Isolation of bacteria and characterization of their enzymes to develop biocatalysts for plastic biodegradation

[B. Pantelić]

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Biologija - Molekularna biologija prokariota / Biology - Molecular biology of prokaryotes

Usled masovne proizvodnje i neadekvatnog korišćenja plastike došlo je do rastućeg globalnog problema akumulacije plastičnog otpada. Različite strategije su primenjene kako bi se ovaj problem rešio, ali upotreba mikroorganizama i njihovih enzima za razgradnju plastičnih polimera bio je fokus istraživača u proteklih nekoliko decenija. Glavni ciljevi ove doktorske disertacije bili su izolacija i karakterizacija bakterijskih sojeva koji razgrađuju plastiku, prevashodno poli(etilentereftalat) (PET) i poli(uretane) (PU), njihov dalji razvoj u efikasne biokatalizatore, identifikacija enzima odgovornih za navedenu aktivnost i istraživanje potencijalnih metoda biološke valorizacije plastičnog otpada. Ispitivanjem laboratorijske kolekcije mikroorganizama tri bakterijska soja su odabrana kao potencijalni biokatalizatori za razgradnju plastike. Bacillus subtilis BPM12 efikasno je formirao biofilm na PET plastici i pokazao izuzetnu aktivnost na bis(2-hidroksietil) tereftalatu i različitim supstratima, strukturno sličnim PET oligomerima i monomerima. Kroz heterolognu i homolognu ekspresiju, potvrđeno je da je enzim Bpm12CE ključan za ovu aktivnost. Delovanjem više različitih enzima, Amycolatopsis mediterranei ISP5501 razgrađivao je različite vrste PU, a upotrebom model jedinjenja PU-7 potvrđena je i hidroliza uretanske veze. Streptomyces sp. PU10 pokazao se kao jedan od najaktivnijih, do sada prijavljenih, sojeva za hidrolizu PU disperzije – Impranil sa preko 96% razgradnje za 72 h. Na osnovu rezultata kvantitativne proteomike zaključeno je da su u razgradnju PU uključene esteraza, amidaza i oksidaza ovog soja, dok se produkti razgradnje inkorporiraju u primarni metabolizam Streptomyces sp. PU10 i usmeravaju ka biosintezi poliketida. Konačno, uspostavljena je i strategija za konvertovanje biorazgradivih skrob/poli(vinil-alkohol) filmova u jedinjenja sa dodatom vrednošću: biopolimer poli(hidroksibutirat) i biološki aktivni biopigment undecilprodigiozin.

Due to mass production and inadequate plastic usage, a growing issue of global plastic waste accumulation has emerged. Various strategies have been employed to address this problem, but using microorganisms and their enzymes for plastic polymer degradation has captivated researchers' attention in recent decades. The primary goal of this doctoral dissertation was the isolation and characterization of bacterial strains capable of degrading plastics, mainly poly(ethylene terephthalate) (PET) and poly(urethanes) (PU), and their further development into efficient biocatalysts, the identification of enzymes responsible for this activity and exploration of potential methods for the biological valorization of plastic waste. From a laboratory collection of microorganisms, three bacterial strains were selected as potential biocatalysts for plastic degradation. Bacillus subtilis BPM12 efficiently formed biofilm on PET plastic, exhibiting exceptional activity on bis(2-hydroxyethyl) terephthalate and structurally similar PET oligomers and monomers. Through heterologous and homologous expression, the enzyme Bpm12CE was confirmed to be crucial for this activity. Amycolatopsis mediterranei ISP5501 degraded various types of PU using multiple enzymes, and the hydrolysis of the urethane bond was confirmed using the model compound PU-7. Streptomyces sp. PU10 demonstrated high activity, with over 96% degradation of PU dispersion - Impranil within 72 hours. Quantitative proteomics results indicated that an esterase, amidase, and oxidase of this strain were involved in PU degradation, with the degradation products being incorporated into the primary metabolism of Streptomyces sp. PU10 and directed towards polyketide biosynthesis. Finally, a strategy was established for converting biodegradable starch/poly(vinyl alcohol) films into value-added compounds: the biopolymer poly(hydroxybutyrate) and the biologically active biopigment undecylprodigiosin.

Plastika, PET, PU, Biodegradacija, Biokataliza, Bacillus, Amycolatopsis, Streptomyces, Undecilprodigiozin

Plastic, PET, PU, Biodegradation, Biocatalysis, Esterase, Bacillus, Amycolatopsis, Streptomyces, Undecylprodigiosin

577.2:577.15(043.3)

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