A novel poly (L-lactide) degrading actinomycetes isolated from Thai forest soil, phylogenic relationship and the enzyme characterization.
Thirteen poly (L-lactide)-degrading microorganisms were isolated and selected based on their ability of clear zone formation on an emulsified PLA agar plate and the enzyme activity in culture broth. According to phenotypic properties and 16S rRNA gene sequence, these strains were classified to various families such as Thermomonosporaceae, Micromonosporaceae, Streptosporangiaceae, Bacillaceae and Thermoactinomycetaceae. Strain T16-1, identified as Actinomadura sp., demonstrated the highest PLA-degrading activity in the liquid culture using PLA film as a carbon source. A PLA-degrading enzyme produced by the strain was purified to homogeneity shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with specific activity of 38.3 unit/mg protein. The optimum pH and temperature were 10.0 and 70 masculineC, respectively, which are higher than previously reported among PLA-degrading enzyme. The enzyme was stable at pH 11-12. However, the enzyme activity remained at 70% when kept at 70 masculineC for 1 h. The molecular weight of purified PLA-degrading enzyme from the strain T16-1 was 30 kDa. The purified enzyme was inhibited by 5 mM EDTA and 5 mM phenylmethylsulfonyl fluoride and diisopropyl fluorophosphates, strongly hydrolyzed Suc-(Ala)(3)-pNA, gelatin and PLA, but showed low activity on casein. The results indicated the PLA-degrading enzyme produced by the strain Actinomadura sp. T16-1 should be classified as serine protease.
Sukkhum
Sukhumaporn
S
Faculty of Science, Kasetsart University, Bangkok, Thailand.
Tokuyama
Shinji
S
Tamura
Tomohiko
T
Kitpreechavanich
Vichien
V
D000192:Actinomycetales / Q000145:classification / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003470:Culture Media; D008855:Microscopy, Electron, Scanning; D010641:Phenotype; D010802:Phylogeny; D011091:Polyesters / Q000378:metabolism*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA; D057057:Serine Proteases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012988:Soil Microbiology; D013171:Spores, Bacterial / Q000648:ultrastructure; D013785:Thailand; D014197:Trees
D001426:Bacterial Proteins; D003470:Culture Media; D011091:Polyesters; D012336:RNA, Ribosomal, 16S; C033616:poly(lactide); D057057:Serine Proteases