Peptide synthesis, enzymatic

At the beginning of this century, Sawyalow gave the name plastein to the insoluble material which appeared upon the incubation of a soluble mixture of enzymatic digestion products of fibrin with rennin (a pepsin-like enzyme from calf-stomach). This reaction, later observed also with enzymes different from rennin was studied more intensively in the 1920s by Wasteneys and Borsook [29] who showed that the products of peptic hydrolysis of egg albumin at pH 1.6 gradually formed a precipitate when the concentrated solution was incubated with pepsin at pH 4. 

This plastein was considered to be a protein largely because of its solubility properties. As mentioned in the Introduction (p. 3-4), at that time the nature of proteins was still far from being defined the formation of a slightly soluble product by pepsin could have been a sort of coagulation without the real formation of peptide bonds. This may be the reason why the plastein reaction was not investigated further for more than twenty years, and completely elucidated only in the 1960s. 

The problem of the enzymatic synthesis of peptide bonds assumed a new aspect in around 1937, when Heinz Fraenkel-Conrat [30], then in the laboratory of M. Bergmann, demonstrated the papain-catalyzed formation at pH 5 of sparingly soluble benzoylglycine anilide from benzoylglycine amide or from benzoylglycine (hippuric acid) and aniline as well as the condensation of benzoylleucine with leucine anilide yielding the nearly insoluble dipeptide benzoyldileucine anilide (Fig. 6). 

By these and similar observations with proteolytic enzymes other then papain it appeared that proteinases established an equilibrium between carboxyl (or 

There is a distinct dependence of the equilibrium constant of these systems on the ionization state (pH) of the components. Mechanistically, and thermodynamically, peptide synthesis occurs by negative free-energy (exergonic) change, if the carboxylate anion is protonated and the ammonium cation is deprotonated. Since, however, in aqueous solution of pH around the neutral point (5-8) the groups are ionized, prior to amide bond formation, energy is consumed by transfer of a proton from — NH3 to —C02 (neutralization). 

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Proteases can be used for the synthesis of peptides in a way analogous to the ester synthesis catalysed by lipases. The most successful industrial example of enzymatic peptide synthesis is described in section 4.6 aspartame synthesis. In the industrial process in Europe the equilibrium position is shifted towards synthesis because the... 

W. Kullmann in Enzymatic Peptide Synthesis, CRCPress, Boca Raton, 1987. 

Kullman, W. (1987) Enzymatic Peptide Synthesis, CRC Press, Boca Raon, Florida... 

A. Fischer, A. S. Bommarius, K. Drauz, and C. Wandrey, A novel approach to enzymatic peptide synthesis using highly solubilizing N -protecting groups of amino adds, Biocatalysis 1994, 8, 289-307. 

Aspartame , Artificial Sweetener through Enzymatic Peptide Synthesis... 

When one is using proteases in a direct reversal of their normal hydrolytic function, the equilibrium position is very important in limiting the attainable yield in equilibrium-controlled enzymatic peptide synthesis. If both reactants and products are largely undissolved in the reaction medium as suspended solids, thermodynamic analysis of such a system shows the reaction will proceed until at least one reactant has dissolved completely, towards either products or reactants ( switchlike behavior). In case of a favorable equilibrium for synthesis, the yield is maximized in the solvent of least solubility for the starting materials (Hailing, 1995). Thermolysin-catalyzed reactions ofX-Phe-OH (X = formyl, Ac, Z) with Leu-NH2 yielded X-Phe-Leu-NH2 with equilibrium yields > 90% over a range of solvents. Some predictions, such as a linear decrease in yield with the reciprocal of the initial reactant concentrations, could be verified (Hailing, 1995). 

S. Gerisch, G. Ullmann, K. Stubenrauch, and H.-D. Jakubke, Enzymatic peptide synthesis in frozen aqueous systems influence of modified reaction conditions on the peptide yield, Biol. Chem. Hoppe Seyler, 1994, 375, 825-828. 

Table 7 Enzymatic Peptide Synthesis With ACV Synthetase... 

There is a general belief that peptides are not thermodynamically stable [99], but the formation of an internal bond in a peptide chain actually corresponds to a process that is not far from equilibrium (Kpep 0.1 M-1) and enzymatic peptide synthesis can yield peptides in satisfactory yield provided that the product is removed from the solution by precipitation or other systems of phase partition [100,101]. 

Clapes P, Torres JL, Adlercreutz P. Enzymatic peptide synthesis in low water content systems preparative enzymatic synthesis of [Leu]- and [Met]-enkephalin derivatives. Bioorg. Med. Chem. 1995 3 245-255. 

Besides being used for the production of the precursors 6-APA and 7-ADCA, penicillin amidases are also able to couple 6-APA and 7-ADCA with D-phenyl-glycine ester or amide in a kinetically controlled enzymatic peptide synthesis forming ampicillin or cephalexin (38, 39). These reactions (Scheme 21) have a great potential for being commercialized in the near future [95]. 

An instructive example of the occurrence of selectivity problems is available from the field of enzymatic peptide synthesis. For instance, the following complex reaction scheme shows where parallel reactions and consecutive reactions occur. It describes the synthesis of Tyr-Arg from the electrophile Tyr-OEt and the nucleophile Arg-NH2 by simultaneous use of Carboxypeptidase Y (CPD-Y) and Peptide amidase (PA) from orange flavedo[54) (Eq. (11)). 

Enzymatic Peptide Synthesis in Frozen Aqueous Systems... 

F. Bordusa, H.-D. Jakubke, Enzymatic Peptide Synthesis, in Synthesis of Peptides and Peptidomimetics (Eds. M. Goodman, A. Felix, L. Moroder, C. Toniolo) Houben-Weyl, Vol. E22a, Georg Thieme Verlag, Stuttgart, 2002. 

Planning and Process Development of Enzymatic Peptide Synthesis 851 12.5.4 Conclusion and Outlook 858... 

Figure 1. Possibilities for engineering enzymatic peptide synthesis at different levels.

H. Kitaguchi and A.M. Klibanov Enzymatic peptide synthesis via segment condensation in the presence of water mimics. Journal of the American Chemical Society 111 (1989) 9272-9273. 

Acyl enzyme, an intermediate in the catalytic mechanism of serine proteases, such as trypsin and chymotrypsin. After the serine protease has bound a peptide substrate to form the Michaelis complex, Ser (in the case of chymotrypsin) nucleophilically attacks the peptide bond in the rate-determining step, forming a transition-state complex, known as a tetrahedral intermediate. The latter decomposes to the acyl enzyme, an extremely unstable intermediate, that bears the acyl moiety at the hydroxy group of Ser . The acyl enzyme intermediate is deacylated by water during proteolysis, or the attacking nucleophile is an amino component in case of kineticaUy controlled enzymatic peptide synthesis. 

Enzymatic peptide synthesis, enzyme-catalyzed synthesis, the application of enzymes for peptide bond formation. The very complex ribosomal peptidyl transferase center is not suitable as a catalyst for simple practical peptide synthesis. However, enzymes which usually act as hydrolases, catalyzing the cleavage of peptide bonds, should be considered as peptide ligases due to the principle of microscopic reversibility. The protease-catalyzed peptide synthesis has been developed as a useful tool in the new catalytic synthetic approach based on protease-engineering and the develop-... 

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