title: Characterization of poly(L-lactide)-degrading enzyme produced by thermophilic filamentous bacteria Laceyella sacchari LP175.
abstract: Eleven strains of poly(L-lactide) (PLLA)-degrading thermophilic bacteria were isolated from forest soils and selected based on clear zone formation on an emulsified PLLA agar plate at 50°C. Among the isolates, strain LP175 showed the highest PLLA-degrading ability. It was closely related to Laceyella sacchari, with 99.9% similarity based on the 16S rRNA gene sequence. The PLLA-degrading enzyme produced by the strain was purified to homogeneity by 48.1% yield and specific activity of 328 U·mg-protein-1 with a 15.3-fold purity increase. The purified enzyme was strongly active against specific substrates such as casein and gelatin and weakly active against Suc-(Ala)₃-pNA. Optimum enzyme activity was exhibited at a temperature of 60°C with thermal stability up to 50°C and a pH of 9.0 with pH stability in a range of 8.5-10.5. Molecular weight of the enzyme was approximately 28.0 kDa, as determined by gel filtration and SDS-PAGE. The inhibitors phenylmethylsulfonyl fluoride (PMSF), ethylenediaminetetraacetate (EDTA), and ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) strongly inhibited enzyme activity, but the activity was not inhibited by 1 mM 1,10-phenanthroline (1,10-phen). The N-terminal amino acid sequences had 100% homology with thermostable serine protease (thermitase) from Thermoactinomyces vulgaris. The results obtained suggest that the PLLA-degrading enzyme produced by L. sacchari strain LP175 is serine protease.
authors.affiliation: Faculty of Science, Kasetsart University.
authors.forename: Srisuda
authors.initials: S
authors.lastname: Hanphakphoom
authors.forename: Narisara
authors.initials: N
authors.lastname: Maneewong
authors.forename: Sukhumaporn
authors.initials: S
authors.lastname: Sukkhum
authors.forename: Shinji
authors.initials: S
authors.lastname: Tokuyama
authors.forename: Vichien
authors.initials: V
authors.lastname: Kitpreechavanich
mesh_terms: D000595:Amino Acid Sequence; D056449:Bacillales / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D001673:Biodegradation, Environmental; D004795:Enzyme Stability; D004798:Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D020459:Genes, rRNA; D006358:Hot Temperature; D008855:Microscopy, Electron, Scanning; D008969:Molecular Sequence Data; D010641:Phenotype; D010802:Phylogeny; D011091:Polyesters / Q000378:metabolism*; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012988:Soil Microbiology; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D004798:Enzymes; D011091:Polyesters; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; C033616:poly(lactide)