PLA降解菌的分离鉴定及其产酶和降解特性

Isolation and Characterization of Polylactic acid-degrading Bacteria

摘要:

以山东潍坊使用聚乳酸/聚己二酸-对苯二甲酸丁二酯共混物(PLA/PBAT)地膜的土壤为样本，采用以聚乳酸(PLA)为唯一碳源的无机盐培养基筛选PLA降解菌。使用形态学观察、生理生化实验、16S rRNA基因序列分析及构建系统发育树等方法鉴定降解菌。通过水解圈法确认产蛋白酶活性高的降解菌为研究对象，并采用福林酚法结合单因素及响应曲面法对外源营养源与产酶条件进行了优化。采用SEM、FTIR和XRD等方法分析了降解前后PLA的形貌和结构变化，研究了降解菌对PLA的降解情况。结果表明：从土壤中筛选获得了一株产蛋白酶的PLA降解菌(门多萨假单胞菌Pseudomonas mendocina)；优化得到的最佳产酶条件为：在100 mL筛选培养基中，加入质量分数为2%的葡萄糖和质量分数为1%的胰蛋白胨作外源营养源，pH为7.4，接种量为1.4 mL及培养温度32 ℃。在此条件下，蛋白酶活性最高为32.15 U/mL，较未优化前提高约26倍；PLA降解菌产蛋白酶可催化降解PLA材料中的酯键，5 d内PLA降解率可达29.35%。

Abstract:

In this study, polylactic acid(PLA)-degrading bacteria were isolated and screened from the soil with PLA/PBAT film（Weifang, Shandong），using inorganic salt medium in which carbon source was the PLA. The PLA-degrading bacteria were identified by morphological observation, physiological and biochemical experiments, 16S rRNA gene sequence analysis and phylogenetic tree construction. The degrading bacteria with high protease activity, a research object, was confirmed by hydrolysis circle method. And the protease-producing conditions of the degrading bacteria was optimized by using the folinphenol reagent method and response surface methodology. Under the optimal conditions, the morphology and structure of PLA before and after degradation were analyzed by SEM, FTIR and XRD. The results showed that the PLA-degrading bacteria (Pseudomonas mendocina) was isolated and screened. After optimization, the optimal culture conditions were pH 7.4, inoculum size 1.4 mL and culture temperature 32 ℃. The highest protease activity was up to 32.15 U/mL. At the same time, the protease of PLA-degrading bacteria could catalyzed the ester bond in the degradation material. The degradation rate of PLA could reach 29.35% within 5 days.