Enzymatic degradation of PET by hydrolase from Brucella intermedia IITR130 and its genomic insights

Abstract

Plastic pollution, particularly from polyethylene terephthalate (PET), has become a significant environmental concern, necessitating innovative and sustainable degradation strategies. The present study provides valuable perspectives on the genomic and functional characteristics of Brucella intermedia IITR130, a bacterium capable of degrading PET. Hybrid genome sequencing of IITR130 resulted in identification of two chromosomes combining 4.59 Mbp size. Genomic annotation revealed occurrence of key enzymes involved in the PET sheet biodegradation pathway, including hydrolases, ring hydroxylating dioxygenases, protocatechuate 3,4 dioxygenases, genes for metabolism of several other natural and synthetic plastic. A hydrolase gene Hy1 of 24 kDa, was identified, expressed, and characterized, demonstrating an optimal catalytic activity at 37 °C and pH 8.5. Scanning electron microscopy (SEM) and fourier-transform infrared spectroscopy (FTIR) confirmed substantial degradation of PET surfaces treated with Hy1 protein, resulted in surface erosion, crack formation, and functional group modifications in the range 2150–2550 cm⁻¹ and 2950–3350 cm⁻¹ suggestive of O=C=O stretching and O–H stretching respectively. Monomethyl terephthalate (MMT) and terephthalic acid (TPA) were identified as PET degradation metabolites formed by strain IITR130. Fluorescence quenching showed higher substrate affinity for bis(2-hydroxyethyl) terephthalate (BHET) (K_d = 148.2) than terephthalic acid (TPA) (K_d = 674). Moreover, phylogenetic analysis of Hy1 protein revealed that Hy1 containing conserved catalytic triad (Ser¹⁰⁸, His¹⁸⁸, Asp¹⁵⁵) belonging to the family III of hydrolase enzyme sharing a clade with PET degrading hydrolase PETase from Ideonella sakaiensis. These results demonstrate the potential of B. intermedia IITR130 as an efficient biocatalyst for PET biodegradation which could be exploited appropriately for plastic waste management.