一株产钙调磷酸酶抑制剂链霉菌CZW-15的

Screening, identification and fermentation conditions optimization of Streptomyces CZW-15 with inhibitory activities on calcineurin

摘要:

【目的】筛选产钙调磷酸酶抑制剂的放线菌菌株。【方法】通过酵母报告基因(CDRE::LacZ)高通量筛选模型与人体钙调磷酸酶试剂盒检测相结合的方法，从土壤放线菌中筛选产钙调磷酸酶抑制剂的菌株；根据形态和培养特征、生理生化实验以及16S rRNA基因序列比对进行菌株鉴定；采用单因素实验和正交实验对发酵条件进行优化。【结果】从土壤中筛选得到一株产高活性钙调磷酸酶抑制剂的放线菌CZW-15，经鉴定属于链霉菌属(Streptomyces)。酵母报告基因检测显示其发酵上清液对依赖酵母钙调磷酸酶的报告基因表达抑制活性IC50为7.4 mL/L，而对照药物FK506的IC50为65 μg/L。该菌的最佳摇瓶发酵配方为(%)：玉米粉2、酵母粉0.1、K2HPO4 0.04、MgSO4 0.04、NaCl 0.05、FeSO4 0.001；发酵条件为：初始pH 7.5，温度28 °C，转速180 r/min，装液量50 mL/250 mL，接种量4%，培养时间4 d。优化后1.5 μL发酵上清液对依赖酵母钙调磷酸酶的报告基因表达抑制活性达到117%，比优化前提高4.5倍。【结论】CZW-15属于链霉菌属，该菌产生高活性的抑制酵母钙调磷酸酶的活性物质。

Abstract:

[Objective] To screen actinomycetes strains with calcineurin inhibitory activity. [Methods] Actinomycetes strains with calcineurin inhibitory activity were screened by a yeast report gene (CDRE::LacZ) based high throughput screening model and a calcineurin phosphatase assay kit. The isolated actinomycetes strains were indentified by morphological, physiological and biochemical characteristics and 16S rRNA gene sequence analysis. And the fermentation conditions were optimized by single factor and orthogonal experiment. [Results] strain CZW-15 with high inhibitory activity on calcineurin was isolated from soil, and it belongs to the Streptomyces spp.. Yeast report gene assay showed that the fermentation supernatant of CZW-15 inhibited yeast calcineurin-dependent gene expression with IC50=7.4 mL/L, and the IC50 of FK506 was 65 μg/L. The optimum fermentation conditions of CZW-15 were as follows: 2% corn flour, 0.1% yeast extracts, 0.04% K2HPO4, 0.04% MgSO4, 0.05% NaCl, 0.001% FeSO4, at initial pH of 7.5, 28 °C, 180 r/min, liquid volume 50 mL in 250 mL flask, inoculation size of 4% for 4 d. At the optimum conditions, the calcineurin inhibitory activity of 1.5 μL fermentation supernatant of CZW-15 reached 117%, increased 4.5 times. [Conclusion] CZW-15 belongs to Streptomyces spp., and produces highly active substances with calcineurin inhibitory activity.