Journal
JOURNAL OF GENERAL AND APPLIED MICROBIOLOGY
Volume 60, Issue 1, Pages 13-22Publisher
MICROBIOL RES FOUNDATION
DOI: 10.2323/jgam.60.13
Keywords
Laceyella sacchari; PLLA-degrading enzyme; Thermoactinomycetaceae; thermophilic filamentous bacteria
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Funding
- Kasetsart University Research and Development Institute (KURDI)
- Center for Advanced Studies in Tropical Natural Resources (CASTNAR),
- international Thai-Japan Academic Cooperation Project
- National Research Council of Thailand (NRCT)
- Japan Society for the Promotion of Science (JSPS), Asian Core Program
- NRU-KU, Thailand
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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 degrees 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 degrees C with thermal stability up to 50 degrees 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.
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