Sukkhum Sukhumaporn, Tokuyama Shinji, Tamura Tomohiko, Kitpreechavanich Vichien
Faculty of Science, Kasetsart University, Bangkok, Thailand.
J Gen Appl Microbiol. 2009 Dec;55(6):459-67. doi: 10.2323/jgam.55.459.
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.
基于在乳化聚乳酸琼脂平板上形成透明圈的能力以及培养液中的酶活性，分离并筛选出了13株聚（L-丙交酯）降解微生物。根据表型特征和16S rRNA基因序列，这些菌株被归类到不同的科，如嗜热放线菌科、小单孢菌科、链孢囊菌科、芽孢杆菌科和嗜热放线菌科。菌株T16-1被鉴定为马杜拉放线菌属，在以聚乳酸薄膜为碳源的液体培养中表现出最高的聚乳酸降解活性。该菌株产生的聚乳酸降解酶经纯化后达到十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示的均一性，比活性为38.3单位/毫克蛋白。最适pH和温度分别为10.0和70℃，高于先前报道的聚乳酸降解酶。该酶在pH 11 - 12时稳定。然而，在70℃保持1小时后，酶活性仍保持在70%。从菌株T16-1纯化的聚乳酸降解酶的分子量为30 kDa。纯化后的酶受到5 mM乙二胺四乙酸、5 mM苯甲基磺酰氟和二异丙基氟磷酸的抑制，能强烈水解琥珀酰-（丙氨酸）3-对硝基苯胺、明胶和聚乳酸，但对酪蛋白活性较低。结果表明，马杜拉放线菌属菌株T16-1产生的聚乳酸降解酶应归类为丝氨酸蛋白酶。
