[P004] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P02-224] Identification of poly(ε-caprolactone)-degrading enzymes from a marine bacterium

○Haruno Kusumoto¹, Shin-ichi Hachisuka², Kyogo Iseki¹, Hiroshi Kikukawa², Ken’ichiro Matsumoto² (1. Graduate School of Chemical Sciences and Engineering, Hokkaido University (Japan), 2. Division of Applied Chemistry, Faculty of Engineering, Hokkaido University (Japan))

Keywords:

degrading enzyme,depolymerase,marine bacterium,poly(ε-caprolactone),cutinase

Poly(ε-caprolactone) (PCL) is attracting attention as a solution to the problem of marine plastic pollution. It is a synthetic aliphatic polyester widely recognized for its biodegradability and is one of the few plastics reported to undergo degradation even in marine environments, where microbial activity is generally limited. This biodegradation is catalyzed by degrading enzymes secreted by degrading bacteria. Therefore, elucidating the functions of PCL-degrading enzymes present in the marine environment may provide valuable insights for the rational design and development of new biodegradable materials, requiring further research on enzyme function. Although several PCL-degrading enzymes have been identified from terrestrial microorganisms, marine-derived PCL depolymerases remain scarce.
In this study, we aimed to identify and characterize PCL-degrading enzymes from the marine bacterium Alloalcanivorax gelatiniphagus JCM 18425, which we found to degrade PCL. A genome-based screening approach was employed to explore candidate hydrolases potentially involved in polyester degradation. Five putative lipase/cutinase genes were selected based on genome annotation and sequence similarity to known hydrolases. These genes were heterologously expressed in Escherichia coli, and crude enzyme extracts were evaluated using a degradation assay with a PCL emulsion. High-performance liquid chromatography (HPLC) analysis detected the release of the PCL monomer, 6-hydroxyhexanoic acid, from one candidate, designated Ag0826, confirming its depolymerase activity toward PCL.
Recombinant Ag0826 was subsequently purified and subjected to biochemical characterization. The enzyme exhibited optimal activity at 35–40°C and pH 8.0. To examine its substrate specificity, its activity was compared with that of leaf-branch compost cutinase (LCC), a well-characterized poly(ethylene terephthalate) (PET) hydrolase. The overall substrate ranges of the two enzymes largely overlapped across the tested substrates. However, LCC efficiently hydrolyzed PET to release ethylene glycol and terephthalic acid, whereas Ag0826 exhibited only marginal PET-degrading activity, suggesting a distinct substrate preference.
Phylogenetic analysis based on amino acid sequences revealed that Ag0826 forms a clade distinct from well-characterized PET hydrolases, including LCC and IsPETase (from Ideonella sakaiensis). At a broader phylogenetic scale, it was positioned near HaloPETase1, a more recently identified PET hydrolase derived from the marine bacterium Halopseudomonas pachastrellae. However, amino acid sequence identity was only about 50%, and several key residues corresponding to substrate-binding subsites differed from those of HaloPETase1.
These findings indicate that Ag0826 is a previously uncharacterized marine polyester-degrading enzyme with unique sequence features. This study expands our understanding of the functional diversity of marine polyester-degrading enzymes.