BIOTECHNOLOGY - V. H. Gerasymenko - 2006

Part II. Special Biotechnologies

Chapter 19. BIOTECHNOLOGIES FOR L-AMINO ACID PRODUCTION

19.1. METHODS FOR L-AMINO ACID PRODUCTION

Given the national economy's demand for Amino Acids, large-scale industrial production has been established. Industrial production of amino acids is based primarily on three processes: Hydrolysis of natural protein-containing raw Materials (of PLANT AND ANIMAL origin), chemical synthesis, and microbiological synthesis. Obtaining amino acids via hydrolysis of protein substances is the oldest and least efficient method. Its main disadvantages include the high cost of the resulting Amino Acids and limited raw material resources.

Among the industrial-scale Methods for producing amino acids, microbiological and chemical methods are of the greatest importance. In modern global practice, microbial synthesis accounts for about 60% of the total volume of manufactured amino acids.

Chemical synthesis of amino acids is quite efficient. It makes it possible to obtain compounds of any Structure and to organize production with a high level of automation. Today, The production of many amino acids via organic synthesis—such as Methionine, Lysine, glutamic acid, Tryptophan, and Glycine—has been developed and implemented on an industrial scale. Modern technologies also enable the synthesis of individual amino acids with high yields and a high degree of purity. However, this method has A number of significant drawbacks.

Specifically, while modern fine organic synthesis methods can yield D- and L-forms of amino acids in any quantities, they exist as racemates—that is, equal-mass mixtures of L- and D-amino acids whose molecules are mirror images of each other. In Chemical Reactions, these isomers are indistinguishable. However, as is well known, animal (human) organisms utilize exclusively L-amino acids. The second isomer (the D-amino acid) lacks biological activity and therefore cannot participate in METABOLISM/35.html">Protein Biosynthesis. Mammalian Proteins are constructed entirely from a single type of amino acid—the L-form. The presence of a D-amino acid in the final product is always undesirable not only because it acts as ballast, but also because Some amino acids exhibit toxic properties. Exceptions include glycine and methionine; the former has no optically active isomers, while for the latter, both D- and L-forms are assimilated equally by Human and Animal organisms.

Furthermore, the production of amino acids via organic synthesis involves A large number of technological operations. Practically every step requires specific equipment. In most cases, the technology relies on sufficiently toxic substances, highly purified Reagents, and a step to separate the resulting racemates. The methods for separating racemic mixtures, in turn, are quite complex and costly, and for Certain amino acids, they have not been developed at all.

Resolution of the racemic mixture into L- and D-amino acids. Among the existing methods for resolving racemic mixtures into their constituent isomers, the enzymatic method based on The Use of fungal aminoacylase is the most suitable. Acylated D- and L-isomers of amino acids obtained via chemical synthesis are used as starting materials and treated with aminoacylase. The enzyme selectively hydrolyzes only the acyl-L-isomer, cleaving off its bulky acyl group. As a result, the corresponding L-amino acid is formed, which has a higher solubility than the unreacted acyl-D-isomer remaining in solution.

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Enzymatic hydrolysis yields a mixture consisting of the L-Amino Acid and the acyl-D-amino acid isomer. It is easily separated because its components have different solubilities, allowing the L-amino acid to be isolated. The remaining acyl-D-amino acid is racemized upon heating—meaning it reverts to a mixture of acylated L- and D-amino acids—and the process is repeated from the beginning. Thus, the sole product is the L-amino acid.

Since aminoacylase exhibits strict Specificity only toward The structure of the acyl moiety and low specificity toward the side chain STRUCTURE OF THE amino acid, it can be used for the resolution of numerous amino acids.

Industrial-scale resolution of a racemic mixture into its constituent isomers was first accomplished in Japan by Tanabe Seiyaku in 1969. Initially, the native enzyme was used, followed later by the immobilized enzyme, which increased the economic efficiency of the process by 1.5 times. Today, the company industrially produces five L-amino acids, four of which are essential (methionine, valine, phenylalanine, tryptophan).

Commercial preparations of aminoacylase are immobilized by adsorption onto DEAE-Sephadex via ionic interactions (by the Japanese company Tanabe Seiyaku) and by entrapping the enzyme within Cellulose triacetate hollow fibers (by the Italian company Snamprogetti).

Microbial synthesis is the most promising and economically viable method for Amino Acid Production, serving as an alternative to chemical synthesis. This method can be used to obtain almost all Proteinogenic Amino Acids. THE PRINCIPLE OF the microbial method involves the aerobic CULTIVATION OF MICROORGANISMS in nutrient solutions containing various sources of carbon, nitrogen, mineral salts, and growth factors. The microbial method has several advantages over chemical synthesis. First and foremost, the enzyme System of the microbial Cell produces exclusively the biologically active L-isomer, which facilitates its Isolation and Purification, enabling the release of low-cost technical preparations in sufficient quantities.

The foundation for establishing large-scale industrial production of L-amino acids via the microbiological method was the discovery by Japanese scientists of the oversynthesis phenomenon regarding extracellular amino acids by auxotrophic bacterial mutants belonging to the genera Brevibacterium, Micrococcus, Corynebacterium, etc.

Auxotrophic mutants are microbial Cells that, on the one hand, have lost The ability to independently synthesize various amino acids necessary for GROWTH AND DEVELOPMENT, but on the other hand, have acquired the capacity for the oversynthesis of a target amino acid. Such mutants can be obtained by exposing the original microbial culture to various Physical and Chemical mutagens followed by strain Selection based on predetermined traits, or via Introduction/32.html">Genetic Engineering METHODS.

Consequently, specially selected, screened, and occasionally genetically engineered producer strains perform what is known as the oversynthesis of L-amino acids during their life cycle (usually at various developmental stages), producing them in quantities far exceeding the cells' own requirements. The excess L-Amino acids are excreted into the culture liquid, from which they are subsequently extracted.

Industrial production of L-amino acids using microorganisms can be carried out according to two technological schemes. They differ primarily in the stage at which the culture liquid is obtained. The first scheme involves a two-stage process for producing the culture liquid, while the second involves a single-stage process.

The two-stage method is based on using a precursor of The biosynthesis of the desired amino acid as a raw material, which can be obtained via chemical or biological methods. Obtaining the precursor is The First stage of production. This also includes

the biosynthesis of the enzymatic preparation (typically of microbial origin) that will mediate the transformation of the precursor into the target amino acid.

The Second Stage is the actual process of transforming the precursor into The amino acid using the enzyme systems of the microorganism cultivated in the first stage.

The single-stage method of Amino acid synthesis is the most widespread and is based on cultivating a well-defined producer strain of the target amino acid in a medium of a specific composition under appropriate Fermentation parameters. Polyauxotrophic mutants are typically chosen for this purpose.

In the production of Amino acids as highly purified crystalline preparations, upon completion of fermentation, the producer cells are separated from the culture liquid. The target amino acid is isolated from the culture liquid using Ion Exchange or precipitation methods. Eluates or mother liquors are concentrated by vacuum evaporation, and the resulting technical crystals are purified by recrystallization from a saturated solution. The process of obtaining the crystalline preparation typically concludes with vacuum drying of the purified crystals and their packaging.

By-products of such production can include various feed additives based on different manufacturing wastes—such as amino acid mother liquors, producer biomass, and wash waters—which are dried to a residual moisture content of about 10 %.

When producing feed additives with a low content of the target amino acid (no more than 10 %), the manufacturing process typically involves only stabilizing the culture liquid solution prior to vacuum evaporation, concentrating the dry matter of the culture liquid, standardizing the solution by adding a filler, drying the final product, and packaging it.

When obtaining technical-grade or feed products with an elevated content of the main substance (amino acid), producer cells are additionally separated, and amino acids in the native solution are partially concentrated using ion exchange or precipitation methods.

The drawbacks of the microbiological method for amino acid production also lie in the inherent specifics of applying microbiological techniques. Furthermore, our understanding of cellular metabolism and its regulatory pathways is not always comprehensive.

When the direct fermentation pathway is insufficiently developed or economically unviable, amino acids are obtained via a combined chemo-microbiological route, where the precursor compound is synthesized through chemical reactions while The final stage is carried out by microorganisms.

Production of amino acids from protein hydrolysates. This method involves the hydrolysis (acid, alkaline, or enzymatic) of some of the most accessible natural proteins. These include meat Processing wastes, milk casein, wheat gluten, and others. The disadvantages of this approach include limited and non-standardized raw material sources, multi-stage chemical processing associated with amino acid isolation and purification, and the fact that hydrolysis using mineral agents leads to the partial destruction of valuable amino acids such as tryptophan, Threonine, Cysteine, and Serine.