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

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.

Key words:Actinomycetes, Calcineurin, Inhibitor, Screening, Fermentation conditions