高浓度氯苯优势降解菌的筛选及其降解酶的纯化

Screening of chlorobenzene-degrading bacterium and purification of its degradation enzyme

摘要:

摘要:【目的】分离纯化出一株高浓度氯苯优势降解菌株，对其所产氯苯降解酶进行分离与纯化，为该菌株及其氯苯降解酶的研究提供理论参考。【方法】利用梯度富集培养技术和无菌滤纸片平板法分离菌株，通过形态特征及16S rRNA基因序列分析初步鉴定菌株，用气相色谱法测定培养液中氯苯浓度，以单位细胞氯苯降解率评价菌株对氯苯的降解能力，以氯苯降解率表示氯苯降解酶的活性。取纯化菌株的发酵酶液制备粗酶液，经硫酸铵梯度盐析、透析脱盐、DE-52离子交换层析、G-100凝胶层析和透析浓缩后，进行SDS-PAGE凝胶电泳检验酶的纯度并测定酶的分子量。【结果】从氯苯长期驯化的成熟期活性污泥中筛选到一株以氯苯为唯一碳源和能源的氯苯优势降解细菌LW13，该菌株在以2000 mg/L 氯苯为唯一碳源的无机盐培养基中仍能正常生长，其单位细胞氯苯降解率可达1.37×10－10。扫描电镜观察到该菌株细胞大小约为2.3×0.8μm，长有数根端生鞭毛。16S rRNA基因序列相似性比较表明该菌株与Lysinibacillus fusiformis(溶藻菌)的相似性达95.5%。所纯化的氯苯降解酶为胞外酶，带正电荷，其分子大小约为57 kDa。整个纯化过程中酶纯化倍数化达8.0倍，酶活回收率达52.51%，酶量回收率达6.57%。纯化后的氯苯降解酶在30℃－55℃和pH在6.0－8.0之间都保持较高的酶活性，其最适反应温度和pH分别在40℃和pH8.0左右。【结论】所分离的氯苯优势降解菌属于Lysinibacillus属菌株，该菌株能有效降解高浓度(500－2000 mg/L)氯苯废水，通过逐级分离纯化，可获得氯苯降解酶纯酶，纯化指标符合分离纯化基本规律，纯化效果较为理想。

Abstract:

Abstract:［Objective］We screened a bacterial strain capable of degrading chlorobenzene，and purified the corresponding degradation enzyme．［Methods］ The strain was screened by gradient enrichment culture and sterile filter paper plate method，and identified by morphology and 16S rRNA gene sequence． Chlorobenzene concentration in the liquid culture was determined by gas chromatography． Degradation capability was assayed by the proportion of chlorobenzene degraded per cell． The purity quotient and molecular weight of the purified degradation enzyme were determined by gel electrophoresis.［Results］The isolated bacterium，LW13，used chlorobenzene in activated sludge as sole carbon and energy source．Cells were 2.3 μm long and 0.8 μm wide，with several terminal flagella． Strain LW13 was 95.5% similar to Lysinibacillus fusiformis，and its degradation enzyme was a positively-charged exoenzyme (molecular weight about 57 kDa)．The optimal temperature and pH of the purified enzyme were approximately 40 °C and 8.0，respectively．［Conclusion］Strain LW13 belongs to genus Lysinibacillus，and can degrade chlorobenzene (500－2000 mg/L)．