RESUMEN
A thermophilic poly(L-lactide)-degrading Gram-stain-positive filamentous bacterial strain that develops single spores on the aerial mycelium was isolated from forest soil at Srinagarind Dam, Kanchanaburi Province, Thailand. The results of a polyphasic taxonomic study showed that our isolate had characteristics typical of members of the genus Polycladomyces. The isolate grew aerobically at an optimum temperature of 50-55 °C and optimal pH 6-7. Meso-diaminopimelic acid was present as the diagnostic diamino acid in the peptidoglycan but no characteristic sugars are detected. The predominant menaquinone was MK-7. The diagnostic phospholipids were phosphatidylethanolamine, phosphatidylmethylethanolamine diphosphatidylglycerol, phosphatidylglycerol and phosphatidylserine. The predominant cellular fatty acid was iso-C15â:â0. The DNA G+C content of strain KSR 13T was 53.4 mol%. The 16S rRNA gene sequence analysis also indicated that strain KSR 13T belonged to the genus Polycladomyces, being most closely related to Polycladomyces abyssicola JIR-001T (99.2â%). The DNA-DNA relatedness values that distinguished KSR 13T from P. abyssicola JIR-001T were 17.8-32.1â%, which were significantly below the 70â% cutoff value recommended for species delineation. Following an evaluation of phenotypic, chemotaxonomic and genotypic studies, the new isolate is proposed as a novel species and named Polycladomyces subterraneus sp. nov. The type strain is KSR 13T (=BCC 50740T=NBRC 109332T).
Asunto(s)
Bacillales/clasificación , Bosques , Filogenia , Microbiología del Suelo , Bacillales/genética , Bacillales/aislamiento & purificación , Técnicas de Tipificación Bacteriana , Composición de Base , Pared Celular/química , ADN Bacteriano/genética , Ácido Diaminopimélico/química , Ácidos Grasos/química , Hibridación de Ácido Nucleico , Peptidoglicano/química , Fosfolípidos/química , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Tailandia , Vitamina K 2/análogos & derivados , Vitamina K 2/químicaRESUMEN
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.
