The sensor retained 95% of its original activity after 400 assays over a period of 3 weeks. The sensor was applied to determine the concentration of L-glutamate in broth samples during L-glutamic acid fermentation. Good correlations were achieved between results obtained with the sensor and by enzymatic analysis using glutamate dehydrogenase.

13C nuclear magnetic resonance studies of glucose metabolism in L-glutamic acid and L-lysine fermentation by Corynebacterium glutamicum.

Glucose metabolism in Corynebacterium glutamicum was investigated using 13C nuclear magnetic resonance (13C NMR) spectroscopy. L-Glutamic acid and L-lysine producers were cultivated in medium containing [1-13C]- or [6-13C] glucose, and the 13C NMR spectrum of the culture filtrate was measured. In each fermentation, the ratio of the contributions of the Embden-Meyerhof pathway (EMP) and the hexosemonophosphate pathway (HMP) (EMP/HMP) was calculated on the basis of the 13C population at each carbon in the products. The EMP/HMP was estimated as 80/20 in glutamic acid fermentation; in contrast, it was 30-40/60-70 in lysine fermentation. These results indicate that HMP contributes much more in lysine fermentation, probably because of the greater requirement of NADPH in lysine formation from glucose.

Electrochemical studies on fermentation. I. Application of electro-energizing method to L-glutamic acid fermentation.

An electro-energizing fermentation (E-E F) method has been developed. In this method, a direct electrical current is applied to a microbial culture to accelerate the reductive metabolism of microorganisms or to impart profitable effects to microbial cells. This E-E F method was applied to L-glutamic acid fermentation by Brevibacteriurn fiavum No. 2247. When glucose was used as a substrate, the addition of 0.01 mi s neutral red (NR), redox dye (electron carrier), to the fermentation broth at the beginning of cultivation was effective for L-glutamate (L-Glu) production. A direct current of 200_??_300 μA/cmz at 1.5 V was applied through out the cul-tivation of this bacterium. This resulted in about a 10% increase in yield of L-Glu.

Electrochemical studies on fermentation. II. Determination of electro-energizing conditions for L-glutamic acid fermentation.

The most effective electro-energizing fermentation (E-E F) conditions for L-glutamate (L-Glu) production by Brevibacterium flavum No. 2247 were determined. The adding of 0.01 mat neutral red at the beginning of cultivation was found most effective. A 1.5 V direct current was applied to the culture broth at 6_??_8 hr after inoculation in the cathode compartment. L-Glu was produced at 51.0mg per ml, and this is about a 15% increase in yield compared to the yield of the not electro-energizing (E-E) control (44.3mg/ml).

The Mechanism of the Formation of γ-Glutamylpeptides duringl-Glutamic Acid Fermentation Contributed Solely by γ-Glutamyltranspeptidase

The enzyme of Corynebacterium glutamicum that catalyzes the formation of various γ-L(D)-glutamylpeptides by the reversal of hydrolysis was found to be γ-glutamyltranspeptidase. The enzyme catalyzed γ-l(d)-glutamyl transfer reaction from various γ-L(D)-glutamyl- peptides including glutathione. The susceptibility of D-isomers was 17% of that of l-isomers. Many kinds of α-l-amino acids and peptides such as γ-l-GIu-l-GIu could be γ-glutamyl acceptors. These findings offered full explanation on the formation of γ-l-glutamylpeptides in the l-glutamic acid fermentation. In the supposed mechanism, γ-l-GIu-l-GIu is formed mainly by the reversal of hydrolysis from l-glutamic acid being richly produced by the bacteria. The other four minor peptides are formed by the γ-glutamyltranspeptidatkm from γ-l-GIu-l-Glu to some α-l-amino acids and to γ-l-GIu-l-GIu itself in the broths. The enzyme was found to be widely distributed among bacteria, especially in some strains of Corynebacterium, Bacillus, Erwinea, Serratia, Pseu...

Studies on the .GAMMA.-glutamylpeptides in L-glutamic acid fermentation broths. III. The mechanism of the formation of .GAMMA.-glutamylpeptides during L-glutamic acid fermentation contributed solely by .GAMMA.-glutamyltranspeptidase.

The enzyme of Corynebacterium glutainicum that catalyzes the formation of various γ-L(D)-glutamylpeptides by the reversal of hydrolysis was found to be γ-glutamyltranspepti-dase. The enzyme catalyzed γ-L(D)-glutamyl transfer reaction from various γ-L(D)-glutamyl-peptides including glutathione. The susceptibility of D-isomers was 17% of that of L-isomers. Many kinds of α-L-amino acids and peptides such as γ-L-Glu-L-Glu could be γ-glutamyl ac-ceptors. These findings offered full explanation on the formation of γ-L-glutamylpeptides in the L-glutamic acid fermentation. In the supposed mechanism, γ-L-GIu-L-Glu is formed mainly by the reversal of hydrolysis from L-glutamic acid being richly produced by the bacteria. The other four minor peptides are formed by the γ-glutamyltranspeptidation from γ-L-Glu-L-Glu to some α-L-amino acids and to γ-L-GIu-L-Glu itself in the broths. The enzyme was found to be widely distributed among bacteria, especially in some strains of Corynebacteriun, Bacillus, Erwinea, Serratia, Pseudomonas, Achromobacter, and Alcaligenes.

Studies on the .GAMMA.-glutamylpeptides in L-glutamic acid fermentation broths. II. .GAMMA.-Glutamylpeptide formative activity of Corynebacterium glutamicum by the reverse reaction of the .GAMMA.-glutamylpeptide hydrolytic enzyme.

To clarify the mechanism of the γ-L-glutamylpeptide formation in the L-glutamic acid fermentation with Corynebacterium glutamicum, γ-glutamylpeptide synthetic activity of the intact cells and the cell extracts of the bacteria was studied. γ-L-Glu-L-Glu and other various γ-glutamylpeptides were formed by these crude preparations under high substrate amino acid concentrations without direct or indirect biological energy supplying systems. It was re-vealed that these enzyme preparations possessed strong hydrolytic activity to γ-glutamyl-peptides, and that notable amounts of these peptides could be formed by the reverse reaction of the hydrolysis. These reactions were found to be catalyzed by an enzyme. To give the evidence, the equilibrium constants of the hydrolysis of γ-L-glutamylpeptides, the substrate specificity and the distribution of the enzyme among various strains of the bacteria were studied. The partial purification of the enzyme gave sufficient proof to the idea. The mecha-nism was thought to contribute to the γ-L-GIu-L-Glu formation in the fermentation.

The studies on the .GAMMA.-glutamylpeptides in L-glutamic acid fermentation broths. I. The isolation and identification of .GAMMA.-glutamylpeptides from L-glutamic acid fermentation broths and their actions to the crystallization of the amino acid.

Five γ-glutamylpeptides were isolated from the L-glutamic acid fermentation broths using Corynebacterium glutamicum, and were identified as γ-L-glutamyl-L-glutamic acid, γ-L-glutamyl-L-glutamine, γ-L-glutamyl-L-valine, γ-L-glutamyl-L-leucine, and γ-L-glutamyl-γ-L-glutamyl-L-glutamic acid, respectively. γ-L-Glutamyl-L-glutamine and the tripeptide are new compounds found in fermentation. It was suggested that these peptides were synthesized by the bacteria. The studies on the crystallization of L-glutamic acid revealed that γ-L-glutamyl-L-glutamic acid was the essential compound that affected the crystallization. More than 0.5% existence of the peptide lowered the crystallization rate, and more than 1.0% existence caused the change of morphological tendency of the crystallization, α-form to β-form. The other peptides did not show any actions to the crystallization by themselves, but when they coexisted with γ-L-glutamyl-L-glutamic acid, they enhanced the action of the peptide.

Irradiation treatment of medium on L-glutamic acid fermentation. II. Gamma-ray inactivation of biotin in dilute aqueous solution.

The relative roles of the radicals produced by water radiolysis in the inactivation of biotin in aqueous solution were investigated. The effects of nitrous oxide and isopropanol used as selective free radical scavengers allowed the inactivation efficiencies per unit G-value of OH, H, and to be estimated; these efficiencies were 0.73, 0.10, and 0.02 in neutral solution, respectively. Hydrogen gas and hydrogen peroxide unaffected the activity of biotin. G0-value for biotin inactivation in oxygen-free neutral solution was 2.08. Under these conditions the hydroxyl radical attack was found to be responsible for the large part of inactivation. On the other hand, in oxygenated neutral solution, G0-value was 4.16. This large increase of inactivation in oxygenated solution suggested that, although hydrated electrons were considerably ineffective as an inactivating species in oxygen-free solution, superoxide ions would be much more effective in causing inactivation of biotin in oxygenated solution.A rate constant f...

Conditions of Oxygen Supply in L-Glutamic Acid Fermentation from Hydrocarbon

A small jar fermentor was developed in order to investigate the effect of oxygen supply on hydrocarbon fermentation. Several indices to oxygen transfer were examined with this small jar fermentor. Conditions for suitable oxygen supply were examined in l-glutamic acid fermentation from hydrocarbon by use of shaking flasks and these small jar fermentors. The data indicated that the rate of oxygen transfer ought to be more than 14.3 × 10−7 mole/ml·min in order to obtain satisfactory results. The coefficient of oxygen transfer rate (KLa/H) decreased as the fermentation went on, so the supply of oxygen enriched gas mixture was effective to increase the production of l-glutamic acid.

L-Glutamic Acid Fermentation with Molasses

An l-glutamic acid (l-GA)-forming bacterium. Microbacterium ammoniaphium was cultured in the molasses medium with or without poiyoxyethylene fatty acid esters to obtain l-GA-accumulating cells or non-accumulating cells, respectively.Then protoplast-like bodies (PLB) were prepared from each group of cells by reacting them with egg white lysozyme.l-GA-accumulating reaction by the PLB was carried out under high and low osmotic pressures.From the results of the experiment, it was shown that the difference in the ability of l-GA accumulation between l-GA-accumulating cells and non-accumulating cells was attributed mainly to the difference in the nature of the cell membrane.Further, the relationship between the molar ratio of saturated fatty acids/unsaturated fatty acids which was reported previously and the nature of the membrane was discussed.The lipid composition of the cell membrane from Microbacterium ammoniaphilum was determined by thin-layer and column chromatographies to make clear the relation between ...