Many poly(lactic acid) (PLA)-degrading microorganisms have been isolated from the natural environment by culture-based methods, but there is no study about unculturable PLA-degrading microorganisms. In this study, we constructed a metagenomic library consisting of the DNA extracted from PLA disks buried in compost. We identified three PLA-degrading genes encoding lipase or hydrolase. The purified enzymes degraded not only PLA, but also various aliphatic polyesters, tributyrin, and p-nitrophenyl esters. From their substrate specificities, the PLA depolymerases were classified into an esterase rather than a lipase. Among the PLA depolymerases, PlaM4 exhibited thermophilic properties; that is, it showed the highest activity at 70 degrees C and was stable even after incubation for 1 h at 50 degrees C. PlaM4 had absorption and degradation activities for solid PLA at 60 degrees C, which indicates that the enzyme can effectively degrade PLA in a high-temperature environment. On the other hand, the enzyme classification based on amino acid sequences showed that the other PLA depolymerases, PlaM7 and PlaM9, were not classified into known lipases or esterases. This is the first report on the identification and characterization of PLA depolymerase from a metagenome.
A bacterium which degrades urethane compounds was isolated and identified as Rhodococcus equi strain TB-60. Strain TB-60 degraded toluene-2,4-dicarbamic acid dibutyl ester (TDCB) and accumulated toluene diamine as the degradation product. The enzyme which cleaves urethane bond in TDCB was strongly induced by acetanilide. The purified enzyme (urethane hydrolase) was found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight was estimated to be 55 kDa. The optimal temperature and pH were 45 degrees C and 5.5, respectively. The enzyme hydrolyzed aliphatic urethane compound as well as aromatic ones. The activity was inhibited by HgCl(2), p-chrolomercuribenzoic acid, and phenylmethylsulfonyl fluoride, suggesting that cysteine and/or serine residues play an important role in the activity. The enzyme catalyzed the hydrolysis of anilides, amides, and esters as well as TDCB. It was characterized as a novel amidase/esterase, differing in some properties from other known amidases/esterases.
The gene encoding a poly(DL-lactic acid) (PLA) depolymerase from Paenibacillus amylolyticus strain TB-13 was cloned and overexpressed in Escherichia coli. The purified recombinant PLA depolymerase, PlaA, exhibited degradation activities toward various biodegradable polyesters, such as poly(butylene succinate), poly(butylene succinate-co-adipate), poly(ethylene succinate), and poly(epsilon-caprolactone), as well as PLA. The monomeric lactic acid was detected as the degradation product of PLA. The substrate specificity toward triglycerides and p-nitrophenyl esters indicated that PlaA is a type of lipase. The gene encoded 201 amino acid residues, including the conserved pentapeptide Ala-His-Ser-Met-Gly, present in the lipases of mesophilic Bacillus species. The identity of the amino acid sequence of PlaA with Bacillus lipases was no more than 45 to 50%, and some of its properties were different from those of these lipases.
