Page 89 - 《精细化工》2020年第3期

第 37 卷第 3 期 精 细 化 工 Vol.37, No.3
202 0 年 3 月 FINE CHEMICALS Mar. 2020

生物工程
聚乳酸降解菌的分离鉴定及其产酶和降解特性

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贾 昊 ，张 敏 ，翁云宣 ，李成涛
（1. 陕西科技大学 环境科学与工程学院，陕西 西安 710021；2. 北京工商大学 塑料卫生与安全质量评
价技术北京市重点实验室，北京 100048）
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）为惟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，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%。
关键词：生物降解；聚乳酸；门多萨假单胞菌；蛋白酶；响应曲面法；生物工程
中图分类号：TB324; Q814.9; TQ925 文献标识码：A 文章编号：1003-5214 (2020) 03-0507-08

Isolation and characterization of polylactic acid-degrading bacteria and
their enzyme production and degradation characteristics

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JIA Hao , ZHANG Min , WENG Yunxuan , LI Chengtao
（1. College of Environmental Science and Engineering, Shaanxi University of Science & Technology, Xi'an 710021,
Shaanxi, China; 2. Beijing Key Laboratory of Plastic Hygiene and Safety Quality Evaluation Technology, Beijing
Technology and Business University, Beijing 100048, China）
Abstract: Polylactic acid (PLA)-degrading bacteria were isolated and screened from the soil containing
PLA/PBAT film (Weifang, Shandong), using inorganic salt medium in which the PLA was used as carbon
source. 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 were confirmed as a research object by hydrolysis circle
method. Furthermore, the protease-producing conditions of the degrading bacteria were optimized by the
folinphenol reagent method combined with single factor and response surface methodology. 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. The
optimal culture conditions were as follows: in the 100 mL screening medium, 2% (mass fraction) glucose
and 1% (mass fraction) tryptone being added as external nutrient sources, pH of 7.4, inoculum size of 1.4
mL and culture temperature of 32 ℃. Under these conditions, the highest protease activity was up to 32.15
U/mL, which was 26 times higher than that before optimization. At the same time, the protease produced by
PLA-degrading bacteria could catalyze ester bonds in PLA degradation materials. The degradation rate of

收稿日期：2019-07-26; 定用日期：2019-10-12; DOI: 10.13550/j.jxhg.20190738
基金项目： 陕西省重点研发计划项目（2018SF-375）；塑料卫生与安全质量评价技术北京市重点实验室（北京工商大学 TQETJP2018004）
作者简介：贾 昊(1994—)，男，硕士生，E-mail：1703044@sust.edu.cn。联系人：张 敏(1958—)，女，教授，电话：029-86132765，
E-mail：yanjiushi206@163.com。