Peptide Synthesis in Organic Solvents Catalyzed by an Industrial Alkaline Protease “Alcalase”

Abstract

An industrial alkaline protease "Alcalase'' is stable (half life> 5 days in ethanol or in r-butanol) and active in alcoholic solvents (except methanol), and usable as a catalyst for kinetically controlled peptide bond formation in these solvents. We give an overview of our use of alcalase as catalyst in peptide synthesis; it includes 1) the stability of alcalase in various organic solvents; 2) the resolution of N-protected amino acids at large concentration of organic solvent; 3) the saponification of Fmoc-peptide esters in high concentration of organic solvent; 4) kinetically controlled peptide bond formation in alcoholic solvent, and 5) enzymatic synthesis of a Cathepsin inhibitor, peptidyl (J-acyl hydroxamates,

Papain Catalyzed Oligomerization in Monophasic Aqueous Organic Media - Synthesis and Characterization of Neutral and Polar Amino Acid Oligomers

“…Because of the increased solubility, higher oligomers are available to act as nucleophile in ACN/ water systems whereas they completely precipitate out in aqueous systems. The obtained result correlates with the well-accepted acylintermediate mechanism for catalytic action of proteases [20,30]. As the size of the oligomer chain increases, it cannot fit into the active site of the enzyme to form acyl-intermediate complex.…”

“…The enzymatic peptide synthesis is a typical two-step process (i.e., formation of an acyl-enzyme complex and nucleophilic attack of the second substrate or water on the complex to form a peptide or hydrolyzed product). Yield of the process is a function of the relative rates of hydrolysis and aminolysis [20]. When the reaction is carried out in monophasic aqueous organic solvent systems, hydrolysis is greatly reduced; it is comparable to bi-phasic and tri-phasic solvent systems the cysteine proteases present in papaya (Carica papaya) which is useful in tenderizing meat and other proteins.…”

“…The low water content media are highly attractive in kinetically controlled peptide synthesis because the secondary hydrolysis of the product peptide is minimized [14]. However, in all monophasic systems, a certain minimum amount of water is essential for the catalytic activity of the enzyme [20]. The effect of water in monophasic reaction mixtures has been quantified in terms of changes in product conversion upon changes in water content [21].…”

“…It is a well-known fact that L and D enantiomers have different biological activity. Enzymatic resolution of L/D forms of amino acids has been studied extensively [20][21][22][23].…”

Thermal Inactivation Kinetics of Suspensions of α-Amylase in Hydrophobic Organic Solvents

“…Results reported in literature indicated that the stability of dried enzymes in organic solvents changed with water content and the nature of the organic solvent used (2,3). However, there are almost no data available in the literature concerning the effect of organic solvents on the temperature sensitivity of the inactivation rate (z value or activation energy); for most proteins studied so far, inactivation experiments were carried out at only one or two different temperatures (2)(3)(4)(5). The activation energy (466.5 kJ/mol) for the inactivation of polyphenol oxidase in toluene with 8 mL/L water was reported (6), but the authors did not study the effect of other water concentrations or the use of other solvents.…”