Carboxylic ester hydrolases from Antarctic psychrophilic Psychrobacter strains: From genome prospecting to biotreatment of polyester plastics

Abstract

Background: Antarctica can serve as the source of novel extremophilic microorganisms and biomolecules, including extremozymes which could find applications in bioremediation processes for the presence of enzymes able to degrade plastic polymers. Carboxylic ester hydrolases (EC 3.1.1) catalyze the hydrolysis of linear and cyclic ester bonds. Among these, esterases (EC 3.1.1.1) and triacylglycerol lipases (EC 3.1.1.3) have been shown effective in degrading polyester polymers.

Investigation and methodologies: Two psychrophilic bacterial strains ASPA161_6 and ASPA161_9 were isolated from environmental samples, and their optimal growth conditions were determined. The marine sediment samples were collected at a depth of 20 m in the Antarctic Specially Protected Area n. 161 (74°42' S, 164°07' E, Ross Sea) during the 2017/2018 XXXIII Italian Antarctic expedition. Genomes were sequenced, assembled and annotated. Genes encoding for proteins involved in cold adaptation were searched with an in-silico search approach. Moreover, the sequences of genes involved in the degradation of polyester plastics were studied through the annotation via PlaticDB tool. Esterase and lipase activities were also detected in-plate and quantitatively evaluated at different temperatures on crude protein extracts from the extracellular fraction of the microorganisms using a specific assay based on the hydrolysis of p-nitrophenyl esters. Furthermore, the same fractions were tested onto the polymer polyethylene adipate as a model of biodegradable polyester plastic. The degradation studies with polyethylene adipate were performed following the reaction progress with ¹H NMR.

Results and discussion: Based on digital DNA-DNA hybridization and average nucleotide identity values, the two new isolates were assigned to the Psychrobacter genus. A comprehensive annotation using PlasticDB revealed substantial degree of similarity in the gene sequences coding for a variety of plastic-degrading enzymes, particularly carboxylic ester hydrolases. However, some differences were observed in the enzymatic profiles of the two bacterial strains, highlighting their versatility and potential for effective biodegradation of a range of plastic polymers. Subsequently, carboxylic ester hydrolases were detected with qualitative in-plate assays with olive oil and tributyrin as substrates. The enzymatic activity of psychrotolerant extracellular esterases and lipases was quantified using chromogenic-spectrophotometric assays. The presence of esterases was assayed against pNP-acetate, and for both strains an optimal activity was detected at 35 °C (0.5 and 0.8 nmol pNP released/mg protein in 30 min for strain ASPA161_6 and strain ASPA161_9, respectively). A significant activity was maintained at lower temperatures, ranging from 50.1 and 71.8 % at 15 and 25 °C for strain ASPA161_6 and from 49.6 and 68.1 % for strain ASPA161_9. These results suggested psychrotolerant characteristics of these enzymes. The presence of lipases was determined with pNP-dodecanoate, resulting in 2.6 and 3.5 nmol pNP released/mg protein at 25 °C in 30 min for the two strains. Most of the activity was retained at 25 °C (73.4 and 66.3 %, respectively), with a decrease at 15 °C (45.3 and 38.6 %), which may be attributed to the reduced kinetic activity. Finally, the degradation experiments with PEA with averaged molar mass 1000 g/mol as a model polyester plastic demonstrated a 55-65 % degradation rate after 12 days of incubation.

Conclusions: The obtained data underlined the ability of the investigated strains to express carboxylic ester hydrolases under the specified growth conditions. These findings highlight their biotechnological potential in bioremediation, particularly for sustainable plastic waste management in cold or temperate environments. Future investigations could focus on optimizing enzymatic activity through heterologous expression or utilizing whole-cell systems for direct polymer degradation.

Keywords: Bioplastic degradation; Cold-active enzymes; Enzymatic degradation; Extremophiles; Genome annotation; Polyester depolymerization; Polyethylene adipate; Psychrotolerant bacteria.