Lysine Fermentation Research Articles

Optimization of Lysine Adsorption Process Using Strong Cation-Exchange Resin

Abstract A simplified method is proposed for determining the optimum conditions of multicolumn adsorption of lysine from a lysine fermentation broth, in which the resin columns periodically move countercurrent to a continuous broth flow. Experimental data on dynamic isotherms in the fixed-bed column are required for the calculations. The recurrence equation involved was solved on a personal computer. Based on this model, the optimum operation conditions of lysine adsorption were determined in order to minimize the production cost of lysine recovery from lysine fermentation broth.

Changes in characteristics of cation exchange resin in cyclic operation for lysine recovery from lysine fermentation broth

The mechanism of deterioration of a cation exchange resin (Diaion SK-IB of ammonium form) was examined by using a life tester operated with a sequential control. Resins sampled at intervals were analyzed for composition, ion exchange capacity and maximum amount of lysine adsorbed. The loss of resin capacity per volume appears to be caused by (1) irreversible loss of -SO 3 − groups, (2) both cleavege of the network structure of resin by oxidation reaction and fouling induced mainly by colored substances derived from lysine fermentation broth. Regenerants such as sodium sulfite, sodium nitrite and sodium hydroxide are not effective for complete regeneration of resin capacity at 60°C. Finally, the optimum number of cycles for which the resin can be used was determined.

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.

An Algorithm for operating a fed‐batch fermentation using quasi‐steady state

AbstractThe determination of an optimum feeding profile of a fed‐batch fermentation requires the solution of a singular optimum control problem, which is often complicated by changes in the process kinetics during the fermentation. The procedure of optimization may be sufficiently simple, if the feeding part of fermentation is carried out in the quasi‐steady state. In this work an algorithm for operating a fed‐batch fermentation using mentioned regime is offered. The algorithm supposes a periodical correction of the feeding strategy. Applying to fed‐batch lysine fermentation demonstrate efficacy of this algorithm over frequently used strategies.

Selectivity coefficient of a colored substance in lysine fermentation broth for a strong cation exchange resin of ammonium form.

A colored substance found in lysine fermentation broth is thought to be derived mainly from the raw materials and nutrient medium used for the fermentation. In this paper, the selectivity coefficient of this colored substance for an ion exchange resin of ammonium form as well as the apparent dissociation constant of the colored substance in solution were determined

Amino acid-dependent transport of sugars by Fusobacterium nucleatum ATCC 10953.

Resting cells of Fusobacterium nucleatum 10953 (grown previously in a medium containing glucose) failed to accumulate glucose under aerobic or anaerobic conditions. However, the addition of glutamic acid, lysine, or histidine to anaerobic suspensions of cells caused the immediate and rapid accumulation of glucose. Except for the amino acid-dependent transport of galactose and fructose (the latter being transported at approximately one-third the rate of glucose), no other sugars tested were accumulated by the resting cells. Amino acid-dependent uptake of sugar(s) by F. nucleatum was abolished by exposure of cells to air, and under aerobic conditions the rates of fermentation of glutamic acid and lysine were less than 15% of the rates determined anaerobically. The energy necessary for active transport of the sugars (acetyl phosphate and ATP) is derived from the anaerobic fermentation of glutamic acid, lysine, or histidine. Competition studies revealed that glucose and galactose were mutual and exclusive inhibitors of transport, and it is suggested that the two sugars (Km = 14 microM) are translocated via a common carrier. The products of amino acid-dependent sugar transport were recovered from resting cells as ethanol-precipitable, high-molecular-weight polymers. Polymer formation by F. nucleatum, during growth in medium containing glucose or galactose, was confirmed by electron microscopy.

The optimization of L‐lysine fermentation and a mass transfer model

AbstractThe efficiency of L‐lysine biosynthesis is essentially determined by the power input and aeration ratio in the stirred fermenter.A mass transfer model was developed by means of the results of lysine fermentations in four geometrically similar fermenters with working volumes of 10 1, 50 1, 100 1 and 2500 1 which allows the optimization of lysine fermentation from the energetical point of view. The usefullness of this kLa‐model is demonstrated with an example where the power input for an unknown fermenter is calculated.

Performance of laying hens on diets containing Eurolysine bacterial protein or Pekilo protein

The investigation was made to determine to what extent soybean meal and fish meal protein in the diet of laying hens can be replaced by Eurolysine bacterial protein, a by-product of lysine fermentation, or with Pekilo protein. In a 24-week laying trial the inclusion levels of Eurolysine in the diet were 0-4.0- 7.9- 11.9 % and those of Pekilo 0-6.3- 12.7 - 18.0 %, or 0-33-66-100 % of the protein supplement. Eurolysine contained 68.5 % crude protein 53.5 % true protein and 6.9 % ether extract in DM and the corresponding values of Pekilo were 43.3 %, 38,8 % and 1.4 %. The average laying rates decreased with increasing inclusion of Eurolysine, and replacement of the protein supplement by Pekilo also lowered egg production, but the differences between the treatments were not significant (P > 0.05). Feed intakes were increased (P < 0.05) by inclusion of SCP. Feed conversion efficiency did not differ significantly among the treatments (P > 0.05). Mortality increased with inclusion of both types of SCP in the diets, but the principal cause of death was cannibalism.

Excretion of lysine byMicrococcus glutamicus

Analysis of intracellular and extracellular lysine concentration during lysine fermentation by Micrococcus glutamicus AEC RN-13-6/1 indicated that lysine excretion occurs against a concentration gradient towards the end of the fermentation period. The capacity to excrete lysine against a concentration gradient may be a factor contributing to the high yield of lysine.

Optimum operating mode for a class of fermentation

AbstractThis paper is concerned with optimization of the operating mode of a fermentor. Combining the various modes of operation—batch, semibatch, and continuous—the operating pattern which maximizes the desired metabolic product in a single fermentor is determined by using Kelley's transformation method with Pontryagin's maximum principle. Kelley's transformation method is a device which avoids the singular situation which occurs when the usual procedure of selecting the optimal control function by the maximum principle breaks down. This is the case in the problem considered in this paper. For lysine fermentation, the best operating mode depends on the fermentor capacity and operating time. The results of this study are summarized thus: (i) when the operating time is “long enough,” optimal conditions require that continuous operation follows either semibatch and/or batch operation, and (ii) when the fermentor capacity becomes “large enough,” semibatch operation becomes important.

Sugar Substrates for l -Lysine Fermentation by Ustilago maydis

The extracellular production of l -lysine in media with cane sugar, blackstrap molasses, or clarified sugar-cane juice by a previously obtained mutant of Ustilago maydis was studied. Enzymatically inverted clarified juice (medium J-3) gave 2.9 g of lysine per liter under the following conditions: inoculum, 5%; p H 5.8; temperature, 30 C; K La in the fermentors, 0.41 mmoles of O 2 per liter per min; fermentation time, 72 hr. The concentrate, obtained by direct evaporation and drying of the fermentation broth, could be used as a possible feed supplement because of its amino-acid and vitamin content.

Purification and properties of a NAD-dependent glutamate dehydrogenase from clostridium SB 4

The purification of a NAD-dependent glutamate dehydrogenase from lysine-fermenting Clostridium SB 4 is described. The enzyme, purified 250-fold with a 40% yield, was found to be homogenous by gel electrophoresis and ultracentrifugation criteria. Sedimentation analysis gave a s° 20,w value of 11.7 and a molecular weight of 275 000. The sedimentation rate is invariant over a concentration range from 50 μg to 5 mg of protein per ml, in contrast to beef liver glutamate dehydrogenase. The enzyme, although isolated from an anaerobic organism is not sensitive to oxygen. It is stable to storage under various conditions, especially in highly dilute solutions. Amino acid composition, gel filtration behavior, substrate specificity towards various α-keto acids, pH optima and kinetic parameters of the enzymatic reaction were determined and compared with corresponding properties of glutamate dehydrogenases from other sources. Initial velocity measurements result in linar double-reciprocal plots and indicate a sequential order of substrate addition, different from that of other glutamate dehydrogenases. Low Michaelis constants for ammonia and a- ketoglutarate , high coenzyme and substrate specificity, lack of sensitivity towards purine nucleotides and high stability suggest the use of the enzyme for analytical determinations of ammonia and a- ketoglutarate . The function of the enzyme in lysine fermentation is discussed.

Formation and Identification of 3-Keto-5-aminohexanoic Acid, a Probable Intermediate in Lysine Fermentation

Abstract A neutral, heat-labile compound (II) has been detected as a product of lysine fermentation by cell-free extracts of Clostridium SB4. A 2 to 5% yield of II from lysine is observed when coenzyme A is omitted from an otherwise complete reaction mixture. 3,5-Diaminohexanoate appears to be the immediate precursor of Compound II since it can be converted to II under conditions not permitting the utilization of lysine or β-lysine. Under favorable conditions, 3,5-diaminohexanoate is enzymically converted to Compound II in 20 to 30% yield. The cofactors required for the reaction are NAD and α-ketoglutarate or pyruvate; the latter is active only when lactic dehydrogenase is also added. The formation of Compound II appears to occur by a reversible NAD-dependent oxidative deamination of 3,5-diaminohexanoate. The keto acids function as oxidants in the reoxidation of NADH by the appropriate dehydrogenases. Although Compound II has not been isolated as a pure compound because of its instability, chemical and enzymic studies indicate that its structure is 3-keto-5-aminohexanoate. Compound II is reduced by borohydride to a heat-stable hydroxyamino acid, and is decarboxylated to a basic methyl ketone. The latter reacts with hypoiodite to give iodoform and β-aminobutyrate; therefore, it appears to be 2-keto-4-aminopentane.

Identification and Cobamide Coenzyme-dependent Formation of 3,5-Diaminohexanoic Acid, an Intermediate in Lysine Fermentation

Abstract When 14C-l-lysine is incubated with cell-free extracts of Clostridium SB4, a new basic amino acid is formed. This compound does not accumulate when lysine is fermented in the presence of intrinsic factor. The basic amino acid has been isolated in radiochemically pure form. It is not oxidized by chloramine-T or periodate, it yields about 1 mole of acetic acid per mole in the Kuhn-Roth C-terminal methyl group determination, and it reversibly forms a lactam. The data support the conclusion that this new amino acid, which requires a cobamide coenzyme for formation, is 3,5-diaminohexanoic acid. The basic amino acid is readily fermented to volatile acids by extracts of Clostridium SB4. It appears to be an intermediate in the fermentation of lysine.

Anaerobic degradation of lysine: IV. Cobamide coenzyme-dependent migration of an amino group from carbon 6 of β-lysine (3,6-diaminohexanoate) to carbon 5 forming a new naturally occurring amino acid, 3,5-diaminohexanoate

Methods are described for the accumulation and detection of 3,5-diaminohexanoate, a new basic amino acid intermediate in the lysine fermentation pathway formed by the reaction sequence α-lysine ⇌ β-lysine ⇌ 3,5-diaminohexanoate. Migration of the terminal amino group of β-lysine to carbon atom 5 in a cobamide coenzyme-dependent reaction results in the formation of 3,5-diaminohexanoate. The dihydrochloride, and the N,N′-dibenzoyl derivative and the δ-lactam (4-amino-6-methyl-2-piperidone) of the new amino acid were prepared and characterized chemically and by infrared and nuclear magnetic resonance spectral analyses. The new amino acid is fermented by crude extracts of Clostridium sticklandii and Clostridium M-E to the same fatty acids that are produced from α- and β-lysine.

Amino acid fermentation by Fusobacterium nucleatum

Fusobacterium nucleatum (Fusobacterium fusiforme) is a weakly saccharolytic gram negative anaerobic rod which is indigenous to the oral cavity of man. This organism fermented aspartate, cysteine, glutamate, glutamine, histidine, lysine, methionine, serine and threonine forming butyrate and acetate as the main acid end products. The presence of glucose in the medium did not prevent amino acid fermentation as the amino acids and glucose were catabolized concurrently. The butyrate and acetate found in spent glucose-trypticase broth were derived primarily from the broth amino acids, whereas lactate and formate arose from glucose catabolism. The absence of glucose catabolite repression could be accounted for by the slow degradation of glucose i.e. 15–25 μg glucose utilized/mg dry cell weight/hr. The fermentation of lysine did not involve significant decarboxylation but rather appeared to be by a pathway which involved cleavage into butyrate and acetate.

Isolation and Identification of β-Lysine as an Intermediate in Lysine Fermentation

Abstract Clostridium SB4 ferments lysine to acetate, butyrate, and 2 moles of ammonia. The fermenting system appears to be typical of those studied previously (1–7), except that α-ketoglutarate is required for fermentation by cell extracts rather than pyruvate. The omission of all required cofactors except α-ketoglutarate and coenzyme A results in the accumulation of a basic amino acid. The amino acid has been isolated in gram quantities and shown to be l-β,e-diaminocaproic acid (l-β-lysine). The conversion of l-lysine to l-β-lysine is readily reversible; the equilibrium constant (k = [β-lysine]/ [lysine]) is 5.7. The level of α-ketoglutarate influences both the rate and extent of conversion of lysine to β-lysine, but the system does not require substrate levels of α-ketoglutarate. Pyridoxal phosphate can be substituted for α-ketoglutarate when crude cell extracts are used, and α-ketoglutarate can be deleted from reaction mixtures without loss of activity when dialyzed extracts are used. A sulfhydryl compound, i.e. glutathione or mercaptoethanol, and coenzyme A are required for maximal rates of conversion of lysine to β-lysine when dialyzed cell extracts are used. Pyridoxal phosphate has a stimulating effect with such extracts. β-Lysine is fermented more rapidly than lysine by Clostridium SB4 extracts, and the addition of lysine does not inhibit β-lysine fermentation. Apparently β-lysine lies on or very close to the path of lysine fermentation.

Lysine Fermentation to Fatty Acids and Ammonia: A Cobamide Coenzyme-dependent Process

Lysine Fermentation to Fatty Acids and Ammonia: A Cobamide Coenzyme-dependent Process