on an emulsified PLLA agar plate at 50 C. Among the isolates, strain LP175 showed the highest PLLAdegrading 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 glycolbis(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.Key words: Laceyella sacchari; PLLA-degrading enzyme; Thermoactinomycetaceae; thermophilic filamentous bacteria IntroductionPoly(L-lactide) (PLLA); (C 3 H 4 O 2 )n is one of the aliphatic biodegradable polyesters derived from renewable resources such as corn, cassava, sugar cane, rice and potato through lactic acid fermentation, and it is also fully degradable by both microbial and enzymatic processes (Tokiwa et al., 2009;Gupta et al., 2007;Jarerat et al., 2006;Tokiwa and Calabia, 2006;Tomita et al., 2004). PLLA, as an environmentally friendly (eco-friendly or green ) product, has been developed on a large scale and is currently used for a wide range of applications, including packaging materials (Bhalla et al., 2007;Nolan-Itu Pty Ltd., 2002), medical applications (Jalil, 1990), agricultural products (Gross and Kalra, 2002;Sakai et al., 2001) and textiles.Most PLLA-degrading microorganisms are found in the bacterial order Actinomycetales, e.g. the families Pseudonocardiaceae (Pranamuda and Tokiwa, 1999;Pranamuda et al., 1997), Thermomonosporaceae (Sangwan and Wu, 2008;Sukkhum et al., 2009b), and Streptosporangiaceae (Sukkhum et al., 2009b). Apart from that, thermophilic bacteria in the families Thermoactinomycetaceae (Sukkhum et al., 2009b) and Firmicutes (Oda et al., 2000;Sakai et al., 2001), filamentous fungi in the genera Tritirachium and Paecilomyces (Sangwan and Wu, 2008), and yeast in the genus Cryptococcus have also been reported to be PLLA-degrading microorganisms.Many different types of enzymes are found in PLLAdegrading microorganisms, such as: protease from Amycola- Full PaperCharacterization of poly(L-lactide)-degrading enzyme produced by thermophilic filamentous bacteria Laceyella sacchari LP175 (Received F...

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-C15 : 0. 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 (99.2 %). The DNA-DNA relatedness values that distinguished KSR 13T from P. abyssicola JIR-001T were 17.8-32.1 %, which were significantly below the 70 % 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).