Amino acids enzymatic synthesis

There are two main routes for the production of D-amino acids chemical synthesis and enzymatic catalysis. As regards conventional chemical synthesis, unless asymmetrical starting compounds or catalysts are used, a mixture of the D- and L-stereoisomers is obtained in equal proportions. The racemic mixture is therefore optically inactive and the stereoisomers must be separated. The separation of the enantiomers by classical crystallization of diastereomeric salts is the most costly production step and in any case this method can only yield 50% of the desired enantiomer [3]. Enzymatic synthesis can solve the above problems, providing optical purity of the D-amino acid and a 100% yield, as well as mild, environment-friendly reaction conditions. 

This book is based predominantly on the patent literature and provides how to data regarding the production, purification, and application of commercial enzymes. Coverage is not limited to food applications, and 70 subjects are grouped as Enzymes, Enzymatic Processing, Enzyme Stabilization, Polymer-Enzyme Products, Cell Culture, Protein Analysis, Nucleic Acids etc.. Amino Acids, Peptide Synthesis, and Applications. Indexing includes U.S. patent number, company and patent assignee, inventor, and subject. 

Four main processes are distinguished in the production of amino acids chemical synthesis, isolation from protein hydrolysates, enzymatic and microbiological methods of production, which is currently the most important. The following sections will further elucidate the important... 

Chiral amines have been attracting attention as an important composition, particularly for pharmaceutical products. The organic synthetic methods of optically active amine compounds have been developed through the traditional resolution of racemic amines with the formation of diastereomer salts using an optically active mandelic acid or tartaric acid. Enzymatic synthesis has mainly used lipase and S- or R-stereoselective amine transaminase (AT) [29-31] (Figure 19.7). Turner et al. successfully synthesized chiral (R)- and (S)-amines by kinetic resolution using a combination of stereoselective AT and d- or L-amino acid oxidase (AAOx) [32] (Figure 19.7). However, the theoretical yield of the products has been limited to 50% in the kinetic resolution. 

Optically pure d- and L-a-amino acids are of increasing interest as precursors for semi-synthetic antibiotics, new herbicides, insecticides, and physiologically active peptides, and as chiral building blocks for chemical synthesis. For the production of optically active a-amino acids, enzymatic processes have been developed other than fermentation to cover a wide range of products, not only natural L-a-amino acids but also unnatural and D-a-amino acids (Table 1). 

In many cases only the racemic mixtures of a-amino acids can be obtained through chemical synthesis. Therefore, optical resolution (42) is indispensable to get the optically active L- or D-forms in the production of expensive or uncommon amino acids. The optical resolution of amino acids can be done in two general ways physical or chemical methods which apply the stereospecific properties of amino acids, and biological or enzymatic methods which are based on the characteristic behavior of amino acids in living cells in the presence of enzymes. 

Enzymatic hydrolysis of A/-acylamino acids by amino acylase and amino acid esters by Hpase or carboxy esterase (70) is one kind of kinetic resolution. Kinetic resolution is found in chemical synthesis such as by epoxidation of racemic allyl alcohol and asymmetric hydrogenation (71). New routes for amino acid manufacturing are anticipated. 

Biotransformations are carried out by either whole cells (microbial, plant, or animal) or by isolated enzymes. Both methods have advantages and disadvantages. In general, multistep transformations, such as hydroxylations of steroids, or the synthesis of amino acids, riboflavin, vitamins, and alkaloids that require the presence of several enzymes and cofactors are carried out by whole cells. Simple one- or two-step transformations, on the other hand, are usually carried out by isolated enzymes. Compared to fermentations, enzymatic reactions have a number of advantages including simple instmmentation reduced side reactions, easy control, and product isolation. 

In this chapter we consider amino acid production by fermentation and by chemo-enzymatic methods. We first consider the stereochemistry of amino adds and the importance of chirality in chemical synthesis. General approaches to amino add fermentation and recovery of amino adds from fermentation broths are then dealt with, followed by a detailed consideration of the production of L-phenylalanine by direct fermentation. Later in this chapter, chemo-enzymatic methods of amino acid... 

Auxotrophic mutant lack one or more enzymes involved in the synthesis of amino acids (such as tyrosine). This prevents accumulation of the amino acid and thus avoids feedback inhibition of enzymatic steps in the L-phenylalanine pathway. 

The synthesis of a-amino acids by reaction of aldehydes or ketones with ammonia and hydrogen cyanide followed by hydrolysis of the resulting a-aminonitrile is called the Strecker synthesis. Enzymatic hydrolysis has been applied to the kinetic resolution of intermediate a-aminonitriles [90,91]. The hydrolysis of (rac)-phenylglycine nitrile... 

The main application of the enzymatic hydrolysis of the amide bond is the en-antioselective synthesis of amino acids [4,97]. Acylases (EC 3.5.1.n) catalyze the hydrolysis of the N-acyl groups of a broad range of amino acid derivatives. They accept several acyl groups (acetyl, chloroacetyl, formyl, and carbamoyl) but they require a free a-carboxyl group. In general, acylases are selective for i-amino acids, but d-selective acylase have been reported. The kinetic resolution of amino acids by acylase-catalyzed hydrolysis is a well-established process [4]. The in situ racemization of the substrate in the presence of a racemase converts the process into a DKR. Alternatively, the remaining enantiomer of the N-acyl amino acid can be isolated and racemized via the formation of an oxazolone, as shown in Figure 6.34. 

Enzyme preparations from liver or microbial sources were reported to show rather high substrate specificity [76] for the natural phosphorylated acceptor d-(18) but, at much reduced reaction rates, offer a rather broad substrate tolerance for polar, short-chain aldehydes [77-79]. Simple aliphatic or aromatic aldehydes are not converted. Therefore, the aldolase from Escherichia coli has been mutated for improved acceptance of nonphosphorylated and enantiomeric substrates toward facilitated enzymatic syntheses ofboth d- and t-sugars [80,81]. High stereoselectivity of the wild-type enzyme has been utilized in the preparation of compounds (23) / (24) and in a two-step enzymatic synthesis of (22), the N-terminal amino acid portion of nikkomycin antibiotics (Figure 10.12) [82]. 

A major difference between the classical transmitters and peptides is that the production of a peptide is quite different since the synthesis of a peptide is in the form of a huge precursor of about 300 amino acids which is produced in the nucleus of the cell and then transported to the terminal being processed en route (Fig. 12.1). The prepropeptide is produced by translation in ribosomes and so occurs only in cell bodies or dendrites while the classical transmitters are produced at the terminal via a short series of enzymatic steps from a simple precursor. The study of the production of the propeptides have revealed a series of principles in that ... 

Enzymes are generally classified into six groups. Table 1 shows typical polymers produced with catalysis by respective enzymes. The target macromolecules for the enzymatic polymerization have been polysaccharides, poly(amino acid)s, polyesters, polycarbonates, phenolic polymers, poly(aniline)s, vinyl polymers, etc. In the standpoint of potential industrial applications, this chapter deals with recent topics on enzymatic synthesis of polyesters and phenolic polymers by using enzymes as catalyst. 

Figure 7.16 Enzymatic synthesis of the a-hydroxy-/3-amino acid moiety of (—)-hestatin... 

Cells grown in the presence of azide analogs of certain amino acids or sugars will incorporate these derivatives into proteins or carbohydrates through enzymatic synthesis using... 

In normal cells, the GDP/GTP-binding proteins, after protein synthesis, move to the cell membrane to which they become hooked by a hydrophobic farnesyl group. The y-subunit is anchored in the membrane by a post-translational modification of the C-terminal CAAX sequence (C - cystein, AA - aliphatic amino acids, X - methionine). This protein is first enzymatically farnesylated by a specific farnesyltransferase, then the AAX part is cleaved by specific proteases and finally the cystein residue is converted to a methyl ester. 

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