Peptide Synthesis Using Proteases as Catalyst

Abstract

Proteolytic enzymes (proteases) comprise a group of hydrolases (EC 3.4, NC-IUBMB) which share the common feature of acting on peptide bonds. Proteases are among the best studied enzymes in terms of structure-function relationship (Krowarsch et al., 2005). Proteases, by catalyzing the cleavage of other proteins and even themselves, have an enormous physiological significance, their coding genes representing as much as 2% of the total human genome (Schilling and Overall, 2008).Proteases, together with lipases, represent the most important family of enzymes at industrial level, accounting for well over 50% of the enzyme market (Feijoo-Siota and Villa, 2011). Proteases have been used industrially since the onset of enzyme technology in the first decades of the 20th century; many of the early patents issued for the use of enzymes with commercial purposes were proteases, mostly from plant (papain, bromelain) and animal (trypsin, pepsin) origin. Intended uses were in brewing and in leather and rubber manufacturing (Neidelman, 1991). In the decades that follow many large-scale industrial processes were developed using now mostly microbial proteases. A common feature of them was the degradation of proteins and most relevant areas of applications were the food and beverage (Sumantha et al., 2006), detergent (Maurer 2004), leather (Foroughi et al., 2006) and pharmaceutical sectors (Monteiro de Souza et al., 2015). Acid and neutral proteases are relevant to the food industry for the production of protein hydrolyzates (Nielsen and Olsen, 2002), beer chill-proofing (Monsan et al., 1978), meat tenderization (Ashie et al., 2002) and above all, for cheese production (Kim et al., 2004). Alkaline proteases are of paramount importance for the detergent industry (Sellami-Kamoun et al., 2008) and also in tannery (Varela et al., 1997; Thanikaivelan, 2004) and fish-meal production (Schaffeld et al., 1989; Chalamaiah et al., 2012). These conventional applications are by no means outside of continuous technological development (Monteiro de Souza et al. 2015). This is illustrated by the optimization of detergent enzyme performance under the harsh conditions of laundry at high and low temperatures, which has been a continuous challenge tackled by the construction of subtilisin (alkaline protease) variants by random and site-directed mutagenesis and by directed evolution (Kirk et al., 2002; Jares Contesini et al., 2017). It is also illustrated by the production of chymosin in microbial hosts by recombinant DNA technology and further improvement by protein engineering (Mohanty et al., 1999). Therapeutic application of proteases acting as protein hydrolases goes from conventional digestive-aids and anti-inflammatory agents to more sophisticated uses as trombolytic drugs (i.e. urokinase and tissue plasminogen activator) and more recently for the treatment of haemophilia. A comprehensive review on the therapeutic uses of proteases is suggested for the interested reader (Craik et al., 2011)The potential of hydrolytic enzymes for catalyzing reverse reactions of bond formation has been known for quite some time. However, its technological potential as catalysts for organic synthesis developed in the 1980s (Bornscheuer and Kazlauskas, 1999) paralleling the outburst of biocatalysis in non-conventional (non-aqueous) media (Illanes, 2016).Proteases can not only catalyze the cleavage of peptide bonds but, in a proper reaction medium, they can also catalyze the reaction of peptide bond formation. Proteases are highly stereo- and regiospecific, active under mild reaction conditions, do not require coenzymes and are readily available as commodity enzymes, these properties making them quite attractive catalysts for organic synthesis (Bordusa, 2002; Kumar and Bhalla, 2005). Such reactions will not proceed efficiently in aqueous medium where the hydrolytic potential of the enzyme will prevail, so reaction media at low, and hopefully controlled, water activity is necessary for peptide synthesis. This is a major threat since proteases, different from lipases, are not structurally conditioned to act in such environments. The use of proteases in peptide synthesis is analyzed in depth in section 3.4.