A novel high-throughput and quantitative method based on visible color shifts for screening Bacillus subtilis THY-15 for surfactin production
A novel high-throughput and quantitative method based on visible color shifts for screening Bacillus subtilis THY-15 for surfactin production
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DOI：
10.1007/s10295-015-1635-4
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发表时间：
2015-08-01
影响因子：
3.4
通讯作者：
Shen, Zhongyao
中科院分区：
文献类型：
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作者：
Yang, Huan;Yu, Huimin;Shen, Zhongyao
以溴百里酚蓝（BTB）为显色剂，氯化十六烷基吡啶（CPC）为介体，建立了一种新的显色筛选方法，用于筛选高效价的表面活性素产生菌。色移的可靠性和定量准确性也得到了证实。从淡黄色绿色到深绿色和亮蓝色的规则色响应反映了表面活性素浓度的不同范围。此外，使用不同的发酵上清液样品通过反相高效液相色谱法（RP-HPLC）验证了表面活性素定量在100-500 mg/L范围内的定量准确度。利用该方法成功地筛选出一株表面活性素上级菌株枯草芽孢杆菌THY-15。该菌株的表面活性素（Srf）效价达到1240 mg/L。THY-15的RP-HPLC分析揭示了四种表面活性素异构体。氨基酸分析和基质辅助激光解吸电离飞行时间质谱分析表明，组分1、2和4中的表面活性素异构体具有相同的环肽序列Glu-Leu-Leu-瓦尔-Asp-Leu-Leu，但不同的iso-C-13、C-14和C-15脂肪酸链，而组分3中的表面活性素异构体具有特殊的环肽序列Glu-瓦尔-Leu-Leu-Asp-Leu-瓦尔。
A novel chromatic visible screening method using bromothymol blue (BTB) as a color indicator and cetylpyridinium chloride (CPC) as a mediator was constructed to obtain the high titer surfactin-producing strains. The reliability and quantification accuracy of color shift were also confirmed. Regular chromatic responses from faint yellow-green to dark green and bright blue reflected the different ranges of surfactin concentrations. Moreover, the quantitative accuracy of surfactin quantification in the range of 100-500 mg/L was verified by reverse-phase high-performance liquid chromatography (RP-HPLC) using different fermentation supernatant samples. Using this CPC-BTB method, a superior surfactin producer, Bacillus subtilis THY-15, was successfully screened. The producer's surfactin (Srf) titer reached 1240 mg/L. RP-HPLC analysis of THY-15 revealed four surfactin isoforms. As identified by amino acid analysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis, the isoforms of surfactin in fraction 1, 2 and 4 had the same circular peptide sequence of Glu-Leu-Leu-Val-Asp-Leu-Leu but different iso-C-13, C-14 and C-15 fatty acid chains, but the isoform in fraction 3 possessed a special peptide sequence of Glu-Val-Leu-Leu-Asp-Leu-Val.