Recent Trends in Protease-Catalyzed Peptide Synthesis

Abstract

Enzymatic peptide syntheses may be either thermodynamically- or kinetically-controlled. The former may be catalyzed by any proteases; the latter is limited to serine and cysteine proteases. This methodology is quite stereospecific and avoids side chain protection but is suffering of some drawbacks. Thus, only two industrial processes are by now developed: the production of aspartame and the conversion of porcine into human insulin. However, recent improvements have been carried out in different directions: 1-Search for proteases with high and/or new P'1 and P1 specificities. 2-Protease engineering to promote synthesis towards hydrolysis and to enlarge specificity. 3-Development of mimetic or "inverse" substrates to limit further hydrolysis of synthesized peptide. 4-Change of the physical state of reactants. Three axes have mainly be explored: solid-solid conversion, use of cross-linked enzyme crystals (CLEC) and enzyme immobilization. 5-Modification of experimental conditions. The principal and recent developments deal with: heterogeneous catalysis, synthesis in low water-containing organic solvents, in ionic liquids or at subzero temperatures. This review will illustrate these new orientations with examples described in the recent literature.

Kinetically controlled enzymatic synthesis of dipeptide precursor of l‐alanyl–l‐glutamine

“…It offers several advantages over chemical methods, such as enantioselective catalysis; racemization-free fragment formation; dispensation of side-chain protection; and use of mild, nonhazardous reaction conditions that protect health and the environment [7], [8]. Proteases are the most widely utilized enzymes in peptide synthesis because they can catalyze the condensation of peptide bonds [9].…”

Applications of Enzymes in Synthesis and Modification of Polymers

“…72 A síntese de polipeptídeos catalisada por proteases pode ocorrer regida por um controle termodinâmico ou cinético. 73 No caso do controle termodinâmico (aplicável a todos os grupos de proteases), a síntese está em equilíbrio com a hidrólise (reação reversa). As desvantagens dessa abordagem são as baixas taxas de reação e rendimentos nos produtos, quantidade grande de enzima necessária, e as condições de reação que devem ser utilizadas de forma a se deslocar a reação em direção à síntese.…”

Proteases as Powerful Catalysts for Organic Synthesis

“…Contrary to TCS, only serine or cysteine proteases can be used to perform KCS, because the enzyme acts in this case as a transferase and catalyzes the transference of an acyl group from the acyl donor to the amino acid nucleophile through the formation of an acyl-enzyme intermediate. However, there are different strategies to overcome such problems, which comprise the engineering of the reaction medium, the biocatalyst and the substrate [156,198]. It also requires lower enzyme: substrate ratios because the acyl donor is now in the form of an activated carboxylic acid [138].…”

“…Their specific activity is very high since there is no inert matrix and the whole mass of biocatalyst is essentially pure enzyme protein. Site-directed mutagenesis has also been employed to improve the properties of trypsin for performing peptide synthesis [198], and directed evolution based on tandem random mutagenesis has been successfully applied to improve the thermal stability of subtilisin [20,270] and increase the activity and selectivity of an endoprotease from Escherichia coli [271]. CLEAs prepared by precipitation with salts, organic solvents, and polymers, and cross-linked with glutaraldehyde have been quite effective for peptide bond formation in the synthesis of b-lactam antibiotics in nonaqueous environments [18,263,264].…”