Lysobacter sp. A03 : uma abordagem genômica e funcional para a busca de peptidases ativas em baixas temperaturas

Abstract

Peptidases são responsáveis pela hidrólise de ligações peptídicas em cadeias polipeptídicas e, além do seu papel vital em todos os organismos vivos, possuem uma longa história de utilização pela humanidade, principalmente no processamento de alimentos. Estima-se que o mercado global de enzimas chegue a US$7,1 bilhões em 2018, dos quais 60$7.1 billion in 2018, of which the peptidases account for approximately 60%. However, the majority of the commercially available peptidases were isolated from thermophilic and mesophilic organisms. Psychrophilic enzymes are a valuable alternative as they are active at low temperatures, reducing the energy costs of its utilization and as they can be easily inactivated, being useful in reactions that have to be performed under mild conditions. Aiming to search for peptidases with keratinolytic activity under low temperatures, Antarctic bacterial strains were isolated and based on its ability of degrading keratinous waste, three of them were chosen and had their biochemical parameters evaluated. Considering its higher keratinase activity, the bacterium Lysobacter sp. A03 was selected to have its genome sequenced and for the construction of a genomic library to search for keratinolytic peptidases with biotechnological and industrial potential. Through this strategy, one serine-endopeptidase, called A03Pep1, was found and structurally and functionally characterized. Its precursor, of 72.5 kDa, was composed by five domains that, by means of autoprocessing, originate the mature enzyme, estimated in 35 kDa. The heterologous expression was performed in pGEX-4T-2 vector and the recombinant peptidase A03Pep1 was recovered mainly in the supernatants of E. coli ArcticExpress cells cultures. The optimal activity of the enzyme was at pH 9.0 and 40°C and was enhanced in the presence of Ca2+ until 25 mM and in the presence of 10 mM Na2+, NH4+, Mg2+ and Ba2+ and was inhibited by 10mM of Zn2+. Based on the 3D structure modelling, was predicted a wider substrate binding site in A03Pep1 when compared with its mesophilic homologous AprV2, probably due to the shortening of a loop region located next to the active site of the enzyme, presumably as a way to enhance the probability of binding of substrates at low temperatures. By discussing the functional and structural characteristics found in the psychrophilic peptidase A03Pep1, the results provide possible approaches in developing new biotechnologically relevant enzymes active under low to moderate temperatures