Identification and expression of MarCE, a marine carboxylesterase with synthetic ester-degrading activity.

Carboxylic ester hydrolases with the capacity to degrade polyesters are currently highly sought after for their potential use in the biological degradation of PET and other chemically synthesized polymers. Here, we describe MarCE, a carboxylesterase family protein identified via genome mining of a Maribacter sp. isolate from the marine sponge Stelligera stuposa. Based on phylogenetic analysis, MarCE and its closest relatives belong to marine-associated genera from the Cytophaga-Flavobacterium-Bacteroides taxonomic group and appear evolutionarily distinct to any homologous carboxylesterases that have been studied to date in terms of structure or function. Molecular docking revealed putative binding of BHET, a short-chain PET derivative, onto the predicted MarCE three-dimensional structure. The synthetic ester-degrading activity of MarCE was subsequently confirmed by MarCE-mediated hydrolysis of 2 mM BHET substrate, indicated by the release of its breakdown products MHET and TPA, which were measured, respectively, as 1.28 and 0.12 mM following 2-h incubation at 30°C. The findings of this study provide further insight into marine carboxylic ester hydrolases, which have the potential to display unique functional plasticity resulting from their adaptation to complex and fluctuating marine environmentsw.

Enrichment of native plastic-associated biofilm communities to enhance polyester degrading activity.

Plastic pollution is an increasing worldwide problem urgently requiring a solution. While recycling rates are increasing globally, only 9% of all plastic waste has been recycled, and with the cost and limited downstream uses of recycled plastic, an alternative is needed. Here, we found that expanded polystyrene (EPS) promoted high levels of bacterial biofilm formation and sought out environmental EPS waste to characterize these native communities. We demonstrated that the EPS attached communities had limited plastic degrading activity. We then performed a long-term enrichment experiment where we placed a robust selection pressure on these communities by limiting carbon availability such that the waste plastic was the only carbon source. Seven of the resulting enriched bacterial communities had increased plastic degrading activity compared to the starting bacterial communities. Pseudomonas stutzeri was predominantly identified in six of the seven enriched communities as the strongest polyester degrader. Sequencing of one isolate of P. stutzeri revealed two putative polyesterases and one putative MHETase. This indicates that waste plastic-associated biofilms are a source for bacteria that have plastic-degrading potential, and that this potential can be unlocked through selective pressure and further in vitro enrichment experiments, resulting in biodegradative communities that are better than nature.

Purification and biochemical characterization of SM14est, a PET-hydrolyzing enzyme from the marine sponge-derived Streptomyces sp. SM14.

The successful enzymatic degradation of polyester substrates has fueled worldwide investigation into the treatment of plastic waste using bio-based processes. Within this realm, marine-associated microorganisms have emerged as a promising source of polyester-degrading enzymes. In this work, we describe the hydrolysis of the synthetic polymer PET by SM14est, a polyesterase which was previously identified from Streptomyces sp. SM14, an isolate of the marine sponge Haliclona simulans. The PET hydrolase activity of purified SM14est was assessed using a suspension-based assay and subsequent analysis of reaction products by UV-spectrophotometry and RP-HPLC. SM14est displayed a preference for high salt conditions, with activity significantly increasing at sodium chloride concentrations from 100 mM up to 1,000 mM. The initial rate of PET hydrolysis by SM14est was determined to be 0.004 s-1 at 45°C, which was increased by 5-fold to 0.02 s-1 upon addition of 500 mM sodium chloride. Sequence alignment and structural comparison with known PET hydrolases, including the marine halophile PET6, and the highly efficient, thermophilic PHL7, revealed conserved features of interest. Based on this work, SM14est emerges as a useful enzyme that is more similar to key players in the area of PET hydrolysis, like PHL7 and IsPETase, than it is to its marine counterparts. Salt-tolerant polyesterases such as SM14est are potentially valuable in the biological degradation of plastic particles that readily contaminate marine ecosystems and industrial wastewaters.

Identification of BgP, a Cutinase-Like Polyesterase From a Deep-Sea Sponge-Derived Actinobacterium.

Many marine bacteria produce extracellular enzymes that degrade complex molecules to facilitate their growth in environmental conditions that are often harsh and low in nutrients. Marine bacteria, including those inhabiting sea sponges, have previously been reported to be a promising source of polyesterase enzymes, which have received recent attention due to their potential ability to degrade polyethylene terephthalate (PET) plastic. During the screening of 51 marine bacterial isolates for hydrolytic activities targeting ester and polyester substrates, a Brachybacterium ginsengisoli B129SM11 isolate from the deep-sea sponge Pheronema sp. was identified as a polyesterase producer. Sequence analysis of genomic DNA from strain B129SM11, coupled with a genome "mining" strategy, allowed the identification of potential polyesterases, using a custom database of enzymes that had previously been reported to hydrolyze PET or other synthetic polyesters. This resulted in the identification of a putative PET hydrolase gene, encoding a polyesterase-type enzyme which we named BgP that shared high overall similarity with three well-characterized PET hydrolases-LCC, TfCut2, and Cut190, all of which are key enzymes currently under investigation for the biological recycling of PET. In silico protein analyses and homology protein modeling offered structural and functional insights into BgP, and a detailed comparison with Cut190 revealed highly conserved features with implications for both catalysis and substrate binding. Polyesterase activity was confirmed using an agar-based polycaprolactone (PCL) clearing assay, following heterologous expression of BgP in Escherichia coli. This is the first report of a polyesterase being identified from a deep-sea sponge bacterium such as Brachybacterium ginsengisoli and provides further insights into marine-derived polyesterases, an important family of enzymes for PET plastic hydrolysis. Microorganisms living in association with sponges are likely to have increased exposure to plastics and microplastics given the wide-scale contamination of marine ecosystems with these plastics, and thus they may represent a worthwhile source of enzymes for use in new plastic waste management systems. This study adds to the growing knowledge of microbial polyesterases and endorses further exploration of marine host-associated microorganisms as a potentially valuable source of this family of enzymes for PET plastic hydrolysis.

Microbial Polyethylene Terephthalate Hydrolases: Current and Future Perspectives.

Plastic has rapidly transformed our world, with many aspects of human life now relying on a variety of plastic materials. Biological plastic degradation, which employs microorganisms and their degradative enzymes, has emerged as one way to address the unforeseen consequences of the waste streams that have resulted from mass plastic production. The focus of this review is microbial hydrolase enzymes which have been found to act on polyethylene terephthalate (PET) plastic. The best characterized examples are discussed together with the use of genomic and protein engineering technologies to obtain PET hydrolase enzymes for different applications. In addition, the obstacles which are currently limiting the development of efficient PET bioprocessing are presented. By continuing to study the possible mechanisms and the structural elements of key enzymes involved in microbial PET hydrolysis, and by assessing the ability of PET hydrolase enzymes to work under practical conditions, this research will help inform large-scale waste management operations. Finally, the contribution of microbial PET hydrolases in creating a potential circular PET economy will be explored. This review combines the current knowledge on enzymatic PET processing with proposed strategies for optimization and use, to help clarify the next steps in addressing pollution by PET and other plastics.

Harnessing the sponge microbiome for industrial biocatalysts.

Within the marine sphere, host-associated microbiomes are receiving growing attention as prolific sources of novel biocatalysts. Given the known biocatalytic potential of poriferan microbial inhabitants, this review focuses on enzymes from the sponge microbiome, with special attention on their relevant properties and the wide range of their potential biotechnological applications within various industries. Cultivable bacterial and filamentous fungal isolates account for the majority of the enzymatic sources. Hydrolases, mainly glycoside hydrolases and carboxylesterases, are the predominant reported group of enzymes, with varying degrees of tolerance to alkaline pH and growing salt concentrations being common. Prospective areas for the application of these microbial enzymes include biorefinery, detergent, food and effluent treatment industries. Finally, alternative strategies to identify novel biocatalysts from the sponge microbiome are addressed, with an emphasis on modern -omics-based approaches that are currently available in the enzyme research arena. By providing this current overview of the field, we hope to not only increase the appetite of researchers to instigate forthcoming studies but also to stress how basic and applied research can pave the way for new biocatalysts from these symbiotic microbial communities in a productive fashion. KEY POINTS: • The sponge microbiome is a burgeoning source of industrial biocatalysts. • Sponge microbial enzymes have useful habitat-related traits for several industries. • Strategies are provided for the future discovery of microbial enzymes from sponges.