L-Isoleucine production

Figure 2 shows the typical pattern of continuous L-isoleucine production with 2-bed reactors. The steady state was maintained for long periods in 1st and 2nd beds, and L-isoleucine concentration reached to 3.6 mg/ml. 

B., Schartges, D., and Wandrey, C. (1996) Integrated development of fermentation and downstream processing for L-isoleucine production with Corynehacterium glutamicum. Appl. Microbiol. Biotechnol, 46, 209-219. 

Purification. The objective of crystallization also can be purification of a chemical species. For example, L-isoleucine (an essential amino acid) is separated by crystallization from a fermentation broth that has been filtered and subjected to ion exchange. The recovered crystals contain impurities deleterious to use of the product, and these crystals are, therefore, redissolved and recrystalHzed to enhance purity. 

Morbach, S., Sahm, H. and Eggeling, L. (1996) 1-Isoleucine Production with Corynebacterium glutamicum further flux increase and limitation of export. Applied and Environmental Microbiology, 62 (12), 4345 -1351. 

One of the first known examples of allosteric feedback inhibition was the bacterial enzyme system that catalyzes the conversion of L-threonine to L-isoleucine in five steps (Fig. 6-28). In this system, the first enzyme, threonine dehydratase, is inhibited by isoleucine, the product of the last reaction of the series. This is an example of heterotropic allosteric inhibition. Isoleucine is quite specific as an inhibitor. No other intermediate in this sequence inhibits threonine dehydratase, nor is any other enzyme in the sequence inhibited by isoleucine. Isoleucine binds not to the active site but to another specific site on the enzyme molecule, the regulatory site. This binding is noncovalent and readily reversible if the isoleucine concentration decreases, the rate of threonine dehydration increases. Thus threonine dehydratase activity responds rapidly and reversibly to fluctuations in the cellular concentration of isoleucine. 

FIGURE 6-28 Feedback inhibition. The conversion of L-threonine to L-isoleucine is catalyzed by a sequence of five enzymes (E, to E5). Threonine dehydratase (E,) is specifically inhibited allosterically by L-isoleucine, the end product of the sequence, but not by any of the four intermediates (A to D). Feedback inhibition is indicated by the dashed feedback line and the symbol at the threonine dehydratase reaction arrow, a device used throughout this book. 

Repression as applied lo biochemical reactions is a process of feedback control whereby a cell limiLs its produedon of the substances produced within it. An example that has been investigated shows the nature and mechanism by which this limitation is effected. Ii has been found that production of the amino acid L-isoleucine by cells of the bacterium Escherichia coll is repressed in the presence of an excess of the product. This excess is obtained experimentally by adding the substance in the culture medium in which the bacterium is grown. A form of the L-isoleucinc is used that has been labeled with a radioactive isotope, so ihat the mechanism of the repression can be followed. 

By this means, it has been found that the excess of L-isoleucine has two distinct effects—one that is relatively slow, and unothcr that is rapid. The slower effect is to repress production by the cell of all the enzymes required io catalyze the series of biochemical reactions in the metabolic pathway by which the cell synthesizes L-isoleucine. The Iasi effect is to inhibit production of the enzyme for the first reaction ill the series. This enzyme is L-thrconinc deaminase, which removes the amino group from L-threonine. as a preliminary step to iis oxidation and reimroduction of (he amino group, in order to produce L-isolcucine from it. 

The first enzyme in the sequence, L-threonine dehydratase, is strongly inhibited by L-isoleucine, the end product, but not by any other intermediates in the sequence. 

Several V- IJ<>c-A-MOM-a-am ino acid derivatives undergo a-methylation in 78% to nearly 93% ee with retention of the configuration upon treatment with KHMDS followed by methyl iodide at —78°C. The substituents of the nitrogen are essential for control of the stereochemistry. How much is the stereochemical course of the reaction affected by an additional chiral center at C(3) of substrates a-Alkylation of A -lioc-A-MOM-L-isoleucine derivative 61 and its C(2)-epimer, D-a/fo-isoleucine derivative 62, were investigated (Scheme 3.16). If the chirality at C(2) is completely lost with formation of the enolate, a-methylation of either 61 or 62 should give a mixture of 63 and 64 with an identical diastereomeric composition via common enolate intermediate K. On the other hand, if the chirality of C(2) is memorized in enolate intermediates, 61 and 62 should give products with independent diastereomeric compositions via diastereomeric enolate intermediates. 

The structures of the natural products, ceratospongamides from marine red alga (Rhodophyta) Ceratodictyon spongiosum, which each consist of two L-phenylalanine residues, one (L-isoleucine)-methyloxazoline residue, one L-proline residue, and one (L-proline)thiazole residue, were established through extensive NMR experiments, including heteronuclear multiple quantum correlation total correlated spectroscopy (HMQC-TOCSY), and... 

A classic example of allosteric inhibition is the case of the enzymatic conversion of L-threonine into L-isoleucine by bacteria. The first of five en mes, threonine dehydratase is inhibited by the end product, isoleucine. This inhibition is very specific, and is accomplished only by isoleucine, which binds to a site on the enzyme molecule called the regulatory, or allosteric, site. This site is different from the active site of the en2yme, which is the site of the catalytic action of the eri2yme on the substrate, or molecule being acted on by the eri2yme. 

Auxotrophic mutants are used in the production of end products of branched pathways, ie pathways leading to more than one amino add at the same time. This is the case for L-lysine, L-methionine, L-threoiune and L-isoleucine in Breoibacterium flavum and Coiynebacteriumglutamicum. 

The economic analysis of MBR processes is often a complex task, because it depends on many variables like the extent of reaction, number and configuration of the separation steps, etc. An interesting study was reported by Weuster-Botz et al [6.17] for the synthesis of L-isoleucine. Isoleucine is an amino acid, which is used in the preparation of infusion solutions in parenteral nutrition therapies its annual production is about 200 tons per year. Weuster-Botz et al [6.17] carried out an economic analysis for the production of... 

Best known are the major component bacitracin A, and bacitracin F, an inactivation product of bacitracin A, through the work of Craig and his co-workers, Abraham and Newton and, to a lesser degree, Porath. The molecular weight of bacitracin A was shown by different methods to be about 1,5001 . Total and partial hydrolysis gave evidence of the presence of three L-isoleucine molecules and one each of L-leucine, L-cysteine, i.-histidine, L-lysine, L-aspartic acid, D-phenylalanine, D-ornithine, D-aspartic acid, D-glutamic acid and ammonia in the molecule. 

Production of L-isoleucine from ethanol and a-keto butyric acid or a-amino butyric acid using a multistep bioconversion with Brevibacterium flavum under native immobilization and biotin-free conditions (Mitsubishi Petrochemical Co., Inc.). Productivity of this system is 200 mmol 1-1 d "1. 

Figure 2. Pattern of continuous production of L-isoleucine using immobilized S. marcescens cells in two-bed reactor. Retention time, 10 hr + 10 hr working volume, 150 mL gel, 50 mL flow rate, 15 mL/hr temperature, 30°C.

A fermentation study [18] using Streptomyces avermitilis culture 5192 and [1- C] acetate and [1- C] propionate precursors, followed by NMR analysis of the products, established the incorporation of seven acetate and five propionate units into the macrocycle. Carbon 25 and its substituents were shown to be derived from L-isoleucine and l-valine following deamination and conversion into the analogs carboxylic acids. That the C-25 substituent is derived from a... 

L-glutamic acid. Today 1.5 Mio mto a of this amino acid are produced worldwide and predominantly used in food as a flavour-enhancing additive. The essential amino acids L-lysine, L-threonine and L-tryptophan are produced by fermentation for the feed additive market. In 2004 worldwide 770,000 mtoa L-lysin and 65,000 mto a L-threonine were produced by bacterial fermentation. According to Ajinomoto sales of feed additive amino acids grow up to 9% per year. Today Evonik Industries produces feed grade L-lysine, L-threonine and L-tryptophan by large-scale fermentation and produces pharma-grade L-alanine, L-aspartic acid, L-isoleucine, L-proline, L-valine and L-tryptophan for applications in the pharma markets. More products from fermentation are shown in Table 12.2. 

Tenuazonic acid (741), a phytotoxin produced by Alternaria spp., is structurally related to the tetramic acid family of compounds, and has been found to exhibit antibiotic activity (511). Since 1964, there have been several publications on the total synthesis of 741 (512-515), including the report by Poncet and his group in 1990 (516) (Scheme 11.10). A general method to synthesize the tetramic acids is an intramolecular Dieckmann cyclization of (V-acyl amino esters. Beginning with methyl L-isoleucinate 780, the A/-acyl compound 782 was obtained through a nucleophilic reaction (512), which then cyclized to tenuazonic acid 741 under basic conditions and neutralization by acidic work-up. The synthetic product showed a diastereomeric excess of 89%, with the major epimer presenting the same configuration as its precursor (517). 

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