Biotehnološki značajni enzimi iz sojeva roda Pseudomonas: identifikacija i rekombinantna ekspresija lakaza i lipaza

Abstract

Bakterije roda Pseudomonas imaju sposobnost produkcije i razgradnje širokog spektra jedinjenja. Cilj ove doktorske teze bio je da se u kolekciji sojeva roda Pseudomonas identifikuju i rekombinantno eksprimiraju nove lakaze i lipaze, i evaluira njihov aplikativni potencijal. Sedam sojeva obuhvaćeno je analizom oksidativne i hidrolitičke aktivnosti prema pet različitih supstrata. Kod sojeva Pseudomonas putida F6, P. putida KT2440 i P. putida CA-3, utvrđeno je prisustvo gena koji kodiraju lakaze. Proteini McoCA3, McoKT, Cbp i CopA heterologo su eksprimirani u E. coli domaćinu, prečišćeni i okarakterisani. Pokazana je njihova aktivnost u širokom pH i temperaturnom opsegu kao i visoka termostabilnost. Ispitana je i sposobnost enzima da razgrade boje koje se koriste u tekstilnoj industriji, potencijalne zagađivače voda. Ćelijski ekstrakt koji je sadržao CopA enzim iz P. putida F6 pokazao je najznačajniju aktivnost razgradivši pet od sedam testiranih boja. Kod sojeva P. aeruginosa PAO1 i P. chlororaphis B-561, detektovana je lipolitička aktivnost i utvrđen potencijal za biorazgradnju polimera kao što su polihidroksialkanoati srednjeg lanca i polikaprolakton, a degradacioni potencijal soja B-561 potvrđen je i u modelu komposta. Homologo su eksprimirana tri proteina – LipA, PlcB i LipA, ali je zaključeno da nije došlo do njihove sekrecije. U ovom radu je pokazano da su Pseudomonas sojevi dobar izvor biotehnološki značajnih enzima lakaza i lipaza. Rekombinantna ekspresija lakaza dala je funkcionalne enzime pogodne za primenu u razgradnji sintetičkih boja iz otpadnih voda. Za primenu rekombinantnih lipaza u biorazgradnji polimernih materijala neophodna je optimizacija sekrecije, ali divlji sojevi koji eksprimiraju ove enzime su dovoljno dobri biokatalizatori za njihovu razgradnju.

Pseudomonas strains have the ability to produce and degrade a wide range of compounds. The main objective of this thesis was to identify, recombinantly express and characterize novel laccases and lipases from Pseudomonas spp. collection, and to evaluate their application potential. Seven strains were analyzed for oxidative and hydrolytic activity towards five different substrates. Genes encoding laccases were detected in P. putida F6, P. putida KT2440 and P. putida CA-3 strains. Proteins McoCA3, McoKT, Cbp and CopA were heterologously expressed in E. coli, purified and characterized, exhibiting broad temperature and pH range and high thermal stability. The ability of enzymes to degrade textile dyes was also examined. All enzymes proved to be very efficient in degradation of synthetic dyes, with the CopA enzyme from P. putida F6 showing the most significant activity, degrading five out of seven tested dyes. Lipolytic activity was detected in two strains: P. aeruginosa PAO1 and P. chlororaphis B-561. The potential for biodegradation of polymeric materials such as medium chain length polyhydroxyalkanoates and polycaprolactone was demonstrated, while degradation potential of B-561 was confirmed in the compost system as well. Three lipases were homologously expressed – LipA, PlcB and LipA, but they were not secreted. This study showed that Pseudomonas genus is a good source of biotechnologically relevant enzymes. Recombinant laccase expression yielded enzymes suitable for application in the degradation of persistent synthetic dyes from wastewaters. For the application of recombinant lipases in the biodegradation of polymers, optimization of the secretion is necessary, however wild type strains expressing these enzymes are good enough biocatalysts for biodegradation purpose.